Relaxin 2008 - University Of Illinoisconferences.illinois.edu/relaxin/relaxin_abstract_book_08final.pdf · and available for discussion during the assigned session. Posters should

Relaxin 2008

Fifth International Conference on Relaxin and Related Peptides

May 18–23, 2008 | Sheraton, Maui | Hawai’i, USA

Organizing Committee Gillian D. Bryant-Greenwood (chair)Carol A. BagnellDaniele BaniRoss BathgateO. David SherwoodJosh SilvertownElaine Wolff (Conference Secretary)

www.conferences.uiuc.edu/relaxin

iii

iii Relaxin 2008

Foreword

As Chair of the organizing committee, I welcome you to Relaxin 2008 and hope that you will learn a lot and also update your knowledge about relaxin and its related peptides, meet new colleagues and old friends, forge new collaborations and at the same time, enjoy the beauty of the Island of Maui.

I am truly grateful for all the assistance, suggestions, and ideas from the organizing committee. Their help with the program was particularly valuable. I have been very fortunate to have Elaine Wolff as the conference secretary and to tap her memory of Relaxin 2004. The sponsorship from U.S. government sources, private foundations, commercial organizations, and private individuals has made it possible to offer some financial support for 20 new investigators to attend and present their data at this conference. The recipients of these awards are listed in this program. This generosity has also allowed us to keep the costs as low as possible. I recognize the difficulty of raising funds for travel and accommodation and particularly thank all the invited speakers for their efforts to come and present their most recent and exciting data. They have made this all worthwhile.

On behalf of the organizing committee, I have great pleasure in welcoming you to Relaxin 2008 and wish you a very productive and pleasant time in our beautiful Hawaiian Islands.

Gillian D. Bryant-Greenwood

Chair

We are pleased to announce the following as Sponsors of Relaxin 2008:

Major SponsorsCorthera, Inc. (formerly BAS Medical, Inc.)

Dr. Peter Bryant-Greenwood gave in memory of his father, Dr. Frederick C. Greenwood

Dr. Bernard and Jane Steinetz gave in memory of their son, Carl Steinetz

University of Guelph, Canada

Department of Obstetrics and Gynecology, University of Hawaii—Sponsor of the Opening Dinner

United States Department of Agriculture (USDA)—New Investigator Registration Grants

SponsorsMarch of Dimes—New Investigator Housing Grants

Phoenix Pharmaceuticals, Inc.

Supporters Pacific Biosciences Research Center, University of Hawaii—Sponsor of the Opening Reception

Johnson & Johnson Pharmaceutical Research and Development, LLC

R&D Systems, Inc.—New Investigator Housing Grant

iv Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 v

New Investigator Award Recipients

Alsadek BogzilUniversity of Manchester, United Kingdom

Gabrielle CallanderHoward Florey Institute, Australia

Joseph ChenRutgers University, USA

Dan DebrahUniversity of Pittsburgh, USA

Amy-Lynn FrankshunRutgers University, USA

Michelle HallsMonash University, Australia

Linda Haugaard-JonssonUniversity of Kalmar, Sweden

Andras KernUniversity of Hawaii, USA

Zhen LiBaylor College of Medicine, USA

Sherie MaHoward Florey Institute, Australia

Priya MaseelallNew Jersey Medical School, USA

Barbara McGowanImperial College London, United Kingdom

Jonathan McGuaneUniversity of Florida, USA

Fazel ShabanpoorHoward Florey Institute, Australia

Sudhir SinghUniversity of Nebraska Medical Center, USA

Craig SmithHoward Florey Institute, Australia

Yu May SohThe University of Melbourne, Australia

Emma van der WesthuizenMonash University, Australia

Lenka VodstrcilThe University of Melbourne, Australia

Su Ee WongHoward Florey Institute, Australia

v Relaxin 2008

vi Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 vii

Social Program

Sunday, May 18, 2008

1:00–6:00 PM Registration: Kahului Foyer

5:00–6:00 PM Welcome Reception: Courtyard

Pacific Biosciences Research Center, University of Hawaii—Sponsor

Includes Accompanying Persons: yellow, green, pink name badges

6:00–7:30 PM Opening Dinner: Courtyard

Department of Obstetrics and Gynecology, University of Hawaii—Sponsor


7:30–8:00 PM Geology of the Hawaiian Islands: Wailuku Room

Dr. Robert Wright, School of Earth, Ocean Science and Technology, University of Hawaii

Monday, May 19, 2008

8:00–9:00 AM Continental Breakfast: Courtyard

Includes Accompanying Persons: green, pink name badges

12:30–1:30 PM Lunch provided: Anuenue Lawn

Includes Accompanying Persons: green name badges

Tuesday, May 20, 2008

7:30–9:00 AM Continental Breakfast: Kahului Room


Afternoon Free—lunch and dinner on your own

Wednesday, May 21, 2008





5:30 PM Buses leave from hotel lobby for “Ulalena” (dinner not included)


Thursday, May 22, 2008




vii Relaxin 2008

Friday, May 23, 2008



12:45 PM Closing Lunch provided: Anuenue Lawn


Key to Accompanying Persons name badge colors:

Accompanying Person Option #1 (yellow name badge): includes Sunday Evening welcome reception and dinner and Wednesday evening excursion to Ulalena.

Accompanying Person Option #2 (green name badge): includes Sunday Evening welcome reception and dinner; Monday, Wednesday, and Friday luncheons; Monday through Friday continental breakfasts; Wednesday evening excursion to Ulalena.

Accompanying Person Option #3 (pink name badge): includes Sunday Evening welcome reception and dinner; Monday through Friday continental breakfasts; Wednesday evening excursion to Ulalena (no luncheons included).

Information for Conference Presenters

Poster Presentations (designated as “P” in the abstracts)The posters will be divided into three sessions. Due to space limitations, posters will not be displayed for the entire conference. Poster Session I will be from 7:30–9:00 AM on Tuesday; Poster Session II will be from 7:30–9:00 AM on Thursday; Poster Session III will be from 7:30–9:00 AM on Friday. The presenting author should be by his or her poster and available for discussion during the assigned session. Posters should be affixed to the poster boards between 7:00 and 7:30 AM on the day of the session. Numbers will be posted on the display boards. Posters should remain in place throughout the rest of the day and removed at the end of the program that day.

A space of 8 feet wide by 4 feet high (244 cm wide x 122 cm high) is available for each poster.

There will be no audiovisual capability in the poster presentation area.

All Oral Presentations (designated as “O” in the abstracts)In order to ensure the seamless progression of talks within each session, our technical staff will need to have all presentations and laptops ready before the session begins. Presentations must be submitted to our technical staff no later than 30 minutes before the beginning of each morning, afternoon, or evening session. Persons wishing to project from their own laptop computer must bring it to the meeting room at least 15 minutes before the beginning of the session in which they will be speaking.

Presenters may bring their talks to our staff on CD-ROM disks or USB flash drives. In addition, we strongly recommend that you also upload a copy of your PowerPoint presentation before coming to the conference. Uploaded files will be tested on the equipment we will use for the sessions. We will let you know if there are any problems. The deadline for presentation uploads is 5:00 PM (Hawaiian Standard Time) Friday, May 16, 2008. After that time, presentations will only be accepted at the Conference.

viii Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 ix

Relaxin 2008 Program The Fifth International Conference on Relaxin and Related Peptides

Sunday, May 18, 2008

1:00–6:00 PM Registration: Kahului Foyer

5:00–6:00 PM Welcome Reception: Courtyard

Pacific Biosciences Research Center, University of Hawaii—Sponsor

6:00–7:30 PM Opening Dinner: Courtyard

Department of Obstetrics and Gynecology, University of Hawaii—Sponsor

7:30–8:00 PM Geology of the Hawaiian Islands: Wailuku Room

Dr. Robert Wright, School of Earth, Ocean Science and Technology, University of Hawaii

Posters will be displayed in the Kihei/Wailea rooms. Platform Presentations will be in the Wailuku Room.

* New Investigators



9:00–10:30 AM Platform Session I: Relaxin and Related Peptides Structure and Function #1 Presentations O1–4

Chairs: C. Schwabe and G. Tregear

9:00 AM 30 min Structure and Activity in the Relaxin Family of Peptides O-1

Geoffrey Tregear, Howard Florey Institute, Australia

9:30 AM 30 min The Chemical Synthesis of Relaxin and Related Peptides: From Yesterday to Tomorrow O-2

John Wade, Howard Florey Institute, Australia

10:00 AM 15 min Development of High Affinity INSL3 Antagonists O-3

*Fazel Shabanpoor, Howard Florey Institute, Australia

10:15 AM 15 min The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of the Different RXFP Receptors

O-4

Mohammed Hossain, Howard Florey Institute, Australia

10:30–11:00 AM Morning Break

11:00–12:30 PM Platform Session 2: Relaxin and Related Peptides Structure and Function #2 Presentations O5–8

Chairs: D. Craik and J. Wade

11:00 AM 30 min Structural Insights into the Function of Relaxins O-5

Johan Rosengren, University of Kalmar, Sweden

11:30 AM 30 min Probing the Functional Domains of Relaxin-3 and the Creation of a Selective Antagonist for GPCR135 over Relaxin Receptor LGR7

O-6

Changlu Liu, Johnson & Johnson Pharmaceuticals, Inc., USA

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12:00 PM (15) 15 min Structural Characterization of the R3/I5 Chimeric Relaxin Peptide O-7

*Linda Haugaard-Jonsson, University of Kalmar, Sweden

12:15 PM (15) 15 min Degradation of Relaxin Family Peptides by Insulin-degrading Enzyme O-8

Robert Bennett, Veterans Affairs Medical Center, Omaha, Nebraska, USA


1:30–3:00 PM Platform Session 3: Reproductive Functions of Relaxin and Related Peptides #1 Presentations O9–12

Chairs: C. Bagnell and G. Bryant-Greenwood

1:30 PM 25 min Relaxin in Human Pregnancy: Consequences of Too Little or Too Much of A Good Thing! O-9

Laura Goldsmith, Department of Obstetrics and Gynecology, New Jersey Medical School of UMDNJ, USA

1:55 PM 25 min Relaxin Acts on Stromal Cells to Promote Epithelial and Stromal Proliferation and Inhibit Apoptosis in the Mouse Cervix and Vagina

O-10

David Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, USA

2:20 PM 25 min The Good, The Bad and The Ugly: Lessons in Female Reproductive Tract Physiology from the Relaxin Gene Knockout (Rln-/-) Mouse

O-11

Laura Parry, Department of Zoology, The University of Melbourne, Australia

2:45 PM 15 min Decreased Hyaluronic Synthase Expression in the Cervix of Pregnant Relaxin-Deficient (Rln-/-) Mice

O-12

*Yu May Soh, Department of Zoology,The University of Melbourne, Australia

3:00–3:30 PM Afternoon Break

3:30–5:15 PM Platform Session 4: Reproductive Functions of Relaxin and Related Peptides #2 Presentations O13–17

Chairs: L. Goldsmith and L. Parry

3:30 PM 25 min Milk-Borne Relaxin and the Lactocrine Hypothesis for Maternal Programming of Neonatal Tissues O-13

Carol Bagnell, Department of Animal Sciences, Rutgers University, USA

3:55 PM 25 min Relaxin and Maternal Lactocrine Programming of Neonatal Uterine Development O-14

Frank Bartol, Department of Animal Sciences, Auburn University, USA

4:20 PM 25 min Relaxin in the Marmoset Monkey: There Are Two Sides to Every Story O-15

Almuth Einspanier, Institute of Physiological Chemistry, Leipzig, Germany

4:45 PM 15 min Expression of LGR7 in the Primate Corpus Luteum Implicates the Corpus Luteum as a Relaxin Target Organ

O-16

*Priya Maseelall, Department of Obstetrics and Gynecology, New Jersey Medical School, USA

5:00 PM 15 min Characterization and Biological Activity of Prorelaxin in Porcine Milk O-17

*Amy-Lynn Frankshun, Department of Animal Sciences, Rutgers University, USA

Dinner on your own

7:00–8:00 PM Meet the Professors for Trainees: Coral Reef Room

x Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 xi


7:30–9:00 AM Continental Breakfast: Kahului Room and Poster Session I Posters P1–20

Regioselective Disulfide Solid Phase Synthesis of Mouse Relaxin P-01

John D. Wade, Howard Florey Institute, Australia

Trafficking of the Relaxin Family Peptide Receptor 3 (RXFP3; GPCR135) P-02

*Emma T. van der Westhuizen, Monash University, Australia

Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodination and Isolation of Peptide by One-step Ether Precipitation

P-03

Suode Zhang, Howard Florey Institute, Australia

Chemical Synthesis and Biological Activity Evaluation of β-Ala and γ-Abu Substituted Insulin-like Peptide 3 Analogs

P-04

Suode Zhang, Howard Florey Institute, Australia

Synthesis, Conformation and Biological Activity of Novel Dicarba Analogs of Human Relaxin-3/INSL7

P-05

Mohammed Akhter Hossain, Howard Florey Institute, Australia

Human Insulin-like Peptide 5 (INSL5): Synthesis and Activity on RXFP3 and RXFP4 Receptors P-06


De novo Design and Synthesis of Cyclic and Linear Peptides to Mimic the B-chain Binding Elements of Human Relaxin Family Peptides

P-07


Investigating the Molecular Mechanics of Relaxin Family Peptide Receptor Function Using Time Correlated Single Photon Counting Confocal Microscopy

P-08

Ross A.D. Bathgate, Howard Florey Institute, Australia

WithdrawnP-09

Identification of the Relaxin-responsive Cells in the Human Chorion and Decidua P-10

Jaime S. Horton, University of Hawaii, USA

RXFP1 Is Expressed on Sperm Acrosome and Relaxin Stimulates the Acrosomal Reaction of Human Spermatozoa

P-11

Lisa Gianesello, University of Padova, Italy

Involvement of Relaxin in Endometriosis P-12

Sara S. Morelli, New Jersey Medical School, USA

Relaxin and Neopterin as Local Ovarian Granulosa Cell Growth- and Colony-stimulating Factors P-13

Elissaveta Borissova Zvetkova, IEMAM – BAS, Bulgaria

The Lethal Phenotype in Relaxin-Deficient (Rln-/-) Mice Is Not Due to Impaired Mammary Gland Development or Milk Protein Synthesis

P-14

Laura J. Parry, The University of Melbourne, Australia

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Laser Microdissection of Neonatal Porcine Endometrium for Tissue Specific Evaluation of Relaxin Receptor (RXFP1) Expression in Response to Perinatal Zearalenone Exposure

P-15

Anne A. Wiley, Auburn University, USA

The Effects of Relaxin-deficiency on Uterine Angiogenesis in Early Pregnancy P-16

Jane E. Girling, Monash University, Australia

The Effects of Fetal and Placental Numbers on Blood Concentrations of Relaxin in the Rabbit P-17

Phillip A. Fields, University of South Alabama, USA

Cervical Softening in the Common Marmoset Monkey (Callithrix jacchus) P-18

Almuth Einspanier, Institute of Physiological Chemistry, Germany

Perinatal Zearalenone Exposure Affects RXFP1, RXFP2 and Morphoregulatory Gene Expression in the Neonatal Porcine Uterus

P-19

*Joseph C. Chen, Rutgers University, USA

The Effects of Blocking Progesterone Action on Relaxin Receptor Expression in the Myometrium of Pregnant Rats

P-20

*Lenka A Vodstrcil, The University of Melbourne, Australia

9:00–10:30 AM Platform Session 5: Relaxin Family Receptors #1 Presentations O18–21

Chairs: A. Agoulnik and J. Rosengren

9:00 AM 30 min Resolving the Unconventional Mechanisms Underlying RXFP1 and RXFP2 Receptor Function O-18

Ross Bathgate, Howard Florey Institute, Australia

9:30 AM 30 min Mechanisms of Relaxin Receptor (LGR7/RXFP1) Expression and Function O-19

*Andras Kern, Pacific Biosciences Research Center, University of Hawaii, USA

10:00 AM 15 min Relaxin Family Peptide Receptor 1 (RXFP1) Activation Stimulates the Peroxisome Proliferator-Activated Receptor γ

O-20

*Sudhir Singh, Department of Biochemistry and Molecular Biology, University of Nebraska, USA

10:15 AM 15 min Development of a High-throughput Receptor-binding Assay Using Time-Resolved Fluorescent Europium Lanthanide for Screening Insulin-like Peptide 3 (INSL3) Analogues for their Receptor Binding Affinity

O-21

*Fazel Shabanpoor, Howard Florey Institute, Australia


11:00–12:45 PM Platform Session 6: Relaxin Family Receptors #2 Presentations O22–27

Chairs: R. Summers and C. Schwabe

11:00 AM 25 min Structure, Function and Evolution of Ligands and Receptors of the Insulin/Relaxin Peptide Family O-22

Pierre De Meyts, Hagedorn Research Institute, Denmark

11:25 AM 20 min Activation of Relaxin Related Receptors by Short, Linear Peptides Derived from a Collagen Containing Precursor

O-23

Ronen Shemesh, Compugen Ltd., Israel

11:45 AM 15 min RXFP1 Couples to the Gαi3-Gβγ-PI3K-PKCζ Pathway via the Final 10 Amino Acids of the Receptor C-terminal Tail

O-24

*Michelle Halls, Department of Pharmacology, Monash University, Australia

xii Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 xiii

12:00 PM 15 min Local Regulation of Relaxin Receptors in the Myometrium of Pregnant Rats O-25

*Lenka Vodstrcil, Department of Zoology, The University of Melbourne, Australia

12:15 PM 15 min The Role of the N-Terminal Leucine Rich Repeat Cap in Mediating INSL3 Specificity to Relaxin Family Peptide Receptor 2

O-26

Daniel Scott (Ross Bathgate), Howard Florey Institute, Australia

12:30 PM 15 min The Relaxin Family Peptide Receptors (RXFPs): A New Subgroup of Dimeric, Cooperative GPCRs O-27

Angela Manegold Svendsen, Hagedorn Research Institute, Denmark


7:00–8:30 PM Platform Session 7: Physiology of Insulin-like Peptide 3 (INSL3) Presentations O28–30

Chairs: S. Sutton and R. Bathgate

7:00 PM 30 min INSL3/RXFP2 Signaling in Testicular Descent: Mice and Men O-28

Alexander Agoulnik, Department of Obstetrics and Gynecology, Baylor College of Medicine, USA

7:30 PM 30 min New Roles for INSL3 in Adults: Regulation of Bone Metabolism and Association of RXFP2 Gene Mutations with Osteoporosis

O-29

Alberto Ferlin, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy

8:00 PM 30 min Regulation of INSL3 Transcription in Testicular Leydig Cells O-30

Jacques Tremblay, Department of Obstetrics and Gynecology, Laval University, Quebec, Canada



9:00–10:30 AM Platform Session 8: Relaxin and Related Peptides: Receptor Signaling Presentations O31–34

Chairs: T. Klonisch and D. Sherwood

9:00 AM 30 min The "Hot Wires" of the Relaxin-like Factor (INSL3) O-31

Christian Schwabe, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, USA

9:30 AM 30 min Roles of the Receptor, the Ligand and the Cell in the Signal Transduction Pathways Utilised by the Relaxin Family Peptide Receptors 1-3 (RXFP1-3)

O-32

Roger Summers, Department of Pharmacology, Monash University, Australia

10:00 AM 15 min Ligand-directed Signalling Pathways at the Relaxin Family Peptide Receptor 3 (RXFP3; GPCR135) Determined Using Reporter Genes

O-33

*Emma van der Westhuizen, Department of Pharmacology, Monash University, Australia

10:15 AM 15 min Relaxin Activates Multiple cAMP Signalling Pathway Profiles in Different Target Cells O-34

*Michelle Halls, Department of Pharmacology, Monash University, Australia


11:00–12:30 PM Platform Session 9: Cardiac and Vascular Actions of Relaxin Presentations O35–38

Chairs: C. Samuel and R. Summers

11:00 AM 25 min Prominent Role of Relaxin in Improving Post-infarction Heart Remodelling. Clues from in vitro and in vivo Studies with Genetically Engineered Relaxin-producing Myoblasts

O-35

Daniele Bani, Department of Anatomy, Histology and Forensic Medicine, University of Florence, Italy

xiii Relaxin 2008

11:25 AM 25 min New Aspects on Cardiovascular Actions of Relaxin: Inotropy and Signaling O-36

Thomas Dschietzig, Department of Cardiology and Angiology, Charité University Medicine, Germany

11:50 AM 25 min Reversal of Cardiac Fibrosis and Related Dysfunction by Relaxin: Experimental Findings O-37

Xiao-Jun Du, Baker Heart Institute, The University of Melbourne, Australia

12:15 AM 15 min Relaxin-Induced Compositional and Geometric Remodeling of Small Renal Arteries O-38

*Dan Debrah, Department of Bioengineering, University of Pittsburgh, USA


1:30–3:30 PM Platform Session 10: Neurobiology of Relaxin and Relaxin-related Peptides Presentations O39–44

Chairs: R. Bathgate and E. Unemori

1:30 PM 25 min Relaxin-family Peptide and Receptor Systems in Mammalian Brain: Recent Insights from Anatomical and Functional Studies

O-39

Andrew Gundlach, Howard Florey Institute, Australia

1:55 PM 25 min Relaxin-3 and its Role in Neuroendocrine Function O-40

*Barbara McGowan, Department of Investigative Medicine, Imperial College London, UK

2:20 PM 25 min Metabolic and Neuroendocrine Responses to RXFP3 Modulation in the CNS O-41

Steven Sutton, Johnson & Johnson Pharmaceuticals, USA

2:45 PM 15 min Relaxin-3 Neurons of the Nucleus Incertus Modulate Septohippocampal Theta Rhythm and Spatial Working Memory in Rats

O-42

*Sheri Ma, Howard Florey Institute, Australia

3:00 PM 15 min Behavioral Phenotyping of Mixed-background (129SV/B6) Relaxin-3 Knockout O-43

*Craig Smith, Howard Florey Institute, Australia

3:15 PM 15 min An in vitro Study of the Protective Effect of Relaxin on Brain Tissue under Ischemic Stress O-44

Brian Wilson, Department of Biology, Acadia University, Canada

5:30 PM Buses leave from hotel lobby for “Ulalena” (dinner not included)


7:30–9:00 AM Continental Breakfast: Kahului Room and Poster Session II Posters P21–40

Relaxin Activation of Water Drinking in the Mouse: Likely Role of RXFP1 in the Subfornical Organ P-21

Andrew L. Gundlach, Howard Florey Institute, Australia

Relaxin-3 Neurons in Nucleus Incertus of the Rat: Effect on Activity of Psychological Stressors and the Light-Dark Cycle

P-22

Pei-Juan Shen, Howard Florey Institute, Australia

Expression and Distribution of the Relaxin Family Peptide Receptors—RXFP1-3—in Primate and Human Brain

P-23

Pei-Juan Shen, Howard Florey Institute, Australia

Relaxin-3 and RXFP3 Gene Expression and Peptide Distribution in the Brain and Ovary of Teleost Fish

P-24

Brian C. Wilson, Acadia University, Canada

xiv Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 xv

Insulin-like Peptide 7 (Relaxin 3): Distribution and Interaction with Two Functionally Distinct Binding Sites in the Rat Brain

P-25

Jaw Kang Chang, Phoenix Pharmaceuticals, Inc., USA

Central Administration of Human Relaxin-2 in Adult Male Rats Inhibits Food Intake P-26

*Barbara M.C. McGowan, Imperial College London, United Kingdom

Verification of a Relaxin-3 Knockout/LacZ Reporter Mouse as a Model of Relaxin-3 Deficiency P-27

*Craig M. Smith, Howard Florey Institute, Australia

Effects of Intracerebroventricular Injection of RXFP3 Agonist and Antagonist Peptides on Behaviour of Wildtype and Relaxin-3 Knockout Mice

P-28

*Craig M. Smith, Howard Florey Institute, Australia

Distribution of Relaxin-3 mRNA, Relaxin-3 Immunoreactivity and RXFP3 Binding Sites in the Brain of the Macaque (Macaca fascicularis)

P-29

*Sherie Ma, Howard Florey Institute, Australia

Relaxin Receptor LGR7 Is Regulated by Estrogen P-30

*Priya Maseelall, New Jersey Medical School, USA

Prolonged RXFP1 and RXFP2 Signalling Is Due to Weak Internalization and a Lack of βArrestin Recruitment

P-31

*Gabrielle E. Callander, Howard Florey Institute, Australia

The Regulatory Action of Insulin, IGF1 and Relaxin on Nitric Oxide Synthase Activity and its Impairment in Rat Skeletal Muscle in Condition of Streptozotocin Diabetes of Type II

P-32

Ludmila A. Kuznetsova, Russian Academy of Sciences, Russia

Synthetic Peptides, Derived from the Third Intracellular Loop of Relaxin Receptor LGR7, as Probes for Study of Molecular Mechanisms of Relaxin Action on Adenylyl Cyclase Signaling System

P-33

Alexander O. Shpakov, Russian Academy of Sciences, Russia

Regulation of Lgr8 (RXFP2) Expression in the Mouse Fetal Kidney by the Transcription Factor Pod1

P-34

Laura J. Parry, The University of Melbourne, Australia

Expression Profile of Insulin-like Peptide-5 (INSL5) and GPCR142—A Perspective on Functional Roles and Future Directions

P-35

Qian Sang, Howard Florey Institute, Australia

Changes in Arterial Function by Chronic Relaxin Infusion Are Mediated by the Leucine Rich Repeat G Coupled Lgr7/RFXP1 Receptor

P-36

Julianna E. Debrah, Magee-Womens Research Institute, USA

Relaxin Promotes Matrix Metalloproteinase-2 and Decreases Wnt/β-Catenin Expression in the Neonatal Porcine Heart

P-37

Teh-Yuan Ho, Rutgers University, USA

Boar Testis Acts as a Source and Target Tissue of Relaxin P-38

Tetsuya Kohsaka, Shizuoka University and Gifu University, Japan

α-Adrenergic Activation Upregulates Expression of Relaxin Receptor RXFP-1 in Cardiomyocytes P-39

XiaoLei Moore, Baker Heart Research Institute, Australia

xv Relaxin 2008

Relaxin/RXFP1 Signaling in Prostate Cancer Progression P-40

Shu Feng, Baylor College of Medicine, USA

9:00–10:30 AM Platform Session 11: Novel Actions of Relaxin Presentations O45–49

Chairs: K. Conrad and D. Bani

9:00 AM 25 min Relaxin’s Induction of Specific MMPs Contributes to Degradation of Cartilage Matrix in Target Synovial Joints: Receptor Profiles Correlate with Matrix Remodeling Responses

O-45

Sunil Kapila, Department of Orthodontics and Pediatric Dentistry, University of Michigan, USA

9:25 AM 25 min Relaxin’s Involvement in ECM Homeostasis: Two Diverse Lines of Evidence O-46

Timothy Cooney, Department of Orthopaedics, Hamot Medical Center, USA

9:50 AM 25 min Relaxin in the Airway and Lung: Potential as a Novel Treatment for Asthma O-47

Mimi L.K. Tang, Department of Allergy and Immunology, Royal Children’s Hospital, Melbourne, Australia

10:15 AM 25 min Scar Reduction and Cosmetic Effects of Relaxin O-48

Dennis Stewart, Corthera Inc. (BAS Medical Inc.), USA

10:40 AM 15 min Role of Relaxin during Human Osteoclastogenesis O-49

Alberto Ferlin, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy


11:15–1:30 PM Platform Session12: Renal Actions of Relaxin Presentations O50–56

Chairs: T. Dschietzig and G. Weiss

11:15 AM 25 min In Search of the Elusive Vasodepressor Agents of Pregnancy O-50

Kirk Conrad, University of Florida, USA

11:40 AM 25 min Relaxin: An Endogenous Renoprotective Factor? O-51

Tim Hewitson, Department of Nephrology, Royal Melbourne Hospital, Australia

12:05 PM 25 min Investigations into the Signaling Mechanisms by which Relaxin Inhibits Renal Myofibroblast Differentiation

O-52

Chrishan Samuel, Howard Florey Institute, Australia

12:30 PM 15 min Relaxin-induced Changes in Renal Function and RXFP1 Receptor Expression in the Female Rat O-53

*Alsadek Bogzil, Faculty of Life Sciences, University of Manchester, United Kingdom

12:45 PM 15 min Vascular Endothelial and Placental Growth Factors: New Players in the Slow Relaxin Vasodilatory Pathway

O-54

*Jonathan McGuane, Department of Physiology and Functional Genomics, University of Florida, USA

1:00 PM 15 min Effects of Relaxin on the Development of Mesangial Proliferative Nephritis O-55

Naoki Ikegaya, Department of Applied Biological Sciences, Shizuoka University, Japan

1:15 PM 15 min Relaxin Affects Endothelial Progenitor Cell Number and Function O-56

Mark Segal, Department of Medicine, University of Florida College of Medicine, USA


xvi Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 xvii

7:00–8:30 PM Platform Session 13: Relaxin and Relaxin-like Peptides in the Blood and Brain Presentations O57–60

Chairs: A. Einspanier and C. Bagnell

7:00 PM 25 min Evaluation of Relaxin Blood Profiles as a Means of Assessing Placental Function and Responsiveness to Therapeutic Strategies in Mares

O-57

Peter Ryan, College of Veterinary Medicine, Mississippi State University, USA

7:25 PM 25 min Relaxin Concentrations in Serum and Urine of Endangered and Crazy Mixed-up Species: New Methods, Uses and Findings

O-58

Bernard Steinetz, Department of Environmental Medicine, New York University School of Medicine, USA

7:50 PM 15 min Development of Adult Neuronal-specific Relaxin-3 Knockout Rats Using Adeno-associated Viral-driven miRNA

O-59

*Gabrielle Callander, Howard Florey Institute, Australia

8:05 PM 15 min Presence and Role of LGR8 (RXFP2) in Thalamo- and Cortico-Striatal Circuits: Effects of Intrastriatal Injection of INSL3 on Stereotypic Behaviour in the Rat

O-60

Andrew Gundlach, Howard Florey Institute, Australia


7:30–9:00 AM Continental Breakfast: Kahului Room and Poster Session III Posters P41–55

Relaxin Reduces Fibrosis in Models of Progressive and Established Hepatic Fibrosis P-41

Robert G. Bennett, Veterans Affairs Medical Center and University of Nebraska Medical Center, USA

Evaluation of Relaxin’s Anti-fibrotic Actions by SELDI-TOF Mass Spectrometry-based Protein Profiling of Relaxin KO Mice, a Model of Progressive Fibrosis

P-42

John Wade, Howard Florey Institute, Australia

Novel EGF and IGF1 Adenylyl Cyclase Signaling Mechanisms with Similar Structural-Functional Organization

P-43

Marianna N. Pertseva, Russian Academy of Sciences, Russia

Investigating the Role of Endogenous Relaxin in Experimental Diabetic Renal Disease P-44

*Su Ee Wong, Howard Florey Institute, Australia

Determinants of Relaxin-induced Changes in Passive Compliance of Small Renal Arteries: Geometric vs. Compositional Remodeling

P-45

*Dan O. Debrah, University of Pittsburgh, USA

Relaxin as a Protective Substance in the Preserving Solution for Liver Transplantation: Spectrophotometric in vivo Imaging of Local Oxygen Supply in an Isolated Perfused Rat Liver Model

P-46

Markus U. Boehnert, University of Bern, Switzerland

Involvement of Relaxin in Salt-sensitive Hypertension P-47

Naoki Ikegaya, Shizuoka University, Japan

Volar Oblique Ligament Fibroblasts Express Estrogen, LGR7 Receptors P-48

Elisa M. Konieczko, Gannon University, USA

Basement Membrane Localization of LGR7 Receptors and TGF-beta in Juvenile Skin Biopsies P-49

Timothy E. Cooney, Hamot Medical Center, USA

xvii Relaxin 2008

Relaxin Differentially Regulates Gelatinase Expression and Activity in Cultured Gingival Fibroblasts P-50

Chrishan S. Samuel, Howard Florey Institute and Department of Biochemistry and Molecular Biology, The University of Melbourne, Australia

INSL3 Plays a Role in the Balance between Bone Formation and Resorption P-51

Anastasia Pepe, University of Padova, Italy

Mutations in INSL3-RXFP2 Genes in Cryptorchid Boys P-52

Alberto Ferlin, University of Padova, Italy

The Mechanisms behind Relaxin on Improving Muscle Healing P-53

Yong Li, Children’s Hospital of Pittsburgh and University of Pittsburgh, USA

Relaxin Has a Role in the Molecular Dynamics of Cellular Movement P-54

Thomas Klonisch, University of Manitoba, Canada

In vitro Relaxin H2 Strengthens Fetal Membranes? P-55

Gillian Bryant-Greenwood, University of Hawaii, USA

9:00–10:20 AM Platform Session 14: Relaxin and Cancer Presentations O61–64

Chairs: P. Ryan and A. Einspanier

9:00 AM 25 min Implications of Leukocyte Responsiveness to Tumor-derived Relaxin O-61

Michael Cox, The Prostate Center, University of British Columbia, Canada

9:25 AM 25 min Relaxin and INSL3 Promote Tumor Formation of Human Thyroid Carcinoma Cells O-62

Thomas Klonisch, Department of Human Anatomy and Cell Science, University of Manitoba, Canada

9:50 AM 15 min Estrogen and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) Modulate RLN2 Promoter Activity in Estrogen-receptor Positive T47D Human Breast Cancer Cells

O-63

Sabine Hombach-Klonisch, Department of Human Anatomy and Cell Science, University of Manitoba, Canada

10:05 AM 15 min Relaxin Signaling in Human Uterine Fibroids O-64

*Zhen Li, Department of Obstetrics and Gynecology, Baylor College of Medicine, USA


11:00–12:30 PM Platform Session 15: Relaxin Clinical Trials Presentations O65–67

Chairs: D. Stewart and M. Cox

11:00 AM 30 min Scientific Rationale and Design of a Phase I Study of Relaxin in Women with Severe Preeclampsia O-65

Elaine Unemori, Corthera Inc. (BAS Medical Inc.), USA

11:30 AM 30 min A Randomized, Double Blind, Placebo Controlled Trial of Relaxin for Cervical Ripening in Post-date Pregnancies

O-66

Gerson Weiss, Department of Obstetrics and Gynecology, New Jersey Medical School, USA

12:00 PM 30 min A Pilot Safety and Dose-finding Trial of Intravenous Recombinant Human Relaxin (rhRlx) in Compensated Congestive Heart Failure

O-67

Thomas Dschietzig, Department of Cardiology and Angiology, Charité University Medicine, Germany

12:30 PM Closing Remarks

12:45 PM Closing Lunch provided: Anuenue Lawn

xviii Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008

Abstracts


2 Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008 3

O-01

Structure and Activity in the Relaxin Family of Peptides

Geoffrey W. Tregear, Ross A.D. Bathgate, Mohammed Akhter Hossain, Feng Lin, Suode Zhang, Fazel Shabanpoor, Daniel J. Scott, Sherie Ma, Andrew L. Gundlach, and John D. Wade

Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

The availability of improved peptide synthesis procedures, convenient and sensitive assays for receptor binding and activation, together with advances in methods for structural characterisation, has enabled the key structural features of the relaxin family of peptides responsible for biological activity to be defined. Not surprisingly, despite the similarity in primary amino acid sequences, different structural domains and residues are involved in the binding and activation at the four known relaxin family peptide receptors (RXFP1-4). Most of our knowledge on structure and function relates to the relaxin-RXFP1, INSL3-RXFP2 and relaxin 3-RXFP3 systems with information accumulating not only on the critical ligand structures, but also the domains and residues on the receptor itself that are required for specificity and activation. These studies provide the framework for the design of small molecule mimetics. While the B-chain cassette R-X-X-X-R-X-X-I defined by Büllesbach and Schwabe is essential for binding and activation at RXFP1, it is now recognised that the A-chain, particularly the N-terminal domain, is also critical for receptor specificity. The inter- and intra-chain links are clearly required for structural integrity but can be replaced with the more stable dicarba residues. Studies of the various endogenous ligand-receptor pairs have led to the design of potent and specific agonists and antagonists. The relaxin 3 A-chain – INSL5 B-chain chimeric peptide and analogues with C-terminal truncations of the B-chain, developed by Liu and colleagues at Johnson & Johnson, have provided selective agonist and antagonist peptides that are proving invaluable for in vivo studies of the relaxin 3 RXFP-3 system.

