Transcript
Page 1: Investigating α7 nicotinic receptor STAT3 signaling and ...1743/fulltext.pdfInvestigating 7 nicotinic receptor STAT3 signaling and cell-dependent expression Thesis Presented By Thomas

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Investigating 7 nicotinic receptor STAT3

signaling and cell-dependent expression

Thesis Presented

By

Thomas M. Koperniak

To

The Bouve Graduate School of Health Sciences

in Partial Fulfillment of the Requirements for the

degree of Doctor of Philosophy in Pharmaceutical Sciences

with specialization in

Pharmacology

NORTHEASTERN UNIVERSITY

BOSTON, MASSACHUSETTS

2012

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Northeastern University

Bouve Graduate School of Health Sciences

Thesis title: Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent

expression

Author: Thomas M. Koperniak

Program: Department of Pharmaceutical Sciences

This project satisfies all research requirements for the Doctoral Degree

Thesis Committee (Chairman) ___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

Director of Graduate School ___________________________Date________

Dean ___________________________Date________

Copy Deposited at Library ___________________________Date________

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Northeastern University

Bouve Graduate School of Health Sciences

Thesis title: Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent

expression

Author: Thomas M. Koperniak

Program: Department of Pharmaceutical Sciences

This project satisfies all research requirements for the Doctoral Degree

Thesis Committee (Chairman) ___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

Director of Graduate School ___________________________Date________

Dean ___________________________Date________

Copy Deposited at Library ___________________________Date________

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LIST OF ABBREVIATIONS

5HT Serotonin

AA Arachidonic acid

BGT -Bungarotoxin

AC Adenylate cyclase

AChBP Acetylcholine binding protein

ACh Acetylcholine

AD Alzheimer’s disease

ADP Adenosine diphosphate

ADSS Aged and diluted sidestream smoke

ANOVA Analysis of variation

AS Antisense

ATP Adenosine triphosphate

BAPTA-AM Bis(2-aminophenoxyl)ethane tetraacetic acid

BATH-42 BTB-associated Traf homology 42

BiP Binding immunoglobulin protein

BLAST Basic Local Alignment Search Tool

bp Base pairs

BSA Bovine serum albumin

CA Constitutively active

cAMP Cyclic adenosine monophosphate

CC Coiled-coil domain

cDNA Complementary DNA

CMV Cytomegalovirus

CNS Central nervous system

CREB cAMP response element-binding

CTZ Coelenterazine

DA Dopamine

DHE Dihydro-beta-erythroidine

DHS Donor horse serum

DMEM Dulbecco’s modified Eagle’s medium

DMSO Dimethyl sulfoxide

DN Dominant negative

dsRNA Double stranded RNA

EGFP Enhanced green fluorescent protein

EGFR Epidermal growth factor receptor

EGTA Ethylene glycol tetraacetic acid

ELISA Enzyme-linked immunosorbent assay

ER Endoplasmic reticulum

ERK Extracellular signal-regulated kinase

FBS Fetal bovine serum

FDA Food and Drug Administration

FGF2 Fibroblast growth factor 2

GABA Gamma-aminobutyric acid

GAPDH Glyceraldehyde phosphate dehydrogenase

GFP Green fluorescent protein

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Glu Glutamate

HBSS Hank’s balanced salt solution

HEK Human embryonic kidney

hRic3 Human Ric3

HRP Horseradish peroxidase

Hsp90 Heat shock protein 90

IBD Inflammatory bowel disease

IDT Integrated DNA Technologies

IL6 Interleukin 6

IP Intraperitoneal

IP3 Inositol phosphate 3

JAK2 Janus kinase 2

KC Keratinocyte

LBD Ligand binding domain

LD-PCR Long distance-polymerase chain reaction

LPS Lipopolysaccharide

MAPK Mitogen-activated protein kinase

MIP2 Macrophage inflammatory protein 2

ML Metridia luciferase

MLA Methyllycaconitine

Mnmc Manumycin A

mRic3 Mouse Ric3

mRNA Messenger RNA

mTOR Mammalian target of rapamycin

NAc Nucleus accumbens

nAChR Nicotinic acetylcholine receptor

NBM Nucleus basilis of Meynert

NFB Nuclear factor kappa B

NMDA N-Methyl-D-aspartate

NMJ Neuromuscular junction

NNK Nicotine-derived nitrosamine ketone

NNN N'-nitrosonornicotine

NNR Neuronal nicotinic receptor

nSCLC Non-small cell lung cancer

p Probability

P9KB pREP9-kanamycin-blasticidin

P9KG pREP9-kanamycin-geneticin

PBS Phosphate buffered saline

PJAK2 Phosphorylated JAK2

PI3K Phosphoinositol-3 kinase

PKA Protein kinase A

PLC Phospholipase C

PM Plasma membrane

PMSF Phenylmethylsulfonyl fluoride

PNS Peripheral nervous system

PPT Pedunculo-pontine nucleus

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PSTAT3 Phosphorylated STAT3

RFP Red fluorescent protein

Ric3 Resistance to inhibitors of cholinesterase-3

RIPA Radio-immunoprecipitation assay

RISC RNAi-induced silencing complex

RNAi RNA interference

ROS Reactive oxygen species

RT Reverse transcriptase

S-rRic3 Serine (absent) rat Ric3

S+rRic3 Serine (present) rat Ric3

SCLC Small cell lung cancer

sdrRic3 Splice-deleted rat Ric3

SEAP Secreted alkaline phosphatase

SEM Standard error of the mean

shRNA Short hairpin RNA

siRNA Small interfering RNA

SMART Switching mechanism at 5’ end of RNA template

SN/SNc Substantia nigra

SOCS3 Suppressor of cytokine signaling 3

SS Signal sequence

STAT3 Signal transducer and activator of transcription-3

STR Striatum

TBS Tris buffered saline

TBS-T Tris buffered saline-Triton

THAL Thalamus

TLR Toll-like receptor

TM Transmembrane domain

TNF Tumor necrosis factor

Tyk Tyrosine kinase

UNC-50 Uncoordinated-like protein 50

UT Untransfected

US United States

Vegf Vascular endothelial growth factor

VGCC Voltage-gated calcium channel

VTA Ventral tegmental area

WT Wild-type

XIAP X-linked inhibitor of apoptosis protein

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ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels

consisting of a combination of at least 17 possible subunits. Although most receptor subtypes

are heteromeric, 7 subunits can assemble into homomeric receptors. 7 nicotinic receptors are

the 2nd

most highly expressed nicotinic receptor in the brain, and their high permeability to

calcium ions allows participation in calcium-dependent processes such as neurotransmitter

release and immune modulation. Although these receptors have been well-studied, 7-mediated

STAT3 signaling and 7 maturation remain unclear. The two major hypotheses we are

investigating stem from these issues. The first hypothesis, regarding 7-mediated STAT3

activation, is that 7 drives STAT3 signaling independently of calcium ion flow. The second

hypothesis, regarding 7 maturation, is that rat pituitary-derived GH4C1 cells possess Resistance

to Inhibitors of Cholinesterase 3 (Ric3) to allow surface 7 expression.

The major advancement of this project is the development of a novel STAT3-Metridia

luciferase (ML) signaling assay using heterologous expression systems. To our knowledge, this

is the only study using heterologous expression systems to investigate such signaling. To study

7-mediated STAT3 signaling, human neuroblastoma-derived SHEP1 or GH4C1 cells co-

expressing7 and STAT3-ML were exposed to nicotine following pretreatment with the 7

antagonist, -bungarotoxin (BGT), JAK/STAT inhibitors, the PKA inhibitor, PKI 14-22 amide,

calcium chelators, or calcium-low media. Results suggest 7-mediated STAT3 signaling can

take place independently of calcium influx. Since 7 maturation is a prerequisite for obtaining

7-mediated STAT3 signaling, the STAT3-ML assay provided insight into 7 receptor

maturation and motivated investigation into cell-dependency of Ric3 transfection for surface 7

expression.

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Two isoforms of rat Ric3 were cloned from rat brain and designated S-rRic3 and

S+rRic3. S-rRic3 differs from S+rRic3 by three bases due to the absence of a serine residue at

position 173 relative to S+rRic3. Our project addressed the functionality of these while

exploring the possibility of currently unidentified 7 chaperones. Initial 125

I-bungarotoxin

binding assays supported our original hypothesis that GH4C1 cells possess Ric3 to allow surface

7 expression. However, RNAi studies performed alongside dot blot analysis using Ric3

antibodies supported an alternative hypothesis that GH4C1 cells possess 7 chaperones aside

from the currently known functional Ric3 splice variants. To screen for additional chaperones in

GH4C1 cell cDNA, a variation of our novel STAT3-ML assay may be useful. By replacing the

Metrdia luciferase reporter with a gene conferring antibiotic resistance, a STAT3 rescue vector

can be generated and allow isolation of cells expressing unknown 7 chaperones.

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Table of Contents Page

List of Abbreviations…………………………………………………………………………… -iv-

Abstract………………………………………………………………………………………….. -vii-

List of Tables………………………………………………………..........................………....... -xi-

List of Figures………………………………………………………………………….……… -xii-

Acknowledgments……………………………………………………………………..……....... -xvi-

Introduction Statement of Problem…………...……………………………………..………….. -1-

Review of the Literature……………………………………...……………………..………….. -7-

A. Nicotinic acetylcholine receptors……………..…………………………………………. -7-

B. 7 nicotinic receptor transcript distribution……………………………………………... -11-

C. Calcium permeability of 7 nicotinic receptors…………………………………………. -12-

D. 7 nicotinic receptors in Alzheimer’s disease…………………………………………… -14-

E. 7 nicotinic receptors in oxidative stress………………………………………………... -18-

F. 7 nicotinic receptors in schizophrenia………………………………………………….. -19-

G. 7 nicotinic receptors in smoking addiction and withdrawal……………………………. -21-

7 nicotinic receptors in cancer………………………………………………………….. -25-

7 nicotinic receptors in anti-inflammation……………………………………………... -28-

J. 7-mediated JAK-STAT signaling …………………………………………………........ -33-

Multiple modes of 7 nicotinic receptor signaling………………………………………. -39-

L. and 42 receptors activate the STAT pathway independently of calcium ion flow.. -41-

M. 7-mediated signaling assay provides insight into cell line-dependency of 7

maturation……………………………………………………………………………….. -45-

N. 7 nicotinic receptor maturation is a process that remains unclear……………………… -46-

O. Ric3 has differential effects on expression of numerous receptors…………………........ -48-

P. Ric3 increases expression of 7 nicotinic receptors……………………………………... -50-

Q. Ric3 mechanism of action and regions of interest……………………………………….. -52-

R. 7 nicotinic receptor expression without Ric3…………………………………………... -57-

S. Exploring the consequences of Ric3 protein knockdown on surface 7 expression……. -59-

Specific AIMS of the thesis…………………………………………………………………….. -62-

Materials and Methods………………………………………………………………………… -64-

Cell culture and transfections……..………………………………………………….………. -64-

Chemicals and plasmids……………………………………………………………..………... -65-

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Ric3 clones……………………………………………………………………………………. -65-

Radioligand binding………………………………………………………………………….. -66-

Dot blot analysis…………………………………………………………………….………... -66-

NFB and STAT3 reporter assays…………………………………………..…….………….. -67-

Data analysis………………………………………………………………………………….. -68-

Results………………………………………………………………………………………........ -69-

Specific AIM #1……………………………………………………………………………… -69-

Aim 1a) Investigate feasibility of using STAT3-MetLuc signaling assay………………………. -69-

Aim 1b) Validate STAT3-ML signaling assay by measuring IL6-driven signaling of secreted

alkaline phosphatase and luciferase in Invivogen’s HEK-Blue IL6 cells. ……………………… -71-

Aim 1c) Determine ideal conditions for nicotine-induced increases in 7-driven luciferase

expression and establish whether increased luciferase expression is STAT3-attributable………. -75-

Aim 1d) Construct cell lines expressing both 7 and STAT3-driven luciferase plasmids and re-

assess ideal conditions for nicotine-induced increases in 7-driven luciferase expression.........

-81-

Aim 1e) Use STAT3-MetLuc assay to study Ca2+

-dependency of 7-mediated STAT3

signaling ………………………………………………………………………………………... -84-

Aim 1f) Investigate the role of Ric3 in allowing 7-driven STAT3 signaling in non-permissive

cell lines…………………………………………………………………………......................... -88-

Specific AIM #2…………………………………………………………………………....... -90-

Aim 2a) Clone rat Ric3 and investigate functionality of protein………………………………… -90-

Aim 2b) Establish selectivity of Ric3 antibodies and demonstrate cell line-dependency of Ric3

transfection for surface 7 expression………………………………………………………….. -92-

Aim 2c) Test whether RNAi methods that block surface 7 expression in non-permissive cells

fails to block expression in permissive GH4C1 cells…………………………………………… -96-

Aim 2d) Obtain shRNA constructs to investigate alternative splicing, and study Ric3 protein

turnover by determining effects of Ric3 knockdown on 7 expression in cell lines stably

expressing Ric3…………………………………………………………………………………..

-104-

Specific AIM #3……………………………………………………………………………… -119-

Searching for unknown 7 chaperones in GH4C1 cDNA: Expression cloning using the

SMARTer (Switching Mechanism at 5’ end of RNA Template) In-Fusion Cloning Kit

(Clontech)…………………………………………………………………………………………

-120-

Aim 3a) Establish method of plasmid recovery by showing recovery of S ic3/pREP4/hygromycin…………………………………………………………………………..

-123-

Aim 3b) Construct S-rRic3/pREP9/KG using In-Fusion cloning kit (Clontech) and recover

plasmid………………………………………………………………………………………….. -125-

Discussion………………………………………………………………………………………. -127-

Future Directions……………………..………………………………………………...…........ -137-

Conclusions……………………………………………………………………………..………. -141-

References……………………………………………………………………………….…….... -144-

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List of Tables

Table Page

1. 7 receptor compounds in clinical development for central nervous

system disorders…………………………………………………………… -24-

2. Examples of conditions in which cholinergic modulation influences

disease state……………………………………………………………….. -31-

3. Ric3 distribution and labeling intensity in rat CNS……………………….. -58-

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List of Figures

Figure Page

1. Nicotinic acetylcholine receptor subunit composition…………………………………… -7-

2. Basic structure of nicotinic receptors…………………………………………………..... -8-

3. Lining of the nicotinic receptor pore…………………………………………………….. -9-

4. Proposed model for nicotinic receptor gating mechanism…………………………….... -10-

5. Distribution of 7 transcripts in rat brain………………………………………………... -11-

6. Nicotine-induced currents in the absence or presence of chloride channel blockers……. -12-

7. Nicotine-induced currents show two components when the Na+ and Cl

- reversals are

separated……………………………………………………………………………….... -13-

8. Calcium responses in astrocytes following nicotinic receptor activation ………………. -14-

9. Dependence of the ACh response on extracellular calcium…………………………….. -15-

10. 125I-BGT binding to 7SK-N-MC membranes is blocked by -amyloid filaments….. -16-

11. A1-42 blocks hippocampal 7 noncompetitively……………………………………….. -17-

12. Hypothesized locations and function of CNS 7 receptors in schizophrenia. ………… -20-

13. Antagonism of 7 in the NAc shell increases motivation to self-administer nicotine… -22-

14. 7 activation in the NAc shell decreases motivation to self-administer nicotine……… -23-

15. The diverse functions of 7 and 42………………………………………………….. -26-

16. Nicotinic receptors in non-small cell lung cancer (nSCLC) cells……………………… -27-

17. Cholinergic agonists inhibit LPS-induced TNF synthesis in human macrophages……. -29-

18. In human macrophages ACh inhibits release of pro-inflammatory but not anti-

inflammatory cytokine release…………………………………………………………… -29-

19. A proposed mechanism of inhibition of cytokine release mediated by 7……………… -31-

20. Two signaling pathways of 7…………………………………………………………… -32-

21. Antisense oligonucleotides against 7 prevent nicotine-induced reduction in LPS-

stimulated TNF release…………………………………………………………………... -34-

22. Increased cytokine production in 7-deficient mice during endotoxaemia…………….. -35-

23. Nicotine increases STAT3 phosphorylation and SOCS3 expression……………………. -36-

24. STAT3 phosphorylation by nicotine is prevented by 7-selective nAChR antagonists.. -37-

25. Nicotine-induced STAT3 phosphorylation occurs through activation of JAK2 that is

recruited to the 7……………………………………………………………………….. -38-

26. Alterations in STAT3 expression in KCs exposed to nicotine………………………….. -39-

27. JAK2 inhibition restores LPS-induced NFB signaling in nicotine-treated h42

SHEP1 cells……………………………………………………………………………… -41-

28. STAT3 inhibition restores LPS-induced NFB signaling in nicotine-treated h42

SHEP1 cells……………………………………………………………………………… -42-

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29. 42-mediated inhibition of LPS-induced IBphosphorylation may be calcium-

independent …………………………………………………………………………….. -43-

30. 7-mediated anti-inflammation involves two modes of signaling……………………… -44-

31. Presence of 7 mRNA in all infected cell lines…………………………………………. -45-

32. Cell line-dependent expression of mature 7 receptors…………………………………. -46-

33. Ric3 effects on expression of 5HT3 and glycine receptors………………………………. -49-

34. Differential effects of hRic3 on expression of various nicotinic receptors………………. -50-

35. Effect of hRic3 on 7 receptor membrane insertion…………………………………….. -51-

36. Effect of hRic3 on 7 receptor turnover…………………………………………………. -51-

37. Whole cell currents in oocytes co-expressing 7 and hRic3…………………………...... -52-

38. Co-expression of 7 and hRic3 in Xenopus oocytes…………………………………….. -52-

39. Two proposed models of Ric3 structure…………………………………………………. -53-

40. Residues 1-168 comprise the minimal functional domain of mRic3…………………..... -55-

41. The coiled-coil domain is essential for self-association of mRic3………………………. -56-

42. Analysis of Ric3 transcript localization and 125

IBGT binding. ……………………….. -59-

43. Mechanism of RNA interference in mammalian systems……………………………….. -60-

44. LPS-induced increase in NFB production is mediated by 7 in SHEP1 and GH4C1

cells………………………………………………………………………………………. -70-

45. Construction of STAT3-MetLuc reporter plasmid………………………………………. -71-

46. In HEK-Blue IL6 cells, IL6 stimulation of the STAT3 pathway leads to SEAP

expression………………………………………………………………………………… -72-

47. HEK-Blue IL6 cells treated with IL6 show dose-dependent increase in SEAP

expression ………………………………………………………………………………... -72-

48. IL6 exposure to HEK-Blue IL6 cells transfected with STAT3-ML increases luciferase

expression.……………………………………………………………………………….. -73-

49. IL6 exposure to HEK-Blue IL6 cells produces similar increases in SEAP and luciferase

expression ……………………………………………………………………..………… -74-

50. Nicotine dose-response curve in SHEP1 cells transfected with STAT3-ML……………. -76-

51. Nicotine time course for driving luciferase expression in SHEP1 cells………………..... -77-

52. BGT pretreatment blocks 7-mediated luciferase expression………………………… -78-

53. JAK2 inhibition reduced nicotine-dependent luciferase expression……………………. -79-

54. STAT3 inhibition reduces nicotine-dependent luciferase expression………………….. -80-

55. Nicotine dose-response in SHEP1 cells previously transfected with 7, Ric3, and

STAT3-ML-P9KB………………………………………………………………………. -82-

56. Nicotine time course in STAT3-ML-P9KB SHEP1 cells……………………………….. -82-

57. Inhibitors decrease both SEAP and MetLuc expression………………………………… -83-

58. BAPTA-AM fails to lower nicotine-induced luciferase expression in SHEP1 cells -85-

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previously transfected with 7, Ric3, and STAT3-ML-P9KB………………………….. 59. BAPTA-AM and EGTA fail to lower nicotine-induced luciferase expression in SHEP1

cells previously transfected with 7, Ric3, and STAT3-ML-P9KB……………………. -86-

60. Nicotine-induced luciferase expression in SHEP1 cells previously transfected with 7,

Ric3, and STAT3-ML-P9KB persists in a low-calcium environment………………….. -87-

61. Nicotine-induced luciferase expression in SHEP1 cells previously transfected with 7,

Ric3, and STAT3-ML-P9KB persists with PKA inhibition.…………………………….. -88-

62. Ric3 transfection is required for 7-mediated STAT3 signaling in SHEP1 cells……..... -89-

63. Ric3 transfection is not required for 7-mediated STAT3 signaling in GH4C1 cells….. -90-

64. Rat Ric3 open reading frame and translated protein…………………………………….. -91-

65. S- and S+rRic3 allow surface 7 expression in HEK cells…………………………….. -92-

66. Rat Ric3 isoforms promote surface expression of 7 in SHEP1 cells………………….. -93-

67. Ric3 transfection is not required for surface 7 expression in GH4C1 cells………….... -94-

68. Santa Cruz Ric3 antibodies are dependent on protein conformation but can be used for

dot blots………………………………………………………………………………….. -95-

69. Rat/human Ric3 siRNA sequence synthesized by IDT…………………………………. -97-

70. Ric3 siRNA treatment lowers surface 7 expression in HEK293 cells………………… -98-

71. Ric3 siRNA treatment lowers surface 7 expression in SHEP1 cells………………….. -98-

72. Ric3 siRNA treatment does not reduce surface 7 expression in GH4C1 cells………… -99-

73. Ric3 siRNA partially reduced 7 expression in SHEP1 cell semi-stably expressing S-

rRic3……………………………………………………………………………………… -99-

74. GH4C1, but not SHEP1, cells generate surface 7 receptors in the absence of Ric3

protein…………….………….………….………….………….………….……………… -101-

75. Ric3 siRNA reduces levels of human and splice-deleted rat Ric3 protein upon

transfection in SHEP1 and GH4C1 cells………………………………………………… -102-

76. Ric3 knockdown in GH4C1 cells does not affect surface 7 expression………………. -103-

77. HuSH 29mer shRNA constructs against rat Ric3 obtained from Origene……………..... -104-

78. Rat Ric3 shRNA against exon 5 and 6 reduces S-rRic3 protein while decreasing surface

7 expression in SHEP1 cells co-transfected with 7 and S-rRic3…………….….……. -105-

79. Rat Ric3 shRNA against exon 5 and 6 together reduce S-rRic3 while decreasing surface

7 expression in SHEP1 cells transfected with 7/S-rRic3…………………….….…… -106-

80. Scrambled shRNA does not reduce surface 7 expression nor decrease Ric3 protein

levels in SHEP1 cells co-transfected with 7 and S-rRic3 or hRic3…………………….

-107-

81. Rat Ric3 shRNA against exon 5 reduces rat Ric3 protein levels while not having an

effect on surface 7 expression…………………………………………………………. -108-

82. Semi-stably transfected 7 GH4C1 cells and 7/S-rRic3 GH4C1 cells generate surface

7 receptors while only semi-stably transfected S-rRic3 GH4C1 and 7/S-rCRic3

GH4C1 cells possess detectable levels of Ric3 protein………………………..............

-110-

83. Semi-stably transfected 7/S-rRic3 SHEP1 cells generate surface 7 receptors and

possess detectable levels of S-rRic3 protein…………………………………………….. -110-

84. Semi-stable shRNA transfection decreases surface 7 expression and Ric3 in SHEP1

cells previously semi-stably transfected with 7 and S-rRic3 together………………...

-111-

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85. Semi-stable shRNA transfection decreases Ric3 protein but not surface 7 expression in GH4C1 cells previously semi-stably transfected with 7/S-rRic3…………………..

-112-

86. shRNA construct against rat Ric3 exon 2…………………………………………...…… -113-

87. shRNA against rat Ric3 exon 2 diminishes surface 7 expression in SHEP1 cells …..... -113-

88. shRNA against rat Ric3 exon 2 reduces surface 7 expression in SHEP1 cells

transfected with either isoform of rat Ric3………………………………………………. -114-

89. shRNA exon lowers Ric3 protein and 7 expression in SHEP1 cells while having no

effect on hRic3 protein or hRic3-facilitated 7 expression……………………………...

-115-

90. 7 expression persists in GH4C1 cells transfected with shRNA exon 2……………….. -115-

91. In GH4C1 cells, surface 7 expression persists following Ric3 knockdown via shRNA

against exon 2……………………………………………………………………………. -116-

92. S+rRic3 protein levels are lowered by rat Ric3 targeting exon 2……………………….. -117-

93. shRNA against rat exon 6 blocks rat Ric3-induced surface 7 expression in SHEP1

cells.…………………………………………………………………..…………………………… -117-

94. Summary of RNAi studies investigating Ric3 transfection dependency for surface 7

expression. ……………………………………………………………………………….. -118-

95. pREP9 vector…………………………………………………………………………….. -120-

96. SMARTer V Oligo allows selective amplification to yield pure cDNA………………… -121-

97. In-Fusion SMARTer cDNA library construction kit overall procedure………………… -122-

98. Proof-of-concept: During overnight incubation, colonies grew only on plates on which

bacteria transformed with S-rRic3/pREP4/KG was spread……………………………... -124-

99. Proof-of-concept: Agarose gels of Ric3/pREP4 cut with KpnI and XhoI…………….... -124-

100. Proof-of-concept: During overnight incubation, colonies grew only on plates on which

bacteria transformed with S+rRic3/pREP9/KG was spread…………………………….. -125-

101. Proof-of-concept: Agarose gels of Ric3/pREP4 cut with HindIII and XbaI……………. -126-

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ACKNOWLEDGMENTS

This project would not have been possible without guidance and support from several people

who graciously extended their expertise and effort in preparation and completion of this

dissertation. First and foremost, I would like to thank my advisor, Dr. Ralph Loring, for his

continuous support, patience, and hard work throughout the research and writing of this project.

Besides my advisor, I would like to thank the rest of my thesis committee: Dr. Richard Deth, Dr.

John Gatley, Dr. Nina Irwin, and Dr. Wendy Smith for their encouragement and valuable insight.

I would like to thank my friends and family for their love and support.

I gratefully acknowledge financial support from a 2009-2010 American Foundation for

Pharmaceutical Education (AFPE) pre-doctoral fellowship.

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INTRODUCTION

Statement of the problem:

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of

neurotransmitter-gated ion channels. Although most nAChRs are heteromeric, 7, 8, and 9

subunits can assemble into homomeric channels (Millar, 2003). The 7 receptor, for which -

bungarotoxin (BGT) is a specific and high-affinity antagonist, is one of the most abundant

receptor subtypes in the mammalian brain (Dominguez del Toro et al., 1994). It has high Ca2+

permeability (Seguela et al., 1993), and is involved in activation of Ca2+

-dependent processes

such as neurotransmitter release, signal transduction, apoptosis, and immune modulation. 7 has

been an appealing target in drug discovery, as it may play roles in Alzheimer’s disease,

schizophrenia, smoking addiction, Parkinson’s disease, and conditions involving inflammation

(Freedman et al., 1994; Lindstrom, 1997; Lloyd and Williams, 2000). Our understanding of the

link between 7 and inflammation, in particular, has steadily advanced with ongoing research.

Borovikova et al. (2000) described a process called the “cholinergic anti-inflammatory

pathway” by which production of several cytokines, including tumor necrosis factor (TNF), is

decreased by activation of cholinergic receptors on macrophages. The identity of the receptors

involved in this pathway was studied in 2003 when Tracey et al. demonstrated that knockdown

of 7, but not 1 or 10, using antisense oligonucleotides makes macrophages less responsive to

nicotine-induced reductions in lipopolysaccharide (LPS)-stimulated TNF release (Tracey et al.,

2003). Although this implicated a specific receptor in the cholinergic anti-inflammatory

pathway, the signaling mechanism accounting for the ability of receptor activation to reduce

cytokine production remained uncertain. In 2005, De Jonge et al. evaluated the involvement of

the transcription factor STAT3 and demonstrated that nicotinic activation of 7 produces anti-

inflammatory effects in peritoneal macrophages through JAK2-STAT3 signaling. In 2006,

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Arredondo et al. used pathway inhibitors to provide evidence supporting that nicotinic activation

of 7 drives STAT3 upregulation through the Ras/Raf/MEK/ERK pathway. In 2012, Andersson

and Tracey hypothesized that the cholinergic anti-inflammatory response involves activation of

adenylate cyclase and protein kinase A. The downstream signaling mechanisms contributing to

7-mediated immune modulation remain unclear, and investigations into these mechanisms

should consider the multiple signaling modes of 7 receptors.

7 signals through two distinct pathways. The first pathway, involving cationic flow

through activated ligand-gated channels, is a well-known event. Binding of agonists such as

nicotine or acetylcholine results in conformational changes that allow the flow of positively

charged ions down their electrical gradient. The resulting inward currents trigger depolarization

that is essential for neurotransmitter release and other cellular responses. An interesting feature

of 7 receptors is the rapid desensitization that occurs upon agonist exposure. Despite

continuous agonist application, 7 desensitization prevents further ion flow. Perhaps rapid 7

desensitization serves as protection against neurotoxicity, owing to the high calcium permeability

of the receptor (Seguela et al., 1993). Although ion influx is a very transient event, 7

participates in an additional mode of signaling that is longer-lasting.

A second, lesser-studied mode of 7 signaling involves JAK2 recruitment to activated

receptors and subsequent STAT3 phosphorylation and activation. STAT3 signaling leads to

modulation of genes involved in cell proliferation, apoptosis, and cytokine production (Johnston

and Grandis, 2011). While it is well-established that Ca2+

influx triggers many downstream

signaling events, it remains unclear whether 7-mediated STAT3 signaling can take place

without such ion flow. We are investigating the hypothesis that 7 drives STAT3 signaling

independently of calcium. This hypothesis is supported by several findings. Suzuki et al. (2006)

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demonstrated that in rat microglia, nicotinic activation of 7 leads to an increase in intracellular

calcium that can take place independently of extracellular calcium levels and can be blocked by

inhibition of Ca2+

release from intracellular stores. This suggested that 7 can drive signaling

involving Ca2+

release from intracellular stores rather than ion flow through open receptors.

Furthermore, Hosur and Loring (2011) showed that in SHEP1 cells expressing 42 nicotinic

receptors, calcium chelation failed to block the effect of nicotine on LPS-induced NFB activity.

However, JAK/STAT inhibition did block the effect of nicotine on LPS-induced NFB activity,

suggesting that the JAK-STAT pathway may play a role in 42-mediated anti-inflammation

independently of calcium (Hosur and Loring, 2011).