O-02

The Chemical Synthesis of Relaxin and Related Peptides: From Yesterday to Tomorrow

John D. Wade, Feng Lin, Akhter Hossain, Suode Zhang, and Geoffrey W. Tregear


Since Sanger’s pioneering discovery that insulin comprises of not one but two peptide chains that were tethered by three disulfide bonds in a unique disposition, considerable effort has been expended upon developing methods for its efficient chemical synthesis. Herculean efforts by Chinese, American and German teams led to the successful acquisition in the mid-1960s of insulin via random combination in solution of the individual S-reduced chains. These findings showed that all the information required for correct alignment and combination of the two chains are conferred by their primary structure. However, soon after the subsequent identification and establishment of the insulin superfamily of peptides, including the relaxins, it became apparent that such methods were not usefully applicable to the chemical assembly of relaxin as a consequence of the peptide’s less favourable physicochemical properties. However, the refinement of the solid phase peptide synthesis methodology together with the development of a variety of cysteine S-protecting groups that were orthogonal to one another soon allowed the successful acquisition of relaxin and related peptides via elegant regioselective and sequential assembly of the three disulfide bonds. Such methods have been instrumental in allowing detailed structure-function and biochemical and physiological studies to be carried out. However, there remains a need for still more efficient means of chemical synthesis of multicystine double-stranded peptides, particularly involving fewer reactions steps. We describe novel approaches that include the use of single mini-proinsulin-like peptides together with one-pot sequential disulfide bond formation.

3 Relaxin 2008

O-03

Development of High Affinity INSL3 Antagonists

Fazel Shabanpoora,b, Richard A. Hughesc, Frances Separovicb, and John D. Wadea

aHoward Florey Institute, bSchool of Chemistry, cDepartment of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia

Insulin-like peptide 3 (INSL3), also referred to as Leydig-insulin-like (Ley-I-L) and relaxin-like factor (RLF), belongs to the relaxin-insulin superfamily of peptides. INSL3 plays important roles in the reproductive system, such as the meiotic progression of arrested oocytes in females and the suppression of male germ cell apoptosis. INSL3 consists of two chains (A and B) connected via three disulfide bonds. Our previous structure-activity relationship studies on INSL3 led to identification of a cyclic peptide mimetic of the INSL3 B-chain which inhibits binding of 33P-relaxin to the receptor RXFP2 in cell culture (pKi=6.65±0.09, n=3). This compound was designed based on the previously identified INSL3 antagonist (pKi=5.86±0.03, n=3) which we showed to block the spermatogenesis in vivo in rats. However, the INSL3 analogues that have been developed to date are active within the micromolar range. Therefore, our aim is to develop specific mimetics of INSL3 with still higher receptor binding affinity for therapeutic applications in the area of fertility control. To further explore the structure-activity relationships for INSL3 antagonists, we have used SYBYL molecular modeling software to design three series of mimetics: [1] disulphide-constrained, [2] lactam-constrained, and [3] those containing helicogenic residues and N-caps. These compounds were synthesized by solid phase methodology and their secondary structure was determined using circular dichroism (CD) spectroscopy. All mimetics showed a low level of α-helicity in phosphate buffered saline (PBS, pH 7.5), but dramatically increased helicity upon addition of 20% trifluoroethanol (TFE). Currently, we are performing competition binding assays to determine the affinity of these analogues for the INSL3 receptor, RXFPR2.

O-04

The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of the Different RXFP Receptors

Mohammed Akhter Hossaina, K. Johan Rosengrenb, Linda M. Haugaard-Jönssonb, Soude Zhanga, Sharon Layfielda, Tania Ferraroa, Geoffrey W. Tregeara, John D. Wadea, and Ross A. D. Bathgatea

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bDepartment of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden

The role of the A-chain in H2 and H3 relaxin activity is currently unknown. A recent study has shown that INSL3 can be truncated at the N-terminus of its A-chain by up to 9 residues without affecting RXFP2 binding affinity while becoming a high affinity antagonist. This suggests that the N-terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study we have synthesized A-chain truncated H2 and H3 relaxin peptides, characterized their structure by CD spectroscopy and NMR and tested their binding and cAMP activities on RXFP1, RXFP2 and RXFP3. In stark contrast to INSL3, A-chain truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP stimulatory activity with truncation past 8 amino acids. Replacement of these residues at the N-terminus with 4 or 5 alanine residues resulted in the partial rescue of binding activity and potency at RXFP1 but not at RXFP2. H3 relaxin A-chain truncated peptides displayed similar properties on RXFP1 highlighting that H3 relaxin binds to RXFP1 via the same mechanism as H2 relaxin. In contrast, A-chain truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. CD and NMR studies demonstrated that removal of the N-terminal A-chain residues resulted in a significant decrease in overall structure of both H2 and H3 relaxin. This study demonstrates the subtle differences in the role of the A-chain in the interactions between relaxin family peptides and their receptors.


O-05

Structural Insights into the Function of Relaxins

K. Johan Rosengrena, Ross A.D. Bathgateb, David J. Craikc, Norelle L. Dalyc, Linda M. Haugaard-Jönssona, Mohammed

Akhter Hossainb, and John D. Wadeb

aSchool of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; cInstitute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia

The relaxin family of peptide hormones share a structural fold that is characterised by two peptide chains that are cross-braced by three disulfide bonds. Onto this framework, which besides the disulfide array has limited conserved structural features, various amino acid side chains are presented for interactions with the relaxin receptors. Relaxins activate two unrelated classes of G-protein coupled receptors. The relaxin and INSL3 receptors (LGR7 and LGR8) belong to the Leucine-rich repeat containing GPCRs, which have a large N-terminal ligand-binding domain, while the smaller relaxin-3 and INSL5 receptors (GPCR135 and GPCR142) are classic peptide GPCRs.

Interestingly relaxin-3 has the ability to activate its own receptor GPCR135, as well as the GPCR142 and LGR7 receptors. To deduce how changes in the primary sequence can fine-tune the overall structure and thus the ability to interact with the various receptors we have studied these peptides using NMR. Full 3D structures of both relaxin-3 and INSL3 have been determined and revealed a number of interesting features. Both peptides show a significant amount of line-broadening in certain regions, in particular around the intra A-chain disulfide bond, suggesting that despite the disulfide bonds the fold is rather dynamic. While the peptides share a common structural core there are significant differences, particularly around the termini. For example in relaxin-3 the A-chain N-terminus is fully structured into a helix while in INSL3 the first five residues are disordered. The structural data in combination with mutational studies provide valuable insights into the structure-activity relationships of relaxins.

O-06

Probing the Functional Domains of Relaxin-3 and the Creation of a Selective Antagonist for GPCR135 over Relaxin Receptor LGR7

Changlu Liua, Chester Kueia, Steven Suttona, Jonathan Sheltona, Jessica Zhua, Diane Nepomucenoa, Mohammed Akhter

Hossainb, John D. Wadeb, Ross A.D.Bathgateb, Pascal Bonaventurea, and Timothy Lovenberga

aJohnson & Johnson Pharmaceutical Research & Development, LLC, San Diego, California 92121, USA; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

Both relaxin-3 and its receptor (GPCR135) are predominantly expressed in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Since relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to study the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide, designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BD23-27)R/I5, consists of a modified relaxin-3 B-chain (a replacement of Gly23 to Arg and removal of Gly24-Trp27) and the A-chain of INSL5. In vitro pharmacological studies show that R3(BD23-27)R/I5 binds to human GPCR135 and GPCR142 with high affinity and is a potent functional GPCR135 antagonist (pA2 = 9.15), but is not a human LGR7 ligand. Furthermore, R3(BD23-27)R/I5 is also a potent selective rat GPCR135 antagonist. It had a similar binding profile at the rat GPCR135 receptor (pA2 = 9.6) and lacked affinity for the rat LGR7 receptor completely.

5 Relaxin 2008

O-07

Structural Characterization of the R3/I5 Chimeric Relaxin Peptide

Linda M. Haugaard-Jönssona, Mohammed Akhter Hossainb, Norelle L. Dalyc, Ross A.D. Bathgateb, David J. Craikc, John D. Wadeb, and K. Johan Rosengrena

aSchool of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; cInstitute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia

Selective agonist and antagonist for each individual relaxin receptor, would be highly desirable for in vivo studies, in order to fully understand the biological significance of the different relaxin ligands. Recent reports demonstrated that such relaxin analogues can be achieved by developing chimera peptides, where the A-chain from one relaxin ligand is combined with the B-chain from another relaxin ligand. Using this strategy a selective peptide for GPCR135 and GPCR142 over LGR7 was generated by the combination of relaxin-3 B-chain with the A-chain from INSL5 (R3/I5). Here we have used NMR spectroscopy to determine the 3D structure of this R3/I5 chimera to gain structural insight into the consequences of combining chains from two relaxins. In particular it was of interest to see if the relaxin-3 B-chain has undergone conformational changes or if additional LGR7 binding sites are located in the A-chain and thereby lost when generating the R3/I5 chimera peptide.

The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B-chain compared to native relaxin-3 and the INSL5 A-chain displays a relaxin/insulin like fold with two parallel helices. These findings indicate that binding and activation of the smaller relaxin receptors GPCR135 and GPCR142 mainly requires the B-chain and that the A-chain function as bulk and support to the structure. In contrast, the larger LGR7 demonstrate a more complex binding mechanism involving both the A-chain as well as the B-chain.

O-08

Degradation of Relaxin Family Peptides by Insulin-degrading Enzyme

Robert G. Bennetta,b,c, Dean G. Heimanna, and Frederick G. Hamela,b

aVeterans Affairs Medical Center, Omaha, Nebraska 68105, USA; bDepartments of Internal Medicine, Pharmacology and Experimental Neuroscience, and cDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA

Insulin-degrading enzyme (IDE) is a ubiquitously expressed metalloproteinase responsible for the intracellular degradation of insulin. IDE also degrades other members of the insulin superfamily, including relaxin. No studies have been reported regarding the interaction of other relaxin-like peptides with IDE. Purified rat liver IDE was utilized to examine the binding and degradation properties of porcine relaxin, or human InsL3, and relaxin-3. First, we assessed the ability of each peptide to competitively inhibit the degradation of radiolabeled insulin. As observed previously, relaxin inhibited insulin degradation less potently than insulin (IC50 182.0nM vs. 36.3nM respectively). In contrast, both InsL3 and relaxin-3 inhibited IDE with potencies approaching that of insulin (IC50 44.4nM and 53.7nM, respectively). Next, to determine the ability to compete with insulin for binding to IDE, covalent cross-linking of radiolabeled insulin to IDE was performed in the presence of excess (1µM) relaxin family peptides. All peptides reduced insulin binding to IDE. Finally, nonreducing SDS-PAGE was performed to assess actual degradation of the peptides. All peptides were degraded but with varying rates (insulin>relaxin>InsL3=relaxin-3). The degradation of all peptides was reduced by the IDE inhibitor 1,10-phenanthroline. In summary, the data suggest that relaxin, InsL3 and relaxin-3 all interact with IDE, and are able to interfere with the binding and degradation of insulin by IDE. The three relaxin family peptides are all substrates for the proteolytic activity of IDE. Therefore, it is possible that in addition to insulin, IDE may be important for the cellular proteolysis of relaxin, InsL3 and relaxin-3.


O-09

Relaxin in Human Pregnancy: Consequences of Too Little or Too Much of a Good Thing!

Laura T. Goldsmith

Department of Obstetrics, Gynecology and Women’s Health, New Jersey Medical School of UMDNJ, Newark, New Jersey 07103, USA

With any bona fide hormone or growth factor, the concentrations in circulation can be critical to their effects. Low concentrations can cause one effect, whereas high concentrations of the same factor can be responsible for an opposite action. Unlike certain other aspects of life, in endocrinology, more is not necessarily better. This frequently complicates our understanding of the actions of a hormone. It appears to be the case with relaxin, for which elucidation of its role in human pregnancy has been very difficult.

Relaxin has beneficial effects upon the endometrium which are responsible for establishment of pregnancy. Relaxin stimulates endometrial decidualization, the structural and biochemical changes in endometrial parenchymal cells, and the accompanying angiogenesis, modulation of matrix metalloproteinase activity and increased concentration in local immune cells required for implantation.

However, our recent data also demonstrate that either too much or too little relaxin can be detrimental. Elevated circulating maternal relaxin concentrations are associated with premature birth. This is likely due to the effects of relaxin at the level of the cervix, via upsetting the balance in the maintenance of cervical connective tissue architecture. In addition, the absence of circulating relaxin during pregnancy or overabundance of circulating relaxin (hyperrelaxinemia) during pregnancy in women may have negative consequences upon glucose metabolism. Furthermore, decreased expression of relaxin mRNA and relaxin receptor LGR7 mRNA are associated with endometriosis, which may impede fertility.

O-10

Relaxin Acts on Stromal Cells to Promote Epithelial and Stromal Proliferation and Inhibit Apoptosis in the Mouse Cervix and Vagina

LiJuan Yaoa, O. David Sherwooda,b, Paul Cookec, Daryl Melingc, and Alexander Agoulnikd

aDepartment of Molecular and Integrative Physiology, bCollege of Medicine, and cDepartment of Veterinary Biosciences, University of Illinois at Urbana-Campaign, Illinois 61801, USA; dDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA

Relaxin promotes growth of the lower reproductive tract in rodents. The objective of this study was to determine whether stromal and/or epithelial relaxin receptor (LGR7) is required for relaxin to promote proliferation and inhibit apoptosis of stromal and epithelial cells in the mouse cervix and vagina. Tissue recombinants were prepared with stroma (S) and epithelium (E) from wild-type (wt) and LGR7 knockout (ko) mice: wt-S+wt-E, wt-S+ko-E, ko-S+wt-E and ko-S+ko-E. Tissue recombinants were grown for 3 weeks as subrenal capsule grafts in intact syngeneic female mice. Then, on day 1 of treatment, the hosts were ovariectomized and fitted with silicon implants containing progesterone and estradiol-17β. Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on day 8 through 0600 h on day 10 of treatment. To evaluate cell proliferation, 5-bromo-2-deoxyuridine (BrdU) was injected sc 10 h before cervices and vaginas were collected at 1000 h on day 10. TUNEL was used to quantify apoptosis. Relaxin markedly increased proliferation and decreased apoptosis of epithelial and stromal cells in tissue recombinants containing wt stroma (P< 0.01), but had no effect on tissue recombinants lacking wt stroma, regardless of whether epithelium was derived from wt or ko mice. In conclusion, this study shows that LGR7-expressing cells in the stroma are both necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in the cervix and vagina, while epithelial LGR7 does not have any effect on these processes.

7 Relaxin 2008

O-11

The Good, The Bad and The Ugly: Lessons in Female Reproductive Tract Physiology from the Relaxin Gene Knockout (Rln-/-) Mouse

Laura J. Parry

Department of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia

Targeted gene deletion in embryonic stem cells to develop a null relaxin (Rln-/-) mouse has proved an effective tool to study the functions of relaxin in female reproductive tract physiology. However, there are a number of discrepancies in the phenotypes reported between different studies. It is worth considering the variable effects of modifier genes among inbred strains, phenotypic robustness and redundancy or compensation caused by genes performing similar functions. We suggest that age is another factor that should be taken into account. The other main limitation of in vivo mouse models of reproductive tract physiology is the extent to which they can increase our understanding of pregnancy in humans. Despite these challenges, the pregnant Rln-/- mouse has provided useful information on the mechanisms of relaxin action in vivo. We have shown that relaxin decreases collagen synthesis but does not increase matrix metalloproteinase (MMP) expression in the cervix at the end of gestation. Relaxin treatment in Rln-/- mice also affects synthesis of hyaluronan, cervical hydration and collagen dispersal. In addition, we have identified an interaction between relaxin and estrogen receptors, and a ligand-specific down-regulation of Lgr7. Our recent studies demonstrate novel phenotypes in the uterus and placenta of Rln-/- mice related to relaxin’s actions as a vascular hormone. There is a reduction in endometrial angiogenesis in early gestation and abnormalities in placental development. The consequence of these phenotypes is that pups born to Rln-/- dams are significantly smaller than their wildtype counterparts, which is indicative of intrauterine growth restriction.

O-12

Decreased Hyaluronic Synthase Expression in the Cervix of Pregnant Relaxin-Deficient (Rln-/-) Mice

Yu May Soha, Lenka A. Vodstrcila,b, and Laura J. Parrya

aDepartment of Zoology and bDepartment of Physiology, The University of Melbourne, Parkville, Victoria 3010, Australia

The extracellular matrix (ECM) of the cervix changes extensively in a process called cervical ripening in late pregnancy. This occurs through a complex series of events leading to collagen fibre degradation and dispersal. We tested the hypothesis that relaxin increases cervical water content by stimulating the hyaluronic synthase (HAS) enzymes that regulate hyaluronic acid (HA) synthesis. HA is thought to recruit water molecules into the spaces between collagen fibrils and causes them to disperse. In the first study, cervices were collected from Rln+/+ and Rln-/- mice on days 14.5, 16.5, 18.5 and 19 gestation, and day 1 postpartum. HAS expression was compared between the genotypes by qPCR analysis. In the second study, Rln-/- mice were implanted with osmotic minipumps on day 12.5 gestation to infuse rhH2 relaxin; cervices were dissected on days 16.5 and 18.5 gestation. has1 and has2 gene expression increased significantly at term and decreased immediately postpartum in the Rln+/+ mice. Although has1 and has2 were expressed in Rln-/- mice, there was no increase in expression on day 18.5 gestation compared with Rln+/+ mice. Similarly, cervix wet weight did not increase in late pregnant Rln-/- mice. Relaxin infusion in pregnant Rln-/- mice significantly increased cervical has2 but decreased has1 expression compared with saline-controls. Relaxin treatment also caused a 6-fold increase in cervix wet weight and a 4-fold decrease in Lgr7 expression. These data suggest that relaxin promotes cervical hydration through an has2-mediated increase in HA, and facilitates cervical ripening by dispersing collagen fibrils in the ECM.


O-13

Milk-borne Relaxin and the Lactocrine Hypothesis for Maternal Programming of Neonatal Tissues

Carol A. Bagnella, Bernard G. Steinetzb, and Frank F. Bartolc

aDepartment of Animal Sciences, Endocrinology and Animal Biosciences Program, Rutgers University, New Brunswick, New Jersey 08901, USA; bNelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987, USA; cDepartments of Animal Sciences and Anatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA

The fact that all newborn mammals drink milk extends the timeframe of maternal influence on development into neonatal life. While nutritional and immunological benefits of milk are clear, the role of milk as a conduit for bioactive factors with the potential to affect neonatal development is less well defined. Porcine and canine milk contain immunoreactive relaxin (RLX) that is transmitted into the circulation of nursing offspring. In the pig, a window of opportunity for transmission of milk-borne RLX from mother to neonate is open at birth and remains so for the first three days of neonatal life. Recent in vitro studies showed that RLX in porcine milk is bioactive. Moreover, RLX receptor (RXFP1) expression is detectable in the porcine female reproductive tract and other somatic tissues at birth. The lactocrine hypothesis for maternal programming of neonatal development was proposed as a mechanism whereby RLX, a prototypical milk-borne growth factor in the pig, is delivered to nursing offspring where it affects development of RXFP1-positive target tissues. Data indicating that treatment of newborn gilts with RLX increased estrogen receptor-α expression in the uterus and cervix by postnatal day 2 supports a role for RLX in lactocrine programming of the female reproductive tract. Effects of RLX on Wnt/β-catenin expression in porcine cardiac tissue during this period support a role for RLX in developmental programming of non-reproductive target tissues as well. Ongoing studies will test the lactocrine hypothesis for maternal programming of development by RLX and related milk-borne factors. (Support: USDA-NRI 2003-35203-1357 and 2007-35203-18098)

O-14

Relaxin and Maternal Lactocrine Programming of Neonatal Uterine Development

Frank F. Bartola,b, Anne A. Wileya, and Carol A. Bagnellc

Departments of aAnimal Sciences and bAnatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA; cDepartment of Animal Sciences, Endocrinology and Animal Biosciences Program, Rutgers University, New Brunswick, New Jersey 08901, USA

In mammals, including the pig (Sus scrofa domesticus), structural patterning and functional programming of female reproductive tract tissues involve events that occur shortly after birth. Porcine endometrial development between birth (postnatal day = PND 0) and PND 15 is estrogen receptor- (ER) dependent and estrogen-sensitive. The endometrium is relaxin (RLX) receptor- (RXFP1) positive and ERα-negative at birth. Uterine expression of RXFP1 and ERα, detectable by PND 2, increases with age from PND 0-14. Estrogen exposure during this period that is sufficient to affect uterine developmental trajectory and adult uterine phenotype alters uterine RXFP1 and morphoregulatory gene expression patterns, including those for Hoxa10, Wnt7a and MMP-9, by PND 14. Data implicate RXFP1 as an element of the uterine developmental program. Uterotrophic effects documented for both estrogen and RLX in the neonatal pig are age-specific and most pronounced after onset of ERα expression when RLX effects can be inhibited by pretreatment with the ER antagonist ICI 182,780. Thus, crosstalk between RLX and estrogen signaling systems evolves with age in the porcine uterus. Given that RLX administered from birth stimulates uterine ERα expression and that estrogen administration during this period stimulates RXFP1 expression by PND 2, a developmentally relevant functional relationship between these signaling systems is envisioned. Evidence that RLX is present in porcine milk and in the circulation of nursing offspring supports the lactocrine hypothesis for maternal programming of uterine tissues in which milk-borne RLX, acting via RXFP1, facilitates ERα expression and porcine endometrial development in the neonate. (Support: USDA-NRI 2003-35203-1357, USDA-NRI 2007-35203-18098 and NSF EPS-0447675)

9 Relaxin 2008

O-15

Relaxin in the Marmoset Monkey: There Are Two Sides to Every Story

A. Einspaniera, K. Eberta, K. Liedera, M. Eberta, S. Lierb, O. Lampa, R. Einspanierb, N. Beindorffc, C. Schlumbohmc, M. Kempera, J. Jacoba, and M. Sohra

aInstitute of Physiological Chemistry, Faculty of Veterinary Medicine, 04103 Leipzig, Germany; bInstitute of Veterinary Biochemistry, Freie Universität Berlin, Germany, CGerman Primate Center, Göttingen, Germany

Many disorders occurring in adult life date back to influences of the intra-uterine environment. Therefore in the first part of this paper, different factors involved in early pregnancy are analysed while in the second part, the effect of stress throughout pregnancy on the endocrine situation and physiological parameter are examined in the marmoset monkey.

The results of part I showed a massive increase in gene and protein expression from the RLX system (RLX, RXFP1) as well as enzymes of estrogen biosynthesis solely throughout the implantation process (day 10/11), which declined immediately around day 14/15 of pregnancy. Steroid receptor expression remained unchanged expressed throughout early pregnancy.

In part II, a short-term application of stress hormone during early gestation induced a significant rise of serum RLX and E2, whereas serum P4 declined throughout the application period. No significant differences compared to uterine growth rate, gestation length, fetal biparietal diameter, or maternal and newborn body weights were observed in all groups. However, the early prenatal stressed females showed a significant increase in body weight (456±86g) compared to controls (344±51g) at 20 months of age, differences in their blood fatty acids levels, and an earlier onset of cycle activity compared to the control group.

In summary, the RLX-system is involved in the process of implantation and with the enzymes of estrogen biosynthesis it orchestrates the remodelling of the uterine tissue. However, a stress-induced significant serum increase in RLX and estrogens during early pregnancy seems to have an epigenetic effect on the female offspring.

Project was kindly supported by DFG (German Research Council) grant EI 333/11-1 and EU (EUPEAH QLR1-CT 2002-02758)

Some of these results are taken from the thesis by K. Ebert, S. Lier, and M. Kemper.

O-16

Expression of LGR7 in the Primate Corpus Luteum Implicates the Corpus Luteum as a Relaxin Target Organ

Priya Maseelall, Aimee Seungdamrong, Gerson Weiss, Andrea Wojtczuk, and Laura T. Goldsmith

New Jersey Medical School, Department of Obstetrics, Gynecology, and Women’s Health, Newark, New Jersey 07103, USA

In primates, the corpus luteum is the source of circulating relaxin. No studies have determined whether the primate corpus luteum is also a relaxin target organ. We had the opportunity to study LGR7 expression in human term pregnancy corpora lutea and non-human primate corpora lutea obtained during the menstrual cycle. Real-time RT-PCR demonstrated LGR7 mRNA expression in human term pregnancy corpora lutea and in rhesus monkey corpora lutea taken at early, mid, and late luteal phases. The relative expression of LRG7 mRNA in rhesus monkey corpora lutea during the mid and late luteal phases of the menstrual cycle was 4 fold higher than in the early luteal phase. Also, since the primate corpus luteum is LH dependent, we assessed LGR7 mRNA expression using corpora lutea from GnRH antagonist treated rhesus monkeys. GnRH antagonist treatment, which decreased circulating LH to undetectable levels, resulted in a five fold increase in luteal LGR7 mRNA expression. Increased luteal expression of LGR7 mRNA in the mid and late luteal phases of the menstrual cycle may suggest a role for relaxin in the demise of the non-pregnant corpus luteum. In addition, these data suggest that luteal LGR7 mRNA expression may be regulated by relaxin and/or LH, and that the primate corpus luteum is a target organ for relaxin.

10 Fifth International Conference on Relaxin and Related Peptides | May 18–23, 2008

O-17

Characterization and Biological Activity of Prorelaxin in Porcine Milk

Amy-Lynn Frankshuna, Teh Yuan Hoa, Bernard G. Steinetzb, Frank F. Bartolc, and Carol A. Bagnella

aDepartment of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA; bNelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987, USA; cDepartments of Animal Sciences and Anatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA

A lactocrine mechanism for delivery of milk-borne relaxin for maternal programming of neonatal development has been proposed. In support of this hypothesis porcine relaxin (pRLX) was detected in colostrum and in the circulation of nursing pigs. While immunoreactive pRLX concentrations are highest in milk during early lactation (9-19 ng/ml), declining to < 2 ng/ml by postnatal day 14, whether milk-borne pRLX is bioactive is unknown. Therefore, objectives were to characterize the molecular size and determine the bioactivity of pRLX in milk using a bioassay validated for use with milk. Sow milk samples collected on lactation days 0 (day of parturition) and 14 were diluted 1:1 with EDTA to solubilize caseins and centrifuged to remove fat. Samples and native pRLX were resolved under non-reducing/denaturing conditions by SDS-PAGE. To monitor pRLX bioactivity RXFP1-transfected HEK293T cells were employed as targets and total cAMP was measured. Immunoblotting revealed a 19 kDa band corresponding to proRLX in day 0 milk. Bioactivity of proRLX in milk was confirmed by an increase in cAMP in response to increasing volumes of day 0 milk. Dilution curves of pRLX standards and day 0 lactation milk were parallel. Incubation with day 14 milk did not increase cAMP. Addition of porcine insulin or human insulin-like growth factor-I (1 µg/ml) to HEK293T-RXFP1 cells or treatment of non-transfected cells with pRLX failed to elicit a cAMP response. These data show that proRLX in porcine milk is bioactive, supporting the lactocrine hypothesis. The extent to which milk-borne pRLX affects development remains to be determined. (Support: USDA-NRI 2003-35203-1357 and 2007-35203-18098)

Abstracts



P-01

Regioselective Disulfide Solid Phase Synthesis of Mouse Relaxin

Feng Lin, Eleni Giannakis, Nicola F. Hanson, Geoffrey W. Tregear, and John D. Wade


Fibrosis is a leading cause of organ failure where normal tissue is replaced by excess connective tissue. Several organs are prone to this process regardless of etiology. The hormone, relaxin, is emerging as a novel antifibrotic therapy. It limits collagen production and reorganization, while stimulating increased collagen degradation. It not only prevents firbogenesis, but also reduces established scarring. Recent studies on relaxin-deficient mice have established relaxin as an important, naturally occurring regulator of collagen turnover and provide new insights into the therapeutic potential of relaxin. Mouse relaxin has an insulin-like structure that consists of 25 residues A-chain and 35 residues B-chain with three disulfide bonds, it differs from other mammalian relaxins is that the A-chain contains an additional residue within its sequence. Mouse relaxin could not be successfully prepared by simple combination of the individual chains due to the very poor solubility of the B-chain. This necessitated recourse to use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S-protected A- and B-chains followed by their purification and subsequent stepwise formation of each of the three disulfides via aeration, thioloysis and iodolysis led to the successful acquisition of mouse relaxin in good overall yield. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product.

P-02

Trafficking of the Relaxin Family Peptide Receptor 3 (RXFP3; GPCR135)

Emma T. van der Westhuizena, John D. Wadeb, Patrick M. Sextona, and Roger J. Summersa

aDepartment of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

The aim of this study was to examine the influence of relaxin family peptides on the trafficking of the H3 relaxin receptor, RXFP3. In CHO-K1 and HEK293 cells expressing RXFP3 (also known as SALPR or GPCR135), H3 relaxin but not H2 relaxin, porcine relaxin or INSL3, competed for [125I]-INSL5/H3 relaxin chimaera binding. RXFP3 was tagged at the carboxy-terminal tail with a variant of the green fluorescent protein, GFP2, and transiently expressed in CHO-K1 and HEK293 cells. Competition for [125I]-INSL5/H3 relaxin chimaera binding was observed with H3 relaxin in CHO-RXFP3-GFP2 (pIC50 = 8.61±0.20) and HEK-RXFP3-GFP2 cells (pIC50 = 8.93±0.50) and was consistent with that observed at wild type RXFP3 (pIC50 = 8.36±0.16 (CHO-K1); 9.04±0.31 (HEK293)). Similarly, potent H3 relaxin-stimulated ERK1/2 activation was observed downstream of RXFP3 (pEC50 = 9.47±0.32 (CHO-RXFP3-GFP2); 8.99±0.40 (HEK-RXFP3-GFP2) 8.81±0.25 (CHO-RXFP3); 9.04±0.31 (HEK-RXFP3)). Together these results suggested that RXFP3-GFP2 maintained the same phenotype as wild type RXFP3. Internalization of RXFP3 was observed using radioligand binding assays and confocal microscopy. Treatment of cells expressing RXFP3 with H3 relaxin but not H2 relaxin, porcine relaxin or INSL3 (all 10nM; n=4) reduced the number of cell surface binding sites. Likewise, loss of cell surface RXFP3-GFP2 binding sites was observed in cells treated with H3 relaxin (10nM) (n=6). RXFP3-GFP2 internalization visualized by confocal microscopy, showed surface-expressed receptors sequestered into vesicle-like structures 5-30 min after H3 relaxin (10nM) treatment. This study demonstrates for the first time that RXFP3 internalizes following treatment with H3 relaxin, but not other related peptides, in CHO-K1 and HEK293 cells transiently expressing the receptor.

13 Relaxin 2008

P-03

Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodination and Isolation of Peptide by One-step Ether Precipitation

Suode Zhang, Feng Lin, Mohammed Akhter Hossain, Fazel Shabanpoor, and John D. Wade


Multiple disulfide bond-containing peptides have long been challenging synthetic targets. The selection of appropriate sulfhydryl protecting groups is crucial for subsequent correct disulfide bond formation. Among various sulfhydryl protecting groups, the S-acetamidomethyl (Acm) protecting group is particularly valuable. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting groups and disulfide formation. This method is now commonly used. Unfortunately, there are often side reactions associated with this reaction, particularly with tryptophan and tyrosine residues, and thus excess iodine should be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation. This is usually achieved by addition of sodium bisulfate, sodium thiosulfate, ascorbic acid, powdered zinc dust, activated charcoal or by dilution with water followed by extraction with carbon tetrachloride. However, the quenching reagents themselves can generate additional side reactions as does lyophilization of the peptide solution. Thus a new method for the rapid quenching of the iodination and isolation of peptides is urgently required. In the present study, it was established that simple ether precipitation can fulfill these requirements. Peptides containing pairs of S-Acm protecting groups are dissolved in 95% acetic acid for iodination. After completion of the disulfide bond formation, ice-cooled ether is simply added to the reaction solution for quenching of iodination and simultaneous precipitation of peptide that can be stored or applied directly onto an HPLC column for purification. This method is particularly beneficial for the disulfide synthesis of insulin-like peptides such as INSL3.

P-04

Chemical Synthesis and Biological Activity Evaluation of β-Ala and γ-Abu Substituted Insulin-like Peptide 3 Analogs

Suode Zhanga, Ross A. Bathgatea, Geoffrey W. Tregeara, Richard A. Hughesb, and John D. Wadea

aHoward Florey Institute and bDepartment of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia

Insulin-like peptide 3 (INSL3, also known as relaxin-like factor, RLF) is now recognized to be a major circulating hormone in the male and female with important fertility regulatory roles. The receptor for INSL3, LGR8, is a member of the leucine-rich repeat containing G-protein coupled receptor family. Solution NMR spectroscopy showed that INSL3 adopts a characteristic relaxin/insulin-like fold but is a highly dynamic molecule. The B-chain contains a long helical segment which spans residues B12-B22. Detailed structure-activity studies using synthetic Ala-substituted analogues of INSL3 have shown that its receptor ectodomain binding site is mediated by five key residues within the B-chain, with four of these HisB12, ArgB16, ArgB20, ValB19 and Trp B27 located on one face of the helix. We designed two INSL3 analogs in which AlaB17 was substituted with the conformationally different β-Ala and γ-Abu which were expected to alter the position of the side chains of the critical ArgB16 and ValB19 as well as to confer protective effects from endoproteolysis. The peptides were prepared by optimized regioselective directed disulfide bond solid phase synthesis and, after purification, testing for their binding affinity for, and cAMP activity on, cells expressing LGR8. It was found that the β-Ala-substituted INSL3 analog had, slightly higher binding affinity together with increased potency in LGR8 expressing cells. In contrast, the γ-Abu-substituted INSL3 analog exhibited similar binding affinity and activity to native INSL3. The observations will provide new directions for the design of novel specific agonists and antagonists of the LGR8 receptor.


P-05

Synthesis, Conformation and Biological Activity of Novel Dicarba Analogs of Human Relaxin-3/INSL7

Mohammed Akhter Hossaina, Ross A.D. Bathgatea, Bianca J. van Lieropb, Andrea J. Robinsonb, K. Johan Rosengrenc, Geoffrey W. Tregeara, and John D. Wadea

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bSchool of Chemistry, Monash University, Clayton, Victoria 3168, Australia; cDepartment of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden

Disulfide bonds are common structural elements in many peptides and proteins where they play a crucial role in maintaining an active conformation. Such bonds can be susceptible to rupture by intracellular enzymes such as disulfide reductase which, in turn, can decrease the half life of the biomolecule rendering it less effective as therapeutic compound. The replacement of disulfide bonds with non-reducible isosteres is a useful means of increasing the in vivo stability of a protein. Consequently, we have replaced the intramolecular disulfide bond of human relaxin-3 (H3 relaxin), a peptide that has potential for the treatment of stress and obesity, with the physiologically stable dicarba bond. Solid phase synthesis was used to prepare an A-chain analogue in which the two cysteine residues that form the intramolecular bond were replaced with allylglycine. On-resin microwave-mediated ring closing metathesis was then employed to install the dicarba bridge. Subsequent cleavage of the peptide from the solid support, purification and B-chain combination via two intermolecular disulfide bonds, then furnished dicarba-relaxin-3. Two isomers (cis and trans) of the dicarba H3 relaxin were isolated and characterized by both CD and NMR spectroscopy which confirmed their structural similarity to the native peptide. Both analogs demonstrated equivalent binding affinity to native H3 relaxin on RXFP1 and RXFP3 expressing cells. However, although the cAMP activity of the analogs on RXFP3 expressing cells was the same as the native peptide, the potency on RXFP1 expressing cells was slightly lower. The in vivo stability of the peptides is currently under investigation.

P-06

Human Insulin-like Peptide 5 (INSL5): Synthesis and Activity on RXFP3 and RXFP4 Receptors

Mohammed Akhter Hossain, Ross A.D. Bathgate, Chze K. Kong, Geoffrey W. Tregear, and John D. Wade


Insulin-like peptide 5 (INSL5) was first identified by searching EST databases. Primary structure analysis showed it to be a prepropeptide that is predicted to be processed in vivo to yield a two-chain structure (A and B) containing the insulin-like disulfide crosslinks. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of INSL5/RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed in order to systematically investigate its structural and biological properties. A combination of solid phase methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to successful synthesis and required highly optimized conditions for their acquisition. In particular, the use of strong tertiary amidine, DBU, as Nα-deprotection base was required for the assembly of the B-chain. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, obtained in good overall yield, was shown to possess similar secondary structure like other insulin-like peptides. The peptide was able to inhibit cAMP activity in SKN-MC cells expressing the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast it had no activity on the human RXFP3 receptor. Synthetic INSL5 therefore demonstrates equivalent activity to the recombinant peptide and will be a useful tool to determine the biological function of INSL5.

15 Relaxin 2008

P-07

De novo Design and Synthesis of Cyclic and Linear Peptides to Mimic the B-chain Binding Elements of Human Relaxin Family Peptides

Mohammed Akhter Hossain, Ross A. D. Bathgate, Geoffrey W. Tregear, and John D. Wade


Human relaxin family peptides contain distinct amino acids in their B-chains that are responsible for the binding specificity to Relaxin Family Peptide Receptors (RXFP1-4). In particular the trivalent structure (R-X-X-X-R-X-X-I) within the B-chain of relaxin is responsible for its specificity to RXFP1. In conjunction with some of these residues Trp-27 in INSL3 and the equivalent Trp-25 in H3 relaxin are important for RXFP2 and RXFP3 activity respectively. In an attempt to mimic these motifs, we have designed six cyclic peptides using regioselectively addressable functionalized template (RAFT) to direct the side chains of Arg, Arg and Ile towards one direction with or without the additional Trp residue. RAFT, which adopts a solution structure based on an antiparallel beta sheet conformation closed by two beta turns centered on Pro-Gly dipeptides, is a means of arranging multiple functional groups in an appropriate orientation. Additionally, two domain minimized linear analogs of the H3 B-chain with internal salt bridges to increase their a-helicity were also made to compare their binding affinity with the RAFTs. None of the RAFT peptides showed any binding affinity for RXFP1, RXFP2 or RXFP3. Circular dichroism spectral analysis showed that all six RAFT molecules exhibited partial β-sheet structure along with two beta turns but no α-helix. This lack of α-helical structure is the likely reason for their lack of activity. The linear analogs showed similar affinity to RXFP3 as the native linear H3 relaxin B-chain suggesting that the internal salt bridges were unable to increase the α-helical structure and increase binding affinity.

P-08

Investigating the Molecular Mechanics of Relaxin Family Peptide Receptor Function Using Time Correlated Single Photon Counting Confocal Microscopy

Daniel J. Scotta, Brian Jonesb, Geoffrey W. Tregeara, and Ross A.D. Bathgatea

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bOlympus Australia, Mt. Waverley, Victoria 3149, Australia

G-protein coupled receptors are dynamic proteins and their cellular signaling properties are influenced by sub-cellular localization, molecular conformation and oligomerization. In order to investigate the molecular mechanics of relaxin family peptide (RXFP) receptors, one must be able to analyze receptor states at a single cell level. Recently developed time-correlated single photon counting (TCSPC) systems enable highly accurate measurement of fluorescence lifetime (FLIM) and fluorescence correlation spectroscopy (FCS), both powerful techniques for investigating protein dynamics and interactions, in single cells via a confocal microscope. TCSPC based confocal microscopy was used to investigate the molecular mechanics of RXFP1 and RXFP2 function, particularly focusing on the role of receptor oligomerization. RXFP1 and RXFP2 were tagged in several positions with various donor and acceptor fluorophores and their cellular localization and functional ability to increase cAMP in response to ligand was tested. Functional tagged receptor pairs were then utilized with TCSPC to measure FLIM-FRET by determining any decrease in fluorescence lifetime of donor fluorophores as a result of energy transfer to acceptor fluorophores. FLIM-FRET analysis in HEK cells expressing both donor tagged and acceptor tagged RXFP1 indicated a significant population of closely interacting receptors in all cellular areas of receptor expression. The signal could be removed by increasing expression of untagged RXFP1, indicating specific oligomerization. Now that the system is optimized, we aim to use FLIM-FRET and FCS to delve further into the molecular mechanics and conformational changes that occur during RXFP1 and RXFP2 activation, with the ultimate goal of aiding agonist and antagonist design.


P-09

Withdrawn

P-10

Identification of the Relaxin-responsive Cells in the Human Chorion and Decidua

Jaime S. Horton, Sandra Y. Yamamoto, and Gillian D. Bryant-Greenwood

Department of Cell and Molecular Biology, University of Hawaii, Honolulu, Hawaii, 96813 USA

The human fetal membranes lie between the myometrium and amniotic cavity forming the fetal container or sac. This complex tissue together with the decidua, forms a large maternal-fetal interface and is the site of the molecular cross-talk which results in human parturition. The decidua is a source of intrauterine relaxin and the choriodecidua expresses its receptor (LGR7). Since these tissues consist of a variety of maternal and fetal cells, we sought to identify the primary cell(s) responsible for LGR7 expression and relaxin responsiveness. Decidual stromal cells and macrophages were isolated by scraping the membranes, followed either by enzymatic digestion and Percoll gradient selection or by FACS cell sorting, based upon the markers CD14 and CD68. Chorionic cytotrophoblasts were isolated by stripping the amnion, enzymatic digestion and Percoll gradient selection. The stromal cells showed low LGR7 expression, but a 4 day treatment with decidualization cocktail significantly increased (p=0.03) LGR7 and prolactin expression. However, their cAMP response to relaxin was reduced. LGR7 expression by decidual macrophages was variable and about 4-fold less than THP-1 cells, correlating with their cAMP responses. On the other hand, cytotrophoblasts consistently expressed LGR7 and demonstrated a strong cAMP dose-response to relaxin. Therefore, the fetal cytotrophoblast is being used to further characterize its response(s) to relaxin. Supported by NIH grant HD24314.

17 Relaxin 2008

P-11

RXFP1 Is Expressed on Sperm Acrosome and Relaxin Stimulates the Acrosomal Reaction of Human Spermatozoa

Lisa Gianesello, Alberto Ferlin, Massimo Menegazzo, Anastasia Pepe, and Carlo Foresta

University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, 35121 Padova, Italy

The function of relaxin in human reproductive processes remain poorly understood. Nevertheless, relaxin is produced by the prostate in men and by the corpora lutea of ovaries and endometrium in women. Previous studies with contrasting results have suggested a possible role of relaxin on fertilization. In this study we analysed the expression of the relaxin receptor RXFP1 on human sperm and the in vitro effect of relaxin on sperm function. Measurement of relaxin concentration in the seminal plasma of 10 normozoospermic men showed that it is present at high levels (1253±445pg/ml). These concentrations are similar to those found in the serum of pregnant women (1000-3500pg/ml), and significantly higher to those found in the serum of the same male subjects (32±17pg/ml). RXFP1 protein was detected by immunofluorescence on the acrosomal cap of human spermatozoa. This data was confirmed by the co-localization of RXFP1 and the specific acrosomal staining Pisum Sativum, and by the disappearance of RXFP1 fluorescence after the induction of the acrosomal reaction by calcium-ionophore. The analysis of acrosome reaction in vitro under different doses of relaxin showed that the hormone increases the percentage of sperm reacted after 1 and 3h. Interestingly, the entity of acrosome reaction induced by relaxin is similar to that obtained with progesterone, the hormone produced by the corpus luteus and considered the physiological inductor of acrosome reaction. These data suggest a physiological role for relaxin produced by the prostate and/or the female genital tract after ovulation.

P-12

Involvement of Relaxin in Endometriosis

Sara S. Morellia, Felice Petragliab, Gerson Weissa, Stefano Luisib, Pasquale Floriob, Jeff Gardnerc, Andrea Wojtczuka, and Laura T. Goldsmitha

aObstetrics, Gynecology and Women’s Health, and cPediatrics, New Jersey Medical School, Newark, New Jersey, 07101, USA; bPediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy

Endometriosis, a disease characterized by the presence of endometrial stromal and glandular tissue at an extrauterine (ectopic) site, affects 10-15% of all reproductive-aged women and is an important contributing factor to chronic pelvic pain and infertility. Matrix metalloproteinases have been implicated in the establishment of endometriotic lesions. Since relaxin is a potent inhibitor of endometrial matrix metalloproteinase expression, we hypothesized that relaxin plays a role in endometriosis. Expression of relaxin mRNA and its LGR7 receptor mRNA in normal human endometrium were compared to expression in samples from patients with endometriosis. Total RNAs, extracted from ectopic (n=8) and eutopic (uterine) (n=11) endometrium of patients with endometriosis, and from endometrium of normal controls (n=12), were subjected to real time RT-PCR. Relaxin mRNA was detectable in normal endometrium from 9 of 12 (75%) control patients and detectable in a lower proportion of samples [9 of 19 (47.4%)] from patients with endometriosis. LGR7 mRNA was detectable in all samples, with lower expression in endometriosis samples than in endometrium from controls. Relaxin receptor LGR7 mRNA levels vary with cycle phase, with greater expression in the secretory phase (SP) than in proliferative phase (PP). In both phases, LGR7 mRNA levels were lower in ectopic samples than in either eutopic samples (6.2-fold lower in PP and 19.0-fold lower in SP) or endometrium from normal controls (6.5-fold lower in PP and 15.6-fold lower in SP). Decreased local expression of relaxin and LGR7 mRNA in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis.


P-13

Relaxin and Neopterin as Local Ovarian Granulosa Cell Growth- and Colony-stimulating Factors

Elissaveta Zvetkovaa, Roumiana Denkovaa, Violetta Bournevaa, Konstantza Balevaa, Yordanka Martinovaa, Boitcho Nikolova, Tanya Timevab, and Dietmar Fuchsc

aIEMAM-BAS, Sofia, Bulgaria; bInstitute of Obstetrics and Gynecology, MU Sofia, Bulgaria; cInnsbruck Medical Universty, Innsbruck, Austria

The influence of relaxin (RLX) and neopterin (NeoPt) on the porcine- and human ovarian granulosa cell proliferation, differentiation and steroidogenesis has been studied in vitro - in conditions of OGCscultures. It was established that two local biologically active substances stimulate in vitro OGCs proliferation /cell colony-formation, differentiation and steroidogenesis (Endocr. Regul., 32, 1998, 33 - 43; Acta morphologica et anthropologica, /Sofia/, 7, 2002, 28 - 31). The similar mitogenic effects of both cell growth factors - relaxin and neopterin, on the in vitro cultured OGCs, could be mediated through increased expression of inducible nitric oxide synthase and by activation of NOSII nuclear transcription factor NF-kappa B. The in vitro model indicating a role of relaxin peptide and of pteridine neopterin as ovarian granulosa cell proliferative and differentiating factors could be applied in further clinical studies on cellular and molecular mechanisms associated with physiological and/or neoplastic cell growth.

P-14

The Lethal Phenotype in Relaxin-deficient (Rln-/-) Mice Is Not Due to Impaired Mammary Gland Development or Milk Protein Synthesis

Laura J. Parrya, Lenka A. Vodstrcila,b, Anna Maddena, Stephanie H. Amira, Katrina Baldwina, Mary E. Wlodekb, and Kevin R. Nicholasa,c

aDepartments of Zoology and bPhysiology, The University of Melbourne, Parkville, Victoria 3010, Australia; cCRC for Innovative Dairy Products, Melbourne, Australia

Relaxin is essential for nipple development during late pregnancy in mice and may also stimulate mammary gland growth. This study tested the hypothesis that relaxin is involved in milk protein synthesis as well as mammary gland development in pregnant mice. Comparisons between wildtype (Rln+/+) and relaxin-deficient (Rln-/-) mice showed no obvious differences in either lobuloalveolar structure or ductal branching in the mammary gland. Mammary explants obtained from Rln-/- mice on day 16.5 gestation expressed β-casein and α-lactalbumin in response to lactogenic hormones at a similar level to Rln+/+ mice, implying normal milk protein synthesis. Exogenous relaxin treatment had no significant effect on milk protein expression in mammary tissue in vitro. These data are explained by a lack of relaxin receptors in mouse mammary tissues. Pregnant Rln-/- mice chronically infused with human relaxin for 6 days gave birth to live pups without difficulty, and 96% pups survived beyond 7 days. However, birth weights of the relaxin-treated Rln-/- young were significantly lower than Rln+/+ pups. Infusion of relaxin for 3 days was not sufficient to prevent pup mortality. The lactiferous ducts of relaxin-treated Rln-/- mice were devoid of milk, but there were no developmental differences in the lobuloalveolar tissue compared with saline-infused controls. In summary, mammary gland development in Rln-/- mice is not different from Rln+/+ mice and relaxin is not critical for β-casein or α-lactalbumin synthesis. The lethal phenotype in Rln-/- mice results from insufficient milk delivery caused by impaired nipple development and inadequate sucking stimulus from the pups.

19 Relaxin 2008

P-15

Laser Microdissection of Neonatal Porcine Endometrium for Tissue Specific Evaluation of Relaxin Receptor (RXFP1) Expression in Response to Perinatal Zearalenone Exposure

Anne A. Wileya, Johannes Kauffoldb, Martin Wähnerc, Bethany Crean-Harrisa, Dori J. Millera, Carol A. Bagnelld, and Frank F. Bartola,e

Departments of aAnimal Sciences and eAnatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA; bFaculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany; cAnhalt University of Applied Science, 06406 Bernburg, Germany; dDepartment of Animal Sciences, Endocrinology and Animal Biosciences Program, Rutgers University, New Brunswick, New Jersey 08901, USA

In the pig (Sus scrofa domesticus), relaxin receptor (RXFP1) expression is detectable in the endometrium at birth (postnatal day = PND 0). Relaxin can stimulate and may support organizationally critical estrogen receptor (ER) -α expression in the endometrium and endometrial RXFP1 expression is estrogen-sensitive in the neonatal pig. Here, objectives were to: (1) employ laser microdissection (LMD) to enable tissue-specific quantification of the effects of perinatal exposure to zearalenone (ZEA), a selective ER modulator, on endometrial RXFP1 expression; and (2) relate such effects to glandular epithelial cell proliferation patterns as reflected by proliferating cell nuclear antigen (PCNA) labeling index (LI) in the neonatal porcine uterus. Litters from pregnant gilts fed ZEA (1500μg ZEA/kg feed/day) or vehicle from 14 days prepartum through postpartum day 21 were used as a source of neonatal tissues. Neonates were cross-fostered at birth to generate four ZEA exposure groups (n=6 neonates/group), including unexposed controls and exposures limited to pre-, post-, or pre- and postnatal periods. Uteri, obtained on PND 20-21, were fixed in Xpress Molecular Fixative (Sakura Finetek, USA) and tissue cross-sections (7μm) were subjected to LMD. Total RNA extracted from excised endometrium (approximately 800ng from 10-14 cross-sections/neonate) was evaluated for RXFP1 mRNA by quantitative RT-PCR. Epithelial PCNA LI was determined using immunohistochemistry and image analysis. Both endometrial RXFP1 expression (P<0.06) and glandular epithelial PCNA LI (P<0.05) were reduced on postnatal day 20-21 in ZEA-exposed neonates. Thus, perinatal ZEA exposure affects molecular and cellular events, including RXFP1 expression, associated with endometrial programming in the neonatal pig. (Support: AiF ‘’Otto von Guericke’’ e.V.; NSF EPS-0447675; USDA 2007-35203-18098)

P-16

The Effects of Relaxin-deficiency on Uterine Angiogenesis in Early Pregnancy

Jane E. Girlinga, Lee Lee Ngb, and Laura J. Parryb

aCentre for Women’s Health Research, Monash Institute of Medical Research and Monash University Department of Obstetrics and Gynaecology, Clayton, Victoria 3168, Australia; bDepartment of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia

The human endometrium undergoes regular periods of growth and regression during the menstrual cycle, including concomitant remodelling of the vasculature. Previous studies have used mouse models to show that oestrogen and progesterone stimulate angiogenesis (new blood vessel growth) within the endometrium. Endometrial angiogenesis also occurs in the early stages of mouse pregnancy, which coincides with an increase in circulating progesterone. Relaxin stimulates VEGF from human endometrial stromal cells in vitro and increases endometrial vascularization in ovariectomized steroid-primed primates in vivo. To date, no studies have investigated the effects of relaxin on endometrial angiogenesis in early pregnancy. Our aim was to test the hypothesis that endometrial angiogenesis would be reduced in relaxin-deficient mice (Rln-/-) in comparison to their wildtype (Rln+/+) counterparts. Uterine tissues were collected from Rln-/- and Rln+/+ mice on days 1 to 4 of pregnancy, before implantation. All mice were treated with BrdU prior to dissection to allow the number of blood vessel profiles containing proliferating endothelial cells (PVPs) to be quantified by double CD31/BrdU immunohistochemistry. Consistent with published studies, PVPs were first observed on days 3 and 4 of pregnancy. However, the percentage of PVPs was reduced in Rln-/- mice compared to Rln+/+ mice (Day 3: median = 4.4% versus 19.6%, Day 4: 9.6% versus 22.2%). We subsequently identified relaxin and relaxin receptors in the mouse endometrium in early pregnancy. Our data suggest that locally synthesized relaxin acts in synergy with progesterone to initiate endometrial angiogenesis in early pregnancy.


P-17

The Effects of Fetal and Placental Numbers on Blood Concentrations of Relaxin in the Rabbit

Phillip A. Fieldsa and Michael J. Fieldsb

a Department of Cell Biology and Neuroscience, University of South Alabama College of Medicine, Mobile, Alabama 36688, USA; b Animal Sciences Department, University of Florida, Gainesville, Florida 32611, USA

The following study was conducted in order to determine the effects of conceptus (fetus and placenta) and/or fetal numbers on the blood concentrations of relaxin in the rabbit. Experiment 1: Day 18 pregnant rabbit conceptuses were surgically removed through incisions in the uterine horns. Rabbits were left with either 7, 5, 3 or 1 intact implantation sites. Blood was collected from the ear vein on days 5, 10, 15, 18, 25, 30, 32 and 35 of pregnancy and relaxin concentrations determined by RIA using porcine relaxin antiserum. There was no statistical difference between relaxin concentrations in rabbits carrying 1 vs 7 conceptuses. Experiment 2: A total fetectomy was conducted on day 18 pregnant rabbits. However, not all placentas were removed. Rabbits were left with either 7, 5, 3 or 1 placentas minus fetuses. Blood was collected from the ear vein on days 5, 10, 18, 20, 25, 30, 32 and 35 of pregnancy and relaxin concentrations determined by RIA using porcine relaxin antiserum. There was no significant difference in the peak concentrations of relaxin observed at day 25. However, there was a significantly sharper decrease in relaxin concentrations after day 25 in rabbits with only 1 placenta vs those carrying 7, 5 or 3 placentas. The results indicate that the number of conceptuses does not influence blood concentrations of relaxin in the rabbit and that there is potentially a compensatory mechanism with regard to placental size and relaxin secretion.

P-18

Cervical Softening in the Common Marmoset Monkey (Callithrix jacchus)

Christina Simon and Almuth Einspanier

Institute of Physiological Chemistry, Faculty of Veterinary Medicine, An den Tierkliniken 1, 04103 Leipzig, Germany

Tissue remodeling of the cervix is required for the last trimester of pregnancy, which is under endocrine control, e.g. relaxin (RLX) and estradiol (E2). The purpose of the present study was to examine the influence of RLX and E2 on the cervical tissue of the marmoset monkey in two experimental designs. Firstly, an in vivo experiment was set up to determine the intracervical diameter under topically applied RLX. Secondly, an in vitro experiment was carried out to investigate cervical tissue of ovariectomized marmosets treated systemically with E2, RLX and a combination thereof. In vivo locally applied RLX induced a distinct significant widening of the cervix (0.84±0.4 mm in diameter). Under in vitro investigation the wet weights of the cervical tissue were significantly increased after all three hormonal treatments (E2: 0.27±0.07g, RLX: 0.25±0.04g, E2+RLX: 0.30±0.11g vs. control: 0.10±0.04g). Furthermore, loosening of the connective tissue structure and an increase in the number of eosinophile cells as well as higher expression of matrix metalloproteinases and the relaxin receptor RXFP1 were detected in the cervical tissue after all hormone treatments compared to controls. However, strongest signals of all parameters were present under combined treatment with E2 and RLX. To sum up, our results demonstrated for both hormones a widening and a remodeling effect on the primate cervix with the most prominent effects under combined application of E2 and RLX, suggesting mutual influences in their effect on cervical tissue.

This project was supported by the DFG (German Research Council) grant Ei 333/11-1.

21 Relaxin 2008

P-19

Perinatal Zearalenone Exposure Affects RXFP1, RXFP2 and Morphoregulatory Gene Expression in the Neonatal Porcine Uterus

Joseph C. Chena, Anne A. Wileyb, Johannes Kauffoldc, Martin Wähnerd, Frank F. Bartolb,e, and Carol A. Bagnella

aDepartment of Animal Science, Rutgers University, New Brunswick, New Jersey 08901, USA; Departments of bAnimal Sciences and eAnatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA; cFaculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany; dAnhalt University of Applied Science, 06406 Bernburg, Germany

The mycotoxin zearalenone (ZEA) is a selective estrogen receptor modulator (SERM) that can contaminate cereal feeds leading to reproductive disorders. ZEA consumption by periparturient females may result in exposure of offspring to SERMs during pre- and postnatal life, the latter as a consequence of consumption of xenoestrogen-contaminated milk. Exposure of gilts to estrogen within 14 days from birth disrupts the uterine developmental program and has lasting effects on adult uterine capacity. Such effects are marked by alterations in normal patterns of uterine relaxin/insulin-like factor 3 receptors (RXFP1/RXFP2) and morphoregulatory gene expression (i.e. Wnt/Hoxa axis). To determine effects of perinatal ZEA exposure on uterine expression of genes associated with endometrial development in the neonate, pregnant sows were fed ZEA (1500 µg ZEA/kg feed/day) or vehicle from 14 days before farrowing through postnatal day (PND) 20-21, when neonatal uterine tissues were collected. At birth, gilts were cross-fostered to generate four ZEA exposure groups (n=5-6/group): unexposed controls or exposures limited to prenatal, postnatal, or pre and postnatal (continuous) periods. Transcripts were measured by quantitative RT-PCR using cyclophilin as a housekeeping gene. At PND 20-21, uterine Wnt7a, Hoxa10 and RXFP2 mRNA levels were decreased in neonates exposed continuously to ZEA (p<0.05). Uterine RXFP1 mRNA levels were decreased in postnatal and continuously exposed groups (p<0.05). Uterine Wnt4 mRNA levels were unchanged. Results indicate that perinatal exposure to milk-borne ZEA can affect gene expression associated with neonatal uterine programming by altering expression of patterning and relaxin family receptor genes. (Support: AiF "Otto von Guericke" e.V; USDA 2007-35203-18098)

P-20

The Effects of Blocking Progesterone Action on Relaxin Receptor Expression in the Myometrium of Pregnant Rats

Lenka A Vodstrcila,b, Laura J Parrya, Stephen J Lyec, Oksana Shynlovac, and Mary E Wlodekb

aDepartments of Zoology and bPhysiology, The University of Melbourne, Parkville, Victoria 3010, Australia; cSamuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario M5G1X5, Canada

The actions of relaxin are mediated by a Type C GPCR, Lgr7 (RXFP1). Myometrial Lgr7 expression is highest in early- to mid- gestation in the rat but decreases in late gestation when serum levels of relaxin are elevated and progesterone (P4) decreases. Therefore, we hypothesized that functional P4 withdrawal is necessary for the down-regulation in myometrial Lgr7 at term. Conversely, preventing P4 withdrawal will inhibit the decrease in Lgr7 expression. We conducted two studies to test these hypotheses. I) Rats were treated with the P4 receptor antagonist, RU486 (10mg/kg), or vehicle on day 16 (n=6) or 19 (n=4) of gestation and myometrial tissue was collected 24 hours later. II) Pregnant rats received 4 daily subcutaneous injections of P4 (16mg/kg) starting on day 20 gestation to prevent P4 withdrawal (n=4/stage; day 20, 21, 22 and 23). Myometrial Lgr7 expression was compared with vehicle treated controls. Administration of RU486 to block the actions of P4 caused a significant decrease in myometrial Lgr7 gene expression in treatment groups at both stages of gestation, compared with controls. In contrast, daily injections of P4 had no significant effect on Lgr7 expression after 1-4 days of treatment. These data demonstrate that functional progesterone withdrawal contributes to the down-regulation in myometrial Lgr7 at the end of pregnancy. However, prolonging the length of gestation with P4 did not compensate for the inhibitory effects of the fetal-placental unit and/or high circulating relaxin at the stages examined.