A primary objective of this project was to create a novel 7-mediated STAT3-Metridia

luciferase (ML) signaling assay for investigating calcium dependency of 7-mediated STAT3

signaling. By using this assay in human neuroblastoma-derived SHEP1 cells and rat pituitary-

derived GH4C1 cells, we tested the hypothesis that 7-mediated STAT3 signaling can proceed

independently of calcium ion flow. Unlike 7 antagonism and JAK/STAT inhibition, calcium

chelation and maintenance in calcium-low media failed to reduce nicotine-induced luciferase

expression, supporting our hypothesis that 7 mediates STAT3 signaling without calcium ion

flow. Since 7 receptor maturation is a prerequisite for 7-mediated STAT3 signaling, studies

investigating the interplay between calcium influx and STAT3 signaling provided insight into

cell-dependency of 7 maturation, in particular, of the requirement of Resistance to Inhibitors of

Cholinesterase 3 (Ric3) transfection for surface receptor expression. Obtaining a greater

understanding of the mechanistic details allowing receptor expression would have several

important implications in drug discovery.

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Although there have been reports of surface 7 expression in some mammalian cell lines

(Valles et al., 2009; Williams et al., 2005), surface receptor expression remains a challenge in

several cell types (Sweileh et al., 2000). Achieving functional expression on the surface of the

cell would aid in the development of novel therapies designed to selectively target 7 receptors.

Strategies aimed at increasing the number of surface receptors have included alteration of cell

culture conditions, generation of 7/5HT3 chimeras, and site-directed mutagenesis. These have

resulted in only modest increases in the number of functional receptors or the generation of non-

native receptors, neither of which are ideal for effective drug discovery efforts. Therefore, there

is continued interest in identifying cellular factors allowing expression of functional 7

receptors. Although the mechanisms regulating the localization of 7 to the postsynaptic

membrane have been fairly well-studied (Sanes and Lichtman, 2001), there is much to learn

about regulation of 7 assembly and surface expression. Our initial hypothesis was that GH4C1

cells already possess Ric3 to allow surface receptor expression.

Ric3 was discovered in 1995 during a genetic screen in C. elegans (Nguyen et al., 1995).

Follow-up studies showed that this protein is needed for the maturation of multiple nicotinic

receptor subtypes in C. elegans and oocytes (Halevi et al., 2002). The most notable effect of the

chaperone was found to be its promotion of surface 7 expression in otherwise non-permissive

cells. In several mammalian cell lines, co-expression of 7 with Ric3 facilitates appropriate

folding and functional expression of the receptor (Valles et al., 2009; Williams et al., 2005).

Two isoforms of rat Ric3 were cloned and termed S-rRic3 and S+rRic3. S-rRic3 differs from

S+rRic3 by three bases due to the absence of a serine residue present in S+rRic3. Initial 125

I-

bungarotoxin binding assays demonstrated the functionality of both isoforms and supported our

hypothesis that GH4C1 cells possess Ric3 to allow surface 7 expression. We next investigated

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whether this discrepancy is due to the presence of endogenous Ric3 in GH4C1 cells or the

promotion of surface expression by unknown 7 chaperones present in this cell line.

To study the necessity of Ric3 transfection for 7 expression, RNAi constructs were

designed against Ric3. Cell line-dependent consequences of Ric3 knockdown justify a search for

novel 7 chaperones in GH4C1 cell cDNA, and we believe a variation of our STAT3 signaling

assay can be useful in searching for such factors. While STAT3-driven luciferase expression

allowed evaluation of 7-mediated STAT3 signaling, an assay utilizing STAT3-driven antibiotic

resistance may facilitate isolation of cells expressing the unknown chaperone. Proof-of-concept

studies involving recovery of transfected cDNA from mammalian cell lines were performed to

test the hypothesis that a variation of the STAT3 reporter assay will be useful for identifying

novel receptor chaperones.

In summary, we tested three hypotheses in this project. The first hypothesis was that 7-

mediated STAT3 signaling takes place independently from calcium ion flow. This hypothesis

rests upon evidence that IL6 receptors can initiate JAK2-STAT3 signaling without calcium

influx, 42 nicotinic receptors can mediate STAT3-driven anti-inflammatory responses without

calcium ion flow (Hosur and Loring, 2011), and 7 receptors activate PLC without ion influx

(Suzuki et al., 2006). Testing this hypothesis provided evidence supporting an additional

hypothesis regarding a separate topic related to 7 receptors. Our second hypothesis was

originally that GH4C1 cells possess an abundance of Ric3 allowing receptor expression. Upon

further investigation, an alternative hypothesis put forward was that GH4C1 cells possess

unknown 7 chaperones aside from the known functional Ric3 splice variants. This alternative

hypothesis is supported by evidence of surface receptors localized in brain regions lacking Ric3

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mRNA (Halevi et al., 2003). Finally, our third hypothesis is that a variation of the STAT3

signaling assay can serve as a tool in the search for novel 7 chaperones in GH4C1 cell cDNA.

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I. REVIEW OF THE LITERATURE:

A. Nicotinic acetylcholine receptors:

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels belonging to

the Cys-loop receptor superfamily. Other members of this family include 5HT3, glycine, and

GABAA receptors. Nicotinic receptors, like other members, are composed of five subunits,

forming a barrel-like transmembrane structure around an aqueous pore permeable to cations.

Each subunit crosses the membrane four times and has a large N-terminal extracellular domain

with a large intracellular loop between the third and fourth membrane-spanning domains.

Receptors can be generated from a wide variety of subunits, and their subunit composition

dictates their pharmacological properties (Millar, 2003). In vertebrates, the assortment of

subunits includes at least 17 types (1-10, 1-4, , , ). Of these, five (1, 1, , , ) can be

found at the neuromuscular junction (NMJ), while the remaining are localized within the central

and peripheral nervous system (Lindstrom, 1997). The neuronal nicotinic receptor subunits (2-

10, 2-4) combine in various ways to form many receptor subtypes (Millar, 2003). The two

main nicotinic acetylcholine receptors found in the brain of vertebrates are 42 and 7 (Herber

et al., 2004). The general subunit composition of these receptors is depicted in Figure 1 (De

Jonge and Ulloa, 2007).

Figure 1: Nicotinic acetylcholine receptor

subunit composition. Receptors may be

composed from a variety of subunits forming

homopentameric receptors, such as 7 receptors

(left), or heteropentameric receptors, such as 42

receptors (right). These possess different

pharmacological characteristics. For example,

-bungarotoxin (BGT) blocks 7 receptors, but

not 42 receptors. Also, nicotine shows low

affinity for 7 receptors and high affinity for

42 receptors (De Jonge and Ulloa, 2007).

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The high density of nicotinic receptors in the Torpedo electric ray has helped to improve our

understanding of these receptors through the use of electron diffraction analysis. X-ray

crystallography of the snail-derived acetylcholine-binding protein (AChBP) gives even more

detailed information about the structurally similar nicotinic receptor extracellular region (Brejc et

al., 2001; Celie et al., 2005; Hansen et al., 2005). Each nicotinic receptor subunit has four

transmembrane domains in which N- and C-termini are believed to be located outside the cell

(Figure 2). Each subunit has a large N-terminal domain, a variable cytoplasmic domain, and 2 or

5 ligand-binding domains (LBDs) depending on receptor subtype. The transmembrane

conduction channel stretches approximately 160 Å and includes a 20 Å-long ligand-binding

domain. Each subunit in the LBD is organized around two beta-sheets connected by a disulfide

bond forming part of the Cys-loop characteristic of nicotinic receptors (Sharma and

Vijayaraghavan, 2008; Miyazawa et al., 2003).

Figure 2: Basic structure of nicotinic receptors. A) Nicotinic receptor subunits have four

transmembrane domains, with both N- and C-tails located extracellularly. B) Nicotinic receptor

model. The large N-terminal region forms much of the extracellular portion of the receptor. The

distance between residues lining the channel pore narrows deeper within the membrane. The

narrowest point in the pore is lined by a ring of leucine residues, later followed by the

intracellular region. Ligand-binding domains are represented as ovals. PM, plasma membrane

(Sharma and Vijayaraghavan, 2008).

A B

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The second membrane-spanning domain, M2, lines the lumen of the ion channel. The M2

helices along this pathway extend 40 Å and are slightly kinked at Leu-251 and Pro-265 residues

(Figure 3). M2 helices traverse the cell membrane with similar amino acids at each level so that

distinct rings facing the lumen of the pore are formed (Hucho et al., 1986; Giraudat et al., 1986).

This pore is symmetrical due to equivalent side-to-side hydrophobic interactions between

equivalent surfaces of homologous amino acids (Figure 3). These rings are critical to receptor

function (Imoto et al., 1998). An example is the ring of leucine residues located within the pore.

In Xenopus oocytes, 7 receptors are inhibited by non-selective antagonists, such as curare and

hexamethonium. However, these ligands act as agonists at 7 receptors in which the point

mutation L247T has been introduced into the leucine ring of the M2 domain (Palma et al., 1999).

Figure 3: Lining of the nicotinic receptor pore. A) Amino acids facing the pore of the M2

domain. B) Molecular surface of the pore domain. Red and blue depict regions of high negative

and high positive charge, respectively; yellow represents the hydrophobic zone of the gate; V

and L identify -Val 255 and -Leu 251, respectively. C) Symmetrical arrangement of residues

lining the gate. The blue sphere represents a sodium ion (Miyazawa et al., 2003).

Upon agonist binding to the LBD, nicotinic receptors promote signaling between nerve and

muscle cells by opening and closing the ion channel pore. The pore is lined by an inner ring of

five -helices, as well as an outer ring consisting of fifteen -helices that protect the inner ring

from lipids. The gate is a constricting hydrophobic girdle in the middle of the plasma membrane

A C B

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that is formed by weak interactions between nearby inner helices. When an agonist interacts

with the ligand-binding domain, a series of rotations of the protein chains on opposite sides of

the entrance to the pore takes place. This conformational change is communicated through the

inner helices, resulting in pore opening. A proposed model for the gating mechanism is shown in

Figure 4.

Figure 4: Proposed model for nicotinic receptor gating mechanism. Acetylchoine-induced

rotations in the subunits are transmitted to the gate through the M2 helices. The rotations

destabilize the gate, causing the helices to become permeable to cations. The helices move freely

during gating because of flexible loops possessing glycine residues. S-S represents the disulfide-

bridge pivot in the ligand-binding domain, which is anchored to the fixed outer shell of the pore.

The relevant moving parts are shaded gray (Miyazawa et al., 2003).

When an agonist enters the ligand-binding domain and triggers conformational changes that lead

to opening of the receptor gate, sodium and calcium ions travel through the pore down their

electrical gradients. This elicits changes in membrane potential and leads to a host of

downstream cellular events, such as neurotransmitter release and gene regulation. Given the

distribution of 7 nicotinic receptors in the brain, these events have implications in several

important processes. 7 subunits, along with 4 and 2, appear to be the most widely expressed

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nicotinic receptor subunits in mouse and rat brain (Wada et al., 1989; Seguela et al., 1993; Marks

et al., 1992).

B. 7 nicotinic receptor transcript distribution:

Studies evaluating 7 transcript distribution via in situ hybridization have shown broad

localization throughout rat brain (Seguela et al., 1993). A summary of the expression pattern of

7 transcripts is presented in Figure 5. Strong hybridization is present in the primary olfactory

cortex, the endopiriform nucleus, and the claustrum, while moderate signals are observed in

superficial and deep layers of the isocortex from the frontal to the occipital areas (Seguela et al.,

1993). 7 receptors are located in areas of the brain important for cognition and working

memory, such as the hippocampus (Fayuk and Yakel, 2007). In this region, intense

hybridization is seen, particularly in the dentate gyrus and pyramidal cell layers (Seguela, et al.,

1993). In the amygdala, abundant 7 mRNA is observed, while in the hypothalamus, signals are

found in periventricular, medial, and lateral regions. There are moderately high transcript levels

in superior and inferior colliculi, respectively, and in the brainstem, 7 is detected in the central

gray, tegmental nuclei, and lateral lemniscus. There is weak hybridization in the Purkinje layer

of the cerebellum and moderate hybridization in substantia gelatinosa, central gray, and ventral

horn of the spinal cord (Seguela et al., 1993).

Figure 5: Distribution of 7 transcripts in

rat brain. 7 transcripts were found in distinct

locations throughout the brain. A-D) 7 is highly

expressed in olfactory regions as well as the

hippocampus in the dentate gyrus, CA4, CA3,

CA2, and CA1. E) In the brainstem, 7 is

expressed in the central gray, tegmental nuclei,

and raphe nuclei. F) In the spinal cord, 7 is

located in the substantia gelatinosa, central, and

ventral horn (Seguela et al., 1993).

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The reason some neurons yield functional receptors while others do not may be attributed to a

wide range of factors, one of which involves molecular chaperone-mediated receptor assembly

and trafficking to the plasma membrane. The topic of 7 chaperone activity contributing to

receptor maturation will be one focus of this project. Among the nicotinic receptor subtypes, 7

receptors have the highest Ca2+

permeability and promote rapid cholinergic synaptic

transmission. Another interesting characteristic of these receptors is rapid desensitization

following activation.

C. Calcium permeability of 7 receptors:

Calcium permeability of 7 receptors was investigated in a study showing that

application of the chloride channel blockers, niflumic and fluflenamic acid, lowers nicotine-

induced currents in Xenopus oocytes (Seguela et al., 1993). This reduction, shown in Figure 6,

may have been attributable to direct antagonism of the chloride channel blockers at 7 receptors.

However, a second possibility was that decreases in currents arose from interference of calcium-

activated chloride channels, which are opened by calcium flow through activated 7 receptors.

Figure 6: Nicotine-induced currents in the absence or presence of Ca2+-

activated chloride

channel blockers. Nicotine-induced currents at a holding potential of -60 mV are shown from a

single oocyte expressing 7. A) Nicotine-induced current without niflumic acid and fluflenamic

acid exposure. B) Nicotine-induced current following application of niflumic acid and

fluflenamic acid (Seguela et al., 1993).

To investigate whether direct antagonism of the chloride channel blocker at 7 lowers currents,

the extracellular NaCl concentration was raised to 350 mM to shift the reversal potentials of

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chloride and sodium in opposite directions. Under these conditions, nicotine-induced activation

of 7 receptors produced an inward current proceeded by a larger outward current (Figure 7).

Treatment with chloride channel blockers only lowered the second outward current, suggesting

that the decrease in current is not due to direct antagonism, but rather to disturbance with

calcium-activated chloride channels. It is likely that 7 receptors are more permeable to calcium

than other subtypes of nicotinic acetylcholine receptors, as 7 receptors have a greater ability to

activate calcium-dependent chloride channels than other subtypes (Decker and Dani, 1990).

Figure 7: Nicotine-induced currents show two components when the Na+ and Cl

- reversals

are separated. A) With increased NaCl outside the cell, nicotine elicits a small inward current

(1) followed by a large outward current (2). Under these conditions, the inward current through

7 activates the outward current through Ca2+

-activated Cl- channels. B) With niflumic acid and

fluflenamic acid pretreatment, the inward current is not diminished (1), but the outward current is

weaker (2). This suggests the nicotine-induced current is mostly a result of activation of Ca2+

-

activated chloride channels (Seguela et al., 1993).

Castro and Albuquerque (1995) also demonstrated that 7 has the highest Ca2+

permeability

among the nicotinic receptors investigated.

Activation of 7 influences a variety of calcium-dependent events, such as apoptosis and

neurotransmitter release (Sharma and Vijayaraghavan, 2001). Mutant 7 that desensitizes less

rapidly may cause abnormal apoptosis due to greater calcium flow through activated receptors

(Orr-Urtregar et al., 2000). To investigate if acetylcholine induces 7-mediated calcium currents

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in astrocytes, Sharma and Vijayaraghavan (2001) loaded cells with fluo-3 acetoxymethyl ester.

As shown in Figure 8, intracellular calcium increased after acetylcholine treatment, then returned

to baseline over a two-minute period of time. The acetylcholine-induced currents were most

likely attributable to 7 activation, as 10 nM MLA reversibly blocked ion flow (Sharma and

Vijayaraghavan, 2001).

Figure 8: Calcium responses in astrocytes following nicotinic receptor activation. A) Fluo-3

fluorescence of a single astrocyte following 100 M acetylcholine treatment. Images were taken

immediately before, and 1 s, 3 s, 30 s, and 65 s following exposure. (Scale bar= 10 m) B)

Averaged calcium transients from 31 cells (1 M ACh) and 42 (100 M ACh) fluo-3-loaded

astrocytes. C) Calcium response with and without 10 nM MLA. D) Changes in calcium

transients with 100 nM Bgt (Sharma and Vijayaraghavan, 2001).

Pretreatment with the antagonist BGT irreversibly blocked acetylcholine-driven calcium

currents. In the same cells, KCl-induced depolarization resulted in calcium transients,

suggesting that 7 blockade contributes to the reduced response. Modulation of extracellular

calcium concentration provided insight into the nature of 7-dependent calcium influx in

astrocytes.

Decreasing levels of extracellular calcium prevented acetylcholine-induced calcium

currents, indicating the requirement for calcium ion flow into the cell. To investigate whether 7

activation contributed to calcium flow through voltage-gated calcium channels (VGCCs),

acetylcholine-induced calcium currents were measured in the presence of CdCl2 (Figure 9).

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Since blocking calcium channels did not affect calcium currents, calcium influx through 7

receptors, not VGCCs, contributes more significantly to observed currents (Sharma and

Vijayaraghavan, 2001).

Figure 9: Dependence of the ACh response on

extracellular calcium. A) Response of a single

astrocyte to a 2-s application of 100 M ACh with

2 mM Ca2+

, where external Ca2+

was chelated with

EGTA, and with ACh in 2 mM Ca2+

. Removing

Ca2+

abolished the ACh-induced response.

B) Responses to 2-s application of 100 M ACh

with 100 M CdCl2. ACh responses were seen

with the VGCC blocker (Sharma and Vijayaraghavan, 2001).

Amplification of calcium influx through activated 7 receptors by release from intracellular

calcium stores was evaluated by blocking microsomal calcium pumps with 1 M thapsigargin.

Findings indicated that 7 stimulation results in calcium release from intracellular stores, mainly

through ryanodine-sensitive stores. The ability of 7 to influence intracellular calcium

concentration has implications for regulation of numerous signal transduction cascades, synaptic

plasticity, and memory processes (Fayuk and Yakel, 2007). For example, functional receptors in

the hippocampus regulate Ca2+

-dependent pathways involved in cognition (Levin et al., 2002).

D. 7 nicotinic receptors in Alzheimer’s disease:

Previous studies have implicated 7 function in the cognitive deficits associated with

Alzheimer’s disease (Oddo and LaFerla, 2006), auditory gating deficits of schizophrenia

(Freedman et al., 1994; De Luca et al., 2004), and Down’s syndrome (Deutsch et al., 2003).

Among the nicotinic receptors, 7 has particularly significant implications for AD, as a decline

in receptor function is associated with disease pathology. AD is characterized by progressive

cognitive deterioration, accompanied by loss of neurons and synapses- especially cholinergic

synapses- in the basal forebrain, cerebral cortex, and hippocampus and by a substantial reduction

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in both muscarinic and nicotinic receptor expression. These deficits appear early in the disease

and correlate with worsening cognitive ability (Nordberg, 1994, 2001; Whitehouse and Kalaria,

1995). In AD patients, 7 protein levels are reduced in the cortex and hippocampus (Burghaus

et al., 2000; Guan et al., 2000).

Although 7 protein loss is evident in AD, reductions in gene expression at the

transcriptional level are less clear, as the 36% reduction in 7 protein levels in the hippocampus

of patients with AD (Guan et al., 2000) does not correlate with a 65% increase in 7 mRNA

expression reported by Hellstrom-Lindahl et al. (1999). Also, 7 contribution to A42 toxicity is

unclear. Wang et al. (2000) report that direct binding of A42 to 7 may be a triggering factor of

neuronal cell death and AD pathology. As shown in Figure 10, A filaments- a hallmark of AD-

bind to 7 receptors with high affinity and displace BGT binding (Wang et al., 2000). While

the manner in which this interaction occurs remains unclear, recent findings, including those in

Figure 11, have suggested that A42 filaments act as non-competitive antagonists (Liu et al.,

2001). This suggests that selective 7 agonists may be neuroprotective and influence memory

and cognition in AD. Since the 7-A42 association may contribute to cell death by inhibiting

acetylcholine release and calcium flux, perhaps this interaction is key to progression of

pathogenesis observed in AD.

Figure 10: 125

I-BGT binding to

7SK-N-MC membranes is blocked

by amyloid peptide. Ligand receptor

binding assay was used to evaluate the

interaction between A peptides and 7.

A1-42, MLA, and epibatidine all blocked 125

I-BGT binding (Wang et al., 2000).

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Figure 11: A1-42 blocks hippocampal 7 noncompetitively. Cells were tested at

concentrations up to 10 mM ACh ACh before and after application of 10 nM A1-42. Increasing

the concentration of ACh did not overcome the partial block achieved with the concentration of

A1-42 used (Liu et al., 2001).

It is unclear whether A filaments activate or inhibit 7 function (Dineley et al., 2001;

Petit et al., 2001; Spencer et al., 2006). Dziewczapolski et al. (2009) demonstrated that 7

deletion in a mouse model overexpressing mutant human amyloid precursor protein defends

against memory loss and synaptic dysfunction. This suggests it may be useful to develop 7

antagonists for treating Alzheimer’s disease. However, several reports indicate receptor agonists

may have pro-cognitive potential in treating AD (Wehner et al., 2004; Keller et al., 2008; Curzon

et al., 2006; Young et al., 2007). For example, 7 is abundant in regions important for memory

and cognition such as the hippocampus, and several receptor knockdown studies have suggested

normal receptor activity is required for proper learning (Wehner et al., 2004; Keller et al., 2008;

Curzon et al., 2006). Furthermore, a wide range of 7-selecive agonists and allosteric

modulators have been shown to slow cognitive deterioration in AD (Young et al., 2007). The

precise role of 7 in AD pathology remains uncertain, and Lamb et al. (2005) and Small et al.

(2007) contest whether A filaments even bind to 7.

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E. 7 nicotinic receptors in oxidative stress:

7 receptors are transmembrane proteins embedded within membrane lipids. Proper

functioning of these receptors, like other neurotransmitter receptors, are highly dependent upon

the surrounding makeup of the lipid bilayer (Fong and McNamee, 1986). Destruction of this

bilayer, due to lipid peroxidation for example, can lead to disruptions in normal receptor

function. Since the brain requires great amounts of oxygen and cell membranes in brain tissue

possess high levels of oxyradical-sensitive fatty acids (Choe et al., 1995), the lipids surrounding

7 in these regions are particularly vulnerable to imbalances between free radical accumulation

and anti-oxidative protective defenses. This imbalance, termed oxidative stress, has been

implicated in a host of disease states, including neurodegeneration associated with Alzheimer’s

disease. Oxidative stress has been demonstrated to lead to deficits in 7 signaling in the central

nervous system.

A filaments, widely considered a hallmark of AD, may cause neuronal cell death by

inducing lipid peroxidation (Pedersen et al., 1998), and perhaps the loss of 7 receptors observed

in the disease is triggered by this process (Guan et al., 2001). In support of this hypothesis, some

studies suggest 7 may have anti-oxidative capacity. In PC12 cells, ethanol application elicits

increases in oxidative stress. However, activation of 7 via the selective agonist, anabaseine,

protected against ethanol-induced oxidative stress. Perhaps anti-oxidative protection provided

through 7 stimulation is partially driven by preventing the generation or buildup of reactive

oxygen species (ROS) (Li et al., 2000). In SH-SY5Y cells, RNAi knockdown of 7 results in

increased lipid peroxidation and augmented A toxicity (Qi et al., 2007), possibly implicating

the receptor in anti-oxidative activity that may help to protect against Alzheimer’s disease.

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F. 7 nicotinic receptors in schizophrenia:

Schizophrenic patients often exhibit cognitive deficiencies in addition to positive

symptoms like delusions and hallucinations. These deficiencies stem from disturbances in a

process called sensory gating, which permits the brain to properly filter sensory stimuli.

Nicotinic receptors play a critical role in controlling sensory gating, and emerging research

exploring their connection to schizophrenia is uncovering a prominent role for 7. Sensory

gating interference in cases of schizophrenia has been linked to chromosome 15q14, near the 7

subunit gene (Freedman et al., 2001). Furthermore, nicotine treatment to patients with

schizophrenia and DBA/2 mouse models improves sensory gating in a mechanism thought to

involve 7 (Adler et al., 1992; Simosky et al., 2003; Levin et al., 1988). Typical antipsychotic

drugs, as well as many atypical antipsychotic drugs, do not have an impact on the P50 Auditory

Gating deficit widely considered to be a trait associated with schizophrenia. However,

clozapine- an atypical antipsychotic- normalizes auditory gating in DBA/2 mice by interacting

with 7 receptors (Simosky et al., 2003).

7 receptors are found in regions of the brain implicated in schizophrenia, such as the

hippocampus, dorsal striatum, nucleus accumbens (NAc), prefontal cortex, the lateral and medial

geniculate nuclei, and reticular nucleus of the thalamus. They have also been implicated in

neuronal populations affected in this disease, such as dopaminergic, serotonergic, glutamatergic,

and GABAergic cells (Kucinski et al., 2011). In the nigrostriatal system in the midbrain,

dopaminergic cells in the SNc project to the dorsal striatum, while ventral tegmental area cells of

the mesolimbic system project to the nucleus accumbens and prefontal cortex (Kucinski et al.,

2011). These regions are negatively affected in schizophrenia, as patients have diminished area

and nerve cell volume in the SNc and VTA (Bogerts et al., 1983, 1992). In the prefontal cortex,

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patients exhibit reductions in the number and density of neurons, as well as diminished dopamine

and glutamate transmission. The presence of cholinergic signaling in these areas may implicate

7 as a potential target in treating schizophrenia.

In the reticular thalamic nucleus, GABAergic neurons are involved in the regulation of

sensory processing, and their disruption may contribute to aspects of schizophrenia (Kucinski et

al., 2011). 7 may control deficits in sensory gating deficits by stimulating the release of

inhibitory GABA onto thalamic relay cells that enhance random background activity. Interaction

of 7 with glutamatergic neuronal populations may also play a role in schizophrenia, as NMDA

receptors are located on GABAergic neurons of the reticular thalamic nucleus, and 7

stimulation may make up for reductions in NMDA activity by increasing GABA release to

thalamocortical cells (Hajos et al., 2005). 7 is also located on glutamatergic neurons affecting

dopamine output, and nicotine-induced dopamine release in the striatum is attenuated when

either glutamate or 7 receptors are blocked. This suggests NMDA and 7 have comparable

roles in controlling dopamine output in the striatum. 7 modulation of glutamate output in the

cerebral cortex may be responsible for pro-cognitive activity of nicotinic agonists in

schizophrenic patients (Levin and Simon, 2008).

Figure 12: Hypothesized locations

and function of CNS 7 receptors

in schizophrenia. NNR- neuronal

nicotinic receptor; Glu- glutamate;

DA- dopamine; P- pyramidal cell;

STR- striatum; THAL- thalamus;

VTA- ventral tegmental area; SN-

substantia nigra; NBM- nucleus basalis

of Meynert; PPT- pedunculo-pontine

nucleus (Kucinski et al., 2011).

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Patients with schizophrenia are much more likely to be heavy cigarette smokers than those

without the disorder. They also have a much lower density of 7 receptors in the hippocampus

and singulate cortex (Freedman et al., 1995; Guan et al., 1999). Decreased 7 activity may not

only be linked to schizophrenia, but also nicotine addiction (Brunzell and McIntosh, 2012).

Genetic studies have reported 7 gene polymorphisms may be a common predisposition to both

health issues (Leonard et al., 1996; Freedman et al., 1997; Saccone et al., 2010).

G. 7 nicotinic receptors in smoking addiction and withdrawal:

Nicotine is a highly addictive drug that is the primary contributor to the addictive appeal

of cigarette smoking (FDA, Federal Register, 1996). To more accurately forecast the efficacy of

smoking cessation strategies, understanding the role of nicotine in the initiation and continuation

of cigarette smoking is important (Henningfield et al., 1998). Increasing evidence implicates 7

activity in nicotine reinforcement. Rat and human 7 transcripts are over 90% homologous, and

similar to humans, rats self-administer nicotine (Seguela et al., 1993; Elliott et al., 1996).

Brunzell and McIntosh (2012) investigated whether reduced 7 activity increases nicotine self-

administration using a progressive ratio schedule of reinforcement (PR) in rats. If 7

antagonism in brain regions implicated in drug abuse corresponds with more active lever

pressing for nicotine and breakpoints, perhaps 7 signaling deficits contribute to increased

propensity for cigarette smoking among those with schizophrenia. The selective 7 antagonist,

ArIB -conotoxin, or the selective agonist, PNU282987, was infused into the nucleus accumbens

(NAc) shell or anterior cingulate cortex immediately before PR. If diminished receptor activity

raises nicotine self-administration, stimulating it may lower motivation for self-administration.

Such a finding would have implications for smoking cessation therapies and suggest a link

between cigarette smoking and schizophrenia.

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Antagonism of 7 in the NAc via ArIB -conotoxin infusion resulted in a dose-

dependent increase in motivation to self-administer nicotine. To control for the potential impact

of 7 activation on locomotor activity, as well as the reinforcing properties of cues, a “CUE

only” control group receiving cues without subsequent nicotine administration was included.

Dose-dependent increases in breakpoint and active lever pressing following ArIB -conotoxin

treatment were only observed in the nicotine group. Since drug exposure did not impact

response accuracy, ArIB -conotoxin-associated increases in lever pressing were not simply

attributable to hyperactive locomotion (Figure 13). Antagonism of 7 in the anterior cingulate

cortex revealed similar dose-dependent findings (Brunzell and McIntosh, 2012).

Figure 13: Antagonism of 7 in the NAc shell increases motivation to self-administer

nicotine. ArIB -conotoxin infusion into the NAc shell led to a dose-dependent increase in A)

breakpoints, or the highest lever depression criterion achieved, and B) active lever pressing.

There was no effect of ArIB -conotoxin infusion on breakpoint or active lever pressing in rats

reinforced with cues but not administered nicotine. C) Response accuracy as measured by %

active lever pressing was not affected by NAc shell infusion of ArIB -conotoxin in either

group. *Significantly different (p<0.05) (Brunzell and McIntosh, 2012).

Activation of 7 in the nucleus accumbens, on the other hand, reduced motivation to self-

administer nicotine. As show in Figure 14, PNU282987 infusion lowered both breakpoint and

active lever pressing during PR in only the group that was administered nicotine. Again, there

was no effect on response accuracy.