O-18

Resolving the Unconventional Mechanisms Underlying RXFP1 and RXFP2 Receptor Function

Ross A.D. Bathgatea,b, Daniel J. Scotta, Gabrielle E. Callandera, Tracey N. Wilkinsona, Emma J. Hopkinsa, Despina E. Ganellaa, Brigham J. Hartleya, Sharon Layfielda, Tania Ferraroa, Paul Gooleyb, Marco Mudac, John D. Wadea, and Geoffrey W. Tregeara,b

aHoward Florey Institute and bDepartment of Biochemistry and Molecular Biology, The University of Melbourne, Victoria 3010, Australia; cSerono Research Institute, Rockland, Massachusetts 02370, USA

The receptors for relaxin and INSL3 are now well characterized as Relaxin Family Peptide (RXFP) receptors, RXFP1 and RXFP2, respectively. They are G-protein coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs). Additionally RXFP1 and RXFP2 are unique in the LGR family in that they contain an LDL class A (LDLa) module at their N-terminus. Studies from our lab and others have demonstrated that activation of RXFP1 and RXFP2 involves a three stage process. Primary ligand binding occurs via interactions between B-chain residues of the peptides with specific residues in the leucine rich repeats (LRRs) of the ectodomain. There is a secondary binding site in the transmembrane (TM) exoloops which may be mediated by A-chain interactions. Receptor signalling through cAMP then requires the unique LDLa module as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR. Furthermore, we have identified truncated RXFP1 splice variants that are secreted proteins containing the LDLa module. These variants act as antagonists of RXFP1 function and may be endogenous regulators of RXFP1 action. Moreover, we have discovered numerous additional human RXFP1 and RXFP2 splice variants that are co-expressed in numerous human tissues and may have important modulatory roles. Current studies are focussed on resolving the sites of secondary binding interaction, the role of the LDLa module in activation and the influence of receptor oligomerization on receptor function.

O-19

Mechanisms of Relaxin Receptor (LGR7/RXFP1) Expression and Function

Andras Kern and Gillian D. Bryant-Greenwood

Pacific Biosciences Research Center, University of Hawaii, Honolulu, Hawaii 96822, USA

The LGR7/RXFP1 and LGR8/RXFP2 receptors are unique receptors among the GPCRs in having an LDL-A module. Their complex gene organization, among the intron richest of the GPCRs, suggests that alternative splicing is a common occurrence. We have therefore investigated the role of the LDL-A module and showed the identity, expression and functions of three LGR7 splice variants in the human decidua. Point mutations of conserved residues or complete deletion of this module resulted in loss of the cAMP response to relaxin. Its glycosylation also impacted LGR7 cell surface delivery and therefore receptor activation. The wild-type (WT) LGR7 was expressed as both precursor and mature forms, but deletion of the LDL-A module resulted in expression of only the mature form. Three new alternatively spliced variants of LGR7 were identified, all containing a truncated extracellular region. Their functional characterization showed them exerting dominant negative effects on the WT-LGR7, by preventing its homodimerization, maturation and subsequent trafficking to the cell surface, resulting in loss of function. The treatment of cells with relaxin induced desensitization and internalization of LGR7 into endosomal vesicles. Internalization was associated more with β-arrestin 2 than β-arrestin 1. In summary, different mechanisms have been identified for controlling the cell surface expression and function of the LGR7 protein, which are likely to be significant for the role of relaxin in human parturition.

Supported by NIH grant HD24314.

23 Relaxin 2008

O-20

Relaxin Family Peptide Receptor 1 (RXFP1) Activation Stimulates the Peroxisome Proliferator-activated Receptor Gamma

Sudhir Singha and Robert G. Bennetta,b,c

aDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA; bDepartments of Internal Medicine and Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA; cVA Medical Center, Omaha, Nebraska 68105, USA

Relaxin has antifibrotic effects in a number of tissues. Many of these effects are similar to those induced by activators of peroxisome proliferator-activated receptorγ (PPARγ), raising the possibility that a mechanism for relaxin’s antifibrotic effects may involve activation of the PPARγ pathway. To test this possibility, PPAR activity was assessed by transient transfection of 293T cells with RXFP1 and a reporter vector with a PPAR response element upstream of luciferase (PPRE-luc). Relaxin increased PPRE activation in a concentration-dependent manner, maximally at 1nM, with an EC50 of 24 pM. In contrast, InsL3 did not activate PPRE, demonstrating that the relaxin effect was RXFP1 specific. The PPARγ agonists rosiglitazone (rosi) and 15-deoxy-δ12,14-prostaglandin J2 (15dPGJ2) increased PPRE activity, but PPARα or PPARδ agonists had little effect. The combined effect of relaxin+rosi and relaxin+15dPGJ2 was more than additive (Relaxin (Rlx) 169.2±9.1%, Rosi 158.7±15.0%, Rlx+Rosi 270±25.9%, PGJ 135.5±22.1%, Rlx+PGJ 248±41.6%). A PPARγ ligand blocker (GW9662) reduced PPRE activation in response to rosi but not relaxin, suggesting that relaxin stimulates ligand-independent PPAR activation. Relaxin’s effect was mimicked by forskolin, but not blocked by pertussis toxin, consistent with a Gαs coupled mechanism. Finally, both relaxin and rosi increased the levels of CD36, a PPARγ target gene, in THP-1 cells, suggesting that relaxin stimulates PPARγ activity. In summary, relaxin activates PPARγ and the combined effect of relaxin and PPARγ agonists was superadditive, suggesting that both agents might be used together for an increased antifibrotic effect. The better understanding of this pathway might help in amelioration of fibrotic diseases.

O-21

Development of a High-throughput Receptor-binding Assay using Time-Resolved Fluorescent Europium Lanthanide for Screening Insulin-like Peptide 3 (INSL3) Analogues for their Receptor Binding Affinity

Fazel Shabanpoora,b, Richard A. Hughesc, Ross A.D. Bathgatea, Frances Separovicb, and John D.Wadea

aHoward Florey Institute, bSchool of Chemistry, cDepartment of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia

The assessment of the receptor binding affinity of analogues of INSL3 typically requires the use of 125I-labelled INSL3 which has a number of disadvantages including its short half-life, high cost, the need to be prepared prior to use and radioactivity safety issues. To overcome these problems, we have developed a high throughput receptor binding assay using a europium-based time-resolved fluorescence (TRF) label. The emitted fluorescent light from this probe has unique characteristics of long fluorescence halftime, a large Stoke’s shift and a sharp emission peak, which allow measurement of signal to be made after the decay of short-lived background fluorescence. Initially, INSL3 was labeled post-synthesis with a europium chelate at free amino groups. Competition binding assays were undertaken to determine the affinity (pIC50) of labeled INSL3 for RXFP2 in HEK 293 cell line expressing RXFP2. The pIC50 of the INSL3 for RXFP2 using Eu-INSL3 was similar to that of 125I-labelled INSL3, 9.45 and 9.35 respectively. The europium TRF receptor-binding assay is quicker than 125I-radioactive assay as it is possible to directly measure the level of binding without the need to transfer the cells. Moreover, the Eu-INSL3 had a low nonspecific binding and high signal to noise ratio. However, post-synthesis labeling of INSL3 has the disadvantage of not knowing where the Eu-chelate binds and the number of chelates per INSL3 molecule. Consequently we have developed efficient solid phase synthesis technique to prepare specifically mono-labelled peptide, which we have shown possesses optimum biophysical properties for receptor binding studies.


O-22

Structure, Function and Evolution of Ligands and Receptors of the Insulin/Relaxin Peptide Family

Pierre De Meytsa, Angela Manegold Svendsena, Waseem Sajida, Milka Vreclb, Anders Hedingc, John D. Waded, Ross A.D. Bathgated, and Jane Nøhra

aReceptor Systems Biology Laboratory, Hagedorn Research Institute, DK-2820 Gentofte, Denmark; bInstitute of Anatomy, Histology and Embryology, Veterinary Faculty, University of Ljubljana, Sl-1000 Ljubljana, Slovenia; c7TM Pharma A/S, DK-2970 Hørsholm, Denmark; dHoward Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Parkville, Victoria 3010, Australia

The insulin/IGF/relaxin peptide family in humans comprises three ligands (insulin, IGF-I and IGF-II) that bind to RTKs, and seven peptides related to relaxin that bind to GPCRs. The recent crystallographic structure of the insulin receptor (IR) extracellular domain has ended decades of speculation regarding the mechanism of IR binding and negative cooperativity. It supports a model whereby two sites on insulin crosslink alternatively two partial binding sites on each IR half. Ligand-induced or -stabilized receptor dimerization or oligomerization is a general feature of RTKs as well as cytokine receptors. Surprizingly, recent studies indicate that constitutive dimerization and negative cooperativity is also an ubiquitous property of GPCRs, showing allosteric mechanisms and negative cooperativity similar to those described for the IR. I will describe the similarities and differences in the insulin and IGF-I binding kinetics and mechanisms of activation of both IR and IGF-IRs and in the binding of H2 relaxin, INSL3, and a chimeric INSL3/relaxin ligand to RXFP1, RXFP2, and RXFP3 respectively.

O-23

Activation of Relaxin Related Receptors by Short, Linear Peptides Derived from a Collagen Containing Precursor

Ronen Shemesha, Chen Hermesha, Amir Toporika, Zurit Levinea, Amit Novika, Assaf Woola, Yossef Kligera, Avi Rosenberga, Ross A.D. Bathgateb, and Yossi Cohena

aCompugen Ltd., Tel-Aviv, Israel; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

In a screening effort, based on algorithmic prediction for novel GPCR peptide activators, we were able to identify and examine two novel peptides, short, linear and derived from a natural, previously unidentified precursor protein, containing a collagen-like repeat. Both peptides seemed to show a significant cAMP stimulatory and inhibitory effect on CHO-K1 cells transiently transfected with both LGR7 (RXFP1) and LGR8 (RXFP2), after treatment with cAMP generating Forskolin, as compared to the same cells treated with Forskolin+Relaxin. This activation was not found for the Relaxin 3 receptor (RXFP3). In a set of follow-up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP by 5uM of Forskolin in cells transfected with either LGR7 or LGR8. In dye-free cell impedance GPCR activation assay analysis, we were able to show that these peptides were also able to activate a cellular responses mediated by these receptors. Although untransfected CHO-K1 cells showed some cellular activation by both Relaxin and at least by one of our newly discovered peptides, LGR7 and LGR8 transfected cells showed a stronger activation indicating a strong activation of a cellular response through activation of these receptors.

In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the Relaxin/Insulin like family, are potential ligands for the Relaxin related family of receptors, and as such might serve as novel candidates for Relaxin related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.

25 Relaxin 2008

O-24

RXFP1 Couples to the Gαi3-Gβγ-PI3K-PKCζ Pathway via the Final 10 Amino Acids of the Receptor C-terminal Tail

Michelle L. Hallsa, Maria Papaioannoua, John D. Wade b, Bronwyn A. Evansa, Ross A.D. Bathgate b, and Roger J. Summersa


The relaxin family peptide receptors, RXFP1 and RXFP2, are highly similar receptors that share approximately 80% amino acid sequence homology. Constitutively active receptors couple to increased cAMP accumulation, important for relaxin-mediated decidualisation and myometrial inhibition. Despite the high homology the receptors couple to different G-proteins to affect cAMP accumulation. Both RXFP1 and RXFP2 couple to Gαs to increase, and GαoB to negatively modulate cAMP accumulation. However, only RXFP1 can couple to Gαi3 with time to activate a Gβγ-PI3K-PKCζ pathway to further increase cAMP accumulation. This study aimed to determine the region of RXFP1 that directs coupling to the delayed Gαi3 pathway using receptor mutagenesis. Receptor chimeras were initially examined: RXFP1/2 (ectodomain, ED, of RXFP1; membrane-anchored domain, MAD, of RXFP2) lacked signalling via Gαi3 whereas RXFP2/1 (ED of RXFP2; MAD of RXFP1) could activate the delayed pathway in response to both H2 relaxin and INSL3. This not only suggests that activation of the Gαi3 pathway is dependent upon the MAD of RXFP1, but that this activation is not unique to H2 relaxin. A truncated RXFP1 receptor (lacking the C-terminal tail) was also unable to activate the Gαi3 pathway, as was a receptor lacking the final 10 amino acids of the RXFP1 C-terminus. Additionally, a receptor construct in which the final 10 amino acids of RXFP1 was added to the C-terminal tail of RXFP2 demonstrated gain-of-function signalling via the Gαi3 pathway. Thus activation of the delayed Gαi3-Gβγ-PI3K-PKCζ cAMP pathway by RXFP1 is dependent upon the C-terminal 10 amino acids of the receptor.

O-25

Local Regulation of Relaxin Receptors in the Myometrium of Pregnant Rats

Lenka A. Vodstrcila,b, Oksana Shynlovac, Stephen J. Lyec, Mary E. Wlodekb, and Laura J. Parrya

aDepartments of Zoology and bPhysiology, The University of Melbourne, Parkville, Victoria 3010, Australia; cSamuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario M5G1X5, Canada

Relaxin’s actions are mediated by a Type C GPCR, Lgr7 (RXFP1). In pregnant mice, there is a significant down-regulation in myometrial Lgr7 expression at term. The regulatory mechanisms are unknown but could involve local, fetal-placental factors. To address this question, we investigated Lgr7 expression in two models of unilateral pregnancy by qPCR. The gestational profile of myometrial Lgr7 expression was first established in non-pregnant (NP), pregnant and postpartum (PP) rats. A second group of rats had one ovarian tube ligated before mating and tissues were collected from gravid and non-gravid horns at various gestational stages. The tammar wallaby has separate gravid and non-gravid uteri, so myometrium was collected from paired uteri at different stages of gestation. Lgr7 was expressed in the rat myometrium at similar levels to NP in early- to mid-gestation, with a significant reduction (70%) in late gestation. Postpartum receptor expression returned to NP levels. A similar pattern of Lgr7 gene expression was seen in the cervix without the increase postpartum. A comparison of Lgr7 expression in different rat tissues on day 20 gestation demonstrated highest expression in the cervix > myometrium = endometrium = vagina >> placenta = fetal membranes. In the two unilateral pregnancy studies, Lgr7 expression was lower in the gravid uterus compared to the non-gravid uterus, demonstrating a local influence of the fetal-placental unit on myometrial Lgr7 expression. Our data suggest that the fetal-placental unit contributes to the down-regulation in myometrial Lgr7 at term, thereby removing the inhibitory effects of relaxin on uterine contractility.


O-26

The Role of the N-Terminal Leucine Rich Repeat Cap in Mediating INSL3 Specificity to Relaxin Family Peptide Receptor 2

Daniel J. Scott, Tracey Wilkinson, Geoffrey W. Tregear, and Ross A.D. Bathgate


We recently defined the leucine-rich repeat (LRR) residues involved in primary INSL3 binding to relaxin family peptide receptor 2 (RXFP2). Many of these residues are conserved in RXFP1 and have been previously implicated in relaxin binding. The ability of RXFP2 to accommodate relaxin and INSL3 binding suggests conservation of hormone binding conformations in these two receptors. However, the inability of INSL3 to bind RXFP1 indicates a degree of specificity in the INSL3/RXFP2 interaction. We postulated that RXFP2 unique residues are responsible for mediating such specificity, and inserting such residues into RXFP1 may rescue INSL3 binding. RXFP2 unique residues were identified using multiple sequence alignments and inserted into RXFP1 to generate RXFP1/2 chimeric receptors. Each RXFP1/2 chimera was expressed in HEK cells and their ability to bind INSL3 tested. Only one RXFP1/2 chimera exhibited significant INSL3 binding, RXFP1/2 βN2, which contained 3 RXFP2 specific residues from the N-terminal LRR cap. Interestingly, replacement of any of these residues with alanine in RXFP2 resulted in no significant loss of INSL3 binding, indicating that these residues do not bind INSL3. Instead, competition binding assays utilizing mutant INSL3 peptides suggested that the residues inserted into RXFP1/2 βN2 induced structural changes in the LRRs of RXFP1, allowing the basic residues from the B-chain of INSL3 to interact with conserved acidic residues in LRR IV and LRR VI. Overall this demonstrated that there are crucial differences between the relaxin/RXFP1 and the INSL3/RXFP2 interactions, which will be important to consider when designing relaxin and INSL3 mimetics.

O-27

The Relaxin Family Peptide Receptors (RXFPs): A New Subgroup of Dimeric, Cooperative GPCRs

Angela Manegold Svendsena, Julie Køniga, Line Eggebrecht Beka, Milka Vreclb, Anders Hedingc, Jesper Bøggild Kristensend, John D. Wadee, Ross A.D. Bathgatee, Pierre De Meytsa, and Jane Nøhra

aReceptor Systems Biology Laboratory, Hagedorn Research Institute, DK-2820 Gentofte, Denmark; bInstitute of Anatomy, Histology and Embryology, Veterinary Faculty, University of Ljubljana, Sl-1000 Ljubljana, Slovenia; c7TM Pharma A/S, DK-2970 Hørsholm, Denmark; dChemical API Supply Isotopes, Novo Nordisk, Novo Nordisk Park, DK-2760 Måløv, Denmark; eHoward Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Parkville, Victoria 3010, Australia

Following the demonstration of negative cooperativity for insulin (and later IGF-I) receptors (which are dimeric RTKs), it was shown over 30 years ago that β2-adrenergic and TSH receptors (now known to be GPCRs) also exhibit negative cooperativity and therefore might function as dimers or oligomers, but the concept of dimerization in GPCRs was only recently experimentally validated. A connection between negative cooperativity and dimerization was recently demonstrated for the glycoprotein hormone receptors, including the TSH and FSH receptors, members of the LGR subgroup of GPCRs.

To study the dimerization and cooperativity of the GPCRs for relaxin-like peptides, RXFP1, RXFP2 (also LGRs), and RXFP3, we investigated the kinetics of ligand binding to these receptors. To further evaluate the dimerization, we performed Bioluminescence Resonance Energy Transfer experiments (BRET).

We showed that dissociation of prebound H2 relaxin, INSL3, and a chimeric INSL5/relaxin ligand from RXFP1, RXFP2, and RXFP3 respectively was accelerated by unlabeled ligand in an ‘’infinite dilution’’ protocol, indicating the presence of negative cooperativity. This suggested that these receptors function as dimers or oligomers. The dose response curve for negative cooperativity for RXFP2 was bell-shaped (like the curve for insulin receptors), whereas it was monophasic in the case of RXFP1 and RXFP3 (like the curve for IGF-I receptors).

BRET experiments for RXFP1 and RXFP2 confirmed that constitutive dimerization occurs through the 7TM domain. The ectodomain stabilized the dimerization in both cases. We furthermore demonstrated the heterodimerization of RXFP1 and RXFP2. BRET experiments are in progress to study the possible homodimerization of RXFP3.

27 Relaxin 2008

O-28

INSL3/RXFP2 Signaling in Testicular Descent: Mice and MenShu Fenga, Alberto Ferlinb, Natalia Bogatchevaa, Anne Truonga, Sean Corbettc, Shuo Hanc, Dolores Lambc, Carlo Forestab, and Alexander I. Agoulnika

aDepartment of Obstetrics and Gynecology, cScott Department of Urology, Baylor College of Medicine, Houston, Texas 77030, USA; bDepartment of Histology, Microbiology and Medical Biotechnologies, Centre for Male Gamete Cryopreservation, University of Padova, Padova, Italy

The mutations of insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. We performed mutation screening in human patients with cryptorchidism from different populations in Europe and USA. Several missense mutations were identified both in INSL3 and RXFP2 genes. The RXFP2 mutation T222P is the only one strongly associated with the mutant phenotype found in more than 1000 patients and controls analyzed. The expression analysis of T222P mutant receptor transfected into 293T cells revealed that the mutation severely compromised receptor cell membrane expression. We have also detected a number of mutations in INSL3 gene including a novel mutation in a family with a history of undescended testis. In mice the Rxfp2 gene expression was markedly increased after birth in male reproductive tissues and was readily detectable in the epididymis, Leydig cells, and germ cells of the testis. The strongest expression was detected in adult cremaster muscle. INSL3 treatment increased cell proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine signaling in these cells. We generated RXFP2-iCre transgenic mice expressing improved Cre recombinase (iCre) under the control of a 2.4-kb mouse RXFP2 promoter. The iCre was expressed in the gubernacular ligament at E14.5, indicating that this promoter is able to drive RXFP2 gene expression during transabdominal testis descent. Finally, we have produced mice with cre/loxP activated shRNA transgene targeting RXFP2. The analysis of the receptor knockdown in different organs and at different developmental stages is currently underway.

O-29

New Roles for INSL3 in Adults: Regulation of Bone Metabolism and Association of RXFP2 Gene Mutations with Osteoporosis

Alberto Ferlina, Anastasia Pepea, Lisa Gianeselloa, Andrea Garollaa, Shu Fengb, Arianna Facciollia, Roy Morelloc, Alexander I. Agoulnikb, and Carlo Forestaa

aUniversity of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, 35121 Padova, Italy; bDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA; cDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA

Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells and acts by binding to its specific G-protein coupled receptor RXFP2 (Relaxin family peptide 2). INSL3 is involved in testicular descent and mutations in INSL3 and RXFP2 genes cause cryptorchidism. The physiological role of INSL3 in adults unknown, although substantial INSL3 circulating levels are present. After extensive clinical, biochemical and hormonal investigation, including bone densitometry by DEXA, on 25 young men (age 27-41) with the well characterized T222P mutation in the RXFP2 gene, we found that 16 of them (64%) have significantly reduced bone density. No other cause of osteoporosis was evident in these subjects, whose testosterone and gonadal function were normal. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures performed by RT-PCR and immunohistochemistry showed the presence of RXFP2 in these cells. Real-time cAMP imaging analysis and proliferation assay of osteoblasts under the stimulus of INSL3 showed a dose- and time-dependent increase in cAMP and cell proliferation, and specific osteoblast gene activation was observed by real-time PCR after INSL3 stimulation. Lumbar spine and femoral bone of Rxfp2-deficient mice were studied by static and dynamic histomorphometry and micro CT respectively, and showed decreased bone mass, mineralizing surface, bone formation and osteoclast surface compared to wild-type littermates, compatible with a functional osteoblast impairment. This study identified for the first time a role for INSL3 in adults, demonstrating a modulating effect on bone metabolism and linking RXFP2 gene mutations with human osteoporosis.


O-30

Regulation of INSL3 Transcription in Testicular Leydig Cells

Jacques J. Tremblay

Division of Reproduction, Perinatal and Child Health, CHUQ Research Centre, Quebec City, Quebec, G1V 4G2, Canada; Centre for Research in Biology of Reproduction, Department of Obstetrics and Gynecology, Faculty of Medicine, Laval University, Quebec City, Quebec, G1V 0A6, Canada

Insulin-like 3 (INSL3) is almost exclusively expressed in the gonads. During fetal life, INSL3 is produced by testicular Leydig cells and it regulates testicular descent. In adults, INSL3 is expressed in the testis and the ovary where it acts as a survival factor for germ and follicular cells. In contrast to its well defined roles in reproductive development and function, very little is known about the molecular mechanisms regulating INSL3 transcription in gonadal cells. In several mammalian species, the entire INSL3 locus is located within the last intron of the JAK3 gene which suggests that INSL3 regulatory elements must be located within a relatively short 5’ flanking region. So far, two transcription factors have been shown to activate INSL3 transcription in Leydig cells: the nuclear receptors SF1 and NUR77. Because they are also found in tissues that do not express INSL3, additional factors must be involved in directing INSL3 expression in Leydig cells. Through 5’ deletions and site-directed mutagenesis, we have mapped a short region that confers about 70% of activity to the human INSL3 promoter. Within this region, we identified a consensus binding site for the Kruppel-like factor KLF6 which we found to be expressed in Leydig cells. KLF6 can activate the INSL3 promoter and this transactivation is blunted when the KLF element is mutated. Furthermore, we found that KLF6 cooperates with SF1 and NUR77 to further enhance INSL3 transcription. Altogether, these data provide new insights into the molecular mechanisms regulating INSL3 expression in Leydig cells. (Supported by CIHR)

Abstracts



O-31

The "Hot Wires" of the Relaxin-like Factor (INSL3)

Christian Schwabe and Erika E. Büllesbach

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA

Peptide hormones regulate important functions in about 10 power 14 cells in a human as they bind to their specific cell surface receptors, yet for decades it has remained unknown what structures within the receptor-bound hormone are initiating a specific response in the target cells.

Structure/function analysis of RLF suggested that binding and signaling are associated with different regions of the surface. Whereas the B chain segment between cysteine 11 and tryptophan 27 mediates binding, signaling is a function of the N-terminal region and involves both chains. Elimination of the N-terminal residues up to B9 or alternately, removal of residues 1 to 9 of the A-chain, do not reduce binding avidity of RLF whereas signaling ceased in either case. This discovery made it possible to explore the structure of the signal initiation site of RLF.

Our work showed that the function of the "hot wires" of RLF is independent of the amino acid side-chains in the signaling region and that trans-membrane signal induction may be a function of peptide bonds of the ligand, forced into the signaling position by the specific interaction of RLF with the leucine-rich repeat G-protein coupled receptor 8 (LGR8). In addition, our observations offer an explanation for ligand crosstalk as well as for the to date inexplicable ability of some anti-receptor antibodies to elicit a specific biological response.

O-32

Roles of the Receptor, the Ligand and the Cell in the Signal Transduction Pathways Utilised by the Relaxin Family Peptide Receptors 1-3 (RXFP1-3)

Roger J. Summersa, Ross A.D. Bathgateb, John D. Wadeb, Emma T. van der Westhuizena, and Michelle L. Hallsa


The relaxin family peptides act on four G protein-coupled receptors now termed relaxin family peptide receptors (RXFP1-4). The leucine-rich repeat containing RXFP1 and RXFP2 and the small peptide-like RXFP3 and RXFP4 are the physiological targets for relaxin, insulin-like (INSL) peptide 3, relaxin-3 and INSL5, respectively. Activation of RXFP1 or RXFP2 causes increased cAMP that results from Gs-mediated activation and GoB-mediated inhibition of adenylate cyclase. With RXFP1, an additional later increase in cAMP involves βγ subunits released from Gi3 and the final 10-amino acids in the C-terminus. The overall cAMP response to activation of RXFP1 is also highly dependent on the cell type expressing the receptor. In contrast, RXFP3 and RXFP4 inhibit adenylate cyclase and RXFP3 causes ERK1/2 phosphorylation. RXFP3 receptors characterised using receptor binding and inhibition of adenylate cyclase show a competition profile of H3 relaxin> H3 relaxin B-chain>> H2 relaxin=porcine relaxin=INSL3. However, examination of signalling using reporter genes in CHO-K1 RXFP3 cells revealed activator protein - 1 (AP-1) activation in response to both H3 and H2 relaxin but not INSL-3. This suggests that RXFP3 activates the c-Jun N-terminal kinase (JNK) pathway but also that H2 relaxin interacts with RXFP3 at a site distinct from that identified by H3 relaxin. These examples from the RXFP receptors demonstrate the importance of the C-terminal tail of GPCRs in signal transduction and also that the ligand and the cell type have important roles in the pathways activated.

31 Relaxin 2008

O-33

Ligand-directed Signalling Pathways at the Relaxin Family Peptide Receptor 3 (RXFP3; GPCR135) Determined Using Reporter Genes

Emma T. van der Westhuizena, Brigham J. Hartleyb, John D. Wadeb, Patrick M. Sextona, and Roger J. Summersa


The H3 relaxin receptor, RXFP3, has been identified and characterized as a Gi/o-coupled receptor that inhibits forskolin-stimulated cAMP accumulation. This study characterizes signalling pathways activated by RXFP3 in CHO-K1 cells stably expressing RXFP3, using reporter genes. Time course studies using 8 reporter gene constructs were carried out in CHO-K1 RXFP3 cells with H3 relaxin, H2 relaxin and INSL3. Robust activator protein (AP)-1 activation was observed in cells treated with both H3 relaxin and H2 relaxin, but not with INSL3 (all 0.1µM), suggesting that RXFP3 may activate the stress-activated protein kinase (c-Jun N-terminal kinase; JNK) signal transduction pathways. H3 relaxin also strongly activated nuclear factor (NF)-κB signalling pathways, that were not activated by either H2 relaxin or INSL3 (all 0.1µM). No reporter gene activation was observed in cells transfected with heat shock element (HSE), cAMP response element (CRE; cAMP/PKA and p38 MAPK/JNK signalling pathways), serum response element (SRE; ERK and JNK signalling pathways), glucocorticoid response element (GRE; cell cycle related pathways), E-box DNA binding element (Myc; cell proliferation related pathways) or nuclear factor of activated T-cells (NFAT; PKC/Ca2+ signalling pathways) when stimulated with H3 relaxin, H2 relaxin or INSL3 (all 0.1µM). This study demonstrated that RXFP3 activates signalling pathways upstream of AP-1 elements, when stimulated with H3 relaxin or H2 relaxin and that RXFP3 can also activate NF-κB signalling pathways when stimulated with H3 relaxin.

O-34

Relaxin Activates Multiple cAMP Signalling Pathway Profiles in Different Target Cells

Michelle L. Hallsa, Ross A.D. Bathgateb, and Roger J. Summersa


The receptor for H2 relaxin, the relaxin family peptide receptor 1 (RXFP1), activates multiple pathways leading to cAMP accumulation when expressed in human embryonic kidney (HEK) 293T cells. In this cell system, RXFP1 initially couples to both Gαs (to increase cAMP) and GαoB (to negatively modulate cAMP), but with time recruits coupling to a Gαi3-Gβγ-PI3K-PKCζ pathway to further increase cAMP. Although cAMP signalling in this system is relatively well defined, the signalling pathways activated by relaxin in its target cells and tissues are still unclear. This study aimed to examine the cAMP signalling of RXFP1 in cells that endogenously express the receptor. Seven cell types derived from various backgrounds were screened for receptor expression. RXFP1 expression was identified in THP-1 (human monocyte cell line), T-47D (human breast cell line), Colo16 (human lung cell line), rat cardiac fibroblasts and rat renal fibroblasts from obstructed kidneys. Although of small magnitude in comparison to THP-1 cells, relaxin-induced cAMP responses were observed. Only in THP-1 cells and rat cardiac fibroblasts was there activation of the Gαi3-Gβγ-PI3K-PKCζ pathway leading to cAMP accumulation. In all other cells there was activation of a combination of the initial pathways to affect cAMP. T-47D cells could activate only Gαs, whereas Colo16 and rat renal fibroblasts from obstructed kidney could activate both Gαs and GαoB pathways. Thus the signalling pathways activated by relaxin are highly dependent upon the cell line under investigation, and this may help to explain the varied physiological responses exerted by relaxin in its different target tissues.


O-35

Prominent Role of Relaxin in Improving Post-infarction Heart Remodelling: Clues from in vitro and in vivo Studies with Genetically Engineered Relaxin-producing Myoblasts

Daniele Bania, Silvia Nistria, Lucia Formiglia, Elisabetta Meaccib, Fabio Francinic, and Sandra Zecchi-Orlandinia

Departments of aAnatomy, Histology and Forensic Medicine, bBiochemical Sciences, cPhysiological Sciences, University of Florence, I-50139 Florence, Italy

Stem cell transplantation and gene therapy are viewed as promising approaches for treatment of the post-infarcted heart and prevention of deleterious cardiac remodelling and heart failure. We explored this issue by transplanting mouse C2C12 myoblasts, transfected to express eGFP or eGFP/RLX, to swine with chronic myocardial infarction. One month later, C2C12 myoblasts selectively settled in the ischemic scar around blood vessels showing an activated endothelium (ICAM-1-,VCAM-positive). They did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix (ECM) remodeling by the secretion of matrix metalloproteases (MMP) and increase microvessel density by VEGF expression. C2C12/RLX myoblasts displayed greater efficacy to engraft the post-ischemic scar and to induce ECM remodelling and angiogenesis than C2C12/GFP. By echocardiography, C2C12-engrafted swine, especially those which received C2C12/RLX, showed better heart contractility than the untreated controls. Hence, the advantage afforded by the grafted myoblasts on cardiac function is primarily dependent on their paracrine effects on ECM remodelling and vascularization.

Another intriguing possibility is that local cardiomyoblasts could be re-activated during adult life to differentiate and regenerate cardiac tissue. Therefore, we studied whether C2C12/GFP and C2C12/RLX myoblasts could influence the differentiation of mouse neonatal cardiac cells in co-culture. We observed that C2C12 cells established close contacts with cardiomyocytes, mediated by the formation of functional gap junctions, and tended to form a sort of structural support guiding the newly formed cardiac cells during their growth and differentiation into a network of spontaneously beating cardiomyocytes. Compared with cardiomyocytes alone, those grown together with C2C12/GFP and, even more, C2C12/RLX showed increased morphological and biochemical signs of differentiation (troponin-T, ANP, cardiac myosin). These findings indicate that C2C12 myoblasts and RLX may favor differentiation of cardiomyocytes and suggest that cell grafting and RLX gene therapy could reduce cardiac remodelling and help myocardial regeneration.

O-36

New Aspects on Cardiovascular Actions of Relaxin: Inotropy and Signaling

Thomas Dschietziga, Cornelia Bartscha, Hans-Tilman Kinkela, Franz Paul Armbrusterb, Christoph Richtera, Gert Baumanna, and Karl Stangla

aDepartment of Cardiology and Angiology, Charité University Medicine Berlin, Campus Mitte, 10117 Berlin, Germany; bImmundiagnostik AG, Bensheim, Germany

We have shown that relaxin acts as compensatory mediator in human heart failure and that the peptide - apart from its signaling via LGR7 -activates the glucocorticoid receptor (GR). Here, we summarize our latest investigations into relaxin’s inotropic effects in human myocardium and into relaxin-GR signaling. To explore inotropy we used human right and left atrial as well as right and left ventricular myocardial samples obtained from donor hearts and from explanted, terminally failing hearts. In right and left atrial preparations from both donor and failing hearts, human relaxin-2 evoked comparable positive inotropic effects which were mediated by protein kinase A and ATP-sensitive potassium channels. On the other hand, a positive inotropic effect of relaxin-2 could be found neither in donor nor in failing right and left ventricular myocardium. The finding of the preserved atrial inotropic effect of relaxin is of potential relevance to the ongoing therapeutical use of the peptide in human heart failure.

We have also investigated the auto-regulation of relaxin in different cell models. Our experiments, which included silencing of GR gene expression, CAT promoter assays,chromatin immuno-precipitation, and immunofluorescence, show that a positive self-regulatory loop of human relaxin-2 gene expression exists and that it critically involves GR and relaxin/GR binding to the relaxin promoter. This finding deepens our understanding of the complex relaxin signal transduction and may also be relevant to the emerging clinical use of the peptide.

33 Relaxin 2008

O-37

Reversal of Cardiac Fibrosis and Related Dysfunction by Relaxin: Experimental Findings

Xiao-Jun Dua, Qi Xua, Edna Lekgabea,b, Xiao-Ming Gaoa, Geoffrey W. Tregearb, Ross A.D. Bathgateb, and Chrishan S. Samuelb

aBaker Heart Research Institute, and bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

Cardiac fibrosis is a hallmark of heart disease, contributes to development of heart failure and arrhythmias, and forms a key therapeutic target. There is a major unmet need for more selective, effective and safe anti-fibrotic drugs. Following earlier findings of cardiac fibrosis phenotype in relaxin-1 deficient (Rln-/-) mice and anti-fibrotic property of relaxin in other organs, we studied anti-fibrotic action of endogenous or exogenous relaxin in diseased hearts. In Rln-/- and wildtype mice subjected to aorta banding (8-weeks), the extent of cardiac hypertrophy, fibrosis and ventricular dysfunction were comparable between both genotypes, suggesting lack of protection in this model by endogenous relaxin albeit its cardiac expression was elevated. Relaxin therapy (via osmotic minipump for 2-weeks) reversed cardiac fibrosis in mice with cardiac-restricted overexpression of β2-adrenergic receptors (β2-TG) or spontaneously hypertensive rats (SHRs, 9-10 mo). Molecular measures suggest inhibited fibroblast activation/proliferation, reduced collagen synthesis and elevated matrix metalloproteinase-2. Enhanced oxidative stress, inflammation and significant matrix remodelling characterize the β2-TG cardiomyopathy model, which responds well to relaxin administration or virally-mediated gene transfection. However, relaxin failed in reversing cardiac fibrosis in 22-mo-old SHRs implying that the efficacy is disease-stage dependent. In mice with surgically induced ischemic heart disease (transmural infarction,ischemia/reperfusion), relaxin therapy inhibited scar formation, increased angiogenesis and alleviated cardiomyocyte apoptosis without adverse effect. In these studies, however, we detected limited functional benefit by the treatment.

Collectively, there is good experimental evidence that relaxin is able to reverse cardiac fibrosis due to distinct mechanisms. Future research needs to explore functional improvement following fibrosis reversal, cellular and molecular mechanisms involved, and changes in relaxin receptor/signalling in diseased myocardium.

O-38

Relaxin-induced Compositional and Geometric Remodeling of Small Renal Arteries

Dan O. Debraha, Julianna E. Debrahb, Sanjeev G. Shroffa, and Kirk P. Conradc

aDepartment of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA; bMagee-Womens Research Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA; cPhysiology and Functional Genomics and Obstetrics and Gynecology, University of Florida, Gainesville, Florida 32610, USA

We have recently shown that administration of recombinant human relaxin (rhRlx) to nonpregnant female rats increases global arterial compliance. Additionally, we found that relaxin alters the passive mechanical properties of small renal arteries (SRA) in mice and rats and that these arteries exhibit an increase in wall area. The goals of the present study were to (1) examine the effects of relaxin on SRA wall composition and (2) characterize the determinants of the relaxin-induced increase in SRA wall area.

Nonpregnant female mice were administered rhRlx for 5 days after which SRA were isolated. We measured arterial collagen, elastin, and total protein using the Sircol collagen assay, the Fastin elastin assay, and the Pierce BCA protein assay, respectively. Additionally, we quantified arterial smooth muscle cell (SMC) size and density using immunofluorescent imaging techniques.

Compared to control mice, SRA isolated from rhRlx-treated mice were characterized by a significant reduction in collagen to total protein ratio (0.13±0.01 vs 0.19±0.01 µg collagen/µg protein; mean±SEM; P<0.02), as well as a significant increase in SMC density (6.1±0.1 vs 5.0±0.1 cells/1000 µm2; P<0.001). In contrast, there were no significant changes in elastin content (104±4 vs 97±14 µg elastin/mg dry weight) or SMC size (111.3±4.3 vs 105.0±3.2 µm2) between the two groups.

We conclude that: (1) relaxin reduces relative collagen content of SRA and (2) the relaxin-induced increase in SRA wall area is primarily due to SMC hyperplasia and not hypertrophy.


O-39

Relaxin-family Peptide and Receptor Systems in Mammalian Brain: Recent Insights from Anatomical and Functional Studies

Andrew L. Gundlach


Significant advances have been made since the Relaxin 2004 conference in our knowledge of the distribution and function of the three major relaxin-family peptide/receptor systems present in the brain, i.e. the relaxin/RXFP1, INSL3/RXFP2 and relaxin-3/RXFP3 pairings. Studies in our laboratory have attempted to construct detailed comparative maps of these systems in major experimental species (rat/mouse) and higher species (primate/human), in order to determine whether subsequent functional experiments in rat and/or mouse will be reflective of human physiology (see Ma et al., Sedaghat et al., Shen et al., this meeting). These studies have so far revealed a degree of variation in the distribution of RXFP1 and RXFP2 mRNA/protein in rat and mouse brain, in contrast to a conserved distribution of RXFP3 seen in rat, mouse, primate and human. In line with this latter data, the distribution of relaxin-3 neurons and projections is similar in rat, mouse and primate. We have also assessed the regulation of expression of the different relaxin-family peptides and receptors - examining the effect of pregnancy and lactation on RXFP1 levels in rat and mouse brain (see Piccenna et al.) and the effect of psychological stressors on relaxin-3 expression (see Banerjee et al.). Important collaborative studies using new, selective agonist and antagonist peptides have revealed the functional importance of RXFP2 in rat basal ganglia (Sedaghat et al.) and endogenous relaxin-3 signaling in the septohippocampal system and associated behaviours (Ma et al., Smith et al.). These and other studies (McGowan et al., Sutton et al.) are now rapidly revealing the precise details of relaxin peptide biology within the brain.

O-40

Relaxin-3 and its Role in Neuroendocrine Function

Barbara M.C. McGowan, Sarah A. Stanley, Mohammad A. Ghatei, and Stephen R. Bloom

Department of Investigative Medicine, Division of Investigative Science, Imperial College London, Hammersmith Campus, London W12 ONN, United Kingdom

The hypothalamus plays a key role in the regulation of energy homeostasis and endocrine function. Relaxin-3 is a hypothalamic neuropeptide belonging to the insulin superfamily of peptides. It is expressed in the nucleus incertus of the brainstem which has projections to the hypothalamus and is thought to act in the brain via the RXFP3 receptor, though the RXFP1 receptor may also play a role. RXFP3 and RXFP1 are present in the hypothalamic paraventricular nucleus (PVN), an area with a well characterised role in the regulation of energy balance and stress. The PVN also modulates reproductive function by providing inputs to hypothalamic GnRH neurons. The physiological roles for relaxin-3 remain to be established. Evidence for a role of relaxin-3 as a hypothalamic orexigenic peptide will be reviewed, including its effects on the hypothalamo-pituitary-thyroid axis and energy expenditure. Studies pointing towards a putative role of relaxin-3 in the hypothalamic-pituitary-gonadal axis will be discussed together with recent work on the effects of relaxin-3 on the stress axis. Central endocrine effects of relaxin-3 will be compared to ‘classical’ relaxin. We conclude that relaxin-3 may act as a hypothalamic signal to co-ordinate appetite, stress and reproductive status. Further studies will be required to determine whether these are physiological roles for relaxin-3 and to determine the receptors involved.

35 Relaxin 2008

O-41

Metabolic and Neuroendocrine Responses to RXFP3 Modulation in the CNS

Steven Suttona, Jonathan Sheltona, Craig Smithb, John Williamsb, Sujin Yuna, Tim Motleya, Chester Kueia, Andrew Gundlachb, Changlu Liua, and Timothy Lovenberga

aJohnson & Johnson Pharmaceutical Research and Development, LLC, San Diego, California 92121, USA; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

Neuroanatomical studies have shown relaxin-3 neurons, primarily found in the rodent nucleus incertus (NI), project widely to a large number of areas expressing the relaxin-3 receptor (RXFP3) and this data suggests relaxin-3/RXFP3 signaling modulates sensory, emotional and neuroendocrine processing. The similar distribution of this receptor-ligand pair in the brain of rat, mouse and monkey suggests that experimental findings obtained in lower species will translate to higher species. A role for relaxin-3 and RXFP3 in modulating stress responses is strongly suggested by the expression of CRF-R1 by NI cells, increased relaxin-3 expression in NI after stress or CRF injection, and hormonal responses to icv relaxin-3 injection. Recent data (Ma et al., this meeting) is consistent with a further role for this ligand-receptor in modulating memory. In addition, relaxin-3 has been reported to modulate feeding and body weight control. Acute or chronic central (icv or iPVN) injections of relaxin-3 have shown a consistent stimulatory effect on food consumption, while relaxin was inactive suggesting the phagic effect of relaxin-3 was mediated by RXFP3. We have confirmed the role of RXFP3 in modulating feeding and body weight using a selective RXFP3 agonist (IR3/I5) and antagonist {R3(D23-27R)/I5}, collecting feeding, body weight, hormone and body composition data. In addition, we have preliminary body weight and fMRI data from relaxin-3 KO mice, which on a 129:B6 background are smaller and leaner than congenic controls. These data suggest relaxin-3, acting through RXFP3, is involved in coordinating stress, learning/memory and feeding responses as predicted on the basis of neuroanatomy.

O-42

Relaxin-3 Neurons of the Nucleus Incertus Modulate Septohippocampal Theta Rhythm and Spatial Working Memory in Rats

Sherie Maa, Angel Nuñezb, Francisco E. Olucha-Bordonauc, Feng Lina, Chester Kueid, Steven W. Suttond, John D. Wadea, Changlu Liud, and Andrew L. Gundlacha

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bDepartamento de Anatomia, Histología y Neurociencia Facultad de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain; cDepartamento de Anatomía y Embriología Humana, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain; dJohnson & Johnson Pharmaceutical Research and Development, LLC, San Diego, California 92121, USA

The neuropeptide relaxin-3 (RLN3) is enriched in GABA neurons of the nucleus incertus (NI), which are highly responsive to psychological stressors and corticotropin-releasing hormone. These neurons innervate many forebrain areas containing high densities of the RLN3 receptor, "GPCR135" or "RXFP3". The septum and hippocampus are two regions that are particularly enriched in RLN3 fibres/terminals and RXFP3 in the rat and mouse brain. High-frequency, rhythmic neuronal activation or ‘theta rhythm’, occurs in the hippocampus during memory processing, particularly during spatial navigation; and GABAergic and cholinergic neurons projecting from the septum to the hippocampus are capable of generating and pacing theta activity. We have shown in the anaesthetized rat that the NI acts as a key relay for the induction of hippocampal theta rhythm activity by pontine and somatosensory activation. In the current study, we therefore determined if endogenous and/or exogenous activation of septal RXFP3 was involved in this process, using anaesthetized and conscious rats. In anaesthetized rats, acute activation of RXFP3 in the medial septum by a specific RXFP3 agonist (R3/I5) increased the level of hippocampal theta rhythm; an effect blocked by prior septal injection of an RXFP3 antagonist (R3(B"delta"23-27)R/I5). In conscious rats, injection of the RXFP3 antagonist into the medial septum inhibited spatial learning performance in the spontaneous alternation test, an effect that was reversible by co-administration of an equimolar amount of the agonist, R3/I5. Our observations reveal an important action of RLN3/RXFP3 in the ‘septohippocampal system’ that potentially links higher cognitive functions with the control of stress and metabolic and neuroendocrine balance.


O-43

Behavioral Phenotyping of Mixed-background (129SV/B6) Relaxin-3 Knockout Mice

Craig M. Smitha, Andrew J. Lawrencea, Steve W. Suttonb, and Andrew L. Gundlacha

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bJohnson & Johnson Pharmaceutical Research and Development LLC, San Diego, California 92121, USA

In order to assess the behavioral phenotype of mice lacking relaxin-3, two independent cohorts of adult male and female relaxin-3 knockout (KO) mice and their wildtype (WT) littermates (n = 8-12) were subjected to a battery of standard behavioral tests. There were no genotype-related deficits in motor coordination (assessed on the accelerating rotarod) or spatial memory (Y-maze), and levels of anxiety were equivalent (in large open-field, elevated plus-maze, and light/dark box tests). Sex-based differences were observed, however. Female KOs displayed a "hypoactive" phenotype in locomotor cells that was also detected in the large-open field and Y-maze. Combined with results from further testing of females in social-interaction and object-recognition paradigms, this data suggests that relaxin-3 deficiency in female mice may result in reduced exploratory drive, possibly due to the hypothesized role for relaxin-3 in hippocampal theta rhythm generation (Ma et al., this meeting). Male KO mice did not display this hypoactivity, although they did display signs of a "depressive-like" phenotype, with a strong trend to spend more time in the Porsolt posture during the forced swim test, after either an acute or chronic stress (15-min restraint, daily for 1 or 7 days). This is consistent with findings that relaxin-3 neuron activity is strongly influenced by CRF and psychological stressors (Banerjee et al., this meeting). Preliminary results from measures of pre-pulse inhibition of acoustic startle in these mice also indicate that relaxin-3 may be involved in sensorimotor gating.

O-44

An in vitro Study of the Protective Effect of Relaxin on Brain Tissue under Ischemic Stress

Brian C. Wilsona, Peter Milnea, Barry O’Connellb, and Tarek Salehb

aDepartment of Biology, Acadia University, Wolfville, NS, B4P 2R6, Canada; bDepartment of Biomedical Sciences, Atlantic Veterinary College, Charlottetown, PE, C1A 4P3, Canada

Relaxin pretreatment reduces the size of the infarct that develops following middle cerebral artery occlusion in anesthetized rats. A nitric oxide synthase III inhibitor completely negates this effect, which suggests that an endothelin-nitric oxide cascade is involved that might improve collateral perfusion of brain parenchyma in the affected zone. We evaluated an organotypic brain slice culture technique to study the direct actions of relaxin on neural cells under ischemic stress. Four coronal brain slices (400µm) at the level of somatosensory cortex were obtained from 21 neonatal rats. Slices were cultured under standard conditions for 14 days. Experimental slices were placed in a deoxygenated, glucose-free balanced salt solution (gfBSS) in 95% N2 (balance CO2) for one hour whereas control slices were transferred to oxygenated BSS with glucose. An additional group of slices were incubated in gfBSS containing 100 nM H2 relaxin for the same period. Subsequently, all three groups of brain slices were transferred back into normal, oxygenated media. Slices were analysed at 0, 4, 8 and 24 hours following ischemic challenge using propidium iodide (PI) fluorescence to highlight cellular damage or death. The percentage of dead and dying cells in a slice served as a measure of the severity of ischemic damage and was compared between groups. Relaxin treatment attenuated PI staining at 4, 8 and 24 hours post-ischemia; this suggests that the progression of cellular death was attenuated. Relaxin may have direct actions on cells improving their chance of survival during an ischemic challenge. Funded by NSERC and Pfizer Canada.

Abstracts



P-21

Relaxin Activation of Water Drinking in the Mouse: Likely Role of RXFP1 in the Subfornical Organ

Loretta Piccenna, Michael J. McKinley, Lesley L. Walker, Andrew J. Lawrence, and Andrew L. Gundlach


The subfornical organ (SFO) is one of a group of nuclei known as the circumventricular organs and is strongly associated with osmoregulatory activity. Neurons in the SFO of adult rat brain express high levels of receptors for the peptide hormone, relaxin; and intracerebroventricular (icv) administration of human relaxin to pregnant and non-pregnant rats produces increased vasopressin secretion, blood pressure and water drinking. Intravenous (iv) administration of relaxin also increases water drinking via activation of SFO neurons. Finally, SFO ablation in adult rats diminishes relaxin-induced drinking, further substantiating the fundamental involvement of the SFO in this behaviour. Recent studies indicate that the native receptor for relaxin is LGR7 or "RXFP1". However, human relaxin has high affinity for both RXFP1 and RXFP2 and both receptors are present in rat and mouse brain. In studies to establish whether relaxin acts through RXFP1 and/or RXFP2 to induce water consumption, we have conducted studies in wildtype (WT) and LGR7-KO/LacZ reporter mice. Following icv injection of human relaxin (100 & 200 ng/µl), LGR7-KO mice drank no water, or drank less than WT mice over a 5-120 min period, suggesting that relaxin acts primarily via RXFP1 to induce drinking. Furthermore, mice display high levels of RXFP1 expression, but not RXFP2, in the SFO. These studies did reveal an apparent behavioural activation of LGR7-KO mice by central injections of human relaxin, including increased grooming, which is thought to reflect activation of RXFP2 (Sedaghat et al., this meeting). This possibility can be tested using an RXFP2 antagonist, and mouse relaxin, which is more selective than the human peptide for mouse RXFP1 cf. RXFP2.

P-22

Relaxin-3 Neurons in Nucleus Incertus of the Rat: Effect on Activity of Psychological Stressors and the Light-Dark Cycle

Avantika Banerjee, Pei-Juan Shen, Ross A.D. Bathgate, and Andrew L. Gundlach


Relaxin-3 (RLN3) is distinguished by its enriched expression in the pontine nucleus incertus (NI) and its broad projections, postulated to participate in ascending neural circuits involved in behavioural activation, biorhythms and stress responses. In studies to investigate this hypothesis, we are examining the regulation of RLN3 expression by different experimental stimuli. Initially, we studied the effect of swim stress on RLN3 expression in NI neurons. Rats (3-5/group) were subjected to two 10-min forced-swim sessions 24-h apart and killed over a time-course of 0.5-24 h. Expression of RLN3 in NI neurons was assessed by ISHH and a rapid and significant increase in both RLN3 hnRNA and mRNA was observed (70-135% at 1-2 h, p ≤ 0.05). Swim stress induced increases in RLN3 expression were blunted by pre-treatment with a CRF-R1 antagonist. We also studied the pattern of RLN3 expression across the light-dark cycle. Rats were housed under 12:12h light/dark conditions for 7 days (lights on at 07:00 - designated as Zeitgeber time [ZT] 0) and were killed at ZT0, 4, 8, 12, 16, 20. RLN3 mRNA and hnRNA levels displayed an upward trend during the dark phase with a peak at ZT 20 that was significantly above the lowest levels of expression during the light phase at ZT8, suggesting a possible interaction between RLN3 neurons and circuits regulating circadian rhythm that warrants further investigation. Studies are now in progress to explore the effect of ‘stressors’ such as foot-shock and restricted food access on RLN3 neuron activity.

39 Relaxin 2008

P-23

Expression and Distribution of the Relaxin Family Peptide Receptors - RXFP1-3 - in Primate and Human Brain

Pei-Juan Shena, Sherie Maa, Ross A.D. Bathgatea, Jose L. Lanciegob, and Andrew L. Gundlacha

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bCenter for Applied Medical Research, University of Navarra, 31008 Pamplona, Spain

Anatomical studies have established the presence and widespread distribution of native receptors for relaxin, INSL3 and relaxin-3 in rat and mouse brain, suggesting important roles for these systems in sensorimotor, autonomic and neuroendocrine function. However, the precise profile of expression and distribution of these neuropeptides and their receptors in higher species including human and their significance for neurological and psychiatric disease is not established. In order to establish which experimental species best models these systems in human brain and in light of some distinctive differences in their relative distributions in rat and mouse brain; this study examined the localization of LGR7 (or RXFP1), LGR8 (or RXFP2) and GPCR135 (or RXFP3) in macaque and postmortem human brain using radioligand binding and autoradiography. Fresh-frozen sections from the brain of macaque (Macaca fascicularis) and sections of cerebral cortex and hippocampus from a neurologically-normal 40 year-old male were incubated with [125I]-h-relaxin to detect RXFP1, [125I]-h-INSL3 to detect RXFP2 and [125I]-R3/I5 to detect RXFP3. In results so far, specific [125I]-h-relaxin binding sites were detected in human cerebral cortex, not hippocampus; [125I]-h-INSL3 sites were detected in macaque cortex; and specific [125I]-R3/I5 binding was detected in the macaque septum, hippocampus and thalamus, areas that express RXFP3 in rat and mouse brain. Studies are continuing to confirm and extend these initial findings using brain sections from additional primate brains and samples from other human brain areas. This comparative mapping will provide an anatomical framework for the interpretation of research in animals to identify the biological roles of relaxin family peptides in normal physiology and in human disease.

P-24

Relaxin-3 and RXFP3 Gene Expression and Peptide Distribution in the Brain and Ovary of Teleost Fish

Brian C. Wilsona, Emily K. Fletchera, Rebecca Rappaporta, Sara Good-Avilaa, and Laura Parryb

aDepartment of Biology, Acadia University, Wolfville, NS, B4P 2R6, Canada; bDepartment of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia

Bioinformatic and phylogenetic analyses suggest that teleost fish possess 5 or 6 relaxin-3 genes with three forms, -3a, 3b, 3c strongly related to mammalian relaxin-3. Teleost genomes also contain predicted sequence for the relaxin-3 receptor RXFP3. RT-PCR was used to determine if relaxin-3a, -3b and -3c and RXFP3 are expressed in brain and gonad of zebrafish, Danio rerio, and immunohistochemistry was used to determine the distribution of relaxin-3 and its receptor in the ovary and through the rostro-caudal extent of the brains of killifish, Fundulus heteroclitus. In addition, the effect of exogenous human relaxin-3 on steroidogenesis was studied using enzyme immunoassays and cultures of killifish ovarian follicles. Relaxin-3a, -3b and -3c genes, and the RXFP3 gene, were expressed in the brain and gonads of male and female fish regardless of reproductive condition. Immunostaining for relaxin-3 and GPCR135 was observed in key brain areas mediating visual perception and visually-guided behaviours. In addition, structures involved with integrating information from the lateral line system displayed relaxin-3 staining. This supports data suggesting that relaxin-3 is a neuropeptide involved in sensory-motor integration and in fish, may play a role in prey capture or predator avoidance. In the ovary, relaxin-3-immunoreactivity was observed in vitellogenic and post-vitellogenic follicles as well as the corpus luteum. Treatment with human relaxin-3 stimulated estradiol production in vitellogenic and post-vitellogenic follicles, but had no effect on the release of the primary progestin, maturation-inducing steroid. This suggests that ovarian relaxin-3 may be involved with yolk deposition and processes of oocyte maturation. Funded by NSERC Canada.


P-25

Insulin-like Peptide 7 (Relaxin 3): Distribution and Interaction with Two Functionally Distinct Binding Sites in the Rat Brain

Jaw Kang Changa, Jun Yanga, Eugen Brailoiub, Siok L Dunb, Jason Grieshoberb, Jin J. Luob,c, G. Cristina Brailoiub, and Nae J. Dunb

aPhoenix Pharmaceuticals, Inc., Burlingame, California 94010, USA; Departments of bPharmacology and cNeurology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA

Immunohistochemical studies by means of a rabbit polyclonal antiserum against the C-connecting peptide of the human insulin-like peptide 7 (INSL7) revealed INSL7-immunoreactive (irINSL7) cell bodies in the pontine nucleus incertus, with few cells scattered in the lateral or ventrolateral periaquaductal gray area. The mean number of irINSL7 neurons in the nucleus incertus from three rats was about 1200. Numerous labeled cell processes were noted in the forebrain areas; with a high density in the septum, hypothalamus, and thalamus. Optical imaging with a voltage-sensitive dye DiSBAC4(3) or a calcium-sensitive dye Fura-2AM was used to assess the effect of INSL7 on dissociated, cultured rat hypothalamic neurons. INSL7 (100 nM) applied by superfusion depolarized or hyperpolarized a population of hypothalamic neurons. INSL7 (100 nM) increased intracellular calcium concentrations [Ca2+]i in a population of hypothalamic neurons with two distinct patterns: 1) a sustained elevation with a plateau phase lasting for minutes; and 2) a fast, transitory rise followed by oscillations. In a Ca2+-free HBSS, the two types of response to INSL7 were not substantially modified, suggesting that the rise in [Ca2+]i is mainly derived from intracellular stores. Our result shows that INSL7 is expressed in neurons of the nucleus incertus, which provide dense innervation to the forebrain including the hypothalamus. Functional studies suggest that INSL7 causes a membrane depolarization or hyperpolarization in a population of rat cultured hypothalamic neurons or two patterns of calcium mobilization, raising the possibility that the peptide may interact with two functionally distinct binding sites.

P-26

Central Administration of Human Relaxin-2 in Adult Male Rats Inhibits Food Intake

Barbara M.C. McGowan, Nicholas E. White, Debabrata Roy, Sarah A. Stanley, Waljit Dhillo, James V. Gardiner, Mohammad A. Ghatei, and Stephen R. Bloom

Department of Investigative Medicine, Division of Investigative Science, Imperial College London, Hammersmith Campus, London W12 ONN, United Kingdom

Relaxin belongs to the insulin superfamily, which includes “classical” relaxin, (relaxin-1 in most species, relaxin-2 in humans) and the recently identified orexigenic hypothalamic neuropeptide relaxin-3. Central administration of relaxin-3 increases food intake in male Wistar rats, an effect thought to be mediated via the RXFP3 receptor expressed in the hypothalamic paraventricular nucleus (PVN). Relaxin is expressed in the rat PVN, arcuate nucleus, and supraoptic nucleus, hypothalamic regions known to play an important role in appetite. These areas express the relaxin receptor RXFP1, also localised to the circumventricular organs where relaxin is known to affect water intake. We hypothesized that central injections of human relaxin-2 in adult male rats may exert an effect on appetite.

Intracerebroventricular (ICV) injections of human H2 (18-540 pmol) to satiated rats significantly decreased food intake 1h post-administration in the early dark phase [2.51±0.24g (vehicle) vs 1.59±0.37g (18pmol H2), 1.59±0.23g (180pmol H2), 1.51±0.26g (540pmol H2), p<0.05 for all doses H2]. IntraPVN administration of H2 (18-540 pmol) significantly decreased 1h food intake in satiated rats in the early dark phase [3.13±0.35g (vehicle) vs 1.35±0.33g (18pmol H2), p<0.001, 1.61±0.31g (180pmol H2), p<0.01, 1.23 ± 0.32g (540pmol H2), p<0.001]. Cumulative food intake was suppressed up to 8h post-injection of H2 (180-540 pmol) compared to vehicle. ICV administration of H2 (180 pmol) decreased feeding behaviour, and increased grooming and head-down behaviour. These results suggest that H2 may be exerting an effect on appetite through RXFP1 in the PVN.

41 Relaxin 2008

P-27

Verification of a Relaxin-3 Knockout/LacZ Reporter Mouse as a Model of Relaxin-3 Deficiency

Craig M. Smitha, Pei-Juan Shena, Sherie Maa, Steve W. Suttonb, and Andrew L. Gundlacha


A relaxin-3 knockout (KO) mouse strain was generated by deletion of exon 2 of the relaxin-3 gene and replacement with a LacZ reporter gene cassette downstream and in-frame with the endogenous relaxin-3 promoter (Lexicon Genetics Inc.). In this study, biochemical and anatomical studies were completed to determine the effectiveness of the gene deletion, levels of reporter gene activity, and the effect of the gene deletion on relaxin-3 receptor (RXFP3) levels. The genotype of mice was routinely determined from tail samples using RT-PCR and gel electrophoresis separation of different-sized DNA fragments corresponding to the WT and/or KO allele (using primers unique for each allele). In immunohistochemical studies using a polyclonal relaxin-3 antibody, KO mice were shown to lack relaxin-3 staining in coronal sections through the medial septum, while strong relaxin-3 immunoreactivity was observed in equivalent sections from WT mice. The activity of the LacZ reporter gene as a marker of relaxin-3 gene expression was confirmed, with strong X-Gal staining detected in sections through the nucleus incertus from KO and heterozygous mice, consistent with this region containing a high density of relaxin-3 positive cells in WT mice. In radioligand binding studies using [125I]-R3/I5 (a chimeric peptide selective for RXFP3) and brain sections from 3 WT and 3 KO mice, we observed equivalent densities of RXFP3 binding sites in a number of brain areas, indicating that the expression of the receptor is not highly dependent upon activation by the ligand and demonstrating the feasibility of future studies to replace the native ligand in relaxin-3 KO mice by central RXFP3 agonist injections or viral-based methods.

P-28

Effects of Intracerebroventricular Injection of RXFP3 Agonist and Antagonist Peptides on Behaviour of Wildtype and Relaxin-3 Knockout Mice

Craig M. Smitha, Natalie L. Downera, Sherie Maa, Akhter Hossaina, Andrew J. Lawrencea, John D. Wadea, Steve W. Suttonb, and Andrew L. Gundlacha


Neuroanatomical studies in rat and mouse have revealed that a discrete population of relaxin-3 neurons in the nucleus incertus (NI) projects to a wide range of forebrain areas expressing the relaxin-3 receptor, RXFP3 - predicting a role for relaxin-3 signaling in sensory, limbic and neuroendocrine processing. In this regard, expression of CRF-R1 by NI cells, increased relaxin-3 in NI after stress or icv CRF injection, and hormonal responses to icv relaxin-3 injection, suggests a role for relaxin-3 in modulating stress responses in rats. In addition, relaxin-3 has been reported to modulate feeding and body weight in rats, with central injections of relaxin-3 producing a consistent stimulation of food consumption and body weight gain. More recent studies have confirmed the role of RXFP3 in feeding, using a selective RXFP3 agonist (R3/I5) and antagonist (R3(Bδ23-27)R/I5) (Sutton et al., this meeting). The availability of specific RXFP3 agonist and antagonist peptides and relaxin-3 KO mice (Smith et al., this meeting) prompted the present study to examine the behavioral consequences of icv injections of R3/I5, (R3(Bδ23-27)R/I5) or both (cf vehicle) in wildtype (WT) and relaxin-3 KO mice. Mice are anesthetized, and a guide cannula is stereotaxically inserted 1 mm above the lateral ventricle. After recovery for 7 days, guide cannula location is checked by an injection of angiotensin II and a predicted drinking response. In subsequent testing, mice are being assessed for levels of feeding, exploratory behaviour, and stress and anxiety responses. Relaxin-3 KO mice will be assessed for differences in response sensitivity to the RXFP3 agonist and for an absence of responses to the antagonist seen in WT mice.


P-29

Distribution of Relaxin-3 mRNA, Relaxin-3 Immunoreactivity and RXFP3 Binding Sites in the Brain of the Macaque (Macaca fascicularis)

Sherie Maa, Pei-Juan Shena, Qian Sanga, Jose L. Lanciegob, and Andrew L. Gundlacha

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bCentre for Applied Medical Research, University of Navarra, 31008 Pamplona, Spain

Relaxin-3 (RLN3) is the ancestral relaxin peptide that preferentially binds and activates ‘GPCR135’ or ‘RXFP3’. In the rat, RLN3 mRNA and immunoreactivity (IR) are abundant in pontine nucleus incertus (NI) neurons that project to numerous forebrain regions, including cingulate cortex, medial septum, lateral preoptic area, lateral/dorsomedial hypothalamus and ventral hippocampus. Functional studies suggest that NI/RLN3 neurons are involved in stress responses, feeding and somatosensory processing. In order to confirm the relevance of this conserved neuropeptide to higher species, this study initially examined the presence and distribution of RLN3-IR elements throughout the brain of Macaca fascicularis, using a RLN3 C-peptide-directed antibody. In perfusion-fixed sections from four brains, RLN3-IR was observed in nerve fibers in various regions, including the central gray, thalamus, and lateral and dorsomedial hypothalamus. Staining was highly enriched in the septum, hippocampus, ventral tegmental area, and interpeduncular and supramammillary nuclei. RLN3-IR was readily observed in nerve terminals and boutons in these areas, indicating that RLN3 is appropriately positioned for synaptic release. Immunostained cell bodies were observed in the pontine central gray. In situ hybridization with an antisense RLN3-riboprobe revealed RLN3 mRNA-containing cells in the pontine central gray. The distribution of RXFP3 protein was determined by labeling fresh-frozen brain sections with [125I]-R3/I5. The distribution of binding sites correlated with the distribution of RLN3-IR. The broad distribution of RLN3-positive axons and terminals and RXFP3 in the forebrain of Macaca fascicularis is consistent with observations in the rat and mouse and predicts an important role for RLN3 signaling in primate and human brain.