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Figure 14: 7 activation in the NAc shell decreases motivation to self-administer nicotine. PNU 282987 significantly decreased A) breakpoints and B) active lever pressing during a

progressive ratio schedule of reinforcement. There was no effect on breakpoint or active lever

pressing in rats reinforced with cues but no nicotine. C) Response accuracy measured by %

active lever pressing was not affected by -conotoxin in either group. *Significantly different

(p<0.05) (Brunzell and McIntosh, 2012).

Preclinical studies exploring the role of 7 in nicotine addiction have yielded mixed findings.

While the selective 7 antagonist methylconitine (MLA) significantly diminished nicotine self-

administration in an FR1 schedule of reinforcement (Markou and Paterson, 2001), Grottick et al.

(2000) report no effect on self-administration when rats had a 2-day wash-out period between

MLA doses. Given the low affinity of nicotine for 7 and the limited number of nicotine

infusions during PR, it is possible that -conotoxin effects arose from blockade of an

endogenous cholinergic signal rather than blockade of nicotine-induced 7 activation

(Wooltorton et al., 2003; Papke et al., 2010). This hypothesis is strengthened by nicotine

conditioned place preference and nicotine administration experiments showing 7 knockout

mice exhibit normal nicotine reward (Walters et al., 2006; Pons et al., 2008). Results from

Brunzell and McIntosh (2012) suggest that activation of 7 may have potential for drug

development efforts aimed at assisting people achieve smoking cessation.

Although the US Food and Drug Administration-approved smoking cessation drug,

varenicline, is marketed as an 42 partial agonist, it is also a full agonist at 7 receptors

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(Mihalak et al., 2006). Evidence presented by Brunzell and McIntosh (2012) support the notion

that the therapeutic strength of varenicline requires 7 activation. On a broader level, their

results identify 7 linkage to nicotine addiction and suggest that 7 deficiency may predispose

certain populations to both cigarette smoking and schizophrenic symptoms. Along with genetic

studies (Freedman et al., 1995, 1997; Guan et al., 1999) these findings imply that developing 7-

selective drugs may be a viable strategy to pursue for aiding smoking cessation efforts.

There is much interest in modulating nicotinic receptors to treat a variety of central

nervous system disorders, such as Alzheimer’s disease, schizophrenia, depression, and attention

deficit disorder. Table 1 lists several companies conducting clinical trials investigating the safety

and efficacy of 7-targeting ligands for the treatment of various central nervous system

disorders.

Table 1: 7 receptor compounds in clinical development for central nervous system

disorders (Taly et al., 2009).

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Cigarette smoking is widely recognized for increasing cancer risk, especially in lung cancer

(Burns, 2003). The finding that 7 receptors govern proliferation, apoptosis, and that nicotine-

derived nitrosamines activate these receptors fueled a fresh approach to conducting cancer

research, and the role of 7 receptors in cancer development and progression is a topic worthy of

discussion (Gotti et al., 1997; Schuller, 1989; Maneckjee and Minna, 1990; Schuller and Orloff,

1998).

H. 7 nicotinic receptors in cancer:

It was previously thought that 7 receptors were only expressed in the central and

autonomic nervous system. However, recent work has shown that these receptors mediate a

number of responses in cancer cells, such as release of angiogenic and neurogenic factors, in

addition to growth and proliferation (Schuller, 2009). The events following receptor activation

in cancer cells are similar to those in healthy cells. Agonist binding elicits conformational

changes, leading to opening of the intracellular gate. Cations flowing through the open channel

into the cell results in membrane depolarization. Membrane depolarization then triggers

subsequent opening of voltage-activated calcium channels, which further reduces the negative

intracellular charge. 7 receptors and voltage-gated calcium channels cooperate to regulate a

broad spectrum of events, such as neurotransmitters release and production of growth factors.

Increased intracellular calcium concentrations brought about by 7 activation and calcium

channel opening leads to signaling cascades implicated in apoptosis, cell proliferation, as well as

other events shown in Figure 15 (Kunzelmann, 2005; Roderick and Cook, 2008).

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Figure 15: The diverse functions of α7 and α4β2. Both receptors promote dopamine release.

α7 also stimulates the release of the excitatory neurotransmitters glutamate and serotonin, as well

as the neuropeptide bombesin and neurotransmitters, adrenaline and noradrenaline. These agents

stimulate cancer risk by directly activating signaling pathways or indirectly by regulating

epidermal growth factor (EGF), vascular endothelial growth factor (Vegf) and arachidonic acid

(AA) release. α7 also has immune modulatory activity. SCLC, small cell lung cancer; TNFα,

tumour necrosis factor-α (Schuller, 2009).

Given the broad network of events 7 governs, it is highly plausible that long-term

nicotine exposure through cigarette smoking contributes to the risk of developing cancer, along

with numerous other lifestyle factors. The finding that the carcinogenic nicotine derivative NNK

activates 7 receptors with an affinity 1,300 times greater than nicotine (Schuller, 2009) supports

this theory, and suggests that the carcinogenic nature of nicotine may greatly depend upon the

action of nitrosamines displacing it from receptors. Like other agonists, including nicotine, NNK

interaction with 7 triggers calcium ion flow into the cells, leading to subsequent membrane

depolarization and stimulation of voltage-gated calcium channels. NNK exposure also

upregulates 7 receptors. In summary, 7 function following NNK-application is similar to that

following nicotine activation, not only in terms of rapid depolarization and ion influx, but also

downstream signaling. While the role of 7 receptors is thought to vary across different forms

of cancer, the role of nicotinic receptors in non-small-cell lung cancer is illustrated in Figure 16.

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Figure 16: Nicotinic receptors in non-small-cell lung cancer (nSCLC) cells. NNK (4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone) and choline bind with high affinity to α7, while

NNN (N-nitrosonornicotine) and nicotine bind to 42. Ca2+

influx leads to EGFR signaling.

This pathway is enhanced by α7-mediated Ras activation through β-arrestin-dependent SRC.

EGFR activates the Akt pathway and downstream effectors, X-linked inhibitor of apoptosis

protein (XIAP), survivin and NFκB. FGF2, fibroblast growth factor 2; Vegf, vascular

endothelial growth factor (Schuller, 2009).

Nicotine also stimulates mesothelioma cells through 7-driven ERK1-ERK2 activation

and inhibition of apoptosis, NFB activation, and BAD phosphorylation (Trombino et al., 2004).

7-mediated activation of ERK1-ERK2 and STAT3 upon nicotine exposure has also been

implicated in bladder cancer cells (Chen et al., 2008). Although the emerging understanding of

nicotinic receptors in cancer opens many new avenues to cancer treatment, the use of nicotinic

antagonists that completely block the receptors is unlikely to succeed, since receptors regulate a

multitude of cellular processes. For example, although application of 7 antagonists to cells in

vitro may effectively block cancer progression, in in vivo models, they may interfere with

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pathways whose normal function is critical for proper regulation of bodily functions.

Unfortunately, such disturbances may trigger symptoms common to disorders linked with 7

receptors, such as the cognitive decline of Alzheimer’s disease (Gotti et al., 1997; Leonard et al.,

2002). In addition to its involvement with neurological disorders and cancer, 7 receptors have

been shown to influence cytokine production through a process called the “cholinergic anti-

inflammatory pathway.”

I. 7 nicotinic receptors in anti-inflammation:

Until recently, it was believed that the immune system functioned separately from

neuronal control. However, it has been established that numerous systems, including the innate

immune response, are regulated by the nervous system (Tracey et al., 2007). Clinical studies had

indicated that nicotine administration can be effective for treating some cases of inflammatory

bowel disease (IBD) (Guslandi, 1999; Sandborn et al., 1997), and that pro-inflammatory

cytokines are significantly decreased in the colonic mucosa of smokers with inflammatory bowel

disease (Sher et al., 1999). Therefore, Borovikova et al. (2000) reasoned that the cholinergic

parasympathetic nervous system (PNS) may modulate the systemic inflammatory response.

Investigations into the mechanism regulating immune homeostasis have involved the pro-

inflammatory protein, tumor necrosis factor (TNF). In primary human macrophages,

acetylcholine blocked LPS-induced increases in TNF release (Figure 17). Since TNF mRNA in

acetylcholine-treated, LPS-stimulated macrophages was not significantly different from vehicle-

treated, LPS-stimulated macrophages, acetylcholine probably suppresses TNF post-

translationally.

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Figure 17: Cholinergic agonists inhibit LPS-induced TNF synthesis in human

macrophages. A) Acetylcholine, muscarine, and nicotine block TNF production following 4

hour LPS application. B) Anti-TNF antibody immunostaining revealed lowered LPS-induced (2

hour) TNF activity in macrophages exposed to 100 M acetylcholine (Borovikova et al., 2000).

To examine whether the release of other cytokines may be influenced by ACh,

Borovikova et al. measured the release of other cytokines in macrophages treated with LPS and

acetylcholine. As shown in Figure 18, acetylcholine dose-dependently reduced LPS-induced

increases in the pro-inflammatory cytokine, IL6, while failing to block production of the anti-

inflammatory cytokine, IL10 (Figure 18).

Figure 18: In human macrophages acetylcholine inhibits release of pro-inflammatory, but

not anti-inflammatory, cytokine release. Acetylcholine was applied to LPS-treated (100

ng/ml) human macrophages for 20 hours at various concentrations. A) ACh dose-dependently

lowers IL6 release. IL1 and IL18 were also lowered (not shown). B) ACh fails to block LPS-

induced IL10 release. Data are mean +/- SEM from four separate experiments (Borovikova et

al., 2000).

A B

A B

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The finding that vagus nerve stimulation influences TNF activity has broad implications. Since

the cholinergic anti-inflammatory pathway proceeds much more rapidly than the humoral anti-

inflammatory cascade, developing compounds to target 7 receptors may be an effective means

of reducing the harmful effects of toxin exposure. Moreover, activation of this endogenous

pathway during times of stress may prevent harmful peripheral immune responses.

Follow-up work showed that vagus nerve-regulation of cytokine production involves 7

receptors more specifically, as 7 knockout mice show less production of cytokines than wild-

type mice in response to nicotine (Tracey et al., 2005). The downstream signaling pathway

responsible for 7-mediated anti-inflammation remains uncertain. Arredondo et al. (2006)

reported that nicotinic activation of 7 on oral keratinocytes leads to STAT3 upregulation

through the Ras/Raf/MEK/ERK pathway, and DeJonge et al. (2005) demonstrated that nicotinic

activation of 7 leads to phosphorylation and activation of STAT3 following interaction between

7 and JAK2. The effects of STAT3 on inflammation are context-dependent, and it possible that

7-mediated STAT3 signaling inhibits inflammatory cytokine production by inhibiting NFB

activity (Johnston and Grandis, 2011). Andersson and Tracey (2012) hypothesized that the 7

anti-inflammatory pathway responsible for reducing TNF generation may involve activation of

adenylate cyclase (AC) and conversion of ATP into cAMP (Figure 19). Increased cAMP

activates PKA, which phosphorylates CREB. Phosphorylated CREB turns on the transcription

of C-fos, which lowers cytokine production by blocking NFB activity. This pathway is

illustrated in Figure 19.

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Figure 19: Proposed mechanism

of 7-driven cytokine inhibition.

In spleen macrophages, 7

stimulation results in a signaling

cascade ultimately causing

diminished production of

cytokines. Activated 7 interacts

with AC, resulting in increased

levels of intracellular cAMP.

cAMP activates CREB, leading to

increased expression of C-fos

and block of NFB activity

(Andersson and Tracey, 2012).

Although the downstream signaling events responsible for 7-driven anti-inflammation are not

fully understood, the discovery of the cholinergic anti-inflammatory pathway has supported the

development of 7 ligands for conditions involving inflammation. Recently, studies

investigating the utility of compounds affecting 7 signaling have been a major focus in the

development of novel drugs. As shown in Table 2, 7 ligands have been shown to influence the

severity of disease state in a variety of conditions.

Table 2: Examples of conditions in which cholinergic modulation influences disease state.

(Andersson and Tracey, 2012).

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Nicotinic receptors are thought to be involved in both the motor reflex and the inflammatory

reflex (Figure 20). Heteromeric nicotinic receptors are abundant at the motor neuron and play a

role in the motor reflex. 7 receptors located on macrophages serve a role in the inflammatory

pathway and reduce pro-inflammatory cytokine activity upon the binding of acetylcholine.

Figure 20: Signaling comparison of motor reflex and inflammatory reflexA motor

reflex requires stimulation of sensory neurons projecting to dendrites of interneurons that relay

signals to muscles. Evidence shows there are nicotinic responses at the motor neuron. B) The

inflammatory reflex requires stimulation of sensory neurons in the vagus nerve by infection

(LPS) or inflammation (cytokines). These neurons project to the brainstem nuclei, where

interneurons relay signals to motor nuclei of the vagus nerve. Efferent signals travel to the celiac

ganglion, whose axons project to the splenic nerve. Norepinephrine elicits acetylcholine release,

whose binding to 7 on macrophages decreases the activity of pro-inflammatory cytokines

(Andersson and Tracey, 2012).

7 activity has been implicated in a range of conditions, and gaining a better understanding of

receptor signaling and expression may be useful in the development of more effective treatment

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strategies. Although 7 receptors have been well-studied, there are several aspects that remain

unclear. Two aspects this project will focus on are: 1) 7-mediated JAK-STAT signaling, and 2)

7 receptor maturation.

J. 7-mediated JAK-STAT signaling:

While ion flow through 7 is a well-established mode of signaling, a less thoroughly

studied signaling pathway involves signal transducer and activator of transcription 3 (STAT3).

STAT3 is a member of a family of 7 proteins that help to communicate signals from activated

cytokine and growth factor receptors on the surface of the cell into the nucleus where gene

transcription occurs (Johnston and Grandis, 2011). STAT3 modulates transcription of genes

involved in regulating a variety of critical functions, such as cell differentiation, proliferation,

apoptosis, angiogenesis, metastasis, and immune responses. Several cancers are linked with

constitutively active STAT3. Since elevated levels of STAT3 hinder apoptosis, allow cell

proliferation and survival, and promote angiogenesis and metastasis, they have been associated

with a poor prognosis for many different cancers (Johnston and Grandis, 2011). Recent studies

have increased our understanding of the anti-inflammatory pathway and implicated 7 and

STAT3 in modulating cytokine production in human and mouse macrophages (Johnston and

Grandis, 2011).

In 2003, Tracey et al. investigated the identity of receptors involved in the cholinergic

anti-inflammatory pathway revealed by Borovikova et al. (2000). To study whether 7 is

required for cholinergic inhibition of TNF release, 7 antisense oligonucleotides were produced,

along with antisense oligonucleotides (AS) to 1 and 10 as controls. Exposure of

lipopolysaccharide (LPS)- a major component of the outer membrane of gram-negative bacteria-

to macrophages elicited an increase in production of the pro-inflammatory cytokine, TNF

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(Figure 21). Pretreating macrophages with nicotine before LPS exposure reduced TNF

production. The ability of LPS to increase TNF production and effect of nicotine on this event

were already known. Tracey et al. showed that AS7 restored macrophage TNF release in the

presence of nicotine, while under similar conditions, AS1 and AS 10 did not significantly

influence nicotine’s impact on LPS-induced TNF release, demonstrating cholinergic inhibition of

macrophage TNF release depends specifically on 7 receptors.

Figure 21: Antisense oligonucleotides against 7, but not 1 or 10, prevent nicotine-

induced reduction in LPS-stimulated TNF release. Nicotine (1 M) exposure took place 5 to

10 minutes prior to LPS (100 ng/ml) treatment. While AS7 restored LPS-induced TNF

production in the presence of nicotine (A), AS1 (B) and AS10 (C) did not change the effect of

nicotine on TNF generation (Tracey et al., 2003).

To evaluate whether 7 receptors are involved in the regulation of inflammatory cytokine

release in vivo, Tracey et al. (2003) measured TNF production in mice lacking the receptor. As

shown in Figure 22, levels of serum TNF in knockout mice following endotoxin treatment was

significantly greater than that seen in wild-type mice exposed to endotoxin. Similarly, TNF

levels in the liver and spleen were also significantly higher in mice lacking 7, as were levels of

other inflammatory cytokines, such as IL6, and IL1.

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Figure 22: Increased cytokine produced in 7-deficient mice during endotoxaemia. Mice

were administered LPS (0.1 mg/kg, IP). A) TNF levels in serum; n=6 per group. B) IL1 levels

in serum; n=8 per group. C) IL6 levels in serum; n=9 per group. Asterisk, p< 0.05 versus wild-

type controls. Horizontal lines indicate group means (Tracey et al., 2003).

Tracey et al. (2003) demonstrated that the ability of nicotine to reduce LPS-induced TNF

production was mediated by 7 receptors. However, the downstream signaling events

responsible for this anti-inflammatory effect remained unclear. Since STAT3 was known for

inhibiting inflammatory pathways, De Jonge et al. (2005) investigated the involvement of this

transcription factor in the 7-mediated anti-inflammatory response.

The effects of STAT3 on the inflammatory process are context-dependent. STAT3 and

the tyrosine kinase JAK2, which phosphorylates STAT3, are required for both IL6 receptor and

IL10 signaling. IL6 contributes to the progression of many inflammatory diseases, while IL10 is

an anti-inflammatory cytokine. IL6 receptor signaling is inhibited by SOCS3, whose activity is

increased by STAT3 activation. SOCS3 binds to the gp130 subunit of IL6R and inhibits

activation of STAT3 by IL6R agonists. In 2005, De Jonge et al. demonstrated that stimulation of

7 via nicotine reduces NFB through STAT3 signaling (De Jonge et al., 2005). Consistent

with results from Tracey et al., De Jonge et al. showed that nicotine dose-dependently lowers

LPS-driven production of TNF, MIP2 and IL6, but not IL10 (Figure 23). Furthermore, nicotine

dose- and time-dependently activated STAT3 and SOCS3 in LPS-stimulated peritoneal

macrophages.

A B C

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Figure 23: Nicotine attenuates peritoneal macrophage activation and induces

phosphorylation of STAT3 and SOCS3 expression. A) ELISA of TNF, MIP2, IL6, and IL10

in macrophages stimulated with LPS in the presence of nicotine. B) Immunoblots for PSTAT3,

STAT3 and SOCS3 in macrophages stimulated with LPS in the presence of nicotine. C)

Immunoblot of PSTAT3 and STAT3 in peritoneal macrophages stimulated with nicotine (De

Jonge et al., 2005).

To determine whether nicotine-induced STAT3 activation required activation of nicotinic

receptors, macrophages were pretreated with nicotinic receptor antagonists before nicotine

exposure. As shown in Figure 24, non-selective antagonists hexamethonium and tubocurarine

blocked nicotine-induced STAT3 phosphorylation. While 7-selective antagonists BGT and

methyllycaconitine diminished nicotine-induced STAT3 activation, DHE, a selective non-7

antagonist, failed to lower STAT3 activation.

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Figure 24: STAT3 phosphorylation by nicotine is prevented by 7-selective nicotinic

receptor antagonists. Macrophages were exposed to d-tubocurarine, BGT, hexamethonium,

or -methyllycaconitine before nicotine application. Protein lysates were collected for PSTAT3,

STAT3, and actin immunoblotting (De Jonge et al., 2005).

STAT3 activation requires JAK2 activity. To evaluate the dependence of JAK2 recruitment to

7 receptors for STAT3 phosphorylation, immunoprecipitation studies were performed using the

JAK2 inhibitor, AG490 (De Jonge et al., 2005). Phosphorylation of STAT3 following nicotine

exposure was blocked by AG490, confirming necessity of JAK2 activity for STAT3

phosphorylation. AG490 also blocked nicotine-induced reductions in IL6 production in

macrophages exposed to LPS, implicating a role of STAT3 in nicotine-induced alterations of

cytokine release. Immunoprecipitation of JAK2 from untreated peritoneal macrophage lysates

revealed weak associations between JAK2 and 7. However, nicotine application increased

levels of 7 in JAK2 and PJAK2 immunoprecipitates, suggesting JAK2 recruitment to the

receptor and subsequent phosphorylation upon receptor activation. When cells were pretreated

with AG490 before nicotine exposure, reduced levels of PJAK2, but not JAK2, were observed in

7 immunoprecipitates (Figure 25). This implicates JAK2 association with nicotine-stimulated

7 receptors.

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Figure 25: Nicotine-induced

STAT3 phosphorylation occurs

through activation of JAK2 that

is recruited to the 7.

A) Immunoblot of PSTAT3 and

STAT3 in peritoneal macrophages

incubated with AG490.

B) Immunoblots of peritoneal

macrophages treated with 1 M

nicotine or 1 M nicotine and

100 M AG490

(De Jonge et al., 2005).

In 2006, Arredondo et al. further explored downstream signaling mediated by 7

receptors in oral keratinocytes (KCs). 7-mediated morbidity of tobacco products was

investigated by using RNAi and pathway inhibitors in KCs exposed to aged and diluted

sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine for 24 hours.

Real-time PCR and In-cell Western blotting revealed that KCs exposed to ADSS or 10 M

nicotine produced significantly greater STAT3 protein and mRNA compared to untreated KCs

(Arredondo et al., 2006). As shown in Figure 26, pretreatment with BGT or 7 siRNA

significantly lowered nicotine-driven upregulation of STAT3 levels. Furthermore, Ras/Raf-

1/MEK1/ERK inhibitors demonstrated a potential pathway through which 7-dependent

upregulation of STAT3 takes place (Arredondo et al., 2006).

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Figure 26: Alterations in STAT3

expression in KCs exposed to nicotine.

The following treatments were

used: 3 µM manumycin A (Mnmc);

3 µM manumycin A on KCs transfected

with constitutively active (CA)-MEK

(CA-MEK+Mnmc); 0.1 µM GW5074;

0.1 µM GW5074 on the CA-MEK

transfected KCs (CA-MEK+GW5074);

1 µM MEK inhibitor I (MEK-Inh);

10 µM U0126; transfection with

dominant negative (DN)-MEK;

transfection with the control, WT

MEK1 mutant (WT-MEK); 1 µM BGT;

transfection with siRNA-7; and co-

transfection with siRNA-7 and CA-MEK

(CA-MEK+siRNA-7), WT-MEK (WT-

MEK+siRNA-7) or DN-MEK (DN-

MEK+siRNA-7)

(Arredondo et al., 2006).

Since STAT3 upregulation in oral keratinocytes treated with nicotine or sidestream nicotine

exposure may rely upon intracellular signaling pathways stemming from 7 activation, targeting

7 receptors may be a promising approach for managing tobacco-related periodontal disease

(Arredondo et al., 2006). Anti-inflammatory effects of 7 suggest that this receptor is a

promising target in a number of additional disorders. For example, inflammatory bowel disease,

osteoarthritis, and sepsis (Marrero and Bencherif, 2009) may be potential candidates for

treatment through modulation of 7 function.

K. Multiple modes of 7 nicotinic receptor signaling

7 nicotinic receptors are among the most extensively studied type of neurotransmitter

receptor, and it is well-established that calcium influx through open receptors triggers a host of

cellular responses. More recently, evidence suggests 7 also drives STAT3 signaling to reduce

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cytokine production. Although emerging research continues to further our understanding of this

anti-inflammatory pathway, there are several issues that remain uncertain. The primary purpose

of our project is to study the interplay between 7-mediated calcium influx and STAT3

signaling. We aimed to study the relationship between the two modes of signaling and

investigate whether 7-mediated STAT3 signaling can proceed independently of calcium. Our

hypothesis that 7 mediates STAT3 signaling is independent of ion flow is supported by a

number of studies demonstrating signal transduction pathways proceeding independently of

calcium.

Shytle et al. (2004) demonstrated that in rat microglia, nicotinic activation of 7 leads to

an increase in intracellular calcium levels. This increase can take place independently of

extracellular calcium levels and can be blocked by inhibition of Ca2+

release from intracellular

stores. This suggested that 7 can drive signaling involving Ca2+

release from intracellular

stores rather than conventional ion flow through activated receptors. Further evidence

supporting the possibility of a Ca2+

-independent 7-STAT3 signaling pathway is provided by

studies performed by Hosur and Loring (2011). Aside from 7 receptors, 42 nicotinic

receptors also lower cytokine production by activating the JAK-STAT pathway. In SHEP1 cells

expressing human 42, calcium chelation and PKA inhibition failed to block the effect of

nicotine on LPS-induced NFB activity. However, JAK/STAT inhibition did block the effect of

nicotine on LPS-induced NFB activity, suggesting that the JAK-STAT pathway may play a role

in 42-mediated anti-inflammation that is independent of calcium and cAMP (Hosur and

Loring, 2011).

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L. 7 and 42 receptors activate the STAT3 pathway independently of calcium ion flow:

Hosur and Loring (2011) investigated whether JAK2 is involved in 42 mediated anti-

inflammation in LPS-treated SHEP1 cells expressing human 42 nicotinic receptors.

Observations that AG490 abolished nicotine-induced decreases in LPS-stimulated NFB activity

suggest JAK2 plays a role in reducing inflammation (Figure 27).

Figure 27: Inhibition of JAK2 restores LPS-induced NFB production in h42 SHEP1

cells exposed to nicotine. A) h42 SHEP1 cells transfected with NFB reporter were

pretreated with 30 nM JAK inhibitor I and 300 nM nicotine before 1 g/ml LPS application. B)

h42 SHEP1 cells transfected with NFB reporter were pretreated with nicotine and indicated

concentrations of AG490 for 30 minutes before 1 g/ml LPS treatment. One-way ANOVA

determined statistical differences with p< 0.05 (Hosur and Loring, 2011).

Active JAK2 leads to phosphorylation and translocation of STAT3 into the nucleus where

binding to promoter elements affects gene transcription (Heinrich et al., 2003). Nicotine dose-

dependently increases levels of the active, phosphorylated form of STAT3 (PSTAT3) (De Jonge

et al., 2005). Therefore, Hosur and Loring (2011) examined the anti-inflammatory role of

STAT3 by testing the effects of the STAT3 inhibitor, NSC74859 (S3I-201), on nicotine-induced

inhibition of LPS-stimulated NFB signaling in SHEP1 cells stably expressing h42. As

shown in Figure 28, 30 M NSC74859 at 30 M prevented the effects of nicotine.

B A

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Figure 28: STAT3 inhibition restores LPS-induced NFB release in h42 SHEP1 cells

exposed nicotine. A) h42 SHEP1 cells transfected with the NFB reporter were exposed to

LPS with or without nicotine and indicated concentrations of NSC74859 pretreatment (Hosur

and Loring, 2011).

LPS-induced cytokine production involves phosphorylation of IB and translocation of NFB

into the nucleus. Since nicotinic activation of 42 reduces LPS-induced NFB activation and

cytokine production, Hosur and Loring (2011) examined whether 42 stimulation impacts

IB phosphorylation. Pretreatment of h42 SHEP1 cells with nicotine by itself lowered

IBphosphorylation, and preincubation with AG490 before nicotine treatment restored NFB

translocation and IB phosphorylation in LPS-stimulated cells, suggesting that nicotine reduces

LPS-elicited inflammation and IB phosphorylation in a JAK2-dependent manner.

Hosur and Loring (2011) then tested the role of calcium on NFB translocation and IB

phosphorylation using BAPTA-AM, an intracellular calcium chelator. As shown in Figure 29,

nicotine reduces LPS-induced IB phosphorylation by approximately 50% of control in

Western blots and by approximately 10% of control in ELISA. The inability of calcium

chelation to diminish the effects of nicotine suggests that 42-mediated reduction of LPS-

induced NFB activation and IB phosphorylation does not depend on calcium.

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Figure 29: 42-mediated inhibition of LPS-induced IBphosphorylation may be

calcium-independent. h42 SHEP1 cells were pretreated with 5 M BAPTA, then exposed to

300 nM nicotine before LPS application. A) Immunoblot of phospho-IB versus IB.

Nicotine partially blocked LPS-induced IB phosphorylation by approximately 50% of control.

BAPTA failed to block the effects of nicotine. B) Luciferase-linked phospho-IB ELISA.

Nicotine partially blocked LPS-induced IkBa phosphorylation by about 10%. BAPTA failed to

block the effects of nicotine (Hosur and Loring, 2011).

Findings that 42 can mediate STAT3 signaling independently of calcium ion flow (Hosur and

Loring, 2011) motivated us to study 7-mediated STAT3 signaling by disrupting normal calcium

flow through the use of calcium chelators and modified media low in calcium levels. Another

aspect of signal transduction worthy of investigating was protein kinase A (PKA). Hosur and

Loring (2011) demonstrated that PKA inhibition via PKI 14-22 amide failed to block the effect

of nicotine on LPS-induced NFB activity, and Liu et al. (2006) reported that PKA inhibition via

10 M H-89 in HEK293 cells prevented phosphorylation of JAK2 and STAT3. Figure 30

presents several signaling pathways relevant to our project while highlighting our hypothesis that

7-mediates STAT3 signaling independently of calcium.

A

B

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Figure 30: 7-mediated anti-inflammation involves two modes of signaling. The endotoxin

LPS elicits an increase in TNF production through activation of Toll-like receptor 4. Activated

IKK phosphorylates the inhibitory protein IB, resulting in its subsequent ubiquitination,

dissociation from NFB, and degradation in the proteasome. NFB is then free to translocate

into the nucleus to drive the transcript of pro-inflammatory cytokines, such as TNF. Nicotinic

activation of 7 stimulates several possible pathways that result in the reduction of LPS-induced

TNF activity. One hypothesis is that calcium influx leads to adenylate cyclase activation and

downstream activation of CREB. CREB drives transcription of Fos, which decreases pro-

inflammatory cytokine production by blocking NFB. Another possibility is that ERK

activation results in STAT3 upregulation. Nicotinic stimulation of 7 drives activation and

phosphorylation of STAT3, whose anti-inflammatory effect may result from inhibition of NFB.

Similar to nicotinic stimulation of 7, the cytokine IL6 activates IL6R to drive JAK2-STAT3

signaling. Our hypothesis is that calcium influx through open 7 (1) is not required for 7-

mediated STAT3 signaling (2).

In addition to 7-mediated STAT3 signaling, another aspect of 7 receptors that remains

unclear is receptor maturation. While studying 7-mediated STAT3 signaling, insight was

provided in cell-dependency of receptor maturation, as proper folding and assembly are

prerequisites for driving STAT3 signaling. We will be presenting data in the “Results” section

suggesting 7 requires folding and assembly to drive JAK/STAT signaling. This raised the issue

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of what is needed for 7 receptor maturation and suggested we could use JAK/STAT signaling

to explore cell-dependent receptor maturation. One aspect of cell-dependent maturation involves

the necessity of molecular chaperones allowing surface receptor expression. In the case of 7, a

chaperone called Resistance to Inhibitors of Cholinesterase 3 (Ric3) has been demonstrated to

have a profound effect on surface expression.