P-30

Relaxin Receptor LGR7 Is Regulated by Estrogen

Priya Maseelall, Gerson Weiss, Andrea Wojtczuk, and Laura T. Goldsmith

Department of Obstetrics, Gynecology, and Women’s Health, New Jersey Medical School, Newark, New Jersey 07103, USA

Relaxin has pronounced effects upon the mammalian reproductive tract and is involved in the maintenance of pregnancy. Relaxin is an important remodeller of female reproductive tract connective tissue. Unfortunately, despite its significance in relaxin action, understanding of LGR7 regulation is limited. Since the actions of relaxin in certain target tissues appear to require estrogen exposure, we hypothesized that relaxin action in the cervix is modulated by estrogen. We tested this hypothesis by determining expression of LGR7 and whether estrogen regulates its expression in a well established, relaxin responsive, in vitro model of human term pregnancy cervix, lower uterine segment fibroblasts (LUSF). LGR7 mRNA was detected in LUSF using quantitative real-time RT-PCR. This was verified by the expected amplicon size of 192 base pairs and a single peak at 83.3°C seen on melt analysis. LGR7 mRNA levels in LUSF were significantly increased by estradiol to mean levels of 146% ± 6.5 (M ± SE, n=2 experiments, each performed using multiple RT-PCR reactions) above those of control, untreated cells (p = 0.04). These data are the first demonstration of estrogen regulation of relaxin receptor LGR7 expression. That estrogen positively regulates relaxin receptor expression in LUSF, suggests estrogen amplification of relaxin action in the cervix.

43 Relaxin 2008

P-31

Prolonged RXFP1 and RXFP2 Signalling Is Due to Weak Internalization and a Lack of βArrestin Recruitment

Gabrielle E. Callandera, Walter G. Thomasb, and Ross A.D. Bathgatea

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bBaker Heart Research Institute, Melbourne, Victoria 3004, Australia

A number of studies have observed that relaxin can induce prolonged physiological responses. This raises the question as to whether RXFP1 is regulated by the same mechanisms described for most G protein-coupled receptors (GPCR). To address this, we investigated the mechanisms that normally contribute to receptor regulation, including internalization, trafficking of βarrestins and receptor phosphorylation of both RXFP1 and RXFP2. In HEK293 cells expressing RXFP1 or RXFP2, and in the absence of IBMX, both receptors still elicit cAMP responses up to 6 hours after initial stimulation. In accord with this result, RXFP1 and RXFP2 did not demonstrate any significant internalization in response to relaxin or INSL3, as compared to the AT1 receptor, which undergoes rapid and robust internalization in response to AngII stimulation. Additionally co-expression of GPCR kinases was found to have no effect on the rate of internalization for either RXFP1 or RXFP2. Confocal microscopy was utilised to follow the trafficking of GFP-labelled βarrestins after receptor activation. Neither RXFP1 nor RXFP2 activation resulted in recruitment of βarrestins to the cell surface, whereas the AT1 receptor rapidly recruits both βarrestins 1 and 2. Receptors immunoprecipitated from [32P] metabolically labelled cells were used to investigate the agonist-specific phosphorylation. Weak receptor phosphorylation was observed only at 30 mins, perhaps explaining the lack of internalization, but indicating the presence of second messenger-dependent phosphorylation. These results suggest that the prolonged actions of relaxin and related peptides reflect the absence of desensitization of RXFP1 and RXFP2 by the classically described mechanisms.

P-32

The Regulatory Action of Insulin, IGF1 and Relaxin on Nitric Oxide Synthase Activity and its Impairment in Rat Skeletal Muscle in Condition of Streptozotocin Diabetes of Type II

Ludmila A. Kuznetsova, Oksana V. Chistyakova, and Marianna N. Pertseva

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223, St. Petersburg, Russia

We investigated the influence of peptides of insulin superfamily on the nitric oxide synthase (NOS) activity in rat skeletal muscle of control and diabetic groups. It was shown that endothelial NOS (eNOS) and neuronal NOS (nNOS) activity is present in the membrane-bound form in the muscles. The stimulation of the NOS activity by insulin, insulin-like growth factor 1 (IGF-1) and relaxin was revealed. The treatment of muscles by antibodies against the nNOS led to disappearance of stimulatory effects of insulin superfamily peptides on nNOS activity. The specific antibodies against the eNOS have no effect on the insulin and IGF-1 NOS-activating action, but blocked the effect of relaxin. This give evidence that the target for stimulating action of the insulin and IGF-1 is nNOS, meanwhile relaxin activates only eNOS. It was revealed that in rat skeletal muscles with experimental diabetes of type II (80 and 180 days) the activity of NOS is decreased and the activating action of the peptides on the membrane-bound forms of NOS is weakened. Reduced NOS activity is associated with a decrease in the regulation of the nNOS and eNOS activities by the peptides. The conclusion was made that in the condition of type II diabetes in the rat muscle tissues the NOS signaling pathway which mediates the action of hormones and growth factor of insulin nature is impaired. The investigation was supported by grants: Russian Foundation for Basic Research 06-04-48809; Basic Research for Medicine, 2007.


P-33

Synthetic Peptides, Derived from the Third Intracellular Loop of Relaxin Receptor LGR7, as Probes for Study of Molecular Mechanisms of Relaxin Action on Adenylyl Cyclase Signaling System

Alexander O. Shpakova, Ivan A. Guryanovb, Ludmila A. Kuznetsovaa, Svetlana A. Plesnevaa, Gennady P. Vlasovb, and Marianna N. Pertsevaa

aI.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, 194223, St. Petersburg, Russia; bInstitute of Macromolecular Compounds of Russian Academy of Sciences, 199004, St. Petersburg, Russia

The synthetic peptides 619-629, 619-629-Lys(Palm) and 615-629, corresponding to the C-terminal region of the third intracellular loop (C-ICL3) of the relaxin receptor of type 1 (LGR7), were used for identification of the types of relaxin receptor participating in the realization of relaxin stimulating effect on adenylyl cyclase (AC) activity in the rat tissues (brain, myocardium, skeletal muscles) and the muscle tissues of invertebrates - the earthworm Lumbricus terrestris, the mollusks Anodonta cygnea and Lymnaea stagnalis. The peptides 619-629-Lys(Palm) and 615-629 in competition manner inhibit the AC stimulating effect of relaxin in the brain and myocardium but don’t change this effect in the other tissues. The data obtained show that receptor LGR7 participates in realization of AC stimulating effect of relaxin in brain and myocardium, meanwhile give evidence in favor of the another AC signaling mechanisms of relaxin action (in particular six-components, discovered by authors) in skeletal muscles and muscles of invertebrates, which did not involve the receptor of LGR7 type. The AC stimulating effect of relaxin in brain and myocardium was decreased in the presence of C-terminal peptide 385-394 of mammalian Gαs-subunit and was blocked by treatment with cholera toxin. Thus, in brain and myocardium the relaxin stimulates AC via receptor LGR7 and Gs-protein. The coupling between them is mediated by the interaction of C-ICL3 of the receptor and C-terminal segment of Gαs. The investigation was supported by grants: Russian Foundation for Basic Research 06-04-48809, Basic Research for Medicine, 2007.

P-34

Regulation of Lgr8 (RXFP2) Expression in the Mouse Fetal Kidney by the Transcription Factor Pod1

Mary Familari, Duc Vu, and Laura J. Parry

Department of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia

Lgr8 (or RXFP2) is expressed in the developing and adult rat kidney. At embryonic stage E18.5, Lgr8 mRNA was localised to putative mesangial cells in the developing glomerulus, suggesting that Insl3-Lgr8 is involved in glomerulogenesis. This process begins just after the renal tubule undergoes segmentation to form a nephron, with the glomerulus at the proximal end, around E12-13 in mouse. One of the main regulatory factors in glomerulogenesis is the basic helix-loop-helix (bHLH) transcription factor Pod1, which is expressed in the stroma. Null Pod1 mice have a marked reduction in the number of nephrons and delayed glomerulogenesis. The promoter region of the Lgr8 gene contains an E-box consensus sequence that can bind bHLH factors, including Pod1. Therefore, we examined the expression of Lgr8 relative to Pod1 in the fetal and neonate kidney and tested the hypothesis that Lgr8 is a downstream target of Pod1. Fetal and neonate kidneys were collected at E14.5, E16.5 and E18.5, and day 1, 4 and 7 postpartum. Lgr8 and Pod1 expression were compared between these stages using qPCR. In the second study, fetal kidneys were dissected from Pod1-/- mice on E18.5 to assess the effects on Lgr8 expression. Lgr8 gene expression was highest on E14.5 and E16.5 but decreased significantly on E18.5 and in the neonate kidney. This was not correlated with increased Pod1 expression. However, there was a significant increase in Lgr8 expression in the E18.5 kidneys of Pod1-/- mice. These data suggest that Pod1 may negatively regulate Lgr8 expression in the developing glomerulus.

45 Relaxin 2008

P-35

Expression Profile of Insulin-like Peptide-5 (INSL5) and GPCR142—A Perspective on Functional Roles and Future Directions

Qian Sanga, Steven W. Suttonb, Ross A.D. Bathgatea, and Andrew L. Gundlacha

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bJohnson & Johnson Pharmaceutical Research and Development, San Diego, California 92121 USA

Insulin-like peptide-5 (INSL5) was first characterized as a member of the insulin gene superfamily by Conklin et al. in 1991. Human INSL5 cDNA was isolated from a library constructed from sigmoid colon tissues from a Crohn’s disease patient and mouse cDNA was obtained from libraries constructed from irradiated colon and thymus. Quantitative RT-PCR analysis of human and mouse tissues revealed that INSL5 was highly expressed in the colon and thymus, but the physiological role of INSL5 is unknown and yet to be explored. In the present study, we examined the presence and distribution of INSL5 mRNA in the adult mouse using in situ hybridization histochemistry. Using a DIG-labeled antisense riboprobe, we observed apparent INSL5 mRNA expression in the large intestine and in the thymus, where specific labeling was present in the cortex and not the medulla. INSL5 expression in the thymus cortex suggest that INSL5 may be involved in cellular immunity processes; and the thymus cortex contains T-cell precursors and is where early events of T-cell development and positive selection occur. Recent bioinformatics and molecular studies have established that the native receptor for INSL5 is GPCR142 or "RXFP4". Unlike other relaxin peptide-receptor pairings, INSL5/ RXFP4 do not appear to be important for brain function, but high levels of RXFP4 mRNA have been detected in human intestine and colon, suggesting that INSL5-RXFP4 signaling may play a role in these tissues. Further studies are planned to localize the expression of INSL5 and RXFP4 in mouse GI-tract, as a basis for functional studies in normal and gene-knockout mice.

P-36

Changes in Arterial Function by Chronic Relaxin Infusion Are Mediated by the Leucine Rich Repeat G Coupled Lgr7/RFXP1 Receptor

Julianna E. Debraha, Alexander Agoulnikb, and Kirk P. Conradc

aMagee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA; bDeparment of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA; Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 32610, USA

Circulating relaxin contributes to renal circulatory adaptations in pregnancy. Chronic administration of recombinant human relaxin (rhRLX) to nonpregnant rodents inhibits myogenic reactivity and increases passive compliance of small renal arteries (SRA), thereby mimicking pregnancy. We hypothesize that these arterial responses to rhRLX are mediated by the Lgr7/RFXP1, and not the Lgr8/RFXP2 receptor. Lgr7 and Lgr8 receptor-deficient, and wild-type virgin mice were investigated. rhRLX or vehicle (VEH) was infused for 5 days. SRA were then isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a 20 mmHg step increase in intraluminal pressure). SRA from rhRLX-infused Lgr7 wild-type mice showed inhibited myogenic reactivity with a 6.1 ± 0.5% increase in diameter whereas the arteries from rhRLX-infused Lgr7 knock-out mice exhibited robust myogenic reactivity with only a 1.2 ± 0.4% change in diameter (p=0.001). In contrast, myogenic reactivity of SRA was comparably inhibited in both the rhRLX-infused Lgr8 knock-out and wild-type mice (p=NS). SRA from VEH-treated mice, regardless of genotype, all exhibited robust myogenic reactivity. rhRLX infusion increased arterial compliance of SRA from Lgr7 wild-type, but not from Lgr7 knock-out mice (p=0.01). In contrast, arteries from rhRLX-infused Lgr8 knock-out mice showed increased arterial compliance relative to VEH-infused knock-out animals (p=0.001). Thus, relaxin-induced inhibition of myogenic reactivity and increase in passive compliance of small renal arteries is mediated by the Lgr7, and not the Lgr8 receptor.


P-37

Relaxin Promotes Matrix Metalloproteinase-2 and Decreases Wnt/β-Catenin Expression in the Neonatal Porcine Heart

Teh-Yuan Hoa, Megan A. Diltsa, Frank F. Bartolb, and Carol A. Bagnella

aDepartment of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA; bDepartments of Animal Sciences and Anatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA

Relaxin (RLX), reported to play an important role in cardiovascular remodeling, is linked to activation of matrix metalloproteinases (MMPs). RLX is found in the circulation of nursing pigs from birth and RLX receptor (RXFP1) transcripts have been identified in the neonatal heart. The neonatal pig heart undergoes remodeling in the first few days of life with left ventricular (LV) hypertrophy and collagen accumulation due to volume overload. Wnt/β-catenin signaling has also been implicated in cardiac remodeling. Therefore, objectives of this study were to determine the effects of RLX on the myocardial Wnt/β-catenin signaling system and MMP expression at postnatal day (PND) 2. The LV myocardium was obtained from gilts on PND 2 after administration of vehicle (PBS) or porcine RLX (20 ug/kg BW/ in PBS every 6h for 48h; n=4/group). In other studies, LV myocardial explants from PND 2 gilts (n = 3) were incubated with vehicle or porcine RLX (1-100 ng/ml) for 24 hr when both medium and tissue were collected. Results showed that myocardial Wnt7a and Wnt4 gene expression decreased following RLX administration in vivo, while there was no effect on Wnt5a expression. Immunoreactive myocardial β-catenin protein was reduced in RLX-treated animals. Zymographic analysis of medium from RLX-treated heart explants showed an increase in proMMP-2, but not proMMP-9 activity. Western blot analysis indicated that RLX increased myocardial MMP-2 protein as compared to controls. Data suggest that the RLX-induced decline in Wnt/β-catenin expression at PND 2, together with increased MMP-2 activity, may be important for neonatal cardiac remodeling. (Support USDA-NRI 2003-35203-1357 and USDA-2007-35203-18098)

P-38

Boar Testis Acts as a Source and Target Tissue of Relaxin

Tetsuya Kohsakaa,b, Shinichi Katoa, Siqinb, Itaru Minagawaa, Keiichiro Yogoa,b, Tatsuo Kawarasakic, and Hiroshi Sasadad

aLaboratory of Animal Reproduction, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan; bDivision of Animal Resource Production, The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan; cShizuoka Swine and Poultry Experimental Station, Kikugawa 439-0037, Japan; dLaboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan

Testicular function is regulated by the complex interplay of many different molecules that includes both endocrine and paracrine signaling, and proper regulation is critical for optimum reproductive capacity. Among molecules, relaxin has also been suggested to be implicated in regulating testicular function in rodents. Also in boars, there is limited evidence that the testis might be a candidate source and target of relaxin, although it is presently unknown whether relaxin actually has any effect on the testis. To identify that the boar testis is a source and target tissue of relaxin, we characterized the expression and cellular localization of both relaxin and its own receptor LGR7 by molecular and immunological approaches. Relaxin gene expression increased through puberty onwards, while LGR7 gene expression changed little during pubertal development. Cellular expression of relaxin gene and protein was restricted to the Leydig cells, whereas LGR7 gene and protein were expressed by both Leydig cells and seminiferous epithelial cells. Interestingly, relaxin protein was only detected in the testis as 18 kDa form (the expected size of prorelaxin), but not as the 6 kDa mature form, during pubertal development because of lacking the processing enzyme for prorelaxin. Because recombinant and native prorelaxin is known to have a high bioactivity, it is possible that the 18 kDa protein expressed in the boar testis also has substantial bioactivity. Thus, we conclude that the testis is both a source and target tissue of relaxin in boars, suggesting that a functional relaxin/LGR7 hormone-receptor network operates within the boar testis.

47 Relaxin 2008

P-39

α-Adrenergic Activation Upregulates Expression of Relaxin Receptor RXFP-1 in Cardiomyocytes

XiaoLei Moore, Alice Hong, and Xiao-Jun Du

Experimental Cardiology Lab, Baker Heart Research Institute, Melbourne, Australia

Relaxin family peptide receptor-1 (RXFP-1) mediates multiple effects of relaxin. However, factors regulating RXFP-1 expression remain largely undefined. We have explored whether activation of α- and β-adrenergic receptors (AR), a class of GPCRs, may regulate expression RXFP-1 in cardioyocytes. Neonate rat cardiomyocytes (NRCM) in culture were treated with α1- or β-AR agonists for 24 hr, in the presence or absence of specific inhibitors. RXFP-1 mRNA level was analysed by real-time qPCR with results normalized by reference genes. Compared with untreated NRCMs, α1-AR agonists (100 μM methoxamine, 5-50 μM phenylephrine or 10 μM norepinephrine) increased RXFP-1 expression by approximately 3 folds (P<0.0001). In contrast, β1-AR agonists (10 μM dobutamine) reduced RXFP-1 expression by 50% (P=0.001) while β2-AR agonist zinterol (1 μM) showed no effect. The upregulation of RXFP-1 mediated by α1-AR agonists was blocked by selective inhibitors, respectively, for protein kinase-C (PKC, 1 μM BIM or 10 μM Ro320432), protein kinase-A (PKA, 2 μM KT5720 or 1 μM H89) or ERK (30 μM PD98059 or 10 μM U0126). In mice with cardiomyocyte-restricted overexpression of α1A- and α1B-AR, RXFP-1 expression, relative to non-transgenic littermates, increased by 3.2-fold (P<0.0001) and 7.6-fold (P<0.02), respectively, in keeping with the in vitro finding. RXFP-1 expression was not altered in hearts of β2-AR transgenic mice.

Conclusion: α1-AR activation up-regulates RXFP-1 gene expression with signal pathways involving PKA, PKC and ERK, while activation of β1-AR, but not β2-AR, down-regulates RXFP-1 expression. This is the first demonstration on distinct regulation by adrenergic signalling of RXFP-1 expression, which links effects of relaxin with sympathoadrenergic activities.

P-40

Relaxin/RXFP1 Signaling in Prostate Cancer Progression

Shu Fenga, Irina U. Agoulnikb, Zhen Lia, Hee Dong Hanc, Gabriel Lopez-Beresteinc, Anil Soodc, Michael M. Ittmannd, and Alexander I. Agoulnika

aDepartments of Obstetrics and Gynecology, bMolecular and Cellular Biology, dPathology, Baylor College of Medicine, Houston, Texas 77030, USA; cDepartment of Gynecologic Oncology, MD Anderson Cancer Center, University of Texas, Houston, Texas 77030, USA

We investigated the role of relaxin/RXFP1 signaling in prostate cancer progression. Quantitative RT-PCR analysis showed that while the mRNA expression of relaxin receptor RXFP1 was maintained at the same level in human prostate cancer and normal samples, relaxin mRNA expression was significantly higher in prostate cancer samples compared with the normal prostate tissues. Relaxin immunohistochemistry using tissue microarrays confirmed higher relaxin protein expression in prostate cancer samples. Stimulation with relaxin increased LNCaP and PC3 cells proliferation, invasiveness, and adhesion in vitro. Alternatively, the suppression of relaxin/RXFP1 expression via short interfering RNAs decreased prostate carcinoma cells invasiveness by 90-95%, cell growth by 10- 25% and increased cell apoptosis about 3 times. The RXFP1 gene suppression in PC3 cells resulted in decreased phosphorylation of protein kinase B (AKT). Illumina microarray analysis of changes in gene expression in PC-3 cells caused by RXFP1 siRNA knock down revealed several potential targets of relaxin signaling in prostate cancer. The effect of activation of the relaxin signaling on prostate cancer progression in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) was analyzed in mice with transgenic relaxin overexpression and Rxfp1-deficient mice. The Tg(Rln1), TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, whereas Rxfp1-/-, TRAMP mice on average lived longer than the wild type TRAMP mice. Suppression of RXFP1 expression in PC3 xenografts in nude mice using siRNA chitosan nanoparticles resulted in decrease of tumor size. Thus, relaxin/RXFP1 might be a potential therapeutic target in the treatment of prostate cancer.


O-45

Relaxin’s Induction of Specific MMPs Contributes to Degradation of Cartilage Matrix in Target Synovial Joints: Receptor Profiles Correlate with Matrix Remodeling Responses

Sunil Kapila and Wei Wang

Department of Orthodontics and Pediatric Dentistry, The University of Michigan, Ann Arbor, Michigan 48109, USA

The long-term goal of our studies is to understand the mechanisms by which relaxin and estrogen potentially contribute to joint diseases particularly those afflicting the fibrocartilaginous temporomandibular joint (TMJ). Previously, we showed that relaxin produces a dose-dependent induction of tissue degrading enzymes of the matrix metalloproteinase (MMP) family, specifically MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (92-kDa gelatinase), and MMP-13 (collagenase-3) in cell isolates and tissue explants from the rabbit and mice TMJ disc fibrocartilage. Further in vitro tissue explant studies demonstrated that the induction of these MMPs is accompanied by loss of tissue collagen and glycosaminoglycans (GAGs), which was blocked by adding a pan-MMP inhibitor to the cultures. We also found the targeted in vivo loss of collagen and GAGs in TMJ discs of ovariectomized rabbits treated with beta-estradiol or relaxin or both hormones together. Progesterone attenuated the induction of MMPs and matrix loss by relaxin and estrogen. The modulation of matrix composition in TMJ fibrocartilage by these hormones was similar to that observed in the pubic symphysis, and differed from that of the knee meniscus, which did not show any changes in its matrix composition. Finally, we found that the two target tissues showing the greatest modulation of MMPs and matrix loss, namely the TMJ disc and pubic symphysis, had similar expression profiles of the estrogen receptors (ER)-alpha and -beta, relaxin-1 (LGR7) receptor, and INSL3 (LGR8) receptor that differed substantially from those in cells from the knee meniscus. Together, these findings suggest a novel model for targeted tissue turnover of cartilages of specific joints through hormone-mediated induction of select MMPs. (Supported by NIH R29 DE11993, KO2 DE00458 and RO1 DE018455)

O-46

Relaxin’s Involvement in ECM Homeostasis: Two Diverse Lines of EvidenceTimothy E. Cooneyb, John D. Lubahnb, Justine M. Schoberb, and Elisa Konieczkoa

aDepartment of Biology, Gannon University, Erie, Pennsylvania 16541, USA; bDepartments of Orthopaedics, Orthopaedic Research, and Urology, Hamot Medical Center, Erie, Pennsylvania 16550, USA

Introduction: Burgeoning evidence suggests that the hormone, relaxin, modulates collagen in the ECM of diverse tissues (Garber et al., 2001; Williams et al, 2001; Samuel et al., 2002; Masterson et al., 2004; Samuel et al., 2004). In separate lines of study, we provide further substantiation of this notion.

Methods: LGR7 Expression in Ligament Fibroblasts: Immunofluorescence (IF) was used to probe isolated fibroblasts derived from explant culture for vimentin, actin, LGR7, and estrogen receptor beta. Ligaments were obtained as surgical waste from thumb reconstruction patients. Four specimens have been examined to-date.

LGR7 Expression in Pediatric Skin Biopsies: Immunohistochemistry (IHC) was used on frozen sections from 24 biopsies from children undergoing genitoplasty. A subset of samples were also probed for TGF-B isoforms. Appropriate controls were used. Finally, a subset of patient blood was assayed for relaxin using an ELISA-based method.

Results: IF data showed that cells expressed vimentin and actin, consistent with fibroblast morphology. Cells derived from two of three female patients expressed LGR7 receptors; the solitary male was negative. Given the small sample, however, the data are preliminary.

IHC data demonstrated LGR7 receptors in the basement membrane of the epidermis, regardless of gender. Most tissue expressed TGF-B. Finally, serology suggested that relaxin was detectable in these children.

Discussion: Our two lines of research provide additional evidence for diverse tissue tropism for relaxin. In particular, connective tissue as diverse as ligaments and basal lamina keratinocytes express LGR7. These data lend support to our contention that relaxin affects ligament integrity and wound healing.

49 Relaxin 2008

O-47

Relaxin in the Airway and Lung: Potential as a Novel Treatment for Asthma

Mimi L.K. Tanga, Chrishan S. Samuelb, and Simon R. Roycea

aDepartment of Allergy and Immunology, Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Victoria 3050 Australia; bHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

We have identified an important role for relaxin in the regulation of collagen deposition and remodeling in the airway/lung, and demonstrate a potential role for relaxin as a novel treatment for asthma, targeting airway fibrosis and airway hyperresponsiveness (AHR) in an animal model of allergic airway disease (asthma). We have previously reported that mice deficient in relaxin develop age-related changes similar to airway remodeling in asthma, including subepithelial fibrosis, epithelial remodeling, and AHR; and that mice deficient in relaxin have markedly increased airway fibrosis in an animal model of allergic airway disease (AAD). It is now demonstrated that relaxin and the relaxin receptor LGR7 are co-localised to cells involved in the remodeling process in asthma – airway epithelium, fibroblasts, smooth muscle cells, and that relaxin expression by these cells is significantly reduced in human asthma and in a murine model of AAD. Together, these findings suggest a role for relaxin as an inhibitor of airway fibrosis and AHR in asthma. The effect of exogenous relaxin treatment on airway remodeling and airway hyperresponsiveness was examined in a chronic model of AAD. Recombinant relaxin reversed collagen deposition in the airways and significantly reduced AHR when compared to vehicle treated control animals. Matrix metalloprotease levels were increased, suggesting a possible mechanism for the antifibrotic effect.

Conclusion: Relaxin plays an important role in the negative regulation of airway fibrosis during homeostasis and in asthma. Relaxin treatment can reverse remodeling changes of airway fibrosis and significantly inhibit AHR in an animal model of AAD (asthma).

O-48

Scar Reduction and Cosmetic Effects of Relaxin

Dennis R. Stewart

Corthera, Inc. (formerly BAS Medical, Inc.), San Mateo, California 94402, USA

Relaxin has previously been tested in rodent wound healing models and been shown to promote angiogenesis and speed healing. However, pigs have been shown to be a better model for human skin in dermatology studies so juvenile pigs were selected for a study of scar reduction and cosmetic appearance. Twelve 20 x 6 mm excisional wounds were created per animal and relaxin was administered systemically and/or by topical application for 6 weeks. Systemic relaxin was administered via infusion pumps at a rate of 125 ug/kg/day. Some wounds were irrigated with relaxin solution during the first week. Topical formulations of relaxin with 0, 0.5 or 5 mg/ml and applied twice daily for weeks 2-3 and then daily for weeks 3-6. Visual assessments of wound closure and scab removal were made on day 7 following wound creation. Assessments of healing and cosmetic appearance were made by a dermatologist at weeks 2, 4 and 6. Wound sites were collected at 6 weeks and evaluated histologically for granulation tissue, inflammation and collagen organization. Wounds in animals receiving systemic relaxin had improved appearance with less redness, reduced granulation tissue and lower amounts of inflammation. They showed a more well knit collagen structure compared to controls. Wounds treated with topical formulations did not show improvement over controls. The topical formulation used was found to have a short residence time which likely limited penetration of relaxin. Reformulated relaxin preparations with improved penetration might be useful as a topical treatment for wounds to prevent or reduce scarring.


O-49

Role of Relaxin during Human Osteoclastogenesis

Arianna Facciolli, Anastasia Pepe, Lisa Gianesello, Carlo Foresta, and Alberto Ferlin

University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, 35121 Padova, Italy

The aim of this study is to characterize the possible role of relaxin of human osteoclasts. In fact, we are currently investigating the effects of relaxin and INSL3 on bone metabolism, and previous studies showed en effect of relaxin on peripheral blood mononuclear cells (PBMCs), the precursors of osteoclasts. Analysis of RXFP1 and RLN-2 mRNA expression in primary cell culture of human osteoclasts obtained from PBMCs by RT-PCR showed only the presence of RXFP1 transcripts. Immunofluorescence analysis confirmed the expression in osteoclasts also of the corresponding RXFP1 protein. We then analysed the in vitro effect of relaxin on osteoclastogenesis (from PBMCs to mature osteoclasts, about 14 days) and activated mature osteoclasts. We added relaxin 50 pM during osteoclastogenesis and collected the cells every 2-3 days. RNA from these cells was used to perform quantitative RT-PCR for the analysis of expression of 4 osteoclast specific genes (TRAP, CTSK, RANK, NFATc1). Relaxin produced a significant increase in CTSK, RANK and NFATc1 gene expression but not on TRAP. However, acute (minutes, hours) and chronic (days) stimulation of the activated mature cells with the same dose of relaxin did not modify gene expression. This study shows, for the first time, a role for the hormone relaxin on human bone metabolism regulating osteoclastogenesis.

O-50

In Search of the Elusive Vasodepressor Agents of Pregnancy

Kirk P. Conrada, Lee A. Danielsonb, Sanjeev G. Schroffc, Dan O. Debrahc, Arundhathi Jeyabalanc, Jacqueline Novakd, John M. Davisone, and Marie Smithe

aUniversities of Florida, bNew Mexico, cPittsburgh, and dWalsh University, USA; eUniversity of Newcastle upon Tyne, United Kingdom

Understanding the mechanisms underlying arterial adaptations to normal pregnancy may lead to novel therapies for the prevention and treatment of preeclampsia, as well as certain renal and cardiovascular diseases in the nonpregnant population. Our early work challenged the prevailing hypothesis that vasodilatory prostaglandins mediate maternal renal and systemic hemodynamic adaptations to pregnancy. Rather, we provided evidence for a major role of NO and its up- and down-stream mediators, the endothelial ETB receptor and cGMP, respectively. In search of reproductive endocrine signals, we postulated a role for the ovarian hormone, relaxin. We subsequently showed that relaxin administration to conscious female and male rats increases renal blood flow and glomerular filtration rate (GFR), and inhibits myogenic reactivity of small renal arteries ex vivo. Identical renal circulatory changes are observed during pregnancy, and they are prevented by relaxin neutralizing antibodies or ovariectomy. In nonpregnant women and men, relaxin is also a potent renal vasodilator, and women lacking circulating relaxin show markedly subdued increases in GFR during early pregnancy. Relaxin administration to conscious male and female rats increases cardiac output and global arterial compliance, and reduces systemic vascular resistance. Identical changes are observed during pregnancy, and they are completely or partly abrogated by relaxin neutralizing antibodies during mid and late gestation, respectively. Investigations of another candidate vasodilator, hCG, suggests that it may play a role in some organ circulations during pregnancy, but not the kidney. Finally, additional players in the relaxin vasodilatory pathway have been identified, including Lgr7/RXFP1, vascular gelatinases and angiogenic growth factors.

51 Relaxin 2008

O-51

Relaxin: An Endogenous Renoprotective Factor?

Tim D. Hewitsona,b and Chrishan Samuelc,d

aDepartment of Nephrology, The Royal Melbourne Hospital, Parkville, Victoria 3050 Australia; bDepartment of Medicine, The University of Melbourne, cHoward Florey Institute and dDepartment of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia

Fibrosis, so called scarring, is a key cause of pathology and dysfunction in a variety of organs. The biology of healing and scarring are similar, albeit with very different outcomes. While the kidney has some capacity for repair after acute injury, renal fibrosis represents a failure of the wound-healing process after prolonged injury. Typical of this process in general, renal fibrosis is the result of a disproportionate accumulation of extracellular matrix that occurs in ongoing kidney disease. Renal scarring is both progressive and multifactorial, ultimately leading to end-stage renal failure and the requirement for dialysis or kidney transplantation.

Regardless of the underlying etiology, renal mesenchymal cells such as the fibroblast are the cellular basis of this process. Activity and differentiation of these cells are regulated by a number of profibrotic cytokines, growth factors, vasoactive mediators and other signals, with transforming growth factor-beta being amongst the most important. However, we now also recognise that there are a number of endogenous antifibrotic factors that are renoprotective by counteracting these signals to limit matrix synthesis and organisation, and reduce net accumulation. Relaxin is one such factor. The absence of endogenous relaxin results in spontaneous fibrosis with aging and also accelerates the pathogenesis of progressive fibrosis after injury. Both effects are reversed by replenishment with recombinant H2- relaxin.

This talk summarises recent developments in the understanding of the cellular and molecular mechanisms in fibrosis, and how these are influenced by relaxin.

O-52

Investigations into the Signaling Mechanisms by which Relaxin Inhibits Renal Myofibroblast Differentiation

Chrishan S. Samuela,b, Ishanee Mookerjeea,b, Michelle L. Hallsc, Roger J. Summersc, Geoffrey W. Tregeara,b, and Tim D. Hewitsond,e

aHoward Florey Institute and Departments of bBiochemistry and Molecular Biology and dMedicine, The University of Melbourne, Parkville, Victoria 3010, Australia; cDepartment of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; eDepartment of Nephrology, Royal Melbourne Hospital, Parkville, Victoria 3050 Australia

Derived from fibroblasts, myofibroblasts are the principal cells that are responsible for the synthesis and reorganization of excess matrix in renal interstitial fibrosis. Recognized from their de novo expression of α-smooth muscle actin (α-SMA), myofibroblast differentiation and activity can be influenced by several factors including a combination of growth factors and other soluble mediators, extracellular matrix components and mechanical stress. Relaxin has previously been shown to inhibit renal myofibroblast differentiation in vitro, an effect partly mediated through its ability to interfere with the TGF-β pathway via inhibition of Smad2 phosphorylation and translocation. Furthermore, endogenous relaxin has been shown to protect the kidney from a myofibroblast-mediated model of injury in vivo. However, the pathways involved in the interaction between relaxin and TGF-β remain unknown. In this study, we therefore sought to further investigate the signaling mechanisms by which human gene-2 (H2) relaxin regulates (myo)fibroblast differentiation in vitro by examining the effect of relaxin on mixed populations of fibroblasts/myofibroblasts propagated from injured rat kidneys. Cultures containing ~70% myofibroblasts were assessed for relaxin receptor mRNA expression and used to determine which i) relaxin receptors (RXFP1, RXFP2), ii) G-proteins and iii) signaling pathways (PI3 kinase, nitric oxide, Smad2) were involved in the regulation of α-SMA by H2 relaxin. Our findings suggest that relaxin protects the injured kidney by signaling through RXFP1 in addition to the nitric oxide and Smad2 pathways to inhibit renal myofibroblast differentiation.


O-53

Relaxin-induced Changes in Renal Function and RXFP1 Receptor Expression in the Female Rat

Alsadek H. Bogzil and Nick Ashton

Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Pregnancy is associated with profound changes in renal structure and function. The aim of this study was to determine whether relaxin induces pregnancy-like changes in renal function in female rats. Female Sprague Dawley rats received human recombinant relaxin (0.4 μg/h or 4 μg/h) or vehicle (20 mM sodium acetate) via an osmotic minipump for 7 days prior to the measurement of renal function using a servo-controlled fluid replacement system to maintain euvolaemic body fluid status. Extracellular fluid volume was determined by calculating the dilution of injected 3H inulin. Kidneys were harvested from relaxin-treated and pregnant rats (day 11, corresponding to maximal plasma [relaxin]) to quantify RXFP1 receptor expression. Seven-day administration of relaxin at 0.4 μg/h resulted in a decrease in plasma osmolality (control 292.6±2.1 vs relaxin 236.9±6.0 mOsm/kg), sodium and chloride concentrations (P<0.05); extracellular fluid volume was increased (control 19.5±0.4 vs relaxin 21.6±0.7 ml/100g bwt, P<0.05). Urine flow rate was unaffected, but sodium (control 4.9±0.5 vs relaxin 2.8±0.4 μmol min-1 100g bwt-1), chloride and osmolar excretion rates were decreased significantly (P<0.01). RXFP1 expression (protein and mRNA) was increased in the kidneys of rats treated with relaxin at 0.4 μg/h and in pregnant rats. Immunohistochemistry showed that RXFP1 was present on the proximal tubules and inner medullary collecting ducts. In contrast, relaxin-treatment at 4 μg/h for 7 days had no effect on renal function in female rats. These data suggest that relaxin may alter renal electrolyte handling in a manner similar to that seen in pregnancy.

O-54

Vascular Endothelial and Placental Growth Factors: New Players in the Slow Relaxin Vasodilatory Pathway

Julianna Debrahb, Jonathan T. McGuanea, Jacqueline Novakd, J. Peter Rubinc, and Kirk P. Conrada

aDepartment of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 32610, USA; bMagee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA; cUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA; dWalsh University, North Canton, Ohio 44720, USA

The mechanism of relaxin’s "slow" vasodilatory effect in rats involves MMP2- or MMP9-mediated conversion of big endothelin (ET) to ET1-32, which activates the endothelial ETB receptor/NO pathway. Because LGR7/RXFP1 is predominantly expressed in the vascular smooth muscle (VSM), we reasoned that this vasodilatory response to relaxin, which mediates renal vasodilation in vivo and reduced myogenic reactivity of small renal arteries ex vivo, is transduced from the VSM to the endothelium, and hypothesized that vascular endothelial and/or placental growth factors (VEGF and PlGF) could fulfill this function.

Small rat and mouse renal and human subcutaneous arteries were studied in a pressure arteriograph. In arteries pre-treated with control antibodies, myogenic reactivity (measured by the percent change in diameter in response to a 20mmHg step increase in intraluminal pressure) was inhibited by relaxin treatment (30ng/ml) for 3 hours. In contrast, pre-treatment with VEGF-neutralizing antibodies blocked the response to relaxin and restored myogenic reactivity in rat and mouse small renal (1.4±0.8 and 0.3±0.9% [VEGF Abs] vs 8.7±1.1 and 5.4±0.4% [control Abs], respectively; p<0.001) as well as human subcutaneous arteries (0.9±0.7% [VEGF Ab] vs 6.4±1.0% [control Ab]; p=0.003). Furthermore, pre-treatment with PlGF-neutralizing antibodies similarly negated the effect of relaxin in rat and mouse small renal (-0.7±0.8 and 1.2±1.1% [PlGF Abs] vs 7.1±0.9 and 5.4±0.4% [control Abs], respectively; p#less than or equal to#0.005) as well as human subcutaneous arteries (-2.7% [PlGF Ab] vs 5.9% [control Ab]).

These data indicate that VEGF and PlGF are essential players in the relaxin vasodilatory pathway, potentially involved in cross-talk between VSM and endothelium.

53 Relaxin 2008

O-55

Effects of Relaxin on the Development of Mesangial Proliferative Nephritis

Naoki Ikegayaa, Tetsuya Kohsakab, Takuya Yoshidac, Hiromichi Kumagaic, Tatsuo Yamamotod, and Akira Hishidad

aMedical Care Center, bDepartment of Applied Biological Sciences, Shizuoka 422-8529, Shizuoka University, cDepartment of Clinical Nutrition, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, d1st Department of Medicine, Hamamatsu University, School of Medicine 431-3192, Hamamatsu, Japan

Relaxin (RLX), belonging to the insulin family, has been known as a hormone of pregnancy. Although potent antifibrotic and vasodilatory properties of RLX are recently reported, the involvement in nephritis has not been fully elucidated. In this study, we examined the expression of RLX receptors and effects of RLX administration in experimental nephritis induced by anti-thymocyte serum (ATS). When examined immunohistochemically the expression of RLX receptors in normal and nephritic kidneys, nephritic glomeruli showed an increased immunostaining compared to normal glomeruli. The administration of RLX by osmotic minipump for 7 days to rats with ATS nephritis did not cause a significant alteration in blood pressure or body weight. On the 7th day, however, a significant reduction in urinary protein (66.6 vs. 35.8 mg/day, p<0.05) was recognized in rats treated with RLX. Histologic studies revealed the amelioration of glomerular accumulation of PAS positive matrix in rats received RLX. A significant reduction of phosphorylated SMAD2 as a marker of TGF-β activation was observed in rats treated with RLX compared to rats without RLX treatment. These results indicate that RLX may play a significant role in the development of mesangial proliferative glomerulonephritis in rats independently of blood pressure, suggesting that RLX can be a potential therapeutic tool for nephritic diseases.

O-56

Relaxin Affects Endothelial Progenitor Cell Number and Function

Mark Segala, Laura Sautinaa, Elaine Beema, and Kirk Conradb

aDepartments of Medicine, bPhysiology and Functional Genomics, and Ob/Gyn, University of Florida College of Medicine, Gainesville, Florida 32610, USA

Relaxin is known to induce vasodilation that is endothelial and nitric oxide (NO)-dependent. Endothelial progenitor cells (EPC) are derived from the bone marrow and can repair vascular endothelium. Since previous studies have shown a profound effect of intracellular NO on the number and function of EPC. We hypothesized that relaxin would increase the number of circulating cells in vivo and stimulate EPC migration in vitro via an NO dependent mechanism. Male mice were implanted with osmotic minipumps containing recombinant human relaxin (rhRLX) or vehicle (VEH). rhRLX was infused at 1µg/hour. After 5 days, the number of EPC colonies and serum levels of rhRLX were determined. Relaxin-treated mice had more EPC colonies derived from blood than VEH-treated mice (3 +/- 0.8 vs 0.7 +/- 0.5, p <0.05), and there was a significant correlation between the number of EPC colonies and serum rhRLX concentrations (R2 = 0.42). In addition, we demonstrated that human EPC migrate equally well towards rhRLX and stromal-derived factor (SDF)-1, the major chemokine for EPC. Interestingly, EPC chemotaxis was inhibited by L-NAME, an inhibitor of NO synthase. Last we demonstrated that addition of rhRLX to human EPC leads to an increase in NO. These results suggest that relaxin can lead to an increase in NO in EPC and modify EPC number in vivo and behavior in vitro. These findings have profound implications for the therapeutic potential of relaxin. On a cautionary note, relaxin may contribute to progression of diabetic retinopathy during pregnancy.


O-57

Evaluation of Relaxin Blood Profiles as a Means of Assessing Placental Function and Responsiveness to Therapeutic Strategies in Mares

Peter L. Ryana, David L. Christiansena, Richard M. Hoppera, Carol A. Bagnellb, Wendy E. Vallac, and Michelle M. LeBlancd

aMississippi State University, College of Veterinary Medicine, Mississippi State, Mississippi 39762, USA; bRutgers University, New Brunswick, New Jersey 08901, USA; Intervet Incorporated, Millsboro, Delaware 19966, USA; cRood and Riddle Equine Hospital, Lexington, Kentucky 40511, USA

Placental insufficiency is regarded as the primary factor contributing to late-term abortion and perinatal death of foals. Often when problems associated with late-term pregnancy in the horse are manifested the condition is well advanced and therapeutic intervention may be ineffective in rescuing the pregnancy. If a compromised pregnancy due to placental insufficiency could be identified early, the pregnancy might be sustained through medical intervention. Since the placenta is the sole source of circulating relaxin in mares we hypothesized that systemic relaxin may serve as a biomarker of placental function, fetal well-being and predictor of pregnancy outcome at delivery. To test this hypothesis we monitored plasma relaxin in mares (light breeds) with normal and problematic pregnancies from clinical cases presented to the veterinary hospital and pregnant mares experimentally inoculated with Streptococcus equi zooeidemicus to induce uterine infection. Upon establishment of placentitis, mares were assigned to different therapeutic strategies and responsiveness monitored. Blood was collected during the third trimester of pregnancy to determine relaxin concentrations using a homologous equine relaxin radioimmunoassay. The results showed a positive relationship between lower circulating relaxin and poor pregnancy outcome in mares with compromised placental function in addition to improved profiles in response to drug therapy. Data support the hypothesis that relaxin has the potential to serve as a useful means of monitoring placental function and fetal-well being in horses. Relaxin, as a diagnostic assay, could assist veterinarians identify mares with at-risk pregnancies in a more timely fashion, evaluate therapeutic efficacy, placental function and fetal well-being.

O-58

Relaxin Concentrations in Serum and Urine of Endangered and Crazy Mixed-up Species: New Methods, Uses and Findings

Bernard Steinetza, Salamia Lasanoa, Florine de Haas van Dorsserb, Stephen Glickmanc, and William Swansond

aDepartment of Environmental Medicine, NYU School of Medicine, Tuxedo, New York 10987, USA; bDepartment of Veterinary Medicine, University of Cambridge, United Kingdom; cDepartment of Psychology, University of California, Berkeley, California 94720, USA; dCenter for Conservation and Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio 45220, USA

The human population explosion has pushed many mammalian wildlife species to the brink of extinction. Conservationists are increasingly turning to captive breeding as a means of preserving the gene pool. Serum immunoactive relaxin provided an early and reliable indicator of true pregnancy in Asian and African elephants and Sumatran rhinoceroses, and is thus an important aid for conservationists studying reproduction in subjects with very long pregnancies. Unfortunately, the immunoassay was not useful in the giant panda, because of its variable implantation time and gestation length. These studies required judicious selection of relaxin antisera and standards developed in domestic species: equine relaxin RIAs for elephants; porcine relaxin RIAs for Sumatran rhinos and pandas.

Our canine relaxin RIA has now been adapted and validated to measure relaxin in the serum and urine of felids, including domestic, Pallas’ and sand cats, Arabian leopards and cheetahs. Moreover, a commercially available canine serum relaxin kit (Witness Relaxin Kit, Synbiotics), has been adapted for reliable detection of relaxin in felid urine.

The porcine relaxin RIA has also been utilized to investigate the role of relaxin in the reproductive processes of the spotted hyena, a species in which the female fetuses are severely masculinized in utero. Indeed, this species might well now be extinct were it not for the timely secretion of relaxin to enable both copulation and birth of young through the clitoris.

Given appropriate immunoassay reagents, relaxin determination thus provides a powerful tool for conservationists and biologists investigating reproduction in endangered and exotic species.

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O-59

Development of Adult Neuronal-specific Relaxin-3 Knockout Rats Using Adeno-associated Viral-driven miRNA

Gabrielle E. Callandera, Walter G. Thomasb, Andrew L. Gundlacha, and Ross A.D. Bathgatea

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bBaker Heart Research Institute, Melbourne, Victoria 3004, Australia

Recent in vivo peptide administration studies suggest a role for relaxin-3 in feeding and stress, however the function of relaxin-3 remains elusive due to the significant challenges of the technique, including limited permeability of the blood-brain barrier and problems with peptide stability, absorption and metabolism. Additionally, results from knockout (KO) mice must be interpreted with caution as KOs often develop functional compensation. In studies designed to avoid such obstacles, we are employing RNA interference and viral gene transfer, to modulate neuronal relaxin-3 expression in the adult rat brain. We have designed, constructed and characterized adeno-associated viral particles containing relaxin-3 (pAAV-Rln3) or microRNA (pAAV-miRNA) targeting relaxin-3. Media collected from HEK293T cells transiently expressing pAAV-Rln3, stimulated RXFP1-mediated cAMP accumulation in a dose-dependent fashion, similar to H3 relaxin, whereas media from cells transfected with a control viral vector had no effect. Three constructs, containing miRNA directed against different targets within the relaxin-3 transcript, were then tested for their ability to reduce the expression of relaxin-3 in pAAV-Rln3 transfected cells. All miRNA constructs significantly reduced relaxin-3-induced cAMP responses, however miRNA499 was most effective. To investigate the role of relaxin-3 in feeding, rAAV-miRNA499 and a control virus rAAV-miRC, were produced and purified for stereotaxic injection into the nucleus incertus of male rats. Post-infection body weight and food consumption are currently being monitored. These viral tools can be readily utilised in vivo to investigate the role of relaxin-3 in other behaviours including arousal/motivation, biorhythms and responses to stress.

O-60

Presence and Role of LGR8 (RXFP2) in Thalamo- and Cortico-Striatal Circuits: Effects of Intrastriatal Injection of INSL3 on Stereotypic Behaviour in the Rat

Katayoun Sedaghata, Suode Zhanga, Ross A.D. Bathgatea, John D. Wadea, David I. Finkelsteinb, and Andrew L. Gundlacha

aHoward Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia; bMental Health Research Institute, The University of Melbourne, Parkville, Victoria 3052, Australia

Leucine-rich repeat-containing G-protein-coupled Receptor 8 (LGR8 or "RXFP2") is the native receptor for the peptide hormone, insulin-like peptide-3 (INSL3), and the aim of these studies was to determine the anatomical distribution of LGR8 (mRNA and protein) and its functional role in rat brain. In situ hybridization of [35S]-labeled oligonucleotides revealed high densities of LGR8 mRNA in the intralaminar thalamus (especially the parafascicular nucleus), distinct layers of frontal cortex and the medial habenula. Autoradiography of [125I]-INSL3 binding sites revealed high densities of LGR8 protein in distinct bands of frontal and motor cortex, in the thalamus and corpus striatum, and in medial habenula-fasciculus retroflexus-interpeduncular nucleus pathway. In conscious rats, bilateral injection of INSL3 (~10-40 pmol/side in 0.5 µl) into the LGR8-rich ventrolateral striatum, via indwelling guide cannulae, produced a rapid and strong behavioural activation, with a distinct upward-sniffing displayed for between 2-40 min post-injection. Injection of the LGR8 antagonist, Ades(1-8)-INSL3 (~40 pmol), did not produce sniffing, but co-injection with an equal amount of INSL3, blunted the effect of the agonist peptide. Co-injection of amphetamine (20 µg) with INSL3 (40 pmol) produced a significant increase in sniffing behaviour cf. INSL3 alone. In light of the anatomical data suggesting the presynaptic location of LGR8 on excitatory thalamo- and cortico- striatal projections, and the behavioural effects observed, we suggest that INSL3-induced sniffing behavior is produced by LGR8-induced activation of glutamate release in the striatum. This in turn may alter dopamine release from nigrostriatal inputs and alter the activity of target medium spiny neurons - ideas that can be tested experimentally.


Notes

Abstracts



P-41

Relaxin Reduces Fibrosis in Models of Progressive and Established Hepatic Fibrosis

Robert G. Bennetta,b,c, Dean G. Heimanna, and Dean J. Tumaa,b

aVeterans Affairs Medical Center, Omaha, Nebraska 68105, USA; bDepartments of Internal Medicine, Pharmacology and Experimental Neuroscience, and cDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA

Relaxin has antifibrotic effects in a number of tissues, including the liver. Previous studies have focused on the effects of relaxin in the prevention of hepatic fibrosis, but not after establishment of the disease. To address this issue, we compared the effects of relaxin treatment either before or after the establishment of hepatic fibrosis. In the progressive fibrosis study, mice were treated intraperitoneally with carbon tetrachloride (CCl4) for 4 weeks, concomitantly with porcine relaxin administered by osmotic pumps at 0.5mg/kg/day. In these animals, relaxin caused a reduction in collagen and smooth muscle actin content, indicating reduced hepatic stellate cell activation. Serum levels of the liver enzymes ALT and AST were significantly reduced in the relaxin-treated animals, suggesting improved liver function. In the second experiment, CCl4 was administered for 4 weeks, at which time mice were treated with both CCl4 and relaxin at 0.5mg/kg/day for 1 week further. In relaxin-treated mice, both liver collagen and smooth muscle actin content were reduced compared to untreated mice. However, in contrast to the experiment above, relaxin treatment did not significantly reduce the ALT/AST levels. In conclusion, the data suggest that relaxin treatment concomitant with liver injury results in reduced collagen deposition and hepatic stellate cell activation, and improved liver function. Relaxin treatment after fibrosis establishment also reduced collagen and hepatic stellate cell activation, but did not improve overall liver function within the 1 week treatment time examined. Studies are underway to determine the effect of extended relaxin treatment on established hepatic fibrosis.

P-42

Evaluation of Relaxin’s Anti-fibrotic Actions by SELDI-TOF Mass Spectrometry-based Protein Profiling of Relaxin KO Mice, a Model of Progressive Fibrosis

Eleni Giannakisa, Mary Macrisa, Geoffrey W. Tregeara,b, Chrishan S. Samuela,b, and John D. Wadea,b,c

aHoward Florey Institute and Departments of bBiochemistry and Molecular Biology and cChemistry, The University of Melbourne, Parkville, Victoria 3010, Australia

Fibrosis (organ scarring) is a leading cause of morbidity and mortality, and is characterized by an overproduction of collagen and causes a hardening of vital tissues leading to a progressive loss of their function. The peptide hormone, relaxin (RLX) has known anti-fibrotic properties and is currently under investigation as a possible therapeutic agent. However, to be most effective, RLX needs to be administered in the early stages of disease progression. The primary aim of this study was to develop diagnostics for the onset of fibrosis to maximize the therapeutic potential of RLX and provide a means to measure the efficacy of therapeutic intervention strategies. The RLX-1 knockout (-/-) mouse, which exhibits age-related fibrosis progression, was used as the model for this study. RLX-/- mice were administered with recombinant human (H2) RLX in citrate buffer (vehicle), which reverses the diseased phenotype, or, as a control, with vehicle alone each day. Sera from healthy and diseased animals were collected at different time points: 9-months (early onset of fibrosis) and 12-months (established fibrosis). These were analysed by Surface Enhanced Laser Desorption Ionisation- Time of Flight- Mass Spectrometry (SELDI-TOF MS). Bioinformatics analysis using Biomarker Wizard© software revealed the presence of a potential biomarker for early onset fibrosis and which is upregulated in the diseased animals. Additional studies will involve assessing tissue from healthy and diseased animals and examining earlier time points before the diseased phenotype has been detected.

59 Relaxin 2008

P-43

Novel EGF and IGF1 Adenylyl Cyclase Signaling Mechanisms with Similar Structural-functional Organization

Marianna N. Pertseva, Ludmila A. Kuznetsova, Alexander O. Shpakov, and Svetlana A. Plesneva

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223, St. Petersburg, Russia

As we have shown for the first time the hormones of insulin superfamily (insulin, relaxin) are capable to activate adenylyl cyclase in muscle tissues of vertebrates and invertebrates via six-component signaling cascade: receptor-tyrosine kinase -> Gi-protein (βγ dimer) -> phosphatidylinositol 3-kinase -> protein kinase C (ζ) -> Gs-protein -> adenylyl cyclase (AC). The aim of the present study is to prove if this AC signaling mechanism (ACSM) is involved in the action of IGF1 and EGF - two unrelated growth factors. The deciphering of ACSM was performed with pharmacological and immunological approach (specific inhibitors and antibodies). It was found that AC activating effect of both growth factors are realized via: tyrosine kinase (tyrphostin 47 and genistein inhibition), Gi protein (pertussis toxin ADP ribosylation); phosphatidylinositol 3-kinase (wortmannin inhibition), protein kinase C (ζ) (specific antibody inhibition), Gs protein (cholera toxin ADP ribosylation). The data obtained and our foregoing results lead to conclusion that IGF-1 and EGF ACSM have a similar architecture, involving six-component signaling which coincides with that of insulin and relaxin. It is supposed that this similarity in ACSM of insulin superfamily peptides and EGF is based on their common growth promoting activity connected with cAMP-system. According to our hypothesis the mitogenic effect of hormones and growth factors of insulin nature and EGF is realized via ACSM.

The investigation was supported by grants: Russian Foundation for Basic Research 06-04-48809; Basic Research for Medicine, 2007.

P-44

Investigating the Role of Endogenous Relaxin in Experimental Diabetic Renal Disease

Su Ee Wonga,b,c, Tim D. Hewitsonc,d, James Beckerd, Geoffrey W. Tregeara,b, Gavin J. Beckerc,d, and Chrishan S. Samuela,b

aHoward Florey Institute and Departments of bBiochemistry and Molecular Biology and dMedicine, The University of Melbourne, Parkville, Victoria 3010; Australia; cDepartment of Nephrology, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia

Pathological complications of diabetes include the accumulation of excess matrix in several organs, including the kidney. Streptozotocin (STZ)-induced diabetic relaxin wild-type and knock-out mice were used to investigate the ability of endogenous relaxin to regulate renal collagen turnover. Renal pathology in untreated control animals was compared to age-matched diabetic animals with a mean blood glucose >14mmol/L after 2 STZ injections (n=6 per genotype and group). Histochemical and biochemical techniques were used to assess collagen content and sub-types, matrix metalloproteinase (MMP) expression and activity, tissue inhibitor of metalloproteinase (TIMP) expression and TGF-β1 activity. At 12 weeks post-STZ administration, diabetic animals had higher levels of glycated haemoglobin (by 1.3-fold; p<0.05), 1.4-fold (p<0.01) higher total renal collagen concentration (hydroxyproline analysis) and >2-fold (p<0.01) more collagen IV (immunohistochemistry; IHC), with no change in collagen I (IHC), III (SDS-PAGE) or tissue TGF-β1 activity (ELISA). For equivalent levels of hyperglycaemia, no differences in collagen concentration, sub-types or distribution were observed between genotypes. Activity of the type IV collagenase, MMP-2 (gelatin zymography) and its inhibitor, TIMP-2 (reverse zymography) were both up-regulated in diabetic mice (p<0.05), with no difference between genotypes. There was no increased expression of MMP-9, while its inhibitor TIMP-1 was only increased in diabetic knock-out mice (p<0.05). Findings in this model of early diabetic renal disease suggest that endogenous relaxin does not alter the pathology of the diabetic mouse kidney. This may be due in part to the normal levels of TGF-β1 activity in this model.


P-45

Determinants of Relaxin-induced Changes in Passive Compliance of Small Renal Arteries: Geometric vs. Compositional Remodeling

Dan O. Debraha, Julianna E. Debrahb, Michael S. Sacksa, Kirk P. Conradc, and Sanjeev G. Shroffa

aDepartment of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA; bMagee-Womens Research Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA; cDepartments of Physiology and Functional Genomics and Obstetrics and Gynecology, University of Florida, Gainesville, Florida 32610, USA

We have recently demonstrated that small renal arteries (SRA) from relaxin-treated mice are characterized by both geometric (increased wall area) and compositional (decreased collagen content) remodeling. Additionally, we found that these arteries also exhibit increased passive compliance following relaxin treatment. The goal of this work was to determine the relative contributions of these two forms of remodeling to the increase in passive compliance.

Nonpregnant female mice were administered recombinant human relaxin (rhRlx) for 5 days after which SRA were isolated. Ex vivo pressure-diameter data, collected at various longitudinal stretches, were analyzed using a biaxial constitutive relationship (7-parameter mixed polynomial-exponential model of strain energy function). Vascular wall mechanical properties at various levels of circumferential and longitudinal strains were quantified in terms of the corresponding strain energy.

There were no significant differences in strain energy between control and rhRLX-treated groups at low strains (longitudinal stretch ≤ 1.5, circumferential stretch ≤1.8). At higher strains, however, arteries from the rhRLX-treated mice exhibited increasingly lower magnitudes of strain energy such that the greatest reduction was observed at the highest strains.

Our data suggest that under low longitudinal and circumferential loads, the relaxin-induced increase in passive compliance of SRA is primarily a result of geometric and not compositional remodeling because vascular wall mechanical properties (strain energy) are unaltered under these conditions. The relaxin-induced compositional remodeling of SRA becomes functionally relevant only under conditions of high longitudinal and circumferential loads.

P-46

Relaxin as a Protective Substance in the Preserving Solution for Liver Transplantation: Spectrophotometric in vivo Imaging of Local Oxygen Supply in an Isolated Perfused Rat Liver Model

Markus U. Boehnerta, Franz Paul Armbrusterb, and Heidegard Hilbigc

aDepartment of Visceral and Transplantation Surgery, University of Bern, Inselspital, 3010 Bern, Switzerland; bImmundiagnostik AG, 64625 Bensheim, Germany; cInstitute of Anatomy, University of Leipzig, 04103 Leipzig, Germany

Reperfusion injury is a well-known problem in organ transplantation. Liver injury caused by ischemia and reperfusion results from multiple pathologic mechanisms including endothelial damage, neutrophil extravasation, platelet and mast cell activation as well as peroxidation of cell membrane lipids. Relaxin is known to cause inhibition of platelet and mast cell activation and vessel dilation. Firstly, the local oxygen supply in liver tissue was investigated by spectrophotometric in vivo imaging. Secondly, the present study aims to assess the protective effect of relaxin in preservation solution on liver tissue against cell damage during organ preservation and reperfusion. We used a model of isolated perfused rat liver, simulating liver transplantation. Organ preservation was performed identically to human transplantations in 30 male Wistar rats. 64ng of Relaxin per ml solution were applied during preservation. Controls (n=15) were performed identically without Relaxin. Spectrophotometric imaging of oxygen supply was done using the Erlangen Micro-Lightguide Spectrophotometer. The oxygen supply was correlated with markers of oxidative cell damage using immunohistochemistry. Determinants of oxidative stress were malonyldialdehyde (MDA), a metabolite of lipid peroxidation and myeloperoxidase activity (MPO), a marker for accumulation of neutrophil granulocytes. Compared to the controls, spectrophotometry qualitatively revealed a better oxygen supply in Relaxin treated liver tissue. Accordingly, administration of Relaxin in the preservation solution decreased MPO and MDA activity in liver tissue. Our data suggest that Relaxin has an influence on oxygen distribution in liver tissue and is a promising agent to reduce cell damage in the liver caused by ischemia and reperfusion injury.

61 Relaxin 2008

P-47

Involvement of Relaxin in Salt-sensitive Hypertension

Naoki Ikegayaa, Tetsuya Kohsakab, Takuya Yoshidac, and Hiromichi Kumagaic

aMedical Care Center, bDepartment of Applied Biological Sciences, Shizuoka 422-8529, Shizuoka University, cDepartment of Clinical Nutrition, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan

Although potent anti-fibrotic and vasodilatory properties of relaxin (RLX) are recently reported, the involvement of RLX in salt-sensitive hypertension has not been elucidated. We investigated effects of RLX on salt-sensitive hypertension. First, we examined RLX and RLX-receptor (LGR7) in the kidney of male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats placed on 8.0% salt diet for 8 weeks. RLX and LGR-7 mRNA was observed in both cortex and medulla of DS and DR rats. Specific binding of RLX in kidneys was observed and the binding sites of RLX were similar to LGR-7 immunostaining. By western blotting, renal LGR7 expression increased after 8% NaCl diet. The increase of LGR7 protein was greater in DS rats than that in DR rats. Next, we examined effects of RLX treatment on blood pressure, renal histology, apoptosis and TGF-β activation in DS and DR rats placed on an 8% NaCl diet. The administration of RLX (4 microg/h) to DS rats for 8 weeks significantly reduced systolic blood pressure. Histologic studies revealed the amelioration of tubulointerstitial fibrosis (-21.8%) and glomerular hypertrophy in rats received RLX. RLX-treated kidneys showed significantly decreased apoptosis (-40.8%), and phosphorylated Smad2 (-71.0%) expression in tubules compared to saline control.

These results indicate that RLX-LGR7 axis exists within kidneys and not only extrarenal but also intrarenal regulation of RLX may work in kidneys. These findings provide background to future clinical trials with RLX as anti-fibrotic therapy in salt-sensitive hypertension.

P-48

Volar Oblique Ligament Fibroblasts Express Estrogen, LGR7 Receptors

Elisa M. Konieczkoa, John D. Lubahnb, Timothy E. Cooneyb, and Michelle Heida

aDepartment of Biology, Gannon University, Erie, Pennsylvania 16541, USA; bDepartments of Orthopaedics and Orthopaedic Research, Hamot Medical Center, Erie, Pennsylvania 16550, USA

Introduction: Basal joint osteoarthritis (BJA) is predominant in females (Newport, 2000). Deterioration of the volar oblique ligament (VOL) precedes BJA (Pelligrini et al., 1992). Thus, sex hormones such as estrogen and relaxin may play a role in ligament integrity. Immunohistochemistry suggests VOL tissue contains estrogen and relaxin receptors (Krushinski et al., 2002; Lubahn et al, 2006); little is known about isolated VOL fibroblasts. We probed explant fibroblasts to determine whether LGR7, the putative relaxin receptor, and estrogen receptor beta are expressed.

Methods: With consent, four VOLs were obtained from surgery patients (3 female, 1 male, ages 51-65 YO). Tissue was minced and cultured in supplemented medium. After seven days, adherent cells were trypsinized, and passaged. Cells from Passage 3-7 were placed on coverslips, fixed, permeabolized, blocked with serum/PBS, and probed with primary antibodies for vimentin, actin, estrogen receptor beta, or LGR7. Fluorescent-labeled secondary antibodies were used for detection. For negative controls, the primary antibody was omitted.

Results: Cells evidenced abundant staining for vimentin and actin, cytoskeletal elements consistent with fibroblast morphology. Cells obtained from two females stained positively for both LGR7 and estrogen receptor beta; remaining specimens were negative. In all cases, control slides were negative.