M. Cell line-dependency of 7 maturation:

Ric3 was discovered in 1995 in a genetic screen of C.elegans that confers resistance to

the inhibitor of acetylcholinesterase, aldicarb (Nguyen et al., 1995). Since its discovery, this

protein has been shown to allow surface 7 expression in otherwise non-permissive cells

(Lansdell et al., 2005; Williams et al., 2005). We originally hypothesized that the cell-

dependency of 7 receptor maturation observed by Sweileh et al. (2000) could be accounted for

by discrepancies in Ric3 content across various cell lines. To test the hypothesis that 7 receptor

maturation is a host cell line-dependent process, Sweileh et al. used recombinant adenoviruses

encoding rat 7 in which expression is under the control of a tetracycline-dependent promoter.

Following infection of five cell lines, including SHEP1 and GH4C1 with the a recombinant

adenovirus encoding 7, all five cell lines expressed a 60 kDa protein stained by anti-7

antibodies representative of unassembled 7 subunits. However, although all five cell lines

expressed mRNA for 7 (Figure 31), only GH4C1 and SHEP1 cells expressed surface 7

receptors that bound 125

IBGT (Figure 32).

Figure 31: All cell lines produce 7 mRNA

when transfected. 2 ug RNA extracted was

electrophoresed, blotted, and probed with the

indicated radiolabeled cDNA. Tubulin

mRNA was a control for RNA loading

(Sweileh et al., 2000).

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Figure 32: Only two cell lines express mature 7

receptors. Specific 125

-BGT binding is indicated.

While only GH4C1 and SHEP1 cells yielded robust

surface binding, GH4C1 cells exhibited ~10-fold greater

surface expression than SHEP1 cells. All five cell lines

infected with 42 expressed mature surface receptors

(not shown)

(Sweileh et al., 2000).

Since radiolabeled BGT lacks the penetrability to go into the cell, it is an ideal compound for

monitoring surface 7 expression. GH4C1 cells expressed over 10-fold more surface 125

I-BGT

binding sites than SHEP1 cells following 7 adenovirus infection, suggesting that the assembly

and membrane insertion of 7 are cell line-dependent. Although GH4C1 cells produced more

surface receptors than SHEP1 cells, the amounts of immunoprecipitable total receptor were

similar, suggesting that GH4C1 cells are more efficient than SHEP1 cells at either assembling

receptor subunits or inserting them into the plasma membrane. If Ric3 allows surface expression

of 7 receptors essential for STAT3-driven luciferase expression in SHEP1 cells, and co-

transfection of GH4C1 cells with 7 and STAT3-ML alone is sufficient to allow robust

luciferase expression upon nicotine application, GH4C1 cells may already possess high levels of

Ric3 promoting 7 expression.

N. 7 nicotinic receptor maturation is a process that remains unclear:

Surface expression of multimeric transmembrane proteins is a multi-step process

involving currently unknown events. The assembly of muscle-type nicotinic receptors is

relatively well-understood and involves a slow process encompassing receptor subunit folding,

assembly, and insertion into the plasma membrane. Before receptor maturation, formation of

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disulfide bonds, N-linked glycosylation, oligosaccharide trimming, and interaction with

molecular chaperones are some of the events known to take place (Blount et al., 1991; Forsayeth

et al., 1993; Merlie et al., 1992; Wang et al., 1996). While less is known about maturation of

neuronal nicotinic receptors, it is presumable that a complex process such as receptor maturation

may exhibit some degree of cell line-dependence. Increasing evidence suggests that surface

expression of 7 is indeed highly cell line-specific. Despite having comparable levels of 7

mRNA, various types of PC12 cells differ in the amount of surface 7 expressed (Blumenthal et

al., 1997), and of the cell lines examined, only those possessing endogenous 7 were capable of

expressing epitope-tagged 7 receptors upon stable transfection (Cooper and Millar, 1997).

Due to their high calcium permeability and roles in transcription factor activation and

programmed cell death, tight regulation of functional 7 expression is essential (Couturier et al.,

1990). 7 subunits fold and assemble into pentamers in the endoplasmic reticulum (ER) before

being transported through the Golgi apparatus to the plasma membrane. The proper folding and

assembly that precedes export from the ER is believed to be host cell line-dependent and involve

currently unknown cellular machinery (Gu et al., 1991; Green and Millar, 1995). Before

achieving functionality, each subunit must fold properly and undergo a battery of post-

translational alterations, such as glycosylation of extracellular regions (Green and Millar, 1995).

In addition to the requirement for subunits to fold into a correct three dimensional conformation,

they must participate in necessary protein interactions. Evidence has suggested that early stages

of receptor folding typically involve several chaperone proteins.

An important chaperone involved in the maturation of 7 receptors is a protein called

Ric3 (Lansdell et al., 2005; Millar, 2008). Before the discovery of Ric3, the only proteins

implicated in the maturation of muscle nicotinic acetylcholine receptors were BiP, calnexin, and

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48

14-3-3n (Gelman et al., 1995; Jeanclos et al., 2001; Blount and Merlie, 1991; Chang et al., 1997).

However, these are required for the maturation of many other proteins and are not specific for

nicotinic acetylcholine receptors (Shaw, 2000; Kleizen and Braakman, 2004). Proteins identified

in C. elegans using a genetic approach are more specific in their effects than those found in

mammalian cells through biochemical testing. Factors allowing successful maturation of

nicotinic receptors probably include both non-specific chaperones, such as BiP and calnexin, and

receptor-specific chaperones similar to those found in C. elegans. However, it is still not known

how many nAChR-specific and 7-specific chaperones exist. In C. elegans, Ric3 is the only

known chaperone to influence expression of all nicotinic receptor subtypes (Halevi et al., 2002).

Mammalian Ric3, like C. elegans Ric3, affects maturation of several mammalian nicotinic

receptor subtypes and 5HT3 receptors, but not other members of the Cys-loop ligand-gated ion

channel family (Treinin, 2008).

O. Ric3 has differential effects on expression of numerous receptors:

While exploring the cell line-dependency of 7 expression, Sweileh et al. (2000)

observed receptor expression in GH4C1 and SHEP1 cells. Although the complex, multi-step

nature of transmembrane maturation was recognized and thought to differ among various cell

lines, at the time it was unclear what might account for cell line-dependency. Perhaps

differences in surface receptor expression in GH4C1 and SHEP1 cells could be attributed to

different post-translational modifications on assembled subunits. Perhaps the turnover rate of 7

receptors in these cells may be lower than that of other cell lines. Another possibility accounting

for cell line-specific 7 maturation is the ability of certain cells to perform some process

downstream from the actual synthesis of receptor subunits. Perhaps cell line-specific molecular

chaperones necessary for such processes are lacking in non-expressing cells.

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The discovery and characterization of Ric3 helped to further our comprehension of the

cell line-dependent nature of 7 expression. Since its discovery, it has been shown that Ric3

influences surface receptor expression in a manner that depends on receptor subtype and host cell

line, and its influence on receptor maturation is not confined to 7. Ric3 either promotes or

inhibits the activity of numerous receptors depending on the experimental system. For instance,

surface expression of 5HT3 receptors in HEK293 cells is significantly reduced when co-

expressed with human Ric3 (hRic3), while co-expression with 1 glycine has no effect on

glycine-evoked currents (Figure 33). In Xenopus oocytes hRic3 facilitates functional expression

of 5HT3 receptors (Cheng et al., 2005, 2007).

Figure 33: Ric3 effect

on expression of 5HT3

and glycine receptors. A) Effects of hRic3

on whole-cell currents

induced by 100 mM

serotonin or 1 mM

glycine in HEK cells

expressing 5HT3

receptors or human 1

glycine receptors.

B) Summary of peak

current amplitudes

observed in (A) (Halevi et al., 2003).

In HEK293 cells, Ric3 co-expression with 34 and 42 enhances surface expression

(Lansdell et al., 2005). However, as shown in Figure 34, co-expression of hRic3 with either

42 or 34 in Xenopus leavis oocytes results in significantly weaker currents compared to the

control condition without Ric3 (Halevi et al., 2003).

A

B

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Figure 34: Differential effects of

hRic3 on expression of various

nicotinic receptors. A) Effects of hRic3 co-expression

on whole-cell currents elicited by

1 mM ACh in oocytes. B) Effects

of hRic3 co-expression on BGT

binding in oocytes expressing 7.

C) Summary of peak current

amplitudes observed in (A)

(Halevi et al., 2003).

The differential effects of human Ric3 on 5HT3 and heteromeric nicotinic receptor expression

may be due to the different expression systems involved.

P. Ric3 increases expression of 7 nicotinic receptor:

Among mammalian receptors that have been examined, Ric3 most prominently affects

the expression of 7. Functional 7 is expressed poorly in transfected mammalian cell lines

lacking endogenous receptor expression (Sweileh et al., 2000), but co-expression of Ric3

dramatically boosts levels of surface 7 receptors (Lansdell et al., 2005; Williams et al., 2005).

In 2008, Castelan et al. used this technique to explore the effects of hRic3 on the delivery of

newly assembled 7 receptors to the surface of the cell membrane, as well as their rate of

turnover from the surface. Their results, illustrated in Figures 35 and 36, demonstrate that hRic3

expedites the arrival of new receptors to the cellular surface and lowers their rate of

internalization (Castelan et al., 2008).

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Figure 35: Effect of hRic3 on 7

receptor membrane insertion. 125

I-BGT

binding assay reveals that hRic3 increases

the rate of arrival of new 7 receptors

to the surface of the cell (Castelan et al., 2008).

Figure 36: Effect of hRic3 on 7 receptor

turnover. 125

I-BGT binding assay shows that

hRic3 lowers the rate of turnover of 7 receptors

from the surface of the cell. Surface turnover

rates were determined by measuring radioligand

released into oocyte culture

medium (Castelan et al., 2008).

Although Ric3 affects surface expression of multiple nicotinic receptor subtypes in C.

elegans, the most profound effect of the chaperone is its ability to allow 7 expression in

otherwise non-permissive cells. HEK293 cells transfected with rat 7 and hRic3 displayed

nicotine-induced inward currents showing the rapid activation and desensitization characteristic

of 7 receptors, while no currents were detected in HEK293 cells transfected with 7 alone

(Williams et al., 2005). Similar findings were observed in HEK293 cells expressing human 7.

As depicted in Figure 37, HEK cells co-transfected with human 7 and hRic3 displayed more

robust currents upon application of 1 mM nicotine than cells transfected with 7 alone (Williams

et al., 2005).

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Figure 37: Co-expression of 7 and

hRic3 in HEK293 cells. Reponses

to 1 mM ACh applied during the time

indicated by the horizontal bar in two

different oocytes injected with rat 7

(black trace) or rat 7 + hRic3 (gray

trace) (Williams et al., 2005).

Ric3 also influences 7 expression in Xenopus oocytes. As shown in Figure 38, oocytes infected

with 7 and hRic3 exhibited more robust currents upon 1 mM acetylcholine application than

oocytes infected with 7 alone (Williams et al., 2005).

Figure 38: Whole cell currents in

oocytes co-expressing 7 and hRic3. Current responses to 1 mM acetylcholine

applied during the time indicated by the

horizontal bar in mock transfected

(black trace) or transfected with hRic3

(gray trace) (Williams et al., 2005).

Despite variable effects of Ric3 on expression of heteromeric nicotinic receptors, Ric3

promotion of surface 7 expression has been consistently demonstrated. The precise mode of

action by which Ric3 promotes receptor expression remains under speculation.

Q. Ric3 mechanism of action and regions of interest:

Ric3 is a highly charged protein, consisting of 8% aspartic acid, 13% glutamic acid, 10%

lysine and 8% arginine (Altschul et al., 1990). The Ric3 transcript contains 6 exons that code for

a variety of isoforms typically containing approximately 350-400 amino acids (Halevi et al.,

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2003). Human and mouse Ric3 both have multiple isoforms and two transmembrane domains-

one of which may be a signal sequence. Also, both human and mouse Ric3 have two isoforms

whose identity hinges upon the presence or absence of a serine residue located at an ambiguous

splice in exon 4. Aside from the ambiguous splice site, Ric3 has other notable features which

require further investigation. For example, there is at least one coiled-coil domain which may

(Wang et al., 2009) or may not (Ben-Ami et al., 2005) be essential for functionality. Also, the

issue of Ric3 transmembrane domains- the first of which may (Castelan et al., 2008) or may not

(Wang et al., 2009) be a signal sequence- represents another point of speculation with two

topology predictions shown in Figure 39.

Figure 39: Two models of the transmembrane topology of mouse Ric3. A) A type III protein

that crosses the membrane twice with the N-terminal and C-terminal regions in the cytoplasm.

B) A type I protein having a single transmembrane domain with the N-terminal region in the ER

lumen and the C-terminal region in the cytoplasm. SS, signal sequence; TM, transmembrane

domain; CC, coiled-coil domain (Wang et al., 2009).

The transmembrane topology of Ric3 is currently just one of several characteristics of the protein

that remain unclear.

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Wang et al. (2009) evaluated several uncertainties regarding mRic3: 1) localization, 2)

transmembrane orientation, 3) the role of the N-terminal sequence in targeting the protein to the

ER, 4) mechanism of facilitating 7 subunit folding and assembly and 5) the role of the coiled-

coil domain. Colocalization studies with ER and Golgi markers demonstrated that in COS cells

mRic3 is localized in the ER (Wang et al., 2009), contesting evidence that Ric3 cycles between

the ER and Golgi (Cheng et al., 2007) or travels to the surface of the cell (Williams et al., 2005).

To investigate the transmembrane orientation of mRic3, Wang et al. (2009) examined whether

consensus sequence sites for N-glycosylation, including a native site and several sites introduced

into various positions, were glycosylated. Neither the native site, nor any site that was

introduced on the C-terminal side of the transmembrane domain, was glycosylated. However, a

site introduced on the N-terminal side of the hydrophobic sequence was glycosylated, indicative

of a single-pass membrane protein whose N-terminal is lumenal.

While studying the role of the N-terminal sequence in targeting mRic3 to the ER, Wang

et al. (2009) demonstrated the first 31 amino acids of the protein act as a cleavable signal

sequence (SS) restricting the protein to ER localization. Elimination of these amino acids

disrupts targeting to the ER and results in diffuse distribution. Furthermore, linking the first 31

amino acids to DsRed, typically found in the cytoplasm, results in ER localization. The finding

that the N-terminal sequence of mRic3 is a signal sequence required for direction to the ER

supports previous findings from Cheng et al. (2007). To define the role of mRic3 domains in

facilitating maturation of 7, mutagenesis studies were carried out. The various Ric3 mutants

investigated are shown in Figure 40.

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Figure 40: Residues 1-168 comprise

the minimal function domain of mRic3.

A) Mutant mouse RIC-3 protein used

for transfection. B) COS cells were

transfected with h7 cDNA either

alone or with wt or mutated mRic3.

Surface receptor expression was monitored

by 125

I-BGT binding assay.

C) Summary of inward currents upon

3 mM ACh application recorded in

BOSC cells either transfected with h7 or

both 7 and mRIC-3 mutants

(Wang et al., 2009).

Mutagenesis studies revealed several findings regarding the roles of mRic3 domains in

promotion of 7 maturation. First, truncating the N-terminal region greatly diminishes mRic3

functionality (Wang et al., 2009). Since this region possesses the signal sequence directing the

protein to the ER, it appears that ER localization is a requirement for protein function. On the

other hand, mutants with C-terminal deletions display similar activity with respect to surface 7

expression compared to the full-length protein, provided the coiled-coil domain remains intact.

Deletion of all or part of the coiled-coil domain significantly reduced Ric3 activity, contrasting

the finding that the coiled-coil domain is not required for expression of certain C. elegans

receptors (Ben-Ami et al., 2005). Second, elimination of the coiled-coil domain does not abolish

A

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56

protein interactions between mRic3 and 7, as similar levels of 7 were co-precipitated with

mRic3 when 7 was co-transfected with either normal or mutant Ric3 without the coiled-coil

domain. Third, a mutant Ric3 lacking the N-terminal domain and largely excluded from the ER

failed to bind 7, suggesting that the coiled-coil domain alone is not sufficient for the binding of

mRic3 to 7. Fourth, to determine whether the coiled-coil domain may be required for Ric3-

Ric3 interactions, mRic3 mutants with partial or complete coiled-coil domain deletions were

investigated using immunoprecipitation studies. A significantly weaker interaction was observed

with mutants missing the second half of the coiled-coil domain, the first half, or the whole

coiled-coil domain (Figure 41). This suggests the coiled-coil domain, expressed in exon 4, is

essential for mRic3 self-association required for functionality (Wang et al., 2009). mRic3

promotes 7 maturation through association between its cytoplasmic coiled-coil domains, and

amino acids 1-168 form the minimal functional domain of mRic3.

Figure 41: The coiled-coil domain is essential for self-association of mRic3. A) Lysates of

cells expressing Ric3 with h7 were immunoprecipitated with Ric3 antibody and probed with 7

antibody. 7 coimmunoprecipitates with both mRic3 and mRic3 (∆ 138-168) but not

mRic3(∆96-367). B) C-terminal myc and GFP tags were fused to mutant Ric3. Lysates of cells

co-expressing tagged proteins were precipitated with myc antibody and probed with anti-GFP

antibody. The myc-tagged and GFP-tagged mRic3 (∆154-168) show weak interaction, and

neither tagged mRic3 (∆138-153) nor tagged mRic3 (∆138-168) co-precipitate (Wang et al.,

2009).

A B

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Studies elucidating the roles of Ric3 exons in allowing surface 7 expression are

important when considering regions to target for knockdown via RNAi. For example, since no

functional splice variants of Ric3 lacking exon 2 have been shown to impact surface 7

expression, this is an ideal region to target for knockdown. Finally, Wang et al. examined the

role of Ric3 7 maturation in HEK293 cells. Although 7 protein is produced in the absence of

Ric3, surface receptors are undetectable by both 125

IBGT binding and electrophysiological

experiments. Less than 15% of 7 subunits demonstrate BGT binding, indicating that most

subunits are unfolded and unassembled. Therefore, Ric3 probably acts before transport of the

fully assembled receptor to the cell surface. Ric3 transfection marginally raises levels of 7

mRNA, but greatly enhances levels of surface receptors, indicating that the major role of Ric3 is

to facilitate folding and assembly of 7 subunits into complete, pentameric receptors that can be

transported to the surface of the cell. Since Ric3 promotes 7 expression, it is presumable that

Ric3 expression correlates closely with that of surface 7 receptors. However, it has been

shown that this is not always the case.

R. 7 nicotinic receptor expression without Ric3:

In several mammalian cell lines, Ric3 co-transfection with 7 enhances surface receptor

expression (Lansdell et al., 2005; Williams et al., 2005). However, GH4C1 cells infected with

7 alone show robust surface expression detected via 125

I-BGT binding (Cooper and Millar,

1997; Sweileh et al., 2000; Quik et al., 1996). We originally hypothesized that GH4C1 cells

already possess Ric3 to promote surface 7 expression. However, since Ric3 mRNA

localization does not always correlate with levels of 125

IBGT binding, we remained open to the

possibility that these cells may possess an unknown 7 chaperone. Castelan et al. (2008) used

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an antibody that cross-reacts with human and rat Ric3 protein to localize sites in the rat central

nervous system. Immunoreactive-like staining demonstrated the presence of Ric3 protein in rat

brain, the results of which are summarized in Table 3 (Castelan et al., 2008).

Table 1: Ric3 distribution and labeling intensity in rat CNS. - not detected; + weak signal; ++

moderate signal; +++ intense signal; s.c. scattered cells (Castelan et et al, 2008). As shown in

Figure 15, a different distribution was observed by Halevi et al. in mouse CNS (Halevi et al,

2003).

Table 3: Ric3 distribution and labeling intensity in rat CNS. – not detected; + weak signal;

++ moderate signal; +++ intense signal; s.c. scattered cells (Castelan et al., 2008).

In rat brain, levels of 125

IBGT binding generally correlate with Ric3 transcript localization.

However, in mouse brain, discrepancies between Ric3 mRNA and 125

IBGT binding have been

observed. In situ hybridization experiments using mouse brain demonstrated low Ric3 transcript

levels in parts of the hippocampus (Halevi et al., 2003) where 125

I-BGT binding is robust

(Whiteaker et al., 1999). Evidence suggesting a discrepancy between Ric3 mRNA and

125IBGT binding is presented in Figure 42.

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Figure 42: Analysis of Ric3 transcript localization and 125

IBGT binding.

A) Bright field image of mouse hippocampus.

B) Dark field image of mouse hippocampus in situ hybridization showing Ric3 mRNA in CA1-

CA3 (red arrow) but not dentate gyrus (Halevi et al., 2003).

C) 125

I-BGT binding in rat dentate gyrus (blue arrow) (Clarke et al., 1985) - similar binding is

found in mouse (Whiteaker et al., 1999).

Evidence of surface 7 expression in a region lacking Ric3 mRNA opposes our initial

hypothesis that GH4C1 cells already possess Ric3 to allow receptor expression and support an

alternative hypothesis that these cells have an unknown 7 chaperone. Since there are multiple

splice variants of Ric3, we used RNAi methods to look at the consequences of knocking down

different Ric3 splice variants on surface 7 expression and determine whether GH4C1 cells may

possess a functional Ric3 splice variant.

S. Exploring the consequences of Ric3 protein knockdown on surface 7 expression:

RNAi is the process by which double-stranded RNA (dsRNA) is processed into small

interfering RNAs (siRNAs) that degrade complementary mRNA transcripts (Kim and Rossi,

2008). The finding that siRNA molecules are endogenously produced and may control gene

expression has led to applications in studies of gene function and disease management. siRNAs

are ~21 bp long sequences that have characteristic 2 nucleotide-long 3’ overhangs enabling them

to be recognized by RNAi processing enzymes. The definitive event in the RNAi process is

homology-dependent degradation of target mRNA (Kim and Rossi, 2008; Ghildiyal and Zamore,

C 125

I-BGT binding

A B C

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2009). In mammalian cells, siRNAs are produced by cleavage of dsRNA precursors by the

RNase III endonuclease Dicer. Dicer is complexed with RNA-binding proteins, the TAR-RNA-

binding protein, PACT, and Ago-2, which transfers siRNA to the RNA-induced silencing

complex- RISC (Naqvi et al., 2009; Kim and Rossi, 2008). When siRNAs are loaded onto RISC

they are still double-stranded. After Ago-2 cleaves and releases the “passenger” strand, the

active RISC contains a single-stranded “guide” RNA sequence that base pairs to complementary

mRNA sequences. The determination of which strand is the “guide” and which is the

“passenger” is dictated by thermodynamic stabilities of siRNA overhangs (Kim and Rossi, 2008;

Durcan et al., 2008). Ago-2 preferentially binds to the less stable end. Transcripts containing

complementary sequences to the “guide” strand are cleaved by the RNase activity of Ago-2.

Therefore, the functionality of siRNA is the consequence of mRNA cleavage and degradation.

The siRNA pathway is shown in Figure 43 (Aigner, 2006).

Figure 43: Mechanism of RNA

interference in mammalian systems.

(Aigner, 2006).

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In studies involving RNAi, an important factor determining the mode of gene silencing is

the desired length of the knockdown effect. Short interfering RNA (siRNA) is an effective

method if the objective is to explore the short-term effects of knocking down a gene. However,

since siRNA does not divide in tandem with the cells, its levels become less concentrated as cells

divide (Sandy et al., 2005). Therefore, the silencing effect typically lasts for a relatively short

period of time, and additional transfections are required to prolong the knockdown effect-

especially for proteins with low rates of turnover. If the aim is to achieve long-term knockdown

of a stable protein upon a single transfection, vector-based short hairpin RNA (shRNA) is a more

suitable approach (Sandy et al., 2005). A major advantage of using shRNA is that since the

vector is passed on to daughter cells, the gene silencing effect is inherited. Scrambled shRNA

can be used as a control to investigate off-target effects.

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II. SPECIFIC AIMS OF THE THESIS

1) Investigate 7-mediated STAT3 signaling in heterologous expression systems.

1a) Investigate the feasibility of using a STAT3-MetLuc signaling assay.

1b) Validate STAT3-ML signaling assay by measuring IL6-driven expression of

secreted alkaline phosphatase and luciferase in Invivogen’s HEK-Blue IL6 cells.

1c) Determine ideal conditions for nicotine-induced increases in 7-driven

luciferase expression and establish whether increased luciferase expression is

STAT3-attributable.

1d) Construct cell lines expressing both 7 and STAT3-driven luciferase

plasmids and re-assess ideal conditions for nicotine-induced increases in 7-

driven luciferase expression.

1e) Use STAT3-MetLuc assay to study Ca2+

-dependency of 7-mediated STAT3

signaling.

1f) Investigate the role of Ric3 in allowing 7-driven STAT3 signaling in non-

permissive cell lines.

2) Test whether Ric3 is the only chaperone for 7 nicotinic receptors by comparing

receptor expression in permissive cells with non-permissive cells before and after knocking

down Ric3.

2a) Clone rat Ric3 from rat brain and investigate functionality of the protein.

2b) Establish selectivity of Ric3 antibodies and demonstrate cell line-dependency

of Ric3 transfection for surface 7 expression.

2c) Test whether RNAi methods that block surface 7 expression in non-

permissive cells fail to block expression in permissive GH4C1 cells.

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2d) Obtain shRNA constructs to investigate alternative splicing, and study Ric3

protein turnover by determining effects of Ric3 knockdown on 7 expression in

cell lines stably expressing Ric3.

3) Establish a proof-of-principle for functionally cloning additional 7 chaperones

3a) Establish method of plasmid recovery by showing recovery of S-

rRic3/pREP4/hygromycin.

3b) Construct S-rRic3/pREP9/KG using In-Fusion cloning kit (Clontech) and

recover plasmid.

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III. MATERIALS AND METHODS

I. Cell culture and transfections

All cell lines were grown at 37˚ C in 5% CO 2. SHEP1 cells were grown in Dulbecco’s modified

Eagle’s medium (DMEM) containing 5% heat inactivated Fetal Bovine Serum (FBS) and 10%

Donor Horse Serum (DHS) supplemented with 1% penicillin-streptomycin. Rat pituitary

GH4C1 cells were grown in Ham’s F10 medium supplemented with 10% FBS and 0.5%

penicillin-streptomycin. HEK293 cells were grown in DMEM containing 10% FBS and 1%

penicillin-streptomycin. Cells grown in 75 cm2 or 25 cm

2 cell culture flasks were passaged onto

96-, 24-, or 6-well plates (BD Falcon, San Jose, CA, USA) at seeding densities of 10,000

cells/well, 50,000 cells/well, and 200,000 cells/well, respectively, at least 3 hours before

transfection and approximately 2-3 days prior to binding assay or protein extraction. Cell

counting was done using a hemacytometer. All transfections were performed with FuGeneHD

(Roche, Indianapolis, IN, USA) as indicated by the manufacturer, using 2 g DNA per condition.

During transfections, DNA constructs (aside from empty pREP4 or pREP9 vectors) were used in

equal amounts across all samples. Transfection efficiency was monitored by expression of either

a green or red fluorescent protein construct. SHEP1 and GH4C1 cell lines semi-stably

expressing S-rRic3 with and without 7 were created by passaging transfected cells into vessels

containing hygromycin (EMD Biosciences, La Jolla, CA, USA). To maintain selection pressure,

cells were maintained in 100 g/ml hygromycin. After sufficient selection, these cells were

either stored for future experiments or used to create cell lines semi-stably transfected with

shRNA plasmids. These cells were passaged into vessels containing hygromycin and puromycin

(MP Biomedicals, Solon, OH, USA). Cell lines semi-stably transfected with S-rRic3, 7, and

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shRNA were maintained in growth media containing 100 g/ml hygromycin and 1 g/ml

puromycin.

II. Chemicals and plasmids

BGT and nicotine tartrate were purchased from Biotoxins Inc. (St. Cloud, FL, USA). Plasmid

expressing human Ric3 was purchased from Origene (Rockville, MD, USA) while the rat Ric3

gene was cloned from total RNA from rat brain prepared by using an Absolutely RNA kit

(Stratagene, Santa Clara, CA, USA). Reverse transcriptase-polymerase chain reaction (RT-PCR)

amplification was performed using the Advantage2 PCR Kit (Clontech Laboratories, Mountain

View, CA, USA) to obtain cDNA which was cloned into the EcoI-XhoI sites of pREP4. siRNA

against human and rat Ric3 was designed and custom synthesized by Integrated DNA

Technologies (Coralville, IA, USA), while rat Ric3 shRNA was purchased from Origene

(Rockville, MD, USA). Human/mouse/rat Ric3 primary antibodies and HRP-conjugated

secondary antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).

Ric3 blocking peptide for competition studies was also obtained from Santa Cruz

Biotechnologies. STAT3-MetLuc and STAT3-EGFP reporter vectors were prepared by Dr.

Ralph Loring and Sharath Chandra Madasu. The STAT3 promoter element was excised from a

STAT3 luciferase reporter vector (Panomics, Fremont, CA, USA) and cloned into AgeI and

BsrG1 restriction sites of the pMetLuc reporter vector.

III. Ric3 clones

Primers for cloning full-length rat ric3 were based on sequence XM_001072861–(PREDICTED:

Rattus norvegicus similar to resistance to inhibitors of cholinesterase 3 homolog (LOC687147)).

The forward primer included a NheI site 5’ to the open reading frame with the sequence

TTTTTAGCTAGCATGGCGTACTCCACAGTACA. The reverse primer included an XhoI site

four bases past the stop codon (5’ to 3’ TTTTTTCTCGAGGCTTTCACTCAAAACCCTGG).

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mRNA purified (Stratagene Absolutely mRNA kit) from flash-frozen rat hippocampus,

cerebellum, and cortex was copied into cDNA using a Superscript II cDNA kit (Invitrogen).

PCR amplicons were cloned into Invitrogen PCDNA3.1/V5-His TOPO TA plasmid, subcloned

into Invitrogen pREP4 episomal plasmid using NheI and XhoI restrictions sites, and then

sequenced. Human Ric3 isoform A was purchased from Origene (Catalog # TC112180) and

subcloned into Invitrogen pREP4 plasmid and sequenced.