Discussion/Conclusions: These limited data show that 67% of female donors had fibroblasts that expressed sex hormone receptors; cells from the solitary male donor did not. This provides evidence that some ligament fibroblasts are competent to respond to relaxin and estradiol. Given our small sample, additional research is warranted to confirm or refute our findings.


P-49

Basement Membrane Localization of LGR7 Receptors and TGF-beta in Juvenile Skin Biopsies

Justine M. Schoberb and Timothy E. Cooneya

aDepartment of Orthopaedic Research; bDepartment of Urology, Hamot Medical Center, Erie, Pennsylvania 16550, USA

Introduction: Pediatric genitoplasty produces dermal scarring, compromising dermal sensation and sexual function in adulthood. Given potential antifibrotic activities, the polypeptide hormone, relaxin, might benefit wound healing. As a first step, we screened skin biopsies for the relaxin receptor LGR7, as well as TGF-beta, a mediator of scarring.

Methods: Twenty-nine pediatric patients, ages 1-12 YO, provided specimens with consent. Fourteen had labial deformities, 15 had circumcisions/reconstructions. Specimens were snap-frozen, fixed with cold acetone, quenched of peroxidase, and blocked with serum. Standard, two-antibody immunohistochemistry localized antigens of interest. Of the 29 specimens, 24 were screened for LGR7, 17 for TGF-B1, and 16 for TGF-B3. Naïve serum was used as a negative control; uterine tissue served as a positive control for LGR7 staining. Additionally, blood samples from 16 female patients were analyzed for relaxin content.

Results: LGR7 was localized to cells lining the basement membrane in most specimens (23/24). As well, TGF-B1 (7/17) and TGF-B3 (15/16) were co-expressed in this area. Positive and negative controls showed the predicted response. Eighty-eight percent (14/16) of blood samples had detectable levels of relaxin, averaging 51.5 pg/ml (range: 0-223).

Discussion/Conclusions: Skin biopsies from children evidenced LGR7 and TGF-B coexpression in the cells that populate the basement membrane of the epidermis, presumably keratinocytes. Systemic relaxin was also present. That basement membranes in other anatomic areas interact with relaxin has been previously demonstrated (Stemmermann et al., 1994; McDonald et al., 2003; Mookerjee et al., 2006). Taken together, these data infer a role for relaxin in wound healing and epithelializaton.

P-50

Relaxin Differentially Regulates Gelatinase Expression and Activity in Cultured Gingival Fibroblasts

Chrishan S. Samuela,b, Ross Bathgatea,b, Chongxin Zhaoa, Sharon L. Layfielda, Geoffrey W. Tregeara,b, and Dennis R. Stewartc

aHoward Florey Institute and bDepartment of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia; cCorthera Inc., San Mateo, California 94402, USA

Many of relaxin’s diverse actions, including its growth- promoting, matrix remodeling and vasodilatory effects are mediated via regulation of the gelatinases, members of the matrix metalloproteinase (MMP) family. In recent pre-clinical studies conducted by Corthera Inc. (formerly BAS Medical), the administration of recombinant H2 relaxin to Beagle dogs was able to prevent orthodontic relapse in a similar manner to that induced by surgical fiberotomy. It was concluded however, that the relaxin-induced effect may have been potentially enhanced with improved dosing schedules. We therefore investigated various treatment-regimes involving relaxin on dog gingival fibroblast-stimulated gelatinase expression and activity under i) basal and TGF-β-stimulated conditions, ii) conditions simulating orthodontic force, and iii) continuous vs intermittent treatment. Relaxin-treatment of normal gingival fibroblasts significantly increased MMP-2 (gelatinase-A) expression and activity and MMP-9 (gelatinase-B) expression (all p<0.05 vs control cell values) at concentrations of 1-100ng/ml, as assessed by gelatin zymography and MMP-2 activity-assay kits. These effects were not specific to gender, but were enhanced when fibroblasts were stimulated by TGF-β1 or centrifugal force. Maximal increases in MMP-2 and MMP-9 expression (both p<0.01) were observed when H2 relaxin was continuously applied to force-activated cells, while intermittent H2 relaxin application only promoted MMP-2 expression and activity. Furthermore, the H2 relaxin-induced increase in gelatinase expression and activity correlated to decreased gingival collagen deposition, only when H2 relaxin was administered to TGF-β1-activated fibroblasts. These findings suggest that relaxin differentially regulates gelatinase expression which may have consequences on matrix remodeling in general.

63 Relaxin 2008

P-51

INSL3 Plays a Role in the Balance between Bone Formation and Resorption

Anastasia Pepea, Lisa Gianeselloa, Arianna Facciollia, Alexander I. Agoulnikb, Alberto Ferlina, and Carlo Forestaa

aUniversity of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, 35121 Padova, Italy; bDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA

We have recently demonstrated that the disruption of the INSL3 hormonal system, as observed in humans with mutations in RXFP2 gene and Rxfp2-KO mice, might have a pathogenic role in osteoporosis. In particular, the low bone mass phenotype in the Rxfp2-deficient mice is not due to a hyper-resorptive state, but to functional alterations in the osteoblast compartment causing little bone formation and ultimately a negative balance between bone formation and bone resorption. In the present study we better characterized the in vitro effect of INSL3 on human osteoblasts and its interaction with testosterone. Stimulation of human primary osteoblasts with INSL3 at serial concentrations, from 0.01nM to 10nM, induced a significant and dose-dependent increase in proliferation. We then investigated the expression of 4 typical osteoblast genes (Osteonectin, M-CSF, Osterix and NFATc1) on cells stimulated with INSL3 and/or Testosterone at different doses (1pM, 1nM, 1uM). Quantitative RT-PCR analyses show that expression of M-CSF, Osterix and NFATc1 genes are positively affected by the synergic effect of INSL3 and Testosterone at the highest concentration used. A significant increase in Osteonectin expression was observed when the osteoblasts were stimulated with INSL3 or Testosterone alone. This data suggest for the first time that INSL3 could act in synergy with the other testicular hormone, Testosterone, on osteoblast physiology and could also affect the osteoblast-osteoclast equilibrium necessary for the development and maintenance of bone tissue.

P-52

Mutations in INSL3-RXFP2 Genes in Cryptorchid Boys

Alberto Ferlina, Daniela Zuccarelloa, Andrea Garollaa, Riccardo Selicea, Cinzia Vinanzia, Francesco Ganza, Carla Cazzadorea, Biagio Zuccarellob, and Carlo Forestaa

aUniversity of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, 35121 Padova, Italy; bPaediatric Surgery, Department of Medical and Surgical Paediatric Sciences, University of Messina, Italy

Cryptorchidism is the most frequent malformation of the urogenital tract in males, with a incidence of 3% at birth, and it is associated with important consequences in adulthood, such as infertility and testicular cancer. Mutations in the INSL3 and RXFP2 genes have been associated with human cryptorchidism, but genetic screening performed in adults with history of cryptorchidism led to contrasting results. In the present study we analysed the frequency of mutations in these genes (and in the androgen receptor gene, AR) in a large cohort of newborn with cryptorchidism. We study 285 boys (age 6 months-3 years) who underwent unilateral (171 subject) or bilateral (114 subjects) orchidopexy. Mutation analysis was performed by PCR, DHPLC and sequencing of the two exons of INSL3, all 8 exons of AR and exon 8 of RXFP2 (where the T222P mutation map). We found 5 RXFP2 mutations in 5 unrelated bilateral cryptorchid boys, 1 INSL3 mutation in a unilateral cryptorchid boy and 3 AR mutation in three unrelated unilateral cryptorchid boy. Overall, the frequency of INSL3-RXFP2 mutations is therefore 6/285 (2.1%), and 5/114 (4.4%) in bilateral cryptorchidism. These data well fit with the observation that the incidence of INSL3-RXFP2 gene mutations in infertile adults with history of cryptorchidism is 4-5%, considering that about 50% of cryptorchid boys will be infertile in adulthood. This study confirmed the association between INSL3-RXFP2 gene mutations and human cryptorchidism and suggests such a genetic screening in cryptorchid boys, given the potential association with additional phenotypes in adulthood, such as osteoporosis.


P-53

The Mechanisms behind Relaxin on Improving Muscle Healing

Yong Lia,b,c, Xiaodong Mua,b, Haiying Pana, Mia Jeffersona, and Johnny Huarda,b,d

aStem Cell Research Center, Children’s Hospital of Pittsburgh; bDepartment of Orthopaedic Surgery; cDepartment of Pathology and dDepartment of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

Injuries to the muscle represent a large portion of all the injuries encountered professional and recreational sports. These injuries occur through a variety of mechanisms including direct trauma, such as muscle lacerations and contusions, and indirect injuries which include strains, ischemia, and neurological dysfunctions. Injured muscle is capable of healing through the regeneration of the damaged myofibers, however, the process is slow and often results in an incomplete recovery. The optimal treatments for these types of injuries are not clearly defined, varying widely and dependent on the type and severity of the injury. Treatments that utilize various growth factors have demonstrated increased muscle regeneration; however, these approaches have been fraught with the deposition of fibrous scar tissue that ultimately infers with the complete functional recovery of the muscle. Our previous studies have determined that relaxin, a polypeptide cytokine/growth factor within the insulin-like growth factor (IGF) family, could improve muscle healing through the prevention of fibrous scar tissue formation after laceration injury. In the current experiment, we able to demonstrate that relaxin was able to promote muscle cell differentiation and fusion in vitro and in vivo. We observed that relaxin, mostly like its’ other family member IGF-1, can stimulate myogenic gene activation in skeletal muscle cells. Our results also implicated relaxin’s receptor (LGR7), which can be found on the cell surface of skeletal muscle cells, in it’s ability to positively affect skeletal regeneration. Further research will explore if relaxin can share some of signaling pathways with IGF-1 which promote myogenic gene expression in muscle cells and improve muscle healing after strain, an extremely common injury encountered in sports medicine.

P-54

Relaxin Has a Role in the Molecular Dynamics of Cellular Movement

Yvonne Radestockb, Sabine Hombach-Klonischa, Joanna Bialekb, Cuong Hoang-Vub, and Thomas Klonischa

aDepartment of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Canada; bClinic of Surgery, Faculty of Medicine, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany

GTPases are upstream regulators of cofilin and various other pathways, including Arp3/4 (nucleation of actin at the plasma membrane), catenins/ E-cadherin (stabilization of adherens junctions), protein kinase C (PKC) isoforms (cell signaling/ polarization), and adducin (cortical actin network). Relaxin-mediated signaling may involve the action of small GTPases as suggested by the relaxin-induced cytosolic to plasma membrane translocation of PKC zeta. Furthermore, the cytoskeletal dynamics associated with the transition from an adherent polarized cell to a motile/ invasive phenotype with ECM-degrading capabilities involves beta-actin polymerization at the leading edge of cellular movement which is governed by the cofilin capping protein and ARP2/3 pathways. Rho family small GTPases, Cdc42, Rac1, and RhoA, are monomeric G-proteins that act as key transducers of extracellular signals to the actin cytoskeleton and are upstream regulators of this process. Stable transfectants of the human thyroid carcinoma cell line FTC-133 secret bioactive relaxin and display increased motility. Here we show that FTC-133-relaxin clones display an up-regulation of total and phosphorylated cofilin and the small GTPase proteins Cdc42, Rac and RhoA. Cofilin has F-actin severing/ depolymerising activities and is phosphorylated by LIM kinase which was also increased in RLN and INSL3 clones. These data provide a first molecular rationale for a novel role of the relaxin-RXFP1 ligand-receptor system as a morpho-functional regulator of cytoskeletal dynamics. The enhanced expression of Rho family GTPases is likely to contribute to an enhanced beta-actin polymerization and changes in vesicular trafficking which would manifest in enhanced motility, altered vesicular secretion profiles, and enhanced ECM-degrading capabilities observed in the FTC-133-relaxin transfectants.

This work was in part funded by Deutsche Forschungsgemeinschaft (DFG), Natural Sciences and Engineering Research Council of Canada (NSERC), Manitoba Medical Foundation (MMSF), and Manitoba Health Research Council (MHRC).

65 Relaxin 2008

P-55

In vitro Relaxin H2 Strengthens Fetal Membranes?Samer Yousfia, James Novaka, Robert Moorea, Deepak Kumara, Maria Pereza, Brian Mercerb, John Moorea,b, and Gillian Bryant-Greenwoodc

aPediatrics, bReproductive Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA; cDevelopmental and Reproductive Biology, University of Hawaii, Honolulu, Hawaii 96813 USA

Relaxin has been shown to increase extracellular matrix (ECM) remodeling and specifically MMP activity in target tissues. One study of the effect of relaxin on fetal membrane biophysical properties showed a biphasic pattern with low doses causing mild weakening and higher doses showing no effect on the tissue. Towards the end of gestation, the fetal membranes undergo a programmed, biochemically-mediated weakening process with concomitant ECM remodeling and cellular apoptosis. In addition, we have reported that in vitro incubation of fetal membrane with TNFα and IL-1β can reproduce the entire weakening and remodeling process. Evidence is less clear for other potential biochemical mediators and we hypothesized that relaxin would weaken human fetal membrane. Therefore, full-thickness membrane fragments were incubated with increasing doses of relaxin H2 (0-100 ng/ml) for 1-3 days. The fragments were then strength tested using our previously published ball-burst methodology and also tested for biochemical markers of remodeling: gelatinase activity and MMP proteins. Results showed that relaxin induced a dose dependent increase in break strength. At the highest dose, a consistent 20% increase was observed (P<0.01) with concomitant decreases in MMP9 gelatinase activity and protein abundance. Therefore, contrary to our expectations, relaxin paradoxically increased fetal membrane strength at high doses. The mechanism(s) for this relaxin effect are unknown. Supported by NIH grants HD048476 (JM) and HD24314 (GBG).


O-61

Implications of Leukocyte Responsiveness to Tumor-derived Relaxin

Kevin A. Figueiredoa, Jodie B. Palmerb, and Michael E. Coxa,c

aThe Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia V6H 3Z6, Canada; bLudwig Institute for Cancer Research, Austin Health, Heidelberg, Victoria 3084, Australia; cDepartment of Surgery, University of British Columbia, Vancouver, British Columbia V6H 3Z6, Canada

Leukocytes are critical effectors of inflammatory and cancer biology. Cancers are thought to produce chemokine-like factors that promote leukocyte recruitment and tumor infiltration and suppress immune surveillance. Relaxin is an endocrine hormone previously implicated in breast and prostate cancer progression. We have shown that relaxin is a cancer-produced factor with properties important for leukocyte recruitment and activation. Our studies demonstrate that relaxin expression is up-regulated during neuroendocrine differentiation of the human prostate cancer model, LNCaP. To examine the impact of relaxin on host cells associated with adenocarcinomas, we generated recombinant 6 His-tagged relaxin (RLXH) in a mammalian expression system. This immunoreactive and biologically active relaxin preparation was used to screen a variety of cell types for cAMP responsiveness. Of the cell types screened, none were more responsive to RLXH than the monocyte/macrophage cell line THP-1 and peripheral blood mononuclear cells (PBMC). Our studies indicate that relaxin promotes cell-cell clustering, substrate adhesion, and migratory capacity of mononuclear leukocytes in a relaxin dose-dependent manner proportional to cAMP accumulation. We demonstrate that these biological responses occur through a relaxin receptor LGR7-dependent mechanism likely involving cAMP-dependent regulation of the small GTPase, Rap1. Relaxin stimulation of monocytes enhanced chemotaxis of THP-1 and PBMCs to monocyte chemoattractant protein-1 and induced cytokine expression profiles indicative of type II, immunosuppressive, macrophage differentiation. Classical roles for relaxin can now be expanded to include a role in targeting mononuclear leukocytes and that its ability to mediate macrophage activation states have important implications in the context of tumor-induced inflammation and immunosuppression.

O-62

Relaxin and INSL3 Promote Tumor Formation of Human Thyroid Carcinoma Cells

Thomas Klonischa, Joanna Bialekb, Yvonne Radestockb, Cuong Hoang-Vub, and Sabine Hombach-Klonischa

aDepartment of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Canada; bClinic of Surgery, Faculty of Medicine, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany

The relaxin/RXFP1 and INSL3/RXFP2 have been established as ligand-receptor systems associated with tumor growth and metastasis. Both relaxin-like ligands, relaxin and INSL3, and their cognate receptors are present in a variety of tumor types, including breast, prostate, endometrial, hematopoietic, and thyroid cancer tissues and cell lines. Here we provide evidence from human thyroid cancer cell lines and nude mouse studies that both relaxin and INSL3 act as autocrine/ paracrine oncogenic factors that can modulate the local tumor environment to facilitate tumor expansion. When injected into nude mice, thyroid carcinoma transfectants expressing H2 relaxin and human INSL3 developed rapidly growing, cell-dense tumor masses rich in vascularization but with a poor interstitial matrix. Cultured transfectants displayed enhanced production of matrix-metalloproteinase 2 (MMP2; gelatinase A) and MT1-MMP. Activation of proMMP2 in part depends on the formation of a trimolecular complex between MT1-MMP, tissue inhibitor of MMP2 (TIMP2) and proMMP2. Relaxin and INSL3 clones displayed increased MMP2 activity as determined by processing of a fluorescent-labelled MMP2 substrate and this coincided with enhanced production of MT1-MMP and the presence of TIMP2. A similar increase in MMP2 and MT1-MMP production was detected when the follicular thyroid carcinoma cell line FTC-133 was incubated with recombinant human relaxin and INSL3. In conclusion, we have identified MMP2 and MT1-MMP as novel target molecules of relaxin and INSL3 in thyroid carcinoma cells and provide a molecular mechanism for rapid tumor growth and angiogenesis of these cell clones in nude mice.

This work was in part funded by Deutsche Forschungsgemeinschaft (DFG), Natural Sciences and Engineering Research Council of Canada (NSERC) and Manitoba Health Research Council (MHRC).

67 Relaxin 2008

O-63

Estrogen and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) Modulate RLN2 Promoter Activity in Estrogen-receptor Positive T47D Human Breast Cancer Cells

Silke Kietza, Thomas Klonischb, and Sabine Hombach-Klonischb

aUniversity Medical Center Goettingen, Pediatric Clinic I, 37075 Goettingen, Germany; bDepartment of Human Anatomy and Cell Science, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba R3E 0W3, Canada

RLN1 and RLN2 transcripts are increased in human breast cancer specimens. We employed the estrogen-responsive human breast cancer cell line T47D to determine the ability of estrogen and TCDD to increase RLN1 and RLN2 transcriptional gene activity. T47D cells expressed ERalpha protein and the 17beta-estradiol (E2) target gene pS2 was induced following exposure to 10nM E2. Arylhydrocarbon receptor (AhR) was present in T47D and exposure to 10nM 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) resulted in induction of the AhR target gene Cyp1A1. RLN1 and RLN2 promoter luciferase constructs were used to test for RLN promoter activation. RLN1 and RLN2 transcripts were quantified using real-time PCR. When exposed to E2 for 12h and 24h, RLN2 promoter activity was enhanced and this effect was not observed in the absence of E2. The estrogen receptor (ER) blocker ICI-182780 completely prevented the E2-induced increase in RLN2 promoter activity indicating that the RLN2 promoter was activated by an ER-dependent mechanism. After 24h exposure 10 nM TCDD alone did not induce RLN2 promoter activity. However, when applied simultaneously with E2, TCDD reduced the E2-mediated increase in RLN2 promoter activity suggesting an anti-estrogenic action in RLN2 transcriptional regulation. Acting alone, TCDD caused an up-regulation of RLN2 transcripts peaking at 8h exposure and preceding the E2-induced RLN2 up-regulation at 12 h. These results were specific for the RLN2 promoter. In conclusion, our results show that TCDD and E2 stimulate RLN2 transcription by different mechanisms and that TCDD acts anti-estrogenic in the presence of E2.

This work was in part funded by Deutsche Forschungsgemeinschaft (DFG), Natural Sciences and Engineering Research Council of Canada (NSERC), Manitoba Medical Foundation (MMSF) and Manitoba Health Research Council (MHRC).

O-64

Relaxin Signaling in Human Uterine Fibroids

Zhen Lia,b, Jennifer K. Burzawaa, Anne Tronga, Shu Fenga, Xiaowen Tongb, Ertug Kovancia, Aleks Rajkovica, and Alexander I. Agoulnika

aDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77025, USA; bDepartment of Obstetrics and Gynecology, Tongji University, 200065 Shanghai, People’s Republic of China

Uterine fibroid tumors (leiomyomata) are the most common gynecological neoplasms clinically affecting up to 30% of all women during reproductive age. The primary characteristics of uterine fibroids are the increased proliferation of myometrial cell, production of the abundant extracellular matrix (ECM), and the downregulation of ECM-degrading enzymes expression. Recent progress in cDNA microarray analysis revealed a distinct similarity of leiomyomata with other fibrotic processes. The crucial role of several cytokines and growth factors is well-recognized in this disease. Relaxin, a member of insulin-relaxin peptide family of hormones is strongly expressed in myometrium. Relaxin signals through its G protein-coupled receptor RXFP1. It regulates ECM remodeling through alterations of collagen deposition, cell migration and proliferation, and overall tissue homeostasis. Relaxin strongly suppresses the effects of TGF-ß induced myofibroblast differentiation, the major cytokine involved in the pathogenesis of uterine fibroids. Examination of public cDNA array gene expression databases revealed a consistent pattern of down-regulation of RXFP1 and several other genes involved in relaxin signaling, suggesting an involvement of relaxin in fibroid etiology. We used 25 matched pairs of leiomyomata and normal myometrium samples to analyze expression of RXFP1, RXFP2, caveolin 1, steroid receptors and their co-regulators at RNA and protein levels using qRT-PCR, immunohistochemistry and Western blot hybridization. The results of this analysis will be presented.


O-65

Scientific Rationale and Design of a Phase I Study of Relaxin in Women with Severe Preeclampsia

Elaine Unemoria, Baha Sibaib, Susy Wooda, and Sam Teichmana

aCorthera, Inc. (formerly BAS Medical, Inc.), San Mateo, California 94402, USA; bDepartment of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA

The pathophysiology of preeclampsia involves profound systemic vasoconstriction. Its etiology may be related to reduced blood flow to the placenta, leading to the elaboration of soluble factors which increase maternal systemic vascular resistance and cause renal dysfunction. Reduced bioactivity of vascular endothelial growth factor (VEGF) may play a central role in this pathophysiology. Previous clinical studies have strongly suggested that relaxin is a systemic and renal vasodilator. In nonclinical studies, relaxin administration to monkeys has been associated with increased vessel density in the endometrium and in previous human trials, relaxin administration has been highly correlated with increased menstrual bleeding in women. VEGF has been proposed as a mechanism for these endometrial effects. Together, these data suggest that relaxin may be able to relieve systemic and renal vasoconstriction and improve placental perfusion in women with preeclampsia. As a first step in the development program for relaxin in this indication, a multi-center, randomized, double blind, placebo-controlled Phase I safety study in women with severe preeclampsia has been launched in the US. Three doses of relaxin, 3, 10, and 30 μg/kg/day, or placebo will be administered for up to 72 hours in women admitted to the hospital for management of their disease. Although the trial is primarily focused on safety, signs of efficacy, such as changes in blood pressure and renal markers, will be assessed.

O-66

A Randomized, Double Blind, Placebo Controlled Trial of Relaxin for Cervical Ripening in Post-date Pregnancies

Gerson Weissa, Sam Teichmanb, Dennis Stewartb, David Naderb, Susy Woodb, and Elaine Unemorib

aDepartment of Obstetrics, Gynecology and Women’s Health, New Jersey Medical School, UMDNJ, Newark, New Jersey 07103, USA, bCorthera, Inc. (formerly BAS Medical), San Mateo, California 94402, USA

It is standard obstetrical practice to deliver all pregnancies by the 42nd week since longer pregnancy is attendant to increased fetal demise. This study had two objectives, to determine if 24 hr infusion of pharmacological concentrations of serum relaxin was safe in pregnant women and to determine whether the elevated relaxin could ripen the cervix or induce labor. After a small dose escalation phase of the study determined that relaxin could safely be elevated to approximately 30x over baseline (normal pregnancy) concentrations, the effects of 24 hr of infusion of relaxin vs placebo on Bishop score, time to dilation, uterine contractions and time to delivery were determined in 25 subjects/group. The study was multi-centered, randomized, double-blind and placebo controlled, in 72 women > 40 weeks of pregnancy. Elevation of serum relaxin to approximately 30x over baseline for 24 hr was safe to mother and fetus. Safety results for treatment and placebo groups were comparable. Infusion of relaxin for 24 hr did not advance cervical ripening as measured by Bishop score and did not alter secondary end points, including time to delivery. Relaxin infusion did result in trends of lower blood pressure and improvements in renal markers. Thus, while short term human relaxin infusion does not advance cervical ripening or labor onset, it was safe in pregnant women with potentially positive affects on blood pressure and renal function. This suggests use in hypertensive diseases of pregnancy.

69 Relaxin 2008

O-67

A Pilot Safety and Dose-finding Trial of Intravenous Recombinant Human Relaxin (rhRlx) in Compensated Congestive Heart Failure

Thomas Dschietziga, Elaine Unemorib, Susy Woodb, Christoph Richtera, Julia Böhmera, Sam Teichmanb, Gert Baumanna, and Karl Stangla

aDepartment of Cardiology and Angiology, Charité University Medicine Berlin, Campus Mitte, 10117 Berlin, Germany; bCorthera, Inc. (formerly BAS Medical), San Mateo, California 94402, USA

We have recently established that relaxin is constitutively expressed in human cardiovascular tissues and that the hormone -upregulated in myocardium - plays a compensatory role in human heart failure (CHF). In this open-label study, 16 patients with compensated CHF were treated with intravenous rhRlx in one of 3 dose cohorts. Patients were monitored for 24 hours in the intensive care unit during the infusion period and for 24 hours post-infusion. Hemodynamic measurements were performed using a Swan-Ganz and arterial catheter. The safety and tolerability demonstrated by rhRlx in the lowest dose group (A; sequential treatment for 8 hours each with dosages equivalent to 10, 30, and 100 µg/kg/day) allowed escalation to Group B (240, 480, and 960 µg/kg/day), and the highest safe dose, 960 µg/kg/day, was then selected for 24 hours of dosing in Group C. In all subjects and at all dosages, rhRlx was shown to be safe and well tolerated. In these well-medicated patients, rhRlx produced relevant improvements in hemodynamic parameters, including pulmonary wedge pressure, cardiac index, and systemic vascular resistance. Markers of renal function showed improvement in all subjects. Transient increases compared to baseline in some of these renal markers were also observed in some subjects post-dosing. Rebound hypertension or changes in heart rate were not observed. No incidences of hypotension were recorded during or post-dosing. Overall, the safety and efficacy profile of rhRlx in patients with compensated CHF indicates that this drug may be of value in the treatment of patients with acute heart failure syndrome and worsening of renal function.


Participants

Agoulnik, AlexanderBaylor College of [email protected]

Amento, EdwardMolecular Medicine Research [email protected]

Bagnell, CarolRutgers, the State University of New [email protected]

Bani, DanieleUniversity of [email protected]

Bartol, Frank F.Auburn [email protected]

Bathgate, RossHoward Florey [email protected]

Bennett, RobertVA Medical Center/University of [email protected]

Boehnert, MarkusUniversity of [email protected]

Bogerd, JanUtrecht [email protected]

Bogzil, AlsadekManchester [email protected]

Bryant-Greenwood, GillianUniversity of [email protected]

Callander, GabrielleHoward Florey [email protected]

Chang, Jaw-KangPhoenix Pharmaceuticals [email protected]

Charles, ChrisUniversity of [email protected]

Chen, JosephRutgers, the State University of New [email protected]

Conrad, KirkUniversity of [email protected]

Cooney, TimothyHamot Medical [email protected]

Cox, Michael E.The Prostate Centre at [email protected]

Craik, DavidUniversity of [email protected]

De Aizpurua, HenryHoward Florey [email protected]

De Meyts, PierreReceptor Systems Biology [email protected]

Debrah, DanUniversity of [email protected]

Debrah, JuliannaMagee Womens Research [email protected]

Dschietzig, ThomasCharité [email protected]

Du, Xiao-JunBaker Heart Research [email protected]

Einspanier, AlmuthInstitute of Physiological [email protected]

Feng, ShuBaylor College of [email protected]

Ferlin, AlbertoUniversity of [email protected]

Fernandes Gonçalves, RoseliSão Paulo [email protected]

Fields, MikeUniversity of [email protected]

Fields, PhillipUniversity of South Alabama College of [email protected]

Foresta, CarloUniversity of [email protected]

Frankshun, Amy-LynnRutgers, the State University of New [email protected]

Girling, JaneMonash Institute of Medical [email protected]

Goldsmith, LauraNew Jersey Medical [email protected]

Good-Avila, SaraAcadia [email protected]

Gundlach, AndrewHoward Florey [email protected]

Halls, MichelleMonash [email protected]

Haugaard-Jonsson, LindaUniversity of [email protected]

Hewitson, TimRoyal Melbourne [email protected]

Ho, Teh-YuanRutgers, the State University of New [email protected]

Hombach-Klonisch, SabineUniversity of [email protected]

Horton, JaimeUniversity of [email protected]

Hossain, MohammedHoward Florey [email protected]

Ikegaya, NaokiShizuoka [email protected]

Kapila, SunilUniversity of [email protected]

Kern, AndrasUniversity of [email protected]

Klonisch, ThomasUniversity of [email protected]

Koester, AnjaEli Lilly and [email protected]

Kohsaka, TetsuyaShizuoka [email protected]

Konieczko, ElisaGannon [email protected]

Layfield, SharonHoward Florey [email protected]

Li, ZhenBaylor College of [email protected]

Liu, ChangluJohnson & Johnson Pharmaceutical Research & [email protected]

71 Relaxin 2008

Lobb, DerekMcMaster [email protected]

Ma, SherieHoward Florey [email protected]

Makineni, [email protected]

Manegold Svendsen, AngelaHagedorn Research [email protected]

Maseelall, PriyaNew Jersey Medical [email protected]

McGowan, BarbaraImperial [email protected]

McGuane, JonathanUniversity of [email protected]

Moore, XiaoBaker Heart Research [email protected]

Morelli, SaraNew Jersey Medical [email protected]

Nøhr Larsen, JaneHagedorn Research [email protected]

O’Byrne, ElizabethRutgers [email protected]

Parry, LauraUniversity of [email protected]

Rajpert-De Meyts, EwaCopenhagen University [email protected]

Rinderknecht, ErnstCorthera, [email protected]

Rosengran, K. JohanUniversity of [email protected]

Ryan, PeterMississippi State [email protected]

Sacher, FrankBayer Schering Pharma [email protected]

Salimova, [email protected]

Samuel, ChrishanHoward Florey [email protected]

Sanborn, BarbaraColorado State [email protected]

Scarpa, Antonio LuigiIBSA Lugano [email protected]

Schwabe, ChristianMedical University of South [email protected]

Segal, MarkUniversity of [email protected]

Shabanpoor, FazelHoward Florey [email protected]

Shemesh, RonenCompugen [email protected]

Shen, Pei-JuanHoward Florey [email protected]

Sherwood, O. DavidUniversity of [email protected]

Singh, SudhirUniversity of Nebraska Medical [email protected]

Slieker, LawrenceLilly Research [email protected]

Smith, CraigHoward Florey [email protected]

Soh, Yu MayThe University of [email protected]

Steinetz, BernardNYU School of [email protected]

Stewart, DennisCorthera, [email protected]

Summers, RogerMonash [email protected]

Sutton, StevenJohnson & [email protected]

Tan, [email protected]

Tang, MimiMurdoch Childrens Research [email protected]

Tregear, GeoffreyHoward Florey [email protected]

Tremblay, JacquesCHUQ Research Centre - Laval [email protected]

Unemori, ElaineCorthera, [email protected]

van der Westhuizen, EmmaMonash [email protected]

Vodstrcil, LenkaUniversity of [email protected]

Wade, JohnHoward Florey [email protected]

Weiss, GersonNew Jersey Medical [email protected]

Wiley, Anne A.Auburn [email protected]

Willcox, JordanUniversity of [email protected]

Wilson, BrianAcadia [email protected]

Wlodek, MaryThe University of [email protected]

Wong, Su EeHoward Florey [email protected]

Yamamoto, SandraUniversity of [email protected]

Yegorov, SergeyAcadia [email protected]

Zhang, SuodeHoward Florey [email protected]

Notes

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Documents

Relaxin 2008 - University Of Illinoisconferences.illinois.edu/relaxin/relaxin_abstract_book_08final.pdf · and available for discussion during the assigned session. Posters should