IV. Radioligand binding

-BGT was prepared by Dr. Ralph Loring and binding to 7 receptors was performed on live

cells. For our purposes, surface expression was investigated using -bungarotoxin dissolved

in Hank’s buffered saline solution (HBSS). Cells grown in 24-well or 96-well plates were

transfected as described above. After a period of at least 48 hours, each well was washed three

times with ice cold phosphate buffer saline (10 mM sodium phosphate, 0.9% NaCl) and

incubated with 10 nM 125

I -bungarotoxin in the presence and absence of unlabeled -

bungarotoxin for 3 hours on ice. Each well was washed three times with ice cold HBSS before

being solubilized in extraction buffer (0.5 M NaOH, 1% Triton X-100). All binding experiments

with 125

I-BGT were performed in HBSS containing 0.1% bovine serum albumin to reduce non-

specific binding. Radioactivity was measured using a Wallac Wizard gamma counter. Non-

specific binding was determined in the presence of 1 M -bungarotoxin.

Specific 125

I--bungarotoxin binding = Total binding minus Non-specific binding

V. Dot blot analysis

Total protein was extracted from cells grown in 6-well or 24-well plates using RIPA buffer

supplemented with PMSF to inhibit protease activity. After lysates were allowed to sit on ice for

30 min, they were centrifuged at 14,000 g for 10 min at 4˚ C. Supernatant was then aliquoted

into microcentrifuge tubes and stored in -20˚ C until further use. The BCA assay was used to

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determine total protein levels prior to blotting on nitrocellulose, to which 500 ng total protein

was blotted per sample. After blotting, the membrane was blocked for at least 2 h at room

temperature with 5% BSA in TBS-T (20 mM Tris HCl, 150 mM NaCl, 0.2% Triton X-100), then

incubated for 1 h with anti-Ric3 antibody diluted 1:400 in 5% BSA in TBS-T. After incubation

with the HRP-conjugated secondary antibody diluted 1:1200 in TBS-T at room temperature for 1

h, blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate Kit

(Thermo Fisher Scientific Inc., Rockford, IL, USA). Luminescence was monitored using a

Kodak In-Vivo Imaging System using exposure times ranging from 20 s to 2 min.

VI. NFB and STAT3 reporter assays

NFB reporter assays were performed using the pNFB-MetLuc-reporter vector included in the

Ready-To-Glow Secreted Luciferase Reporter Kit (Clontech Laboratories, Mountain View, CA,

USA). Cells were transfected in a 96-well plate as described above with reporter plasmid, 7,

and Ric3. Approximately 48 h after transfection, cells were exposed to growth media containing

0.3 ng/l lipopolysaccharide (LPS). Nicotine-pretreated cells were exposed to 10 M nicotine 1

h prior to LPS treatment, while BGT-pretreated cells were exposed to the 7 antagonist 1 h

prior to nicotine exposure, followed by LPS exposure 1 h afterwards. After 24 h, 50 l

supernatant was collected from each well, to which 5 l substrate/reaction buffer from the

Ready-To-Glow Secreted Luciferase Reporter Kit was added according to the manufacturer’s

instructions. STAT3 reporter assays were performed using the pMetLuc reporter vector

containing the STAT3 promoter element and prepared by Dr. Ralph Loring and Sharath Chandra

Madasu as described above. Cells were transfected as described above with reporter plasmid,

7, and Ric3. Approximately 48 h after transfection, cells were treated with 10 M nicotine

following exposure to the appropriate STAT3 or JAK2 inhibitor for 1 h. Cells exposed to BGT

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were also pretreated for 1 h prior to nicotine addition. After 24 h, 50 l supernatant was

collected from each well, to which 5 l substrate/reaction buffer was added as described for

NFB reporter assays. For some STAT3 reporter assays, rather than using substrate/buffer from

Ready-To-Glow Secreted Luciferase Reporter Kit, coelenterazine (CTZ) (NanoLight

Technology, Pinetop, AZ, USA) was used. For each sample, 20 l substrate/reaction buffer (1 l

CTZ:49 l PBS) was added. For both NFB and STAT3 reporter assays, substrate reactions

were carried out in Microlite 1 Luminescence Microtiter 96-well flat-bottom, opaque white

plates (VWR Scientific Products, Batavia, IL,USA) and luminescence was quantified using a

BioTek Synergy MX Microplate Reader.

VII. Data analysis:

SPSS was used for data analysis and results are presented as mean ± standard error of the mean.

Probability (p) <0.05 was considered statistically significant. ANOVA was used for

comparisons among groups.

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IV. RESULTS

AIM 1

Aim 1a) Investigate the feasibility of using a STAT3-MetLuc signaling assay.

7 receptors signal via influx of calcium ions. Another mode of signaling, involving

STAT3 activation was demonstrated in macrophages (De Jonge et al., 2005) but has not been

studied in heterologous systems. Whether the two signaling pathways proceed independently of

one another is unclear. To explore calcium-dependency for 7-mediated STAT3 signaling, a

novel 7 signaling assay involving a STAT3-driven Metridia luciferase reporter was developed.

Reporter construction began with two commercially available plasmids: an NFB-MetLuc

reporter from Clontech’s Ready-To-Glow Secreted Luciferase Reporter System, and a STAT3-

firefly luciferase plasmid from Panomics. The Ready-To-Glow Secreted Luciferase Reporter

System has several advantages. Since Metridia luciferase is secreted into medium surrounding

cells, the same set of transfected cells can be used for multiple sets of analysis. The assay also

has low background and is more sensitive than fluorescence-based reporter assays (Ready-To-

Glow-Secreted Luciferase Reporter Systems User Manual. 2009. Clontech).

To explore the feasibility of using a STAT3-MetLuc reporter to study 7-mediated

STAT3 signaling, we first monitored levels of NFB in response to the endotoxin

lipopolysaccharide (LPS) using NFB-MetLuc already included in the kit. This plasmid

contains an NFB promoter element upstream of the Metridia luciferase gene (Ready-To-Glow-

Secreted Luciferase Reporter Systems User Manual. 2009. Clontech) and allows LPS-induced

increases in NFB to be monitored by collecting supernatant, adding substrate, and measuring

luminescence. This vector was used by Hosur and Loring (2011) during investigations into

42-mediated effects on NFB activity. NFB-MetLuc-transfected SHEP1 and GH4C1 cells

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*

*

*

*

*

were exposed to nicotine in the presence of various concentrations of -bungarotoxin. In both

cells, nicotine blocked LPS-induced NFB activation, and 1-hour BGT pretreatment made cells

less responsive to nicotine-induced decreases in LPS-driven NFB production (Figure 44).

Overall, findings supported the viability of a MetLuc reporter for monitoring STAT3 activation.

Figure 44: LPS-induced increase in NFB is mediated by 7 in SHEP1 cells (A) and

GH4C1 cells (B). LPS-induced increase in NFB was blocked by 10 M nicotine, and BGT

blocked nicotine-induced decrease in NFB activity dose-dependently.

A

B

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Excising the firefly luciferase gene from the Panomics vector and replacing it with the MetLuc

gene from the Clontech plasmid yielded a novel reporter in which STAT3 activation drives

secretion of Metridia luciferase into the medium surrounding transfected cells. The NFB

promoter within NFB-MetLuc has a SacI restriction site before and a HindIII restriction site

after the promoter element. Since these sites are also located before and after the STAT3

promoter element within the vector purchased from Panomics, replacing the NFB promoter in

NFB-MetLuc with a STAT3 promoter was relatively straightforward. A basic diagram of this

procedure- performed by Dr. Ralph Loring and Sharath Chandra Madasu- is shown in Figure 45.

Figure 45: Construction

of STAT3-MetLuc

reporter plasmid.

Replacement of the NFB

promoter in the Clontech

MetLuc plasmid with a

STAT3 promoter from the

Panomics firefly luciferase

plasmid yielded a STAT3-

MetLuc reporter. In this

reporter the Metridia

luciferase reporter gene is

under the control of a

STAT3 promoter.

After constructing the STAT3-ML reporter vector, we needed to validate its ability to monitor

activation of the JAK2-STAT3 pathway. To carry out this validation, we obtained HEK-Blue

IL6 cells from Invivogen.

Aim 1b) Validate STAT3-ML signaling assay by measuring IL6-driven signaling of

secreted alkaline phosphatase and luciferase in Invivogen’s HEK-Blue IL6 cells.

HEK-Blue IL6 cells are a validated reporter system using HEK293 cells stably

transfected with human IL6R cDNA and a STAT3-inducible secreted alkaline phosphatase

(SEAP) reporter. Exposure to IL6 stimulates IL6R and drives activation of STAT3, which

X

X Ligate Cut & discard

Cut & discard

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0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35

Rela

tive

Lu

min

es

ce

nc

e

IL6 (ng/ml)

results in release of SEAP into media surrounding the cells. SEAP can be monitored adding

QUANTI-Blue to collected supernatant and measuring absorbance at 630 nm (Figure 46).

Figure 46: In HEK-Blue IL6 cells, IL6R activation

of the STAT3 pathway leads to SEAP expression.

IL6 is a pro-inflammatory cytokine implicated in the acute

immune response. Upon IL6 binding to the IL6 receptor,

the complex of IL6 and IL6R interacts with and leads to

dimerization of gp130. This leads to the activation and

phosphorylation of JAK1, JAK2, and Tyk2. Activated

JAKs induce the phosphorylation, dimerization, and

translocation of STAT3 into the nucleus, where it drives

expression of secreted alkaline phosphatase (HEK-Blue

IL-6 Cells: Interleukin 6 Sensor Cells, Catalog # hkb-il6).

Preliminary experiments to validate the IL6 reporter system showed that IL6 concentrations of 3

and 10 ng/ml produce the greatest increases in IL6-driven SEAP expression in cells exposed for

24 hours (Figure 47). This was in agreement with the manufacturer’s recommendations.

Furthermore, 24-hour exposure to nicotine up to 100 M did not induce SEAP expression,

demonstrating the lack of nicotinic receptor contributions (not shown).

Figure 47: HEK-Blue IL6 cells treated with IL6 show dose-dependent increase in SEAP

expression. Untransfected HEK-Blue IL6 cells were treated with varying concentrations of IL6

for 24 hours. Following supernatant collection, SEAP activation was monitored via absorbance

at 630 nm (n=4).

**

** **

*

* p < 0.01; ** p < 0.001

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After confirming the ability of IL6 to increase SEAP expression in the HEK-Blue system, we

sought to validate our own signaling assay by comparing it against Invivogen’s product.

Transfecting HEK-Blue IL6 cells with STAT3-ML allowed comparisons between

STAT3-driven SEAP expression- monitored through absorbance- and STAT3-driven MetLuc

expression- monitored through luminescence. Again, a prime advantage of both signaling assays

is the lack of need to lyse the cells. Since both SEAP and MetLuc are secreted in medium

surrounding cells upon STAT3 activation, supernatant can be collected from the same well of

transfected cells. This allows direct comparison of STAT3-driven MetLuc and SEAP

expression. HEK-Blue IL6 cells transfected with STAT3-ML were exposed to IL6 for 24 hours.

Supernatant was collected and exposed to QUANTI-Blue before monitoring absorbance for

SEAP detection. Additional supernatant from the same wells was collected and treated with

coelenterazine (CTZ) before measuring luminescence to detect MetLuc expression. Initial

experiments using HEK-Blue IL6 cells transfected with STAT3-ML showed IL6-driven

increases in luciferase expression (Figure 48), justifying further validation studies using this cell

line.

Figure 48: IL6 exposure

to HEK-Blue IL6 cells

transfected with STAT3-

ML increases luciferase

expression. HEK-Blue IL6

cells were transiently

transfected with STAT3-ML

approximately 48 hours

before 24-hour application

of IL6. In transfected, but

not untransfected cells, IL6

treatment increased

luciferase expression (n=4).

Transfection: - - STAT3-ML STAT3-ML IL6 (ng/ml): - 10 - 10

*

*p < 0.05

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0

0.5

1

1.5

2

2.5

3

Re

lati

ve

Ab

so

rba

nc

e (

63

0 n

m)

After observing IL6-elicited increased luciferase expression in HEK-Blue IL6 cells transfected

with STAT3-ML, completion of assay validation was carried out with 7 antagonists and

JAK/STAT inhibitors. If SEAP and luciferase expression increases are attributable to STAT3,

JAK/STAT inhibitors would be expected to diminish both IL6-induced SEAP and luciferase

expression in HEK-Blue IL6 cells transfected with STAT3-ML. As shown in Figure 49, JAK

inhibitior I and the STAT3 inhibitor, S3I-201, lowered IL6-driven SEAP and luciferase

expression to comparable extents. As expected, BGT did not lower reporter expression,

suggesting that IL6-induced reporter expression is attributable to IL6R, not 7, activation.

Figure 49: IL6 exposure to

HEK-Blue IL6 cells produces

similar increases in SEAP and

luciferase expression. HEK-

Blue IL6 cells were transiently

transfected with STAT3-ML

approximately 48 hours before

24-hour application of IL6.

Supernatant from identical

wells was assessed for both

luciferase (A) and SEAP

expression (B). BGT and

JAK/STAT inhibitors were

added 1 hour before nicotine

exposure. JAK/STAT

inhibitors reduced expression

of both reporters, while BGT

had no effect. In UT cells,

IL6 produced increases in

SEAP (bottom) expression but

not luciferase (top) expression

(n=4).

0

0.5

1

1.5

2

2.5

3

3.5

Re

lati

ve

Lu

min

es

ce

nc

e

Transfection: - - STAT3 STAT3 STAT3 STAT3 STAT3 STAT3 IL6 (ng/ml): - 10 - 10 10 10 10 10 [Inhibitor]: - - - - JAK Inh. I AG490 S3I-201 BGT (150 nM) (30 M) (30 M) (20 nM)

Transfection: - - STAT3 STAT3 STAT3 STAT3 STAT3 STAT3 IL6 (ng/ml): - 10 - 10 10 10 10 10 [Inhibitor]: - - - - JAK Inh. I AG490 S3I-201 BGT (150 nM) (30 M) (30 M) (20 nM)

* *

* * *

*p < 0.05

*p < 0.05

A

B

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By transfecting HEK-Blue IL6 cells with STAT3-ML, treating them with IL6, and monitoring

STAT3-driven increases in SEAP and MetLuc, the effectiveness of the STAT3 luciferase assay

was measured against a previously validated, commercially available system. Exposure to 10

ng/ml IL6 for 24 hours showed comparable increases in SEAP and MetLuc expression.

Furthermore, IL6-driven increases in SEAP and MetLuc were lowered by pretreatment with

JAK/STAT inhibitors. Since STAT3-driven reporter expression was driven by IL6R activation

and not 7-mediated, BGT pretreatment had no effect on expression of either SEAP or

MetLuc. Following validation of the STAT3-ML signaling assay, we determined ideal

conditions for nicotine-induced, STAT3-driven luciferase expression.

Aim 1c) Determine ideal conditions for nicotine-induced increases in 7-driven luciferase

expression and establish whether increased luciferase expression is STAT3-attributable.

In the STAT3-ML signaling assay, nicotinic activation of surface 7 receptors is the first

event in a pathway leading to secretion of luciferase enzyme into the surrounding media.

Therefore, when optimizing this signaling assay, two important parameters to consider were

concentration of nicotine and duration of nicotine exposure. The concentration of nicotine in

smokers’ blood is typically on the order of 200-300 nM (Henningfield et al., 1998). In a mouse

model of multiple sclerosis, similar concentrations of nicotine diminished autoimmune responses

(Shi et al., 2009). At concentrations achieved in smoker’s blood, nicotine attenuates CNS

inflammation as well as autoimmune responses in experimental autoimmune encephalomyelitis,

a mouse model of multiple sclerosis (Shi et al., 2009). Hosur and Loring (2011) reported that

100-300 nM nicotine decreased LPS-induced NFB activity and IL6 generation in SHEP1 cells

expressing human 42. In the current project, initial experiments to determine optimal

concentrations of nicotine for inducing STAT3-driven MetLuc expression showed that 10 M

nicotine induced the greatest increase in luciferase expression following 24-hour exposure.

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0

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ela

tive

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escen

ce

Treatment

Since 100 M nicotine did not induce a significantly greater increase compared to 10 M

nicotine, we concluded that 10 M was a suitable concentration to use in future experiments.

STAT3-driven luciferase expression appeared to be 7 attributable, as cells transfected with

STAT3-ML alone failed to show increased MetLuc expression upon nicotine application (Figure

50).

Figure 50: Nicotine dose-

response curve in SHEP1

cells transfected with

STAT3-ML. Cells

transfected with 7, S-rRic3

and STAT3-ML were

exposed to nicotine.

Following 24-hour exposure,

10 and 100 M yielded the

greatest increases in

luciferase expression.

STAT3-ML transfection in

the absence of 7 does not

allow nicotine-dependent

luciferase expression (n=4).

After determining the ideal nicotine concentration for inducing STAT3-driven luciferase

expression, time course experiments were performed to investigate whether exposure periods

longer or shorter than 24 hours may yield improved signals. As shown in Figure 51, 24-hour

nicotine exposure evoked the greatest rise in luciferase expression, again resulting in a ~2-fold

increase compared to untreated and untransfected conditions.

Transfection: - 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 STAT3 STAT3 STAT3 STAT3 STAT3 STAT3

Nicotine: - - 0.1 M 1 M 10 M 100 M 10 M

** **

*

*p < 0.05; ** p < 0.01 compared to untreated

7/Ric3/STAT3 (blue)

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0

0.5

1

1.5

2

2.5

UT UT/nic24 hr

a7 10min

a7 1 hra7 2 hra7 4 hra7 8 hr a7 12hr

a7 24hr

Rela

tive L

um

inescence

Nicotine exposure

Figure 51: Nicotine time course for driving luciferase expression in SHEP1 cells. 24-hour

continuous 10 M nicotine exposure yielded the greatest increase in STAT3-driven luciferase

expression. 48-hour exposure resulted in luminescence approximately half that of 24-h condition

(not shown).

After determining that 24-hour exposure to 10 M nicotine yielded the greatest increases in

luciferase expression, experiments were performed to confirm that increased expression was 7-

and STAT3-attributable.

To evaluate whether increased luciferase expression is attributable to 7 activation and

subsequent STAT3 signaling, various 7 and JAK/STAT blockers were used. The first step in

the signaling assay involves the binding of nicotine to 7 receptors. Presumably, if 7 is

blocked and nicotine binding is hindered, STAT3 activation will drop and result in reduced

luciferase expression. Pretreatment with varying concentrations of BGT 1 hour before nicotine

exposure resulted in a dose-dependent decrease in nicotine-induced luciferase expression (Figure

52). Furthermore, nicotine failed to activate the JAK-STAT pathway in cells transfected with

STAT3-ML alone, suggesting that luciferase expression relies on 7 activation. To confirm the

Transfection: - - 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 STAT3 STAT3 STAT3 STAT3 STAT3 STAT3 STAT3 Duration: - 24 h 10 m 1 h 2 h 4 h 8 h 12 h 24 h nicotine exposure

*

**

** *p < 0.05; ** p < 0.01 compared to untransfected, 24 hour nicotine exposure (blue)

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0

0.5

1

1.5

2

2.5

3

UT/UT a7/UT a7/nicnic/2 nM aBGTnic/20 nM aBGTnic/200 nM aBGTnic/2000 nM aBGTSTAT3-MetLuc

Rela

tive L

um

inescence

Treatment

necessity of STAT3 activation for reporter secretion, cells were pretreated with JAK/STAT

inhibitors.

Figure 52: BGT pretreatment blocks 7-mediated luciferase expression. SHEP1 cells

transfected with 7, Ric3, and STAT3-ML were pretreated with BGT for 1 hour before 24-

hour exposure to 10 M nicotine. BGT dose-dependently reduces nicotine-mediated STAT3-

driven luciferase expression (n=4).

Two JAK inhibitors- AG490 and JAK inhibitor I- were used to test JAK2 dependency for

nicotine-mediated STAT3-driven luciferase signaling. Since JAK2 activation drives

phosphorylation and activation of STAT3, inhibiting JAK2 should lower the signal elicited by

nicotine application. SHEP1 cells transfected with 7, Ric3, and STAT3-ML were treated with

10 M nicotine for 24 hours in the presence and absence of JAK inhibitor I or AG490. Hosur

and Loring (2011) showed that JAK2 inhibition using 30 M AG490 restored LPS-induced

NFB signaling in nicotine-treated h42 SHEP1 cells. As shown in Figure 53, 30 M AG490

interfered with nicotine-driven luciferase expression, as luminescence was lowered to levels

similar to those seen for untransfected and untreated conditions.

Transfection: - 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 STAT3 STAT3 STAT3 STAT3 STAT3 STAT3 STAT3

Nicotine: - - 10 M 10 M 10 M 10 M 10 M 10 M BGT: - - - 2 nM 20 nM 200 nM 2 M -

**

* *

*p < 0.05; ** p < 0.01

compared to 7/Ric3/STAT3 transfected cells

exposed to 10 M nicotine without BGT (blue)

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0

0.5

1

1.5

2

2.5

3R

ela

tive

Lu

min

esc

en

ce

Figure 53: JAK2 inhibition reduces nicotine-dependent luciferase expression. SHEP1 cells

transfected with 7, Ric3, and STAT3-ML were exposed to 10 M nicotine for 24 hours.

Nicotine exposure resulted in a ~2-fold increase in luciferase expression compared to

untransfected and untreated conditions. Cells pretreated with 30 M AG490 1 hour before

nicotine exposure showed reduced nicotine-induced luciferase expression (n=4).

Activated JAK2 induces STAT3 phosphorylation, and phosphorylated STAT3 dimerizes

and translocates to the nucleus to bind promoter elements of DNA to induce gene transcription

(Heinrich et al., 2003). De Jonge et al. (2005) demonstrated that nicotine dose-dependently

induces STAT3 phosphorylation, and STAT3 is an essential component of 7-mediated anti-

inflammation. S3I-201 is a STAT3 inhibitor that blocks STAT3 dimer formation and STAT3-

DNA binding. While examining the role of NFB activity in SHEP1 cells stably expressing

human 42, Hosur and Loring (2011) reported that 150 nM S3I-201 (NSC74859) restores LPS-

induced NFB signaling. We demonstrated that the same concentration of S3I-201 lowers

luciferase expression in nicotine-treated SHEP1 cells transfected with 7, Ric3, and STAT3-ML

(Figure 54). Also, DMSO had no effect on reporter activation.

Transfection: - - 7/Ric3 7/Ric3 7/Ric3 7/Ric3 STAT3-ML STAT3-ML STAT3-ML STAT3-ML Treatment: - Nicotine - Nicotine Nicotine Nicotine AG490 DMSO

** **

** p < 0.01 compared to transfected untreated cells (blue)

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80

Figure 54: STAT3 inhibition reduces nicotine-dependent luciferase expression. SHEP1 cells

transfected with 7, Ric3, and STAT3-ML were exposed to 10 M nicotine for 24 hours.

Nicotine exposure resulted in a ~2-fold increase in luciferase expression compared to

untransfected and untreated conditions. Cells pretreated with 150 nM S3I-201 1 hour before

nicotine application showed reduced luciferase expression (n=4).

In summary, a novel STAT3 reporter plasmid was created by replacing the NFB

promoter in a Clontech NFB-MetLuc vector with a STAT3 promoter obtained from a Panomics

STAT3-firefly luciferase vector. Validation was carried out using HEK-Blue IL6 cells

expressing IL6R and SEAP under the control of a STAT3 promoter. In SHEP1 cells transfected

with 7, Ric3, and STAT3-ML, nicotine increases luciferase expression. This increase is likely

attributable to 7 activation of the JAK2-STAT3 pathway, since SHEP1 cells transfected with

STAT3-ML alone fail to display nicotine-dependent luciferase expression, the 7 antagonist

BGT dose-dependently reduces luciferase expression in nicotine-treated cells, and JAK2 or

STAT3 inhibitor pretreatment reduces nicotine-driven luciferase expression. The level of

nicotine-driven luciferase expression was typically ~2-fold greater than untreated conditions.

Transfection: - 7/Ric3/STAT3 7/Ric3/STAT3 7/Ric3/STAT3 7/Ric3/STAT3 Treatment: - - Nicotine Nicotine/S3I Nicotine/DMSO

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

UT/UT a7/UT Nicotine(10 uM)

S31-201 DMSO

Rela

tive

Lu

min

esce

nce

Treatment

** **

** p < 0.01 compared to transfected untreated cells (blue)

Transfection: - 7/Ric3/STAT3 7/Ric3/STAT3 7/Ric3/STAT3 7/Ric3/STAT3

Treatment: - - Nicotine Nicotine/S3I Nicotine/DMSO

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81

We next investigated whether ligating the STAT3-ML sequence into a vector with antibiotic

resistance and selecting transfected cells before nicotine application would increase the signal.

Aim 1d) Construct cell lines expressing both 7 and STAT3-driven luciferase plasmids and

reassess ideal conditions for nicotine-induced increases in 7-driven luciferase expression.

Since the original STAT3-ML plasmid did not have a gene for antibiotic resistance the

studies shown up to this point have involved transient transfections. To create a STAT3-ML

vector possessing antibiotic resistance, Anagha Sawant and Sheba Goklany excised the RFP

gene in a pREP9 vector and replaced it with STAT3-ML, resulting in STAT3-ML-P9KB

(P9=pREP9; K=kanamycin; B=blasticidin). Using this novel plasmid, we reassessed ideal

conditions for obtaining nicotine-induced STAT3 activation. Previous experiments using the

original STAT3-ML plasmid demonstrated that 10 M nicotine applied for 24 hours yields the

largest increase in luciferase expression. Using SHEP1 cells transfected with 7, Ric3, and

STAT3-ML-P9KB before cell selection in antibiotics, two concentrations of nicotine- 10 and

100 M- were tested. As shown in Figure 55, 24-hour treatment of both concentrations yielded

similar increases in reporter activation. Furthermore, the magnitude of nicotine-induced

luciferase expression was increased after cell selection. While increases using the original

STAT3-ML plasmid were roughly 2-fold, the increases using STAT3-ML-P9KB were typically

10-fold following cell selection. To confirm the presence of 7 receptors on the surface of

SHEP1 cells, 125

I-BGT binding assays were carried out. As shown in Figure 56, transfected

cells possessed significantly more binding sites than untransfected cells.

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0

2

4

6

8

10

12

14

UT UT/nic STAT3-ML STAT3-ML/10 uMnic

STAT3-ML/100uM nic

Rela

tive L

um

inescen

ce

Treatment

0

2

4

6

8

10

12

14

16

UT a7 2 h 8 h 24 h 48 h

Rela

tive L

um

inescence

Treatment Group

Figure 55: Nicotine dose-response in SHEP1 cells previously transfected with 7, Ric3, and

STAT3-ML-P9KB. SHEP1 cells were transfected with 7, Ric3, and STAT3-ML-P9KB and

selected in antibiotics prior to experimentation. A) Cells were treated with nicotine for 24 hours

at indicated concentrations (n=4). B) 125

I-BGT binding assay showing surface 7 in

transfected SHEP1 cells (n=4).

To investigate whether an alternative duration of nicotine exposure may be optimal, time course

experiments were carried out. Previously transfected SHEP1 cells were exposed to 10 M

nicotine for various periods of time. Again, 24-hour exposure appeared to be ideal, as after this

period of time the signal dropped off, and significant increases were not observed until 8 hours

of exposure (Figure 56).

Figure 56: Nicotine time

course in STAT3-ML-P9KB

SHEP1 cells. SHEP1 cells

transfected with 7, Ric3,

and STAT-ML-P9KB were

selected in antibiotics prior

to experimentation. Results

show 24-hour exposure to

10 M is optimal for

nicotine-induced

STAT3 activation (n=4). Transfection: - 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 STAT3 STAT3 STAT3 STAT3 STAT3

Duration: - - 2 h 8 h 24 h 48 h

B

A **

**

*

*

*

**

*p < 0.05; ** p < 0.01 compared to transfected untreated cells (blue)

*p < 0.05; ** p < 0.01 compared to transfected untreated cells (blue)

Transfection: - - 7/Ric3 7/Ric3 7/Ric3 STAT3 STAT3 STAT3

Nicotine: - - - 10 M 100 M

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0

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6

8

10

12

14

16

18

Re

lati

ve

Lu

min

es

ce

nc

e

Treatment

After determining optimal concentration and duration of agonist exposure, the necessity of 7

and STAT3 activation for increased luciferase expression was verified.

SHEP1 cells previously transfected with 7, Ric3, and STAT-ML-P9KB were exposed to

nicotine for 24 hours in the presence and absence of JAK/STAT inhibitors. Experiments

involving dot blots performed alongside reporter assays revealed that JAK inhibitor I lowered

both nicotine-dependent luciferase expression and protein levels of PSTAT3. At 30 M the

STAT3 inhibitor S3I-201 blocks phosphorylated STAT3 from dimerizing and translocating into

the nucleus. Zhang et al. (2011) demonstrated that S3I-201 requires over 100 M to

significantly block STAT3 phosphorylation by IL6. This may account for the presence of

PSTAT3 protein with reduced luciferase expression. The 7 antagonist, BGT, lowered both

luciferase expression and PSTAT3 protein levels. Again nicotine treatment produced a 10-fold

increase in luciferase expression, possibly due to selection of transfected cells using antibiotics

(Figure 57).

Figure 57: JAK inhibitor I

and BGT block nicotine-

induced increase in

luciferase expression while

reducing activation of

STAT3. SHEP1 cells stably

expressing 7, S-rRic3, and

STAT3-ML-P9KB were

treated with nicotine for

24 hours following exposure

to inhibitor. Protein

extraction was performed

following supernatant

collection from the same

cells (n=4).

Transfection: - - 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 STAT3 STAT3 STAT3 STAT3 STAT3 STAT3 Nicotine: - 10 M - 10 M 10 M 10 M 10 M 10 M Inhibitor: - - - - JAK inh. I S3I-201 BGT DMSO (150 nM) (30 M) (20 nM)

STAT3:

PSTAT3:

* * *p < 0.05

compared to transfected untreated cells (blue)

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84

Aim 1e) Use STAT3-MetLuc assay to study Ca2+

-dependency of 7-mediated STAT3

signaling.

Following development and validation of the novel STAT3-ML signaling assay, it was

utilized for investigating several questions regarding 7 receptors. One issue to address was the

dependency of calcium influx for STAT3 signaling. 7 receptors signal via two pathways: 1)

ion flow through activated channels, and 2) JAK-STAT signaling through a less-understood

mechanism. Whether these two pathways can take place independently of one another has not

been addressed using heterologous expression systems. Perhaps calcium flow is required for or

enhances JAK-STAT signaling. On the other hand, perhaps JAK-STAT signaling can take place

independently of ion flow. The signaling assay was used to explore if JAK-STAT3 signaling can

take place in conditions unsuitable for proper calcium ion flow. For this purpose, two calcium

chelators were used: BAPTA-AM and EGTA.

Hosur and Loring (2011) used BAPTA when studying calcium-dependency of 42-

mediated anti-inflammation. Bis(2-aminophenoxyl)ethane tetraacetic acid (BAPTA) is very

selective for Ca2+

over Mg2+

and can be used to control calcium levels (Hyrc et al., 2005). The

membrane-permeant version ester- BAPTA-AM- can be used to lower intracellular Ca2+

availability. Ethylene glycol tetraacetic acid (EGTA) is a chelating agent similar to EDTA, but

with a much higher affinity for calcium than magnesium. Unlike BAPTA-AM, which chelates

intracellular calcium, EGTA chelates calcium outside the cell. Using our 7 signaling assay to

investigate calcium dependency for STAT3 signaling, we found that nicotine-treated SHEP1

cells previously transfected with 7, Ric3, and STAT3-ML-P9KB generated greater luciferase

expression compared to untransfected or untreated conditions. Since Hosur and Loring (2011)

showed 5 M BAPTA exposure for 30 minutes was sufficient to block ionomycin-induced Ca2+

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85

release, we used these conditions. Pretreatment of 5 M BAPTA-AM for 30 minutes prior to

nicotine exposure had no effect on STAT3-driven luciferase expression (Figure 58).

Figure 58: BAPTA-AM fails to lower nicotine-induced luciferase expression in SHEP1 cells

previously transfected with 7, Ric3, and STAT3-ML-P9KB. Cells were exposed to 10 M

nicotine for 24 hours with and without pretreatment of 5 M BAPTA-AM for 30 minutes.

BAPTA-AM is a membrane-permeant ester capable of chelating intracellular calcium. DMSO

vehicle had no effect on luciferase expression (n=4).

To investigate whether calcium flow into the cell may be needed for 7-mediated STAT3

signaling, stably transfected cells were maintained in EGTA-containing media before nicotine

application.

Under the same conditions as the previous study involving BAPTA-AM, EGTA exposure

failed to lower nicotine-induced STAT3 driven luciferase expression. Cells plated in EGTA-

containing media 24 hours prior to 10 M nicotine exposure exhibited similar increases in

luciferase expression compared to cells maintained in regular media. Furthermore, cells

maintained in EGTA-containing media and exposed to BAPTA-AM before nicotine exposure

exhibited levels of luciferase expression similar to nicotine-treated cells maintained in regular

media (Figure 59).

Transfection: - STAT3 STAT3 STAT3 STAT3

7/Ric3 7/Ric3 7/Ric3 7/Ric3

Nicotine (M): - - 10 10 10 Chelator: - BAPTA-AM - BAPTA-AM DMSO

** ** **

**p < 0.01

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86

Figure 59: BAPTA-AM and EGTA fail to lower nicotine-induced luciferase expression in

SHEP1 cells previously transfected with 7, Ric3, and STAT3-ML-P9KB. Cells were

maintained in 2 mM EGTA and exposed to 10 M nicotine for 24 hours with and without

pretreatment of 5 M BAPTA-AM for 30 minutes (n=4).

Experiments with calcium chelators suggest that 7-mediated STAT3 signaling can take place in

the absence of calcium ion flow. To further test this hypothesis, cells were maintained in

modified media low in calcium prior to treatment with calcium chelators and nicotine. As shown

in Figure 60, luciferase expression persisted even under these conditions. In summary, in our

signaling assay SHEP1 cells previously transfected with 7, Ric3, and STAT3-ML-P9KB

exhibited increased luciferase expression in response to 10 M nicotine for 24 hours. This signal

could be blocked by the 7 antagonist, BGT, the STAT3 inhibitor S3I-201, and JAK inhibitors,

AG490 and JAK inhibitor I, suggesting luciferase expression is 7- and STAT3-attributable.

However, exposure to calcium chelators failed to lower nicotine-induced luciferase expression.

7 receptors signal through two modes. One is the canonical pathway requiring ion influx

through opened channels. A second involves STAT3 phosphorylation and activation. Data

Transfection: - - - - STAT3 STAT3 STAT3 STAT3 STAT3

7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3

Nicotine (M): - - - - 10 10 10 10 10 Chelator: - BAPTA-AM EGTA BAPTA-AM - BAPTA-AM EGTA BAPTA-AM EGTA

EGTA DMSO

* * * * *

*p < 0.05

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87

STAT3-ML assay using STAT3-ML-P9KB/

a7/Ric3 SHEP1 cells in a 96-well plate

0

2

4

6

8

10

12

14

UT re

gular

UT re

gular/n

ic

UT H

BSS

UT H

BSS/n

ic

STAT3-M

L-P9K

B regu

lar

STAT3-M

L-P9K

B regu

lar/n

ic

STAT3-M

L-P9K

B HBS

S

STAT3-M

L-P9K

B HBS

S/nic

STAT3-M

L-P9K

B HBS

S/BAPTA

STAT3-M

L-P9K

B HBS

S/BAPTA/n

ic

Treatment Group

Rela

tive L

um

inescen

ce

suggests these pathways may be capable of proceeding independently of one another in

heterologous expression systems.

Figure 60: Nicotine-induced luciferase expression in SHEP1 cells previously transfected

with 7, Ric3, and STAT3-ML-P9KB persists in a low-calcium environment. Cells were

maintained in substituted HBSS low in calcium and exposed to 10 M nicotine for 24 hours with

and without pretreatment of 5 M BAPTA-AM and 2 mM EGTA for 30 minutes (n=4).

Andersson and Tracey (2012) hypothesized that the 7 anti-inflammatory pathway leading to

reduced TNF production may involve PKA activation. However, Hosur and Loring (2011)

demonstrated that PKA inhibition via PKI 14-22 amide, unlike JAK/STAT inhibition, failed to

block the effect of nicotine on LPS-induced TNF production in SHEP1 cells expressing human

42 receptors. Therefore, we used PKI 14-22 amide to investigate whether 7-mediated

STAT3 signaling may be dependent upon cAMP. SHEP1 cells transiently transfected with 7,

Ric3, and STAT3-MetLuc were pretreated with the PKA inhibitor prior to nicotine exposure. As

shown in Figure 61, luciferase expression persisted despite PKA inhibition.

Transfection: - - - - STAT3 STAT3 STAT3 STAT3 STAT3 STAT3

7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3 7/Ric3

Nicotine (M): - 10 - 10 - 10 - 10 - 10 Media: Regular Regular HBSS HBSS Regular Regular HBSS HBSS HBSS HBSS Chelator: - - - - - - - - BAPTA-AM BAPTA-AM EGTA EGTA

*

* *

*p < 0.05

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0

0.5

1

1.5

2

2.5

3

UT UT/nic a7/nic a7/Ric3/nicSTAT3/nic

Re

lative

Lu

min

esce

nce

Treatment Group

0

0.5

1

1.5

2

2.5

3

3.5

UT UT/nic STAT3 STAT3/nicSTAT3/nic/PKI 14-22

Rel

ativ

e Lu

min

esce

nce

Figure 61: Nicotine-induced luciferase expression in SHEP1 cells previously transfected

with 7, Ric3, and STAT3-ML-P9KB persists with PKA inhibition. Cells were exposed to

10 M nicotine for 24 hours with and without 1-hour pretreatment of 30 M PKI 14-22 amide

(n=4).

In summary, we tested whether SHEP1 cells expressing 7 and the STAT3 reporter allow

nicotine-driven signaling in environments with low Ca2+

and Mg2+

. Taken together, evidence

suggests calcium ion flow may not be essential for 7-mediated, STAT3 signaling.

Furthermore, such signaling may take place independently of PKA. These findings support two

separate signaling mechanisms for 7 receptors- one ionotropic and the other metabotropic.

Aim 1f) Investigate the role of Ric3 in allowing 7-driven STAT3 signaling in non-

permissive cell lines.

Aside from allowing investigation of 7-mediated STAT3 signaling, the STAT3-ML

assay provides insight into cell line-dependency of receptor maturation- a process that is a

prerequisite for nicotine-mediated luciferase expression. Binding assays have verified that

STAT3-ML/7/Ric3 SHEP1 cells possess surface 7 receptors. Importantly, co-transfection of

Ric3 was required with 7 to elicit nicotine-induced STAT3 signaling in SHEP1 cells. SHEP1

* *

*p < 0.05

Transfection: - - 7/Ric3/STAT3 7/Ric3/STAT3 7/Ric3/STAT3

Nicotine (M): - 10 - 10 10

PKI 14-22 (M): - - - - 30

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cells co-transfected with only 7 and STAT3-ML failed to elicit increases in luciferase

expression upon 24-hour exposure to 10 M nicotine (Figure 62). Inclusion of Ric3 with 7 and

STAT3-ML resulted in increased luciferase expression when the cells were treated with nicotine.

Figure 62: Ric3 is required for 7-mediated STAT3 signaling in SHEP1 cells. Transiently

transfected SHEP1 cells were exposed to 10 M nicotine for 24 hours. Nicotine-driven

luciferase expression depends upon transfection of both 7 and Ric3 with STAT3-ML,

suggesting the requirement of Ric3 for surface 7 expression in SHEP1 cells (n=4).

Since Ric3 transfection is required for STAT3-driven luciferase expression in SHEP1 cells, it is

also required for surface expression. As shown in Figure 63, co-transfection of 7 and STAT3-

ML in GH4C1 cells is sufficient to allow nicotine-induced luciferase expression, suggesting that

GH4C1 cells either already possess Ric3 allowing 7 surface expression or express another 7

chaperone aside from Ric3.

Transfection: - - 7/STAT3R 7/Ric3/STAT3R STAT3R Treatment: - Nicotine Nicotine Nicotine Nicotine

*

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Figure 63: Ric3 is not required for 7-mediated STAT3 signaling in GH4C1 cells.

Luciferase activity, expressed as percentage of constitutive luciferase expression from positive

controls, is dose-dependently driven by nicotine in GH4C1 cells transfected with only 7 and the

STAT3 reporter. At the highest nicotine concentration, 30 M S3I-201 prevents luciferase

expression (Abishek Chandrashekar).

The STAT3-ML signaling assay allowed investigations into calcium dependency of 7-mediated

STAT3 signaling and supported our hypothesis that this signaling can proceed independently of

calcium. It also provided insight into cell-dependent 7 maturation. While transfection of Ric3

appears to be required for surface 7 expression in SHEP1 cells, it may not be necessary in

GH4C1 cells. In Aim 2 we tested the hypothesis that GH4C1 cells possess Ric3 to allow

receptor expression. This work entailed cloning rat Ric3 isoforms and testing their functionality

via 125

IBGT binding assays, followed up with the design of RNAi constructs targeting different

Ric3 exons to investigate the relationship between levels of Ric3 protein and surface 7

expression in SHEP1 and GH4C1 cells. This provided further insight into whether GH4C1 cells

possess unknown chaperone factors allowing surface 7 expression.

AIM 2

Aim 2a) Clone Ric3 from rat brain and investigate functionality of the protein.

We sought to test the hypothesis that GH4C1 cells express more Ric3 protein than

SHEP1 cells to allow greater levels of surface 7 expression. Since the predicted rat Ric3 gene

+ S3I-201

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91

had not been previously shown to be functional, we cloned the rat Ric3 gene. Cloning Ric3 from

rat brain was carried out with the assistance of Jay Boltax and Brijesh Garg. The two cloned

isoforms of rat Ric3 correspond to accession numbers AM422212 and AM422213. AM422212

(S-rRic3) encodes a 366-amino acid protein that differs from AM422213 (S+rRic3) by three

bases due to an ambiguous splice site between exons 4 and 5, resulting in the loss of a serine

residue at position 173 relative to AM422213. The rat Ric3 open reading frame is illustrated in

Figure 64.

Figure 64: Rat Ric3 open reading frame and translated protein. Underlined amino acids

indicate transmembrane domains 1 and 2 and the coiled-coil domain, which are each labeled

above the corresponding DNA. Underlined DNA indicates the shRNA and siRNA sequences

used in Aim 2. The six exons are labeled above the DNA, and inverted triangles indicate the

splice boundaries between exons. The box indicates the serine residue and corresponding DNA

at the ambiguous splice site between exons 4 and 5. A dagger (†) indicates the location of the

splice site between exons 5 and 6 in human and mouse Ric3 (Jay Boltax and Brijesh Garg). Rat

Ric3 protein shares 94% homology with mouse Ric3 and 87% homology with human Ric3.

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0

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cifi

c b

ind

ing/

we

ll

7/GFP 7/S-rRic3 7/S+rRic3

Although Roncarati et al. posted accession numbers AM422212 and AM422213 in 2007, no

published evidence shows these splice variants are functional in heterologous expression

systems. Using 125

IBGT binding to measure surface 7 expression, Brijesh Garg showed that

both rat Ric3 isoforms promote rat 7 surface expression in HEK293 cells (Figure 65).

Figure 65: S- and S+rRic3 both allow surface 7 expression in HEK cells. 125

I-BGT

binding assay was performed in 24-well plates to show surface toxin binding when transfected as

indicated with rat -pREP4 plus either green fluorescent protein (GFP) in pREP4 or S+ or S-

rRic3 in pREP4 in each well. Subsequent experiments failed to show any consistent differences

in surface expression promoted by S+ and S-rRic3 transfections (Brijesh Garg).

Aim 2b) Establish effectiveness of Ric3 antibodies and demonstrate cell line-dependency of

Ric3 transfection for surface 7 expression.

To investigate cell-dependency of 7 maturation, 7 was transfected with and without rat

Ric3 isoforms into SHEP1 and GH4C1 cells before assessing surface 7 expression via

125IBGT binding. As shown in Figure 66, SHEP1 cells transfected with 7 alone yield levels

of 125

IBGT binding comparable to untransfected cells. However, co-transfection with either of

two rat Ric3 isoforms resulted in significant increases in surface binding sites.

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93

Figure 66: Rat Ric3 isoforms promote surface 7 expression in SHEP1 cells. SHEP1 cells

were transfected with 7 alone, or 7 with either rat Ric3 isoform. 125

I-BGT binding assay

was performed ~72 hours after transfection. Cells transfected with 7 alone exhibit similar 7

expression compared to untransfected cells, while Ric3 co-transfection increased surface

expression significantly (n=4).

While Ric3 appeared to be necessary for surface expression of 7 in SHEP1 cells, in GH4C1

cells 7 expression was detected without Ric3 transfection. As shown in Figure 67, GH4C1

cells transfected with 7 alone produce significantly greater levels of 125

I-BGT binding sites

than untransfected cells, and Ric3 co-transfection in these cells does not enhance 7 expression.

Initial binding assays studying cell line-dependency of Ric3 transfection for 7 expression

supported findings from STAT3-ML experiments. In SHEP1 cells, the requirement of Ric3

transfection for surface 7 expression was in agreement with findings obtained in STAT3-ML

studies in which Ric3 transfection was necessary for nicotine-induced luciferase expression. In

GH4C1 cells, binding assays showing surface receptor expression without Ric3 transfection were

in agreement with STAT3-ML studies showing robust luciferase expression in this cell line

without Ric3 transfection.

*p < 0.05

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94

Figure 67: Ric3 transfection is not required for surface 7 expression in GH4C1 cells.

GH4C1 cells were transfected with 7 alone, or 7 with either Ric3 isoform. 125

I-BGT binding

was performed ~72 hours following transfection. Cells transfected with 7 alone show robust

surface receptor expression (n=4).

Initial binding assays supported our results from STAT3-ML reporter assays. In GH4C1 cells,

co-transfection of 7 with STAT3-ML was sufficient for obtaining nicotine-induced luciferase

expression. In this cell line, transfection with 7 alone was sufficient for detecting surface 125

I-

BGT binding sites. In SHEP1 cells, transfection of Ric3 was required for observing both

nicotine-induced luciferase expression and surface 125

I-BGT binding. If surface receptor

expression is a prerequisite for driving STAT3 signaling, it appears that GH4C1 cells, unlike

SHEP1 cells, do not require Ric3 transfection for generating surface 7 receptors. This result,

along with findings that GH4C1 cells infected with recombinant adenovirus encoding 7

generated significantly greater 125

I-BGT binding sites than SHEP1 cells (Sweileh et al., 2000),

supported our initial hypothesis that GH4C1 cells possess more Ric3 chaperone than SHEP1

cells to allow surface 7 expression. To explore this further, we investigated the relationship

between levels of 7 expression- detected via 125

I-BGT binding- and levels of Ric3 protein-

detected via Ric3 antibodies obtained from Santa Cruz Biotechnology.

*p < 0.05

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95

Tests to evaluate the viability of the two antibodies (W-16 and H-282) revealed that

binding of both to rat Ric3 is conformationally-dependent (Figure 68). Boiling protein extracts

from cells transfected with S-rRic3, S+rRic3, or full-length human Ric3 destroyed labeling of

dot blots by both antibodies. Furthermore, under conditions that a glyceraldehyde phosphate

dehydrogenase (GAPDH) antibody shows prominent bands on Western blots, neither Ric3

antibody shows detectable staining.

Figure 68: Santa Cruz Ric3 antibodies are dependent on protein conformation but can be

used for dot blots. A) Protein lysates were boiled at 95˚C for 5 minutes before blotting. For

each Ric3 isoform, boiling samples before blotting abolished the signal. B) Using protein lysates

from SHEP1 cells, under Western blotting conditions in which bands are observed for GAPDH,

no bands are detected for Ric3. C) Ric3 protein is detected in transfected SHEP1 or GH4C1

cells.

Ladder GAPDH Ladder S-rRic3 S+rRic3 Ladder

UT 7 7 7 UT 7 7 7 Buffer S-rRic3 S+rRic3 S-rRic3 S+rRic3

A

B

C SHEP1 GH4C1

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96

Therefore, Ric3 protein levels were monitored through dot blot hybridization, rather than

Western blotting. After establishing effectiveness of Ric3 antibodies and demonstrating the cell-

dependent requirement of Ric3 transfection for surface 7 expression, we obtained Ric3 RNAi

constructs to study the effects of knocking down Ric3 protein on receptor expression. The first

RNAi construct we obtained was an siRNA sequence designed to target h/rRic3 exon 4.

Aim 2c) Test whether RNAi methods that block surface 7 expression in non-permissive

cells fails to block expression in permissive GH4C1 cells.

An advantage of siRNA is that since its chemical synthesis is relatively straightforward,

constructs can usually be purchased from a number of vendors (Sandy et al., 2005). Moreover,

several providers are capable of altering them in ways that augment their stability and selectivity.

Before purchasing an siRNA sequence, it is important to perform a homology search of the

predicted target sequence against the GenBank or UniGene databases. For our purposes, the

homology search tool Basic Local Alignment Search Tool (BLAST) was used. This step is

necessary for lessening the likelihood of off-target effects, as it is best to use sequences having

numerous mismatches with genes that are not supposed to be targeted. In designing siRNA

against both human and rat Ric3, a comparison of both genes found a single, common sequence

spanning 25-27 nucleotides that was deemed a suitable target for siRNA knockdown based on

traits associated with siRNA functionality: low G/C content and lack of inverted repeats, for

instance (Reynolds et al., 2004; Ui-Tei et al., 2004). Furthermore, this sequence was predicted to

be capable of knocking down human and both rat Ric3 isoforms by Integrated DNA

Technologies (IDT) RNAi design. The sequence of the human/rat Ric3 siRNA is shown below,

in Figure 69.

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97

Figure 69: Rat/human Ric3 siRNA sequence synthesized by Integrated DNA Technologies.

BLAST search yielded a single unique sequence that was common to human and rat Ric3 and

potentially capable of targeting both isoforms. The target sequence is located immediately

upstream from the ambiguous splice site in exon 4.

The efficacy of siRNA hinges, in part, on efficient uptake into the cell. To determine

efficiency of transfection, cells were transfected with EGFP in the presence and absence of

EGFP-siRNA. Fluorescence microscopy revealed a knockdown effect in cells co-transfected

with EGFP and EGFP-siRNA compared to those transfected with EGFP only (not shown). In

non-permissive HEK293 and SHEP1 cells, co-transfection of Ric3 siRNA with splice-deleted rat

Ric3 (S-rRic3) and 7 resulted in a decrease of specific 125

I-BGT binding compared to cells

that were not transfected with Ric3 siRNA. This is indicative of a reduction in the surface

expression of 7 receptors, potentially due to the silencing effect of RNAi on Ric3. The results

of studies using HEK293 and SHEP1 cells are depicted below, in Figures 70 and 71,

respectively. Since S-rRic3 has been shown to allow higher levels of surface 7 expression than

full-length rat Ric3, we chose to work primarily with the splice-deleted isoform.

AGC

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98

BGT binding assay using SHEP1 cells in a 96-well plate

Figure 70: Ric3 siRNA treatment lowers

surface expression of 7 in HEK293 cells. 125

I-BGT binding assay in HEK293 cells

showing the effects of Ric3 siRNA on 7

surface expression following transient

transfection. Surface expression of the

receptor was lowered by Ric3 siRNA in a

dose-dependent manner (n=4).

sdrRic3=S-rRic3

Figure 71: Ric3 siRNA treatment

lowers surface expression of 7 in

SHEP1 cells. 125

I-BGT binding assay

in SHEP1 cells following transient

transfection with 7 and S-rRic3- with

or without 30 nM Ric3 siRNA. Similar

to results seen in HEK293 cells, this

concentration of siRNA reduced specific

binding of radioligand to 7 receptors

(n=4).

A decrease in surface expression of 7 receptors was observed following exposure to Ric3

siRNA in HEK293 and SHEP1 cells. However, in permissive GH4C1 cells that have been

shown to carry endogenous Ric3 mRNA, Ric3 siRNA treatment had no effect on 125

I-BGT

binding. This was indicated by 125

I-BGT binding assays in which cells were transfected with

7 in the presence and absence of Ric3 siRNA. Results from these studies, shown in Figure 72,

do not reveal a significant difference in specific binding in the presence or absence of siRNA.

This may indicate that GH4C1 cells possess additional factors allowing 7 receptors to arrive at

the cellular surface. An alternative possibility is that endogenous Ric3 protein persists after

siRNA treatment.

*p < 0.05; ** p < 0.01

*p < 0.05

Transfection: 7 7/Ric3 /Ric3/siRNA

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99

Figure 72: Ric3 siRNA treatment does

not reduce surface expression of 7 in

GH4C1 cells. 125

I-BGT binding assay

following transient transfection of GH4C1

cells. Unlike what was observed in SHEP1

cells, 30 nM Ric3 siRNA did not decrease

125I-BGT binding to 7 receptors (n=4).

To investigate rat Ric3 protein stability and explore the possibility that endogenous Ric3

protein may persist after siRNA treatment, SHEP1wt cells were transfected with S-rRic3 in an

episomal pREP4 plasmid expressing hygromycin resistance. One day after transfection, cells

were treated with 1000 g/ml hygromycin and maintained in hygromycin-containing growth

media for three days prior to 7 and siRNA co-transfection. Three days after transfecting

surviving cells with plasmid and Ric3 siRNA, binding assays revealed that Ric3 siRNA only

partially blocked 7 expression (Figure 73). Receptor expression in siRNA-treated cells was

approximately 15% lower than both the wild-type control and scrambled siRNA-treated groups.

This suggests that Ric3 is a relatively stable protein whose endogenous levels persist in the cell

and may allow receptor expression despite the silencing effects elicited by RNAi. If Ric3 is a

stable protein not subject to rapid turnover, it may not be an ideal target for knockdown via

siRNA.

Figure 73: Ric3 siRNA partially reduces

7 expression in SHEP1 cells semi-stably

expressing S-rRic3. 125

I-BGT binding

assay in SHEP1 cells semi-stably expressing

rat Ric3 and transiently transfected with

7 in the presence and absence of siRNA.

Semi-stably transfected SHEP1 cells were

transfected with a pREP4 plasmid

expressing S-rRic3 and containing a gene

for hygromycin resistance. Specific binding

is presented as counts per minute (n=4).

*p < 0.05

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100

Given the transient nature of siRNA knockdown and the potentially stable nature of Ric3 protein,

it was desirable to obtain a longer-lasting silencing effect. To accomplish this, cells were

transfected with rat Ric3 shRNA expressed from semi-stably transfected plasmids. However,

before this, it was important to use Ric3 antibodies to determine the effects of siRNA on Ric3

protein levels in a more direct manner, and determine whether 7 surface expression could be

detected in the absence of Ric3 protein.

Radioligand binding assays serve as an indirect means of monitoring Ric3 expression.

BGT is known to specifically bind to 7 receptors with high affinity. Therefore, the problem is

not measuring surface receptor expression, but rather being able to attribute the presence or

absence of such expression with Ric3 protein. Assuming that Ric3 is sufficient to allow 7

surface expression, a lack of specific binding may be correlated with a lack of Ric3 protein. A

more direct approach is to use an antibody specific for the protein. A series of experiments was

performed in which cells derived from a single flask were seeded onto 2 plates. Cells from both

plates were co-transfected with Ric3 and 7 in the presence or absence of siRNA. However,

while cells from one plate were used to perform binding assays, cells from the other were

extracted for dot blot analysis. By comparing levels of Ric3 protein seen in dot blot analysis

with the levels of 7 expression observed in binding assays, an investigation into the necessity of

Ric3 for surface receptor expression was performed.

Initial studies not involving siRNA revealed a contrast between levels of specific BGT

binding and Ric3 protein, especially in GH4C1 cells, in which no protein was detected under

conditions that were permissive to 7 surface expression. As shown in Figure 74, SHEP1 cells

not transfected with Ric3 do not display levels of the protein sufficient for detection. This

correlated to lower specific binding compared to SHEP1 cells that were transfected with 7 and

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101

7

Ric3:

Ric3: (blocking peptide)

Ric3. Both human and rat Ric3 were detected via dot blot analysis in these cells, and this

correlated with a significantly greater level of specific binding (Figure 74). However, this was

not the case for 7-transfected GH4C1 cells, which exhibited similar levels of specific binding in

the absence of Ric3 protein. Before doing similar studies involving RNAi, it was necessary to

evaluate the efficacy of our siRNA sequence.

Figure 74: GH4C1, but not SHEP1, cells generate surface 7 receptors in the absence of

detectable Ric3 protein. A) 125

I-BGT binding assay results. Binding assay and protein

extraction were performed three days following transient transfection (n=4). B) Ric3 dot blot

analysis (top), including blocking peptide in 100-fold excess to primary antibody (bottom) (n=4).

To establish the effectiveness of the siRNA sequence against human and rat Ric3, non-

permissive SHEP1 and permissive GH4C1 cells were transfected with human or S-rRic3 in the

presence and absence of Ric3 siRNA. After protein extraction was performed two days

following transfection, dot blot analysis revealed that cells transfected with siRNA possessed

less human and splice-deleted rat Ric3 protein than those not transfected with siRNA (Figure

75). This suggested that Ric3 siRNA was targeting both human and rat Ric3 and effectively

A

B

Transfection: 7 7 7 7 S-rRic3 hRic3 Cell line: SHEP1 SHEP1 SHEP1 GH4C1

*p < 0.05

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102

S-rRic3

in SHEP1

S-rRic3

in GH4C1

hRic3 in

SHEP1

hRic3

in GH4C1

+ siRNA

- siRNA

knocking down both isoforms following transfection into either GH4C1 or SHEP1 cells. After

establishing siRNA efficacy, the consequences of Ric3 protein knockdown on surface 7

expression were investigated using radioligand binding assays.

Figure 75: Ric3 siRNA (30 nM) reduces levels of human and splice-deleted rat Ric3 protein

upon transfection in SHEP1 and GH4C1 cells. A) SHEP1 cells transfected with S-rRic3 with

siRNA (top) and without siRNA (bottom). B) GH4C1 cells with S-rRic3 and siRNA (top) and

without siRNA (bottom). C) SHEP1 cells transfected with human Ric3 with siRNA (top) and

without siRNA (bottom), and D) GH4C1 cells transfected with human Ric3 with siRNA (top)

and without siRNA (bottom). Blots were performed 2 days following transient transfection.

It is not possible to perform radioligand binding assays and protein extraction on identical wells

of transfected cells. Therefore, two plates of SHEP1 and GH4C1 cells were transfected- one pair

to be used for 125

I-BGT binding assays and the other to be used for dot blot analysis. Results

from these studies, shown in Figure 76, reveal two important findings. First, in SHEP1 cells, a

decrease in Ric3 protein levels corresponds to a reduction in 7 surface expression. Second, in

GH4C1 cells, a decrease in Ric3 protein levels does not significantly affect 7 surface

expression. This discrepancy may be explained by unknown chaperone proteins in GH4C1 cells

allowing 7 surface expression in the absence of Ric3. Another possibility is the existence of

inhibitory factors in SHEP1 cells not present in GH4C1 cells.

A B C D

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103

Figure 76: Ric3 knockdown in SHEP1 and GH4C1 cells has cell-dependent consequences

on surface 7 expression. 125

I-BGT binding assays (n=4) and Ric3 dot blot analysis in

SHEP1 (A) and GH4C1 (B) cells. Binding assays and protein extractions were performed two

days following transient transfection.

Initial RNAi studies may justify a search for novel 7 chaperones in GH4C1 cells. This search

will be carried out by screening a cDNA library obtained from this cell line for expression of

surface receptors. To screen the expression library for 7 expression, it will be necessary to

Ric3:

Ric3:

*p < 0.05

A

B

SHEP1

GH4C1

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104

create conditions under which a non-permissive cell line is semi-stably transfected with RNAi

against Ric3, resulting in long-term knockdown of the protein and preventing re-cloning of rat

Ric3. This can be accomplished using rat Ric3 shRNA constructs. First, it is necessary to

confirm efficacy of the constructs by repeating the siRNA experiments with shRNA. Obtaining

shRNA constructs against other Ric3 exons will also allow investigation into unknown Ric3

splice variants.

Aim 2d) Obtain shRNA constructs to investigate alternative splicing, and study Ric3

protein turnover by determining effects of Ric3 knockdown on 7 expression in cell lines

stably expressing Ric3.

Before selecting candidate rat Ric3 shRNA sequences purchased from Origene, a

homology search was done to establish which ones contain the greatest number of mismatches to

human Ric3. Two candidate shRNA sequences, shown in Figure 77, were found to possess at

least four mismatches. It is desirable to use sequences having at least three mismatches to any

gene other than the one being targeted (Sandy et al., 2005). Also, it is important to avoid

stretches of four or more thymine nucleotides to avoid early termination of transcription by

Polymerase III (Sandy et al., 2005).

shRNA against exon 5: GGCAAGTTCATTGACACATCTCCAGAGAA

shRNA against exon 6: GCAGATGGCTACAGTGAGCAAGAGGAAGC

Figure 77: HuSH 29mer shRNA constructs against rat Ric3 obtained from Origene.

To validate the selectivity and efficacy of shRNA silencing, SHEP1 cells were

transfected with 7 and human or splice-deleted rat Ric3 in the presence or absence of rat Ric3

shRNA constructs against exon 5 or 6. Scrambled shRNA was included as a control. Three days

following transfection, 125

I-BGT binding assays and dot blot analysis were performed. Results

from studies utilizing shRNA targeting exon 5 and 6 are shown in Figure 78. Dot blots involving

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105

8

A

7/S-rRic3

7/hRic3/

shRNA

7/hRic3

Buffer 7

7/S-rRic3/

shRNA

7/S-rRic3/

shRNA

7/S-rRic3

7/hRic3/

shRNA

7/hRic3

Buffer 7

7 7 7 7 7 7 7

hRic3 hRic3 hRic3 S-rRic3 S-rRic3 S-rRic3

shRNA scr.shRNA shRNA scr.shRNA Transfection:

7 7 7 7 7 7 7

hRic3 hRic3 hRic3 S-rRic3 S-rRic3 S-rRic3

shRNA scr.shRNA shRNA scr.shRNA Transfection:

the scrambled shRNA treatment groups were performed in a later experiment, the results of

which are shown in Figure 80.

Figure 78: Rat Ric3 shRNA against exon 5 (top) or exon 6 (bottom) reduces S-rRic3 protein

while decreasing surface expression of 7 in SHEP1 cells co-transfected with 7 and S-

rRic3. SHEP1 cells were transfected with human or splice-deleted rat Ric3 in the presence or

absence of rat Ric3 shRNA. A, C) 125

I-BGT binding assay. Binding assay and protein

extraction were performed three days following transient transfection (n=4). B, D) Dot blot

analysis.

*p < 0.05; **p < 0.01

*p < 0.05; **p < 0.01

A

C

B

D

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106

Buffer

7

7/hRic3

7/hRic3/

shRNA

7/S-rRic3

7/S-rRic3/

shRNA

7/S-rRic3

7/hRic3/

shRNA

7/hRic3

7/S-rRic3/

shRNA

Buffer

7

Transfection:

7 7 7 7 7 7 7

hRic3 hRic3 hRic3 S-rRic3 S-rRic3 S-rRic3

shRNA scr.shRNA shRNA scr.shRNA

Rat Ric3 shRNA against either exon 5 and 6 reduced surface 7 expression in SHEP1 cells

transfected with splice-deleted rat Ric3, but did not have an effect on expression in cells

transfected with human Ric3. Scrambled shRNA did not decrease 7 expression in either

treatment group. This suggests that both rat Ric3 shRNA constructs selectively target rat Ric3

and not human Ric3.

SHEP1 cells were transfected with 7 and human or rat Ric3 in the presence or absence

of a combination of shRNA constructs. Again, scrambled shRNA was included as a control.

Three days following transfection, 125

I-BGT binding assays and dot blot analysis were

performed, the results of which are shown in Figure 79.

Figure 79: Rat Ric3 shRNA against exons 5 and 6 together reduce S-rRic3 protein while

decreasing surface expression of 7 in SHEP1 cells co-transfected with 7 and S-rRic3. SHEP1 cells were transfected with human or splice-deleted rat Ric3 in the presence or absence

of rat Ric3 shRNA against exon 5 or 6. A) 125

I-BGT binding assay. Binding assay and protein

extraction were performed three days following transient transfection (n=4). B) Dot blot

analysis. C) Dot blot analysis with blocking peptide in 100-fold excess to primary antibody.

A *p < 0.05

B

C

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107

A

7 7 7 7 7 7 7 7 7

S-rRic3 S-rRic3 S-rRic3 S-rRic3 hRic3 hRic3 hRic3 hRic3

shRNA ex5 shRNA ex6 scr.shRNA shRNA ex5 shRNA ex6 scr.shRNA

A combination of shRNA sequences targeting exons 5 and 6 reduced surface expression of 7 in

SHEP1 cells transfected with splice-deleted rat Ric3 without affecting expression of 7 in cells

transfected with human Ric3. As shown in Figure 80, scrambled shRNA did not lower receptor

expression or Ric3 protein levels in either treatment group. Results suggest that both rat Ric3

shRNA constructs, or a combination of the two, selectively target rat Ric3 and not human Ric3.

Figure 80: Scrambled shRNA does not reduce surface expression of 7 receptors nor

decrease Ric3 protein levels in SHEP1 cells co-transfected with 7 and S-rRic3 or hRic3.

A) 125

I-BGT binding assay. Binding assay and protein extraction were performed three days

after transient transfection (n=4). B) Dot blot analysis. C) Dot blot analysis including blocking

peptide in 100-fold excess to primary antibody.

7/S-rRic3/

scr. shRNA

7/hRic3/

scr. shRNA

7 7/S-rRic3

7/S-rRic3/

shRNA

exon 5

7/S-rRic3/

shRNA

exon 6

7/hRic3/

shRNA

exon 6

7/hRic3/

shRNA

exon 5

7/hRic3

B

C 7/S-rRic3

7/S-

rRic3/

shRNA

exon 5

7/S-rRic3/

shRNA

exon 6

7/hRic3

7/hRic3/

shRNA

exon 5

7/hRic3/

shRNA

exon 6

7 7/hRic3/

scr. shRNA

7/S-rRic3/

scr. shRNA

Blocking peptide

*p < 0.05

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108

B

EGFP

7/S-rRic3

7/S-rRic3/

shRNA

C EGFP 7/S-rRic3 7/ S-rRic3/

shRNA

Taken together, the series of shRNA transient transfections in SHEP1 cells shows that both

constructs selectively reduce protein levels of rat Ric3 while lowering surface 7 expression.

Furthermore, shRNA against exon 5 may diminish rat Ric3 levels to a greater extent than shRNA

targeting exon 6, while also having less of an impact on human Ric3. Unlike SHEP1 cells,

GH4C1 cells are a permissive cell line that has been shown to possess Ric3 mRNA. GH4C1

cells were transiently transfected with 7 and splice-deleted rat Ric3 in the presence or absence

of Ric3 shRNA targeting exon 5. As shown in Figure 81, shRNA did not have a significant

effect on surface expression of 7, despite reducing levels of Ric3 protein. After establishing the

ability of shRNA to knock down Ric3 protein without affecting 7 expression in GH4C1 cells

transiently co-transfected with Ric3 and 7, the next step was to repeat this study using a cell

line already generating Ric3 protein, as well as one in which Ric3 protein production is

disrupted. Given the potentially stable nature of Ric3 protein, studies performed on cells already

containing the protein could provide further insight into its stability. If surface 7 receptors are

detected in cells lacking Ric3, it may suggest the involvement of additional chaperones allowing

receptor expression.

Figure 81: Rat Ric3 shRNA against exon 5 reduces rat Ric3 protein levels while having no

effect on surface 7 expression. GH4C1 cells were transfected with S-rRic3 in the presence or

absence of rat Ric3 shRNA exon 5. Cells in the EGFP control group were transfected with

EGFP only. A) 125

I-BGT binding assay. Binding assay and protein extraction were performed

three days after transient transfection (n=4). B) Dot blot analysis. C) Dot blot analysis with

blocking peptide in 100-fold excess to primary antibody.

A *p < 0.05

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109

To create cell lines already possessing Ric3 protein, GH4C1 and SHEP1 cells were

transfected with either: 1) 7, 2) splice-deleted rat Ric3, or 3) 7 and splice-deleted rat Ric3

together. Both 7 and S-rRic3 are expressed within a pREP4 vector containing a gene of

resistance against hygromycin. The pREP4 vector is episomal and replicates when the cell

divides but is seldom incorporated into the genome. Transfected cells were exposed to 400

g/ml hygromycin before being maintained in 100 g/ml solution to sustain selection pressure.

These semi-stably transfected cells were plated for radioligand binding assays and dot blot

analysis. As shown in Figure 82, GH4C1 cells semi-stably transfected with 7 alone or with 7

and S-rRic3 together produced surface 7 receptors, while wild-type cells and those semi-stably

transfected with S-rRic3 alone did not. Dot blot analysis revealed the presence of Ric3 protein in

cells semi-stably transfected with either S-rRic3 alone or with 7 and S-rRic3 together. By

demonstrating that it is possible to detect surface 7 receptors in GH4C1 cells in the absence of

Ric3, this study may support the hypothesis that GH4C1 cells possess factors aside from Ric3

allowing surface expression of receptors. As shown in Figure 83, SHEP1 cells semi-stably

transfected with 7 and S-rRic3 together showed significantly greater 125

I-BGT specific

binding than both wild-type cells and cells that were semi-stably transfected with S-rRic3 alone.

Dot blot analysis revealed the presence of Ric3 protein in cells semi-stably transfected with

either S-rRic3 alone, or with 7 and S-rRic3 together. The next objective was to transfect these

semi-stably transfected, hygromycin-resistant SHEP1 and GH4C1 cells with either rat Ric3

shRNA exon 5 or scrambled shRNA.

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110

SHEP1 UT SHEP1 S-rRic3 SHEP1

7/S-rRic3

Ric3:

GH4C1 UT GH4C1 GH4C1 GH4C1

7 S-rRic37/S-rRic3

Ric3:

Figure 82: Semi-stably transfected 7 GH4C1 cells and 7/S-rRic3 GH4C1 cells generate

surface receptors, while only S-rRic3 GH4C1 and 7/S-rRic3 GH4C1 cells possess

detectable levels of Ric3 protein. A) 125

I-BGT binding assay (n=4). B) Dot blot analysis.

Figure 83: Semi-stably transfected 7/S-rRic3 SHEP1 cells generate surface 7 receptors

and possess detectable levels of S-rRic3 protein. A) 125

I-BGT binding assay (n=4). B) Dot

blot analysis.

shRNA exon 5 and scrambled shRNA are expressed in vectors conferring puromycin

resistance. Hygromycin-resistant SHEP1 and GH4C1 cells from the studies involved in Figures

82 and 83, as well as wild-type GH4C1 and SHEP1 cells, were transfected with either of these

constructs and exposed to 10 g/ml puromycin and 400 g/ml hygromycin to select for

successfully transfected cells. These cells were grown in puromycin- and hygromycin-

containing media for eight days before being plated for radioligand binding assays and dot blot

analysis. Selection pressure was maintained by growing cells in media containing 100 g/ml

A

B

A

B

*p < 0.05

*p < 0.05

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111

S-rRic3

S-rRic3/

scr. shRNA

UT

scr. shRNA

7/S-rRic3

7/S-rRic3/

scr. shRNA

shRNA

exon 5

S-rRic3/

shRNA

exon 5

7/S-rRic3/

shRNA

exon 5

hygromycin and 1 g/ml puromycin. Red fluorescent protein expressed by shRNA vectors was

monitored to assess transfection efficiency, and a control group exposed only to hygromycin and

not containing an shRNA construct was included for comparison. As shown in Figure 84,

specific binding of 125

I-BGT to SHEP1 cells semi-stably transfected with 7 and S-rRic3 was

significantly greater than specific binding to cells semi-stably transfected with 7, S-rRic3, and

shRNA. However, results shown in Figure 85 illustrate that GH4C1 cells semi-stably transfected

with 7 and shRNA or with 7, S-rRic3, and shRNA retained the ability to produce surface

receptors.

Figure 84: Semi-stable shRNA transfection decreases both surface 7 expression and Ric3 protein

levels in SHEP1 cells previously semi-stably transfected with 7 and S-rRic3 together. A) 125

I-

BGT binding assay (n=4). B) Dot blot analysis. sdrRic3=S-rRic3

A

B

*p < 0.05

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112

Figure 85: Semi-stable shRNA transfection decreases Ric3 protein levels but not 7 surface

expression in GH4C1 cells previously semi-stably transfected with 7 and S-rRic3 together.

A) 125

I-BGT binding assay (n=4). B) Dot blot analysis.

Overall, RNAi constructs targeting either rat Ric3 exon 4, 5, or 6 yielded similar findings. In

SHEP1 cells, Ric3 knockdown corresponded with a reduction in surface 7 expression.

However, in GH4C1 cells, surface receptors were detected under conditions in which Ric3

7/S-rRic3

S-rRic3/

shRNA

exon 5

7/shRNA

exon 5

7

S-rRic3 UT

shRNA

exon 5

7/S-rRic3/

scr. shRNA

S-rRic3/

scr. shRNA

7/scr.

shRNA

scr. shRNA

B

7/S-rRic3/

shRNA exon 5

A *p < 0.05

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113

protein was knocked down. Although a plausible explanation was the presence of unknown

chaperone factors in GH4C1 cells allowing receptor expression, the possibility remained that a

functional splice variant of Ric3 not detectable by available Ric3 antibodies promotes 7

expression in this cell line. To further investigate this possibility, shRNA was designed against

rat Ric3 exon 2. Exon 2 codes for transmembrane domain that is required in C. elegans for

expression of 7-like receptors (Halevi et al., 2002). The shRNA construct against exon 2,

shown in Figure 86, contains blasticidin and kanamycin resistant genes, as well as GFP for

monitoring transfection efficiency.

Figure 86: shRNA construct against rat Ric3 exon 2.

Notable features include GFP under the control of a

CMV promoter, a blasticidin gene for mammalian

resistance, and a kanamycin gene for bacterial

resistance. This construct was obtained from Origene

and is designed to target rat, and not human, Ric3.

Initial experiments using shRNA against exon 2 were performed in SHEP1 cells. Approximately

72 hours following transfection, 125

I-BGT binding assays were carried out to investigate RNAi

functionality. As show in Figure 87, no 7 expression was detected in untransfected cells or

those transfected with 7 alone. As expected, co-transfection of S-rRic3 with 7 increased

BGT binding. When shRNA exon 2 was transfected with S-rRic3 and 7, levels of surface 7

receptors were significantly reduced compared to conditions involving only 7 and S-rRic3.

Figure 87: shRNA against rat Ric3

exon 2 diminishes surface 7 expression

in SHEP1 cells. 125

I-BGT binding assay

(n=4).

*p < 0.05

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114

Since most experiments have involved the use of S-rRic3 rather than S+rRic3, we repeated this

experiment while including S+rRic3. Findings showed that co-transfection of either S-rRic3 or

S+rRic3 with 7 significantly increased surface 7 expression in SHEP1 cells, and that shRNA

against exon 2 lowered BGT binding when transfected with 7 and either isoform of rat Ric3

(Figure 88).

Figure 88: shRNA against rat Ric3 exon 2 reduces surface 7 expression in SHEP1 cells

transfected with either isoform of rat Ric3. Binding assay was performed three days

following transient transfection. 125

I-BGT binding assay (n=4).

Rat Ric3 shRNA against exon 2 was not designed to target human Ric3. To establish selectivity

of this RNAi construct, SHEP1 cells were co-transfected with 7 and either S-rRic3 or hRic3 in

the presence and absence of shRNA. Approximately 72 hours following transfection, binding

assays and dot blotting were carried out to investigate the relationship between Ric3 knockdown

and receptor expression. As shown in Figure 89, rat Ric3 shRNA against exon 2 simultaneously

lowered S-rRic3 protein and surface 7 levels, while having no effect on hRic3 protein levels or

7 expression in hRic3-transfected cells. Results suggest shRNA against exon 2 selectively

targets rat and not human Ric3.

*p < 0.05

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115

Figure 89: shRNA exon lowers Ric3 protein and 7 expression in SHEP1 cells while having

no effect on hRic3 protein or hRic3-facilitated 7 expression. 125

I-BGT binding assay was

performed three days following transient transfection (n=4).

After performing RNAi studies in SHEP1 cells to establish efficacy and selectively of rat Ric3

shRNA against exon 2, experiments were executed to study the effects of protein knockdown in

GH4C1 cells. First, acute experiments were carried out in which binding assays were performed

72 hours after transfection. As shown in Figure 90, in GH4C1 cells, 7 expression persisted

even in the presence of shRNA.

Figure 90: 7 expression persists

in GH4C1 cells transfected with

shRNA targeting exon 2. 125

I-BGT binding assay was

performed three days following

transient transfection (n=4).

*p < 0.05

*p < 0.05

Ric3:

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116

Ric3:

After establishing the ability of shRNA exon 2 to knock down Ric3 protein without affecting 7

expression in GH4C1 cells transiently co-transfected with Ric3 and 7, the next step was to

repeat this experiment in a cell line already generating Ric3 protein, as well as one in which

protein production is prevented. To create a GH4C1 cell line already expressing Ric3 protein,

cells were transfected with either: 1) S-rRic3, 2) S-rRic3 and 7, 3) S-rRic3, 7, and scrambled

shRNA expressing GFP and blasticidin resistance, or 4) S-rRic3, 7, and shRNA expressing

GFP and blasticidin resistance. Since both 7 and S-rRic3 are expressed in a pREP4 vector

possessing a hygromycin gene of resistance, all cells were exposed to hygromycin (100 g/ml).

Since shRNA constructs contain a blasticidin gene of resistance, cells with shRNA were exposed

to 10 g/ml blasticidin in addition to hygromycin for cell selection. Following 10 days in

antibiotics, virtually all surviving cells transfected with shRNA constructs exhibited robust green

fluorescence, indicating survival of properly transfected cells and destruction of non-transfected

cells. Surviving cells were plated for binding assay and dot blot hybridization. As shown in

Figure 91, Ric3 protein was detected in all conditions except where shRNA against exon 2 was

transfected. Furthermore, aside from untransfected S-rRic3 GH4C1 cells, all conditions showed

7 expression, including shRNA-treated cells lacking detectable Ric3 protein.

Figure 91: In GH4C1 cells, surface 7

expression persists following Ric3

knockdown via shRNA against exon 2.

GH4C1 cells previously transfected with

S-rRic3 were transfected with indicated

components and maintained in hygromycin

before being subjected to binding assay and

dot blot analysis. Detectable Ric3 protein

was observed in each condition aside from

shRNA-treated cells, yet 7 expression

persisted in all conditions with 7

transfection (n=4).

*p < 0.05

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117

0

0.2

0.4

0.6

0.8

1

1.2

1.4

fMo

le b

ou

nd

/we

ll

Transfections:

Ric3

dot blots:

*

*

Since most RNAi studies investigated the ability of siRNA or shRNA to target S-rRic3, the

effects of shRNA on S+rRic3 protein levels was also examined. SHEP1 cells were transiently

transfected with S+rRic3 with or without shRNA targeting exon 2. As shown in Figure 92,

shRNA reduced S+rRic3 protein levels, suggesting that RNAi effects on 7 expression would

probably not be unique to cells transfected with S-rRic3.

Figure 92: S+rRic3 protein levels are lowered by rat

Ric3 targeting exon 2. Protein was extracted from

SHEP1 cells two days following transfection. RIPA

buffer was included as a control.

Finally, shRNA targeting exon 6 was used to study the consequences of knocking down rat Ric3

protein on surface 7 expression. SHEP1 cells were transfected with 7 and Ric3 with and

without shRNA constructs. Co-transfection with shRNA blocked surface 7 expression only in

cells co-transfected with S-rRic3, but not hRic3. Dot blots confirmed that S-rRic3 protein is

knocked down by shRNA. However, scrambled shRNA had no effect on surface 7 expression

in cells transfected with 7 and S-rRic3 (Figure 93).

Figure 93: shRNA against rat

exon 6 blocks rat Ric3-induced

surface 7 expression in SHEP1 cells.

Cells transfected with 7 and hRic3 or

S-rRic3 support surface 7

expression measured by 125

IBGT

but not in the absence of Ric3.

Co-transfection with anti-exon 6

shRNA blocks surface 7 expression

only in cells co-transfected with

S-rRic3 but not hRic3. Dot blots

confirm shRNA knocks down S-rRic3.

Scrambled shRNA has no effect on

surface 7 expression. *p 0.001

between data sets as shown.

Buffer S+rRic3 S+rRic3/shRNA

7 7/hRic3 7/hRic3 7/hRic3 7/S-rRic3 7/S-rRic3 7/S-rRic3 shRNA scr.shRNA shRNA scr.shRNA

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118

Major findings of Aim 2 are summarized in Figure 94. Results demonstrate successful

RNAi knockdown of Ric3 protein by constructs targeting exons 2, 4, 5, and 6. Ric3 protein

knockdown showed cell line-dependent consequences on 7 expression. In SHEP1 cells, Ric3

knockdown following treatment with RNAi targeting either exon 2, 4, 5, or 6 brought about

concomitant decreases in 7 expression. Under similar conditions in GH4C1 cells, surface 7

expression persists. Scrambled constructs had no effect on either protein or surface 7

expression.

Figure 94: Summary of RNAi studies investigating Ric3 dependency for surface 7

expression. In SHEP1 cells, Ric3 protein knockdown following treatment with RNAi targeting

exons 2, 4, 5, and 6 correlated with reductions in 7 surface expression. In GH4C1 cells, Ric3

protein knockdown under similar conditions did not correlate with reductions in surface 7

expression.

shRNA exon 6

Cell line Ric3 protein

7 expression

SHEP1 ↓ ↓

GH4C1 ↓ -

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119

Taken together, results suggest the possibility of chaperone factors aside from currently known

Ric3 splice variants allowing surface 7 expression in GH4C1 cells. Aim 2 has provided data

justifying a search of novel 7 chaperones from a GH4C1 cDNA library. Aim 3 involves proof-

of-concept studies to investigate the feasibility of using a variation of the STAT3-ML assay to

identify 7 chaperones.

AIM 3

In Aim 1, a novel 7-mediated STAT3 signaling assay was developed and utilized to

investigate calcium dependency of STAT3 signaling. It also provided evidence suggesting cell

line-dependence of Ric3 for surface 7 expression in SHEP1 and GH4C1 cells. To further

investigate the necessity of Ric3 for 7 expression, Aim 2 involved designing RNAi constructs

against Ric3 and investigating the relationship between Ric3 protein levels and 7 surface

expression. Results suggested the possibility of additional 7 chaperones aside from Ric3 in

GH4C1 cells. We believe a variation of our STAT3-ML signaling assay- one in which STAT3

activation drives antibiotic resistance rather than luciferase expression- will be helpful in

identifying the unknown chaperones that are suggested by the results of Aim 2. There are

numerous possible techniques for identification of unknown chaperones, such as siRNA library,

differential display, yeast-two-hybridization, and expression cloning. All of these techniques

have advantages and disadvantages. For example, the expensive nature of using an siRNA

library is one of the factors prohibiting us from choosing this method, and yeast-two-

hybridization requires knowledge of the important parts of the molecule. In our case, searching

for unknown 7 chaperones will utilize expression cloning.

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Searching for unknown 7 chaperones in GH4C1 cDNA: Expression cloning using the

SMARTer (Switching Mechanism at 5’ end of RNA Template) In-Fusion Cloning Kit

(Clontech).

Expression cloning allows identification of a gene within a cDNA library based on its

function or phenotype without awareness of its nucleic acid sequence. Therefore, quality cDNA

expression libraries lacking the non-coding and regulatory elements characteristic of genomic

DNA are valuable tools for functional analysis. For our purposes, mature mRNA isolated from

GH4C1 cells will serve as the template for cDNA library construction using the SMARTer In-

Fusion Cloning Kit from Clontech. This PCR-based method has several qualities that are

advantageous for this project. First, the SMARTer In-Fusion Cloning Kit will allow cDNA

library fragments to be inserted into pREP9 vectors, shown in Figure 95, possessing a neomycin-

resistant gene without using restriction enzymes. Avoiding the use of restriction enzymes is

necessary due to the possibility of restriction sites within the unknown gene of interest.

Figure 95: pREP9 vector. This is the

vector into which cDNA fragments from

the cDNA library will be directionally

inserted by the In-Fusion SMARTer cDNA

Library Construction Kit (Clontech).

Another benefit of the SMARTer In-Fusion Cloning Kit is selective amplification.

During PCR amplification reactions, if reverse transcriptase dissociates from RNA before the

whole mRNA sequence is transcribed, the 5’ ends will not be represented in the cDNA library.

This scenario is particularly relevant with longer mRNA sequences (Gubler et al., 1983; Zhu et

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121

al., 2001). The In-Fusion SMARTer method overcomes this by incorporating synthetic adaptors

on the 5’ and 3’ ends of cDNA during first-strand synthesis. Only cDNA sequences possessing

these adaptors at the 5’ end are capable of undergoing amplification in subsequent PCR

reactions, and since the adaptor is only incorporated if reverse transcriptase reaches the 5’ end of

the mRNA sequence, amplification of truncated transcripts is prevented (Chenchik et al., 1998;

Zhu et al., 2001). The result of this selective amplification is a cleaner and more fully

representative cDNA library. The use of the adaptors, called SMARTer V Oligos, is illustrated

below in Figure 96 (In-Fusion SMARTer cDNA Library Construction Kit User Manual. 2010.

Clontech).

Figure 96: SMARTer V Oligo allows selective amplification to yield a pure cDNA product.

An oligo(dT) primer is involved in the first-strand cDNA synthesis reaction, and the SMARTer

V Oligo serves as a short, extended template at the 5’ end of the mRNA. When RT reaches the

5’ end, the enzyme’s terminal transferase adds a few nucleotides to the 3’ end of the cDNA. An

extended template is generated when the SMARTer V Oligo base-pairs with these nucleotides.

RT then switches templates and replicates to the end of the oligonucleotide. The resulting full-

length ss cDNA contains the complete 5’ end of the mRNA, as well as the sequence

complementary to the SMARTer V Oligo, which is a priming site during LD-PCR (In-Fusion

SMARTer cDNA Library Construction Kit User Manual. 2010. Clontech).

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Although other methods are available to avoid the issue of truncated fragment amplification,

many involve extensive manipulation of mRNA before cDNA construction and require a large

quantity of starting material- sometimes on the order of 5-100 g. The In-Fusion SMARTer

method allows cDNA construction beginning with 0.002-1 g of total RNA. A basic procedural

outline for cDNA construction via the SMARTer cDNA Library Kit is provided in Figure 97 (In-

Fusion SMARTer cDNA Library Construction Kit User Manual. 2010. Clontech).

Figure 97: In-Fusion SMARTer cDNA

library construction kit overall procedure.

With less than 1 g total RNA, the LD-PCR

method can be performed. Along with elimination

of contamination by genomic DNA or poly-A

RNA, the low required starting material is a major

advantage of using this cDNA construction kit

(In-Fusion SMARTer cDNA Library

Construction Kit User Manual. 2010. Clontech).

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In summary, the In-Fusion SMARTer cDNA Construction Kit enables the generation of a cDNA

library containing a high yield of full-length clones without the use of restriction enzymes and

beginning with a small quantity of starting material. These qualities make this construction

method an ideal technique to use for screening GH4C1 cDNA for unknown 7 chaperones.

However, before cDNA construction and screening can take place, proof-of-concept studies must

be performed to assess the feasibility of using the 7 signaling assay to identify chaperones.

These studies were performed as Aim 3 experiments.

Aim 3a) Establish a method of plasmid recovery by showing recovery of S-

rRic3/pREP4/hygromycin.

Identification of unknown chaperone(s) will require recovery of cDNA following a

screen for 7 expression. Therefore, it was necessary to demonstrate the feasibility of

recovering transfected plasmid DNA allowing antibiotic survival in mammalian cells. To

accomplish this, GH4C1 cells were transfected with readily available S-rRic3/pREP4 possessing

bacterial ampicillin and mammalian hygromycin resistance. Untransfected cells were included

as a negative control. Transfected cells were maintained in antibiotics for selection, after which

cDNA was extracted using a modified miniprep method that has been shown to yield miniprep

products in similar quantities and less time than traditional time- and resource-extensive methods

(Hirt, 1967; Siebenkotten et al., 1995; Bowers et al., 1999). When miniprep product was

separated on an agarose gel following restriction digestion with KpnI and XhoI, no bands were

observed. E. coli was transformed with 5 l or 10 l miniprep product from either untransfected

or transfected conditions before plating on ampicillin plates. This large volume was used

because it was so dilute compared to normal. During overnight incubation, colonies grew only

on plates that had been spread with bacteria transformed with miniprep product from transfected,

but not wild-type cells (Figure 98).

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Figure 98: During overnight incubation,

colonies grew only on plates on which

bacteria transformed with S-rRic3/pREP4/

KG was spread. A) Kanamycin plate on which

5 l miniprep product from transfected cells was

spread. B) Kanamycin plate on which 10 l

miniprep product from transfected cells was spread.

C) Kanamycin plate on which 5 l miniprep

product from UT cells was spread. D) Kanamycin

plate on which 10 l miniprep product from UT

cells survived.

Two colonies from each plate were picked, and E. coli was once again transformed before DNA

extraction via miniprep. Restriction digestion of miniprep product, once again with KpnI and

XhoI, yielded bands of the expected size (Figure 99). Additional sequencing confirmed recovery

of transfected plasmid DNA from mammalian cells that provided antibiotic resistance.

Figure 99: Agarose gels of Ric3/pREP4 cut with KpnI and XhoI. Restriction digestion using

KpnI and XhoI failed to show bands following first miniprep procedure (left). However,

following further amplification steps, restriction digestion using the same enzymes yielded band

of the expected size (right). sdrRic3=S-rRic3

Screening for novel 7 chaperones will rely on the ability of unknown chaperones to

promote expression of surface 7 receptors that will mediate STAT3-driven antibiotic survival.

Success of this screen requires the ability to recover plasmid DNA from a cDNA library

containing the chaperone of interest. Like the S-rRic3/pREP4 vector previously recovered, the

A B

C D

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125

gene-of-interest will allow survival of transfected cells in antibiotics. Therefore, by

demonstrating the recovery of S-rRic3/pREP4 transfected into GH4C1 cells, an aspect of the

screening process has been validated. S-rRic3/pREP4 was initially used since it was readily

available. However, actual screening will involve SHEP1 cells transfected with pREP9

expressing cDNA fragments obtained from GH4C1 cells. Since construction of the cDNA

library will involve the In-Fusion Cloning Kit (Clontech), the next proof-of-concept study began

with construction of S+rRic3/pREP9 using this kit. This vector has genes for bacterial

kanamycin resistance and mammalian geneticin resistance. By demonstrating the recovery of

this plasmid following transfection into GH4C1 cells, cDNA library construction using this kit

was justified.

Aim 3b) Construct S-rRic3/pREP9/KG using In-Fusion cloning kit (Clontech) and recover

plasmid.

Following transfection of GH4C1 cells with S+rRic3/pREP9, geneticin was applied for

cell selection. Untransfected GH4C1 cells, treated and untreated with geneticin, were included

as controls. cDNA from surviving cells was extracted using the same modified miniprep method

used to recover S-rRic3/pREP4. Restriction digestion of miniprep product using HindIII and

XbaI revealed no bands. Since both 5 l and 10 l of miniprep product of cells transfected with

Ric3/pREP4 yielded similar results, only 5 l of miniprep product was used for transformation of

E.coli. After incubation, bacteria was spread on kanamycin plates. During overnight incubation,

colonies grew only on the plate on which bacteria transformed with S+rRic3/pREP9/KG was

spread (Figure 100).

Figure 100: During overnight

incubation, colonies grew only on

plate on which bacteria transformed

with S+rRic3/pREP9/KG was

spread. A) UT B) S+rRic3-P9KG

A B

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126

Three colonies were selected from this plate and once again transformed into E.coli. Restriction

digestion of extracted DNA using HindIII and XbaI yielded bands of the expected size (Figure

101). Plasmid DNA conferring survival in antibiotics had been successfully recovered.

Figure 101: Agarose gels of Ric3/pREP9/KG cut with HindIII and XbaI. Restriction

digestion using HindIII and XbaI failed to show bands following first miniprep procedure (left).

However, following further amplification steps, restriction digestion using the same enzymes

yielded band of the expected size (right).

By demonstrating the recovery of S+rRic3/pREP9/KG from GH4C1 cells, another aspect of the

screening method- the use of the In-Fusion cloning kit to construct a cDNA library and insert

fragments of this library into pREP9- was demonstrated.

In summary, Aim 3 studies represent a starting point for establishing the feasibility of

screening novel chaperones using the 7 signaling assay developed during Aim 1. During Aim

1 studies, nicotine-induced, 7 mediated, STAT3-driven increases in luciferase expression using

a MetLuc plasmid containing a STAT3 promoter was exhibited. To recover 7 chaperones, a

STAT3 rescue vector in which nicotine induces STAT3-driven antibiotic resistance will be

constructed. Before this can be performed, additional proof-of-concept studies must be carried

out. The first will demonstrate the feasibility of STAT3-driven antibiotic survival using a

STAT3 rescue vector. This will involve construction of a STAT3 rescue vector followed by its

transfection into SHEP1 cells. After exposure to antibiotics, cDNA from surviving cells will be

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127

extracted and the STAT3 rescue vector recovered. The second and final proof-of-concept

experiment will require recovery of hRic3/pREP9 diluted into varying concentrations of pREP9.

The gene-of-interest allowing 7 expression may be a poorly expressed gene, and by recovering

hRic3 diluted into pREP9, a threshold for being able to recover rare genes will be investigated.

V. DISCUSSION

Calcium is one of the most versatile signal transduction components, and its intracellular

concentration impacts a wide variety of cellular processes including cell cycle, differentiation,

and proliferation. Regulation of calcium levels is an intricate process involving balances

between cation release through ryanodine receptors and inositol triphosphate receptors on the

ER, and calcium influx through several voltage-gated ion channels and ligand-gated receptors.

Calcium pumps and ion exchangers also aid in regulating intracellular calcium concentration.

Since Ca2+

controls many cellular signaling cascades, imbalances in its homeostasis may be

present in a variety of disorders. For example, several forms of cancer are associated with

abnormal activity of Ca2+

channels and binding proteins important for maintaining normal

intracellular Ca2+

levels (Schuller, 2009). Calcium levels are also implicated in neurological

disorders, as ion flow into the neuron triggers depolarization essential for neurotransmitter

release. Ca2+

influx through 7 in chick ciliary ganglion or cultured rat hippocampal neurons

initiates Ca2+

-dependent signaling pathways while altering gene expression and synaptic

plasticity (Chang and Berg, 2001). Among the neuronal nicotinic receptors, 7 receptors are the

most highly permeable to Ca2+

. This characteristic contributes to its appeal as a target for drug

development efforts.

By influencing intracellular Ca2+

levels, 7 receptors help to regulate numerous signaling

pathways, synaptic plasticity, and cognitive processes (Quick and Lester, 2002). Although

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128

nicotinic receptors are traditionally recognized as ligand-gated ion channels, nicotine-activated

7 receptors participate in STAT3 signaling that is part of the cholinergic anti-inflammatory

pathway (De Jonge et al., 2005). STAT3 modulates the transcription of genes involved in

regulating cell differentiation, proliferation, apoptosis, angiogenesis, metastasis, and immune

modulation. Elevated levels of activated STAT3 have been associated with a poor outcome in

cancer, as STAT3-activated genes block apoptosis and inhibit antitumor events while promoting

angiogenesis and metastasis (Johnston and Grandis, 2011). Modification of STAT3 activity is a

widely accepted anticancer strategy.

Observations that metabotropic IL6 receptors mediate STAT3 signaling independently of

ion flow and that nicotinic 42 receptors can mediate such signaling without Ca2+

influx (Hosur

and Loring, 2011) fueled our interest in studying the dependency of ion flow for 7-mediated

STAT3 signaling. Using a custom reporter with a Metridia luciferase reporter gene downstream

from a STAT3 promoter, we investigated whether 7 heterologously expressed in mammalian

cells can drive STAT3 signaling in an environment lacking normal Ca2+

, Mg2+

, and Na+ flow.

Validation of the STAT3-ML assay involved transfection into Invivogen’s HEK-Blue IL6 cells

driving secreted alkaline phosphatase (SEAP) upon IL6 application. Since IL6 simultaneously

raised expression of SEAP and luciferase, and IL6-driven reporter expression was blocked by

150 nM JAK inhibitor I, 30 M AG490, or 30 M S3I-201, it was likely that nicotine-driven

luciferase expression was attributable to STAT3 activation. Following assay optimization and

validation, experiments were performed to study whether 7-mediated, nicotine-driven STAT3-

driven signaling can take place in heterologous expression systems modified to possess greatly

reduced ion flow.

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To study the necessity of cation influx for-mediated STAT3 signaling, experiments

were performed with reduced calcium and magnesium ions. To lower Ca2+

levels, cells were

pretreated with the intracellular calcium chelator, BAPTA-AM, and the extracellular calcium

chelator, EGTA, prior to nicotine application. Under these conditions, luciferase expression

indicative of STAT3 activation was similar in amplitude compared to conditions with normal

levels of calcium. Maintenance of cells in substituted HBSS lacking calcium before chelator

application did not abolish STAT3-driven luciferase expression, providing further evidence that

7-mediated STAT3 signaling may not require calcium ion flow. Further work is required for

determining if 7-STAT3 signaling is metabotropic and occurs in the absence of ion flow.

If no ion flow is required for 7-mediated STAT3 signaling, 7 nicotinic receptors may

have two distinct signaling mechanisms: one ionotropic, and the other metabotropic. This would

have several implications for drug discovery. For example, since STAT3 activity is elevated in

many cancers, it is possible that nicotine increases cancer risk in a different way than

contributing to the widely-established addictive nature of cigarette smoking. Results may also

have implications for desensitized 7 receptors in drug development. Previous reports show

nicotinic receptors interact with G proteins, arrestins, and protein kinases (Lee et al., 2008;

Buccafusco et al., 2009; Lefkowitz et al., 2006; Kihara et al., 2001; Buckingham et al., 2009).

These findings, in addition to our results, suggest that in the case of 7, there may be more to

desensitization than solely preventing further ion flow. Perhaps slower metabotropic signaling

trails an rapid initial stage of ionotropic signaling after desensitization.

Findings from this project further highlight the signaling diversity associated with

nicotinic activation of 7 receptors. The anti-inflammatory role of 7 demonstrated by Tracey

et al. (2003) was found to similarly occur in mouse brain via microglial 7 receptors (Shytle et

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al., 2004). Additionally, nicotine-driven, 7-mediated protection of cortical neurons was

reported to occur via the PI3K/Akt pathway (Kihara et al., 2001), and 7 signaling in microglia

may involve PLC activation and increased intracellular calcium levels through ion release from

intracellular storage compartments rather than canonical ion flow through activated receptors

(Suzuki et al., 2006). The wide variety of potential 7 signaling modes may be partially due to

multiple receptor isoforms. At least two isoforms of rat 7 receptors have been reported

(Severance et al., 2004), and additional isoforms may be involved in mediating the lesser-known

signaling pathways elicited by nicotine binding.

One drawback of the STAT3-MetLuc assay is that it does not offer insight into whether

open or desensitized receptors are responsible for mediating downstream signaling. It is likely

that JAK2 binding to 7 depends upon conformational changes in the receptor following

activation, since competitive and noncompetitive antagonists block nicotine-induced decreases in

NFB and cytokine activity (Hosur et al., 2009). However, whether JAK2 associates with the

open or higher-affinity desensitized form of the receptor is undecipherable from the STAT3-

MetLuc assay. Despite this shortcoming, there are many facets of 7 signaling that can be

investigated using this assay. For example, whether other subtypes of nicotinic receptors, or

even other types of Cys-loop receptors, possess immune modulatory capability can be

investigated. Although 7 and 42 share an ability to mediate STAT3 signaling, they share

little homology, particularly in the large intracellular loop between the third and fourth

transmembrane domains of receptor subunits where JAK2 interaction may occur (Hosur and

Loring, 2011). The most highly homologous regions are the intracellular loop bridging the first

and second transmembrane domains of subunits, and the end of the intracellular loop between

the third and fourth transmembrane domains (Hosur and Loring, 2011). With this in mind,

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perhaps other receptors can bind to and activate JAK2 despite differences in amino acid

sequence. Also, the STAT3-MeLuc assay can be used to examine if 7-mediated STAT3

immune suppression takes place in other types of cells. Studies have suggested the 42-driven

anti-inflammatory response is limited to peripheral macrophages (Matsunaga et al., 2001; van

der Zanden et al., 2009), but the localization of these receptors to neuronal and non-neuronal

cells may implicate them in immune modulation effects in the brain (Gahring et al., 2004).

Findings demonstrating 7 involvement with anti-inflammatory pathways in the brain would

have numerous implications in further justifying drug therapies aimed at targeting this receptor.

Although JAK2-STAT3 signaling has typically been associated with decreasing

generation and activity of pro-inflammatory cytokines, in some cases it may actually enhance

production of pro-inflammatory cytokines (Kox et al., 2009). JAK2-STAT3 signaling has also

been shown to either increase or decrease NFB activity. While De Jonge et al. (2005)

demonstrated STAT3 is a component of 7-mediated anti-inflammatory effects in macrophages,

and Chen et al. (2008) showed that nicotine-driven STAT3 activation leading to increased NFB

activation, Marrero and Bencherif (2009) reported that in PC12 cells, 7-dependent STAT3

signaling increases NFB activity. Another unresolved aspect of STAT3 signaling is the role of

phosphorylated versus nonphosphorylated STAT3. Although phosphorylated STAT3 has

commonly been regarded as the active form, some reports suggest the nonphosphorylated protein

modulates the immune response in macrophages (Pena et al., 2010). Further research is needed

to elucidate how STAT3 signaling elicits both pro-inflammatory and anti-inflammatory effects

depending on the context. Findings from the current project demonstrate that 7 can promote

anti-inflammatory events following nicotine application in our particular heterologous

expression system. However, it is possible that 7-mediated pro-inflammatory effects may be

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observed in a different system. The context-dependence of STAT3 signaling warrants further

investigation.

The initial objective of the current project was to study 7-mediated STAT3 signaling.

While investigating calcium dependency of7-mediated STAT3 signaling, we observed cell

line-dependent requirements of Ric3 transfection for nicotine-induced 7-mediated STAT3

expression, indicative of surface 7 expression. Ric3 was discovered during a genetic screen in

C. elegans for mutants that could survive in the acetylcholinesterase inhibitor, aldicarb (Nguyen

et al., 1995). It promotes surface expression of several nicotinic receptor subtypes in C. elegans

and allows surface 7 expression in oocytes and several mammalian cell lines (Halevi et al.,

2002). However, it does not affect surface expression of glutamate or GABA receptors (Lansdell

et al., 2005). The most prominent effect of Ric3 activity appears to be its enhancement of surface

7 expression. Non-permissive HEK293 cells fail to generate surface 7 receptors detected by

125I-BGT binding without Ric3 transfection (Williams et al., 2005). However, co-transfection

of Ric3 with 7 significantly enhances toxin binding. While SHEP1 and HEK293 cells

transfected with 7 alone produce low levels of surface receptors (Sweileh et al., 2000) that can

be increased by Ric3 co-transfection, GH4C1 cells produce surface 7 receptors without Ric3

transfection. It was initially postulated that greater levels of 7 receptors in GH4C1 cells could

be accounted for by greater amounts of Ric3. Our original hypothesis was that knocking down

Ric3 protein in GH4C1 cells lowers surface 7 expression. To test this hypothesis, the

relationship between Ric3 protein levels- determined using Santa Cruz antibodies- and surface

receptor levels was investigated.

Findings that SHEP1 and GH4C1 cells (Williams et al., 2005) express low levels of Ric3

mRNA, and that neither shows significant Ric3 antibody staining without prior transfection

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suggest there may be additional chaperones allowing 7 expression. The Santa Cruz antibodies

against Ric3 recognize proprietary epitopes in rat Ric3 exons 5 and 6 (Santa Cruz Biotechnology

Technical Support) and our findings suggest that binding of these antibodies to Ric3 is

conformation dependent. Although antibodies exhibit staining in lysates from Ric3-transfected

cells that is prevented by a blocking peptide, the existence of multiple human and Drosophila

Ric3 splice variants (Halevi et al., 2003; Seredenina et al., 2008) raises the possibility that our

antibodies may not recognize all functional rat Ric3 splice variants. To investigate the

possibility of unknown rat Ric3 splice variants and explore whether specific exons may facilitate

maturation, we tested the effects of knocking down each targetable rat Ric3 exon. Several

exons targeted for knockdown coded for functionally significant regions of the protein.

The sequence and short length of rat Ric3 exon 1 renders it unsuitable for RNAi

knockdown. However, exon 2- a region encoding a critical transmembrane domain present in all

currently known Ric3 splice variants that influence 7 expression- is a suitable region to target

for knockdown. In SHEP1 cells transfected with 7 and rat Ric3, but not hRic3, shRNA against

rat Ric3 exon 2 prevented surface 7 expression and knocked down Ric3 protein. However,

under similar conditions in GH4C1 cells, shRNA against exon 2 had no effect on surface 7

expression despite Ric3 protein knockdown. Another important region of Ric3 is the coiled-coil

domain in exon 4 of mouse, rat, and human isoforms, as all known functional Ric3 splice

variants possess this domain. Using siRNA against exon 4, Ric3 knockdown correlated with

reductions in 7 expression in SHEP1 cells co-transfected with 7 and Ric3. However, under

similar conditions in GH4C1 cells, 7 expression persisted despite Ric3 knockdown. shRNA

constructs against exons 2, 5, and 6 were also obtained. Each construct lowered 7 expression in

SHEP1 cells co-transfected with 7 and rat Ric3, but failed to affect surface 7 expression in

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GH4C1 cells. RNAi targeting a broad range of the Ric3 transcript yielded similar findings.

Taken together, results suggest that while Ric3 protein is obligatory for robust 7 expression in

SHEP1 cells, GH4C1 cells do not need currently known splice variants of rat Ric3 protein to

yield surface 7 receptors.

The major finding from Ric3 knockdown experiments is the cell line-dependent

requirement for Ric3 facilitation of surface 7 expression. However, these studies also provided

insight into the stability of Ric3 protein, suggesting a half-life of a few days. A ten-day period in

antibiotics was the minimum time to allow sufficient selection against cells not expressing

shRNA. Furthermore, it was unlikely that cell line-dependent consequences of Ric3 protein

knockdown on 7 expression arose from cell line-dependent discrepancies in susceptibility to

RNAi, as both SHEP1 and GH4C1 cells pre-transfected with rat Ric3 and maintained in

antibiotics for 10 days after transfection with shRNA, but not scrambled shRNA, did not possess

detectable Ric3 protein. Although protein was not detectable, it is important to note that the

sensitivity of detection for Ric3 was not established.

While evidence suggests GH4C1 cells have unknown 7 chaperones aside from Ric3,

other scenarios involving protein-protein associations are possible. For example, perhaps Ric3

interactions with other proteins in GH4C1 cells allow low amounts of Ric3 to promote surface

7 expression. In oocytes, Ric3 is one of three chaperones necessary for expression of C.

elegans levamisole-sensitive nicotinic receptors (Boulin et al., 2008). Although Ric3 does not

appear to influence expression of nicotinic receptors by itself, surface expression is increased

when it is combined with Unc-50 and Unc-74 (Eimer et al., 2007; Lewis et al., 1980). Also, it is

possible that proteins may interact with Ric3 to interfere with its ability to promote 7

maturation in GH4C1 cells. In C. elegans, BATH-42 blocks Ric3 promotion of nicotinic

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receptor expression by targeting it for degradation following strong interaction with the C-

terminus of the 7 chaperone (Shteingauz et al., 2009).

While knocking down Ric3 reduces surface 7 expression in non-permissive SHEP1 and

HEK293 cells, overexpressing Ric3 has been shown to be detrimental to receptor expression as

well. Results from Shteingauz et al. (2009) indicate that Ric3 expression leads to accumulation

of protein aggregates that may associate with nicotinic receptor subunits and reduce the amount

of subunits free to be assembled into mature receptors. BATH-42 regulates levels of Ric3 by

targeting excess protein for proteasomal degradation, and loss of BATH-42 actually interferes

with 7 activity (Shteingauz et al., 2009). While the implications of controlling 7 expression

have been major topics underscoring the significance of our project, it is also important to

acknowledge the importance of regulating levels of Ric3. The 7 chaperone is not exclusive in

its overexpression being detrimental to functionality. Overexpression of UNC-45, a myosin

chaperone, results in reduced activity and disturbance with myosin construction (Hoppe et al.,

2004). Although Ric3 is necessary for surface 7 expression in some cases, Ric3 aggregates

stemming from overexpression may pull receptor subunits away from the assembly process.

Therefore, normal receptor signaling may require proper Ric3 regulation.

Experiments investigating cell line-dependency of surface 7 expression have broader

repercussions beyond the mechanistic details of receptor function. Evidence supporting cell line-

dependency of unknown 7 chaperones for successful surface expression has implications

stretching across the entire nervous system. If neurons that do not express surface receptors can

somehow be engineered to generate functional receptors, the ability of altering 7 activity by

targeting chaperone activity may be of interest for a wide range of drug development efforts. If

all neurons already possess an intrinsic, controllable ability to produce surface receptors, the

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regulation of expression in a novel fashion may be of particular interest to those attempting to

manage diseases by altering receptor activity. Results implicating receptor signaling through

both ionotropic and metabotropic modes further strengthens the utility of developing 7-specific

agents.

Ongoing research continues to improve our understanding of the roles of 7 receptors in

the development and progression of a wide range of disorders. As more work is done, it appears

that solely activating or inhibiting 7 to treat diseases may be an overly simplistic approach,

given the multitude of cellular pathways affected by these receptors. Targeting voltage-gated

calcium channel blockers and developing allosteric modulators may be more promising

strategies (Schuller, 2009). For some cancers, determining 7 expression may be useful,

especially if supplemented with knowledge of cAMP levels. Drugs that raise cAMP, like

phosphodiesterases, may help people with elevated 7 expression and low cAMP. An

alternative strategy would be to compensate for high 7 expression by adjusting receptor

expression. Our project supports the potential of modulating receptor levels by targeting

molecular chaperones. Recent work has led to the emergence of molecular chaperones as targets

for drug discovery. One example is heat shock protein 90 (Hsp90).

Hsp90 is a molecular chaperone involved in the folding of many proteins. Several Hsp90

inhibitors have been developed and studied in clinical trials (Petrikaite and Matulis, 2011). Most

candidates are being examined for anti-cancer effects (Ortiz and Salcedo, 2010; Banerji, 2009).

However, there are also ongoing clinical trials investigating their potential for treating

neurodegenerative diseases, such as Alzheimer’s disease (Luo et al., 2010), Parkinson’s disease,

multiple sclerosis, Huntingtons’s disease (Peterson and Blagg, 2009), rheumatoid arthritis (Rice

et al., 2008), and cystic fibrosis (Jouret and Devuyst, 2009). Although Hsp90 inhibitors target a

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specific protein, their action affects numerous signaling pathways. This may be particularly

useful for treating cancer, as Hsp90 inhibitors may circumvent the genetic plasticity that lets

cancer cells evade the cytotoxicity of other compounds. The development of drugs targeting

Hsp90 and other chaperones, including Ric3, requires a thorough understanding of their actions.

Although Ric3 transfection was necessary for robust 7 expression in non-permissive

HEK293 and SHEP1 cells, its requirement for surface 7 expression in GH4C1 cells was not

observed. Since Ric3 knockdown achieved through RNAi constructs targeting several exons

along the transcript failed to reduce 7 expression in GH4C1 cells, 7 expression in this cell line

may rely on an unknown functional Ric3 splice variant or an alternative chaperone present in

GH4C1 cells but absent in SHEP1cells. In mouse dentate gyrus, 125

I-BGT binding in the

absence of Ric3 mRNA expression suggests additional factors may promote 7 maturation

(Halevi et al., 2003). Our findings suggest that mouse models lacking Ric3 may still show

BGT binding in this region. Results also justify future screens for novel 7 chaperones in a

cDNA library obtained from GH4C1 cells using a variation of the 7 signaling assay initially

developed to explore calcium dependency of STAT signaling. In our STAT3-ML assay, nicotine

application to cells expressing surface 7 receptors produced robust increases in STAT3-driven

luciferase expression that were blocked by 7, JAK2, and STAT3 inhibitors. An assay utilizing

a STAT3 rescue vector in which nicotine treatment allows 7-expressing cells to survive in the

presence of antibiotics through STAT3 activation will be useful for identifying unknown 7

chaperones.

VI. FUTURE DIRECTIONS

There are two major routes of future research our project can open. One area of future

work involves using the current STAT3-ML assay to further explore 7 signaling. Another

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possibility involves using a modified version of the STAT3-ML signaling assay to search for

unknown 7 chaperones in GH4C1 cell cDNA. A variation of this assay in which 7-dependent

STAT3 activation drives antibiotic resistance, rather than luciferase expression, may be useful

for screening a cDNA expression library for unknown 7 chaperones. Initial proof-of-concept

studies were carried out and supported the feasibility of constructing a cDNA library with the In-

Fusion Cloning kit (Clontech) and screening it for 7 chaperones. Before pursuing these

endeavors, it may be useful to carry out several experiments that would have implications for

both the current STAT3-ML signaling assay and a modified version for chaperone screening.

Both avenues of future research would benefit from greater signal-to-noise ratio of our

assay. Experiments using the original STAT3-ML plasmid without cell selection yielded ~2-fold

increases in luciferase expression, while studies using STAT3-ML-P9KB with cell selection

showed ~10-fold increases. Perhaps designing a positive feedback loop by which STAT3

activation leads to more STAT3 activation will further increase the signal. The signal-to-noise

ratio may also be improved by using a suppressor of cytokine signaling 3 (SOCS3) inhibitor.

STAT3 activation increases SOCS3 activity, which acts in a negative feedback loop to suppress

further STAT3 activation. Perhaps inhibiting this protein would increase the signal-to-noise

ratio further. Increased STAT3-driven luciferase expression would be a useful improvement of

the current signaling assay, while increased STAT3-driven antibiotic resistance would make

isolation of cells expressing unknown 7 chaperone cDNA more effective.

Most studies performed in this project involved either SHEP1 cells maintained in DMEM

or GH4C1 cells maintained in Ham’s F10. Future studies should be carried out to investigate the

possibility that differences in media are biasing the results. Ham’s F10 contains various

components, such as hypoxanthine, that are not found in DMEM. Maintaining cells in modified

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media prior to experimentation may provide insight into the impact media components may have

on the results. Another factor worthy of investigation is which 7 agonist to use for driving

STAT3 activation. Although nicotine was used to stimulate 7-driven STAT3 signaling

throughout this project, it may be useful to explore the use of alternative agonists, such as

acetylcholine. The cell permeability of nicotine may skew the results somewhat by binding to

unassembled 7 subunits within the cell. A non-permeant agonist may be better suited for

exploring activation of only fully assembled receptors that have arrived at the cellular surface.

After addressing the signal-to-noise ratio, media bias, and type of agonist, further investigations

into 7 signaling can be carried out.

Future studies using the current STAT3-ML assay can address a host of features

surrounding 7-dependent STAT3 signaling, such as the interaction between the receptor and

JAK2. Perhaps conformational changes in the intracellular loops, communicated across the

plasma membrane after agonist interaction with the ligand-binding domain, trigger JAK2

interaction and activation. JAK2 mediates signaling stemming from activation of G-protein

coupled receptors (Ahr et al., 2005) and receptor tyrosine kinases (Pelletier et al., 2006).

Another aspect of 7-driven STAT3 signaling that warrants further investigation is the

involvement of open versus desensitized receptors. A disadvantage of the current STAT3-

MetLuc signaling assay is that it does not decipher whether it is the open, or the high-affinity

desensitized receptor that is responsible for interacting with JAK2. Perhaps co-transfecting cells

with STAT3-ML and mutated 7 less inclined to desensitize would provide insight into whether

desensitized receptors can drive STAT3 signaling. It would also be worthwhile to examine if

other types of receptors in the Cys-loop family of receptors are capable of eliciting pro-

inflammatory or anti-inflammatory responses. Whether 7 has the capacity to influence immune

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processes in other cell lines is another question worthy of investigation. If 7-mediated anti-

inflammatory effects occur in the brain, this may have major implications for drug development.

While the current STAT3-ML assay has further utility, another major route of future work

involves constructing a STAT3 rescue vector in which STAT3 activation drives antibiotic

resistance. This will serve as tool for screening GH4C1 cell cDNA for 7 chaperones.

Following cDNA library construction, screening for 7 chaperones will involve several

transfections. First, non-permissive SHEP1 cells will be transfected with a HuSH 29mer short

hairpin RNA (shRNA) construct against rat Ric3 in a pRFP-C-RS vector. This will prevent the

re-cloning of Ric3 protein. The pRFP-C-RS vector has a CMV promoter region that

constitutively drives the expression of red fluorescent protein (RFP) in mammalian cells (HuSH

shRNA Plasmid Panels Application Guide. 2009. Origene), as well as a puromycin-resistant

gene. Following shRNA transfection, dot blot analysis will confirm the absence of Ric3 and red

fluorescent protein will indicate the extent of transfection. Transfected cells will be grown and

maintained in media containing puromycin to sustain selection pressure. Cells lacking Ric3

protein will then be transfected with three constructs: 1) an /pREP4 vector possessing a

hygromycin-resistant gene, 2) a cDNA library containing pREP9 episomal replicating vector

with a neomycin-resistant gene, and 3) a STAT3-driven selection vector conferring zeocin

resistance. Transfected cells will be grown and maintained in media containing hygromycin,

neomycin, and puromycin to sustain selection pressure. Cells possessing functional 7 receptors

will be selected using the STAT3-driven selection vector, whether it involves EGFP for cell

sorting, or survival via zeocin resistance. In the latter case, cells will be treated with 10 M

nicotine, while being exposed to zeocin at an appropriate time following nicotine exposure, to

stimulate 7 receptors to activate STAT3-driven antibiotic resistance and allow the survival of

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7-producing cells. Under these conditions, cells that survive in the presence of antibiotics each

vector confers resistance to are those that have been successfully transfected and are activating

STAT3 via stimulation of surface receptors in the absence of Ric3 or genes that directly

activate STAT3. A second screen will remove those genes directly activating STAT3 using 125

I-

-bungarotoxin binding assays for 7 expression in the SHEP1 cell subclones. The number of

false positives may be previewed by treating transfected cells with zeocin without a nicotine

pretreatment. Cells surviving zeocin in the absence of nicotine are likely to represent false

positive, but those genes should be eliminated by the binding screen. Therefore, the remaining

cells should contain additional chaperones aside from Ric3 allowing 7 expression. cDNA

plasmids from these cells will be extracted, transformed into bacteria, purified, sequenced, and

BLAST screened.

VII. CONCLUSIONS

7 nicotinic receptors participate in two distinct modes of signaling. One involves

calcium flow into the cell that triggers a host of cellular responses but is transient due to rapid

desensitization (Seguela et al., 1993). The second is a longer-lasting process by which 7

receptors mediate STAT3 signaling (Borovikova et al., 2000; Tracey et al., 2003; De Jonge et al.,

2005). The primary purpose of this project was to develop a signaling assay for investigating the

relationship between these two modes of signaling. To accomplish this, a novel STAT3-ML

reporter plasmid was constructed and co-transfected with 7 into SHEP1 or GH4C1 cells.

Pretreating cells with calcium chelators prior to nicotine exposure allowed investigations into

calcium-dependency of 7-driven STAT3 signaling. To our knowledge, this is the only study

investigating nicotinic STAT3 signaling in a heterologous expression system.

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This project was driven by recent work showing IL6-driven STAT3 signaling can take

place independently of calcium flow, and that activation of 42 nicotinic receptors can reduce

NFB activity by activating STAT3 without calcium (Hosur and Loring, 2011). Furthermore,

microglial 7 activation leads to increased intracellular Ca2+

that takes place independently of

extracellular Ca2+

and that can be blocked by inhibition of Ca2+

release from intracellular stores

(Suzuki et al., 2006). Studies using the STAT3-ML signaling assay provided further evidence of

calcium-independent signaling, as pretreatment with calcium chelators and maintenance in

calcium-low media failed to reduce nicotine-induced STAT3 activation. Aside from allowing

investigations into 7-mediated STAT3 signaling, the STAT3-ML assay provided insight into

cell-dependency of 7 maturation, as the 7 chaperone Ric3 was required for 7-driven STAT3

activation in SHEP1 but not GH4C1 cells. These findings motivated the design of RNAi

constructs to explore the necessity of Ric3 for surface 7 expression.

A series of RNAi constructs were designed to target various exons along the Ric3

transcript. In SHEP1 cells, Ric3 protein knockdown by each RNAi construct tested resulted in

reduced surface 7 expression. However, in GH4C1 cells, Ric3 protein knockdown by each

construct had no impact on receptor expression. Taken together, results suggest the possibility

that GH4C1 cells possess 7 chaperones aside from the known functional Ric3 splice variants.

To investigate the identity of these chaperones, a variation of the 7 signaling assay used to

study 7-STAT3 signaling can be developed. In this assay, 7 activation would drive antibiotic

resistance, rather than luciferase expression, and allow 7 producing cells lacking Ric3 to

survive in the presence of antibiotics. GH4C1 cDNA fragments obtained from surviving cells

can be further analyzed for the unknown chaperones. Proof-of-concept studies were carried out

to assess the feasibility of using this method to screen for 7 chaperones and provided evidence

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supporting the practicality of using the In-Fusion Cloning Kit (Clontech) to generate a cDNA

library from GH4C1 cells and screen it for 7 chaperones using a STAT3 rescue vector.

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