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Probing Circadian Clock-Steroid Receptor Interactions in GnRH
Neurons
Victoria Grimwood1 , Patrick Chappell2
Department of Bioresource Research, Oregon State University, Corvallis OR1
Department of Veterinary Medicine, Oregon State University, Corvallis OR2
Anterior Pituitary
GnRH
LH
Hypothalamus
Hypothalamic-pituitary-gonadal (HPG) axis regulation
Pulse vs. Surge
Steroid Hormones
+
GnRH NeuronsLocation
Approximately 2000 in hypothalamusScattered cell bodies
Axons release neuropeptide outside of brainPulsatile secretionsPossess many receptors
Pacemaker clockActivated by lightCoordination peripheral clocksSecretes neuropeptides
Composed of heterogeneous mixture of cell typesOscillation at molecular, cellular, and tissue levels
Suprachiasmatic nuclei of the hypothalamus (SCN)
KisspeptinSecreted by Kiss-1 neuronsPowerful stimulator GnRH releaseEssential to pubertal progression and feedback to steroid
hormonesMay be essential to GnRH pulsatile secretions
When pulse comes to surge: Do endogenous oscillators play a role in modulation of
GnRH secretion patterns in response to estradiol?
GnRH
DNSSCN
Kiss1?
• We use our in vitro GnRH cell models (GT1-7 cells) to examine direct dose- and time-dependent effects of E2 as well as circadian genes.
Proestrus
DNS
E2 GnRH
SCN
E 2
Kiss1?
When pulse comes to surge: Do endogenous oscillators play a role in modulation of
GnRH secretion patterns in response to estrogen?
Gpr54/Kiss1-R expression in GT1-7 cells is rhythmic in the presence of 17β-E2
hours
Fold
ch
an
ge f
rom
0h
24 36 48120
EtOH
24 36 48120
100pM E2
13
20
Circadian RhythmsBiological process containing an endogenous
approximately twenty-four hour oscillation patternRegulates many bodily functions
Sleep-wake cycleHormonal secretionCore body temp, hunger, heart rate, ect.
Occur throughout many taxonomic levelsProkaryotes, basic eukaryotes, and multicellular organismsAll organisms have highly conserved clock
Molecular ClockCoordinate circadian feedback
Can be entrained by extracellular signalingInitiate intracellular response
Regulate transcription patterns of specific genesClock, Bmal1, Period, and CryptochromeIn response to cell signaling
E-box Per1/Per2
E-box Cry1/Cry2
E-box CCG
B Cl
Cy
P
B Cl
P
Cy
CyP
B ClOUTPUT
Nuc Cyto
bHLH-PAS txn factorsPeriod
CryptochromeBMAL1
CLOCK
Gpr54-luciferase expression is significantly higher whenco-transfected with CLOCK/BMAL1
pcDNA3.1 CLOCK/BMAL10
10
20
30
40*
rela
tive
lu
cife
rase
GPR54-luc + CLOCK/BMAL10
2
4
6
8
average fold induction compared toGPR54-luc + pcDNA3.1
luci
fera
se f
old
in
du
ctio
n
GPR54 promoter luciferase LUC
B Cl?
Gpr54-luciferase expression is depressed when treated with E2, even when co-transfected with CLOCK/BMAL1
mock E2 mock E2 0
10
20
60
70
80
rela
tive
lu
cife
rase
mock E2 mock E20
10
20
60
70
80
GPR54-luc+ pcDNA3.1GPR54-luc + CLOCK/BMAL1
rela
tive
lu
cife
rase
B Cl?
GPR54 promoter luciferase LUCE2
Estrogen Receptors (ER)Nuclear hormone family of intracellular receptorsMultiple isoforms of estrogen receptors exist
Estrogen receptor alpha (ERα) Estrogen receptor beta (ERβ)
LegendA. Estrogen receptorB. EstrogenC. Estrogen helper
proteinsD. Cell nucleusE. DNA
Estrogen Receptor AlphaBelieved essential to fertilityPrior study demonstrated that E2 in ERα breast cancer
cell lines induces Per2 mRNA levels in mammary epithelial cells
Physical interaction between Per2 and ERα Why we looked at ERβ
Not expressed by GnRH neurons
Estrogen Receptor BetaPresent in GnRH neuronsE2 binding appears to down-regulate Erβ expression
Negative feedback mechanismE2 possibly interacts with endogenous circadian
oscillators at ERβ
Nuc
E-box Per1/Per2
E-box Cry1/Cry2 Cy
PB Cl
B Cl
E-box GPR54B Cl
GPR54
?
?
ERβ?
E2
B Cl
identify protein-protein interactions between ERβ and BMAL1 in multiple in the absence and presence of E2
We believe direct protein-protein interactions exist between clock component BMAL1 and ERβ in GT1-7 and SCN cell lines, modulated by presence of E2
-steroid hormone modulation of the cellular clock
Materials and MethodsMaintanence of Cell Lines
-GT1-7, E2 GT1-7, SCN Cell Lines-Immortalized neuronal cell line
Protein extraction and quantification-Cell lysis and protein extraction-Quantification BCA Assay
Western Blot-Identify protein by size-Anti-estrogen receptor beta primary antibody (rabbit)-Anti-BMAL1 primary antibody (rabbit)-Anti-rabbit secondary antibody
Co-Immunoprecipitation-isolate specific protein and complexes with direct protein-antibody interactions
Co-ImmunoprecipitationPrimary antibody coupled to
columnPre-cleared lysate in control
columnLysate incubated in antibody
coupled columnWash of unbound proteinElution of protein complex
associated with column
Western BlotWestern blot utilized to produced visual results of Co-
ImmunoprecipitionRun products of Co-IP through polyacrylamide gelBind products with target primary and secondary
antibodies
SCN 2.2 Co-IP Blot
Bands perceived in antibody verification, unbound protein, elution, and SCN protein lanes
Bands only appeared just above 50kDa in the elution lanes.
GT1-7 Co-IP Blot
Bands in antibody verification, unbound protein, elution, and GT1-7 lanes Elution bands at ~53kDa in each Unexpected
Fewer bands in BMAL coupled blot
GT1-7 BMAL1 coupled, Erβ probedOnly blot supporting hypothesisEvidence of Erβ protein in elution lanes
Theoretically contain only protein associated with anti-BMAL1 antibody
Band at 53kb appears in each lane except negative control and blank Expected
Estradiol exposed GT1-7 Co-IP Blot
Both blots exhibit lack of association in elution lanesBMAL1 probed possesses more bands in protein and unbound protein lanesUnexpected
Estradiol ModulationEstradiol down-regulates positive clock elements
BMAL1 is positive clock transcription factorNo indication of BMAL1-Erβ protein interactionsStrong band in lysate lanes at 53kDa
Should see 53kDa in Erβ probed elution lane
Original HypothesisDirect protein-protein interactions between BMAL1 and
Erβ in GT1-7 (in presence or absence of estradiol) and SCNcell lines appear unclearPositive Erβ and BMAL1 bands expressed in lysate did not
show in altogher appropriate positions of elutionOnly bands of roughly Erβ size appeared in elution lanesConfirmatory band in BMAL1 coupled column when probed
with ERβ
?
Generation of an effective BMAL1or ER beta antibody in species other than rabbit necessary
Future ResearchChromatin Immunoprecipitation (chIP)
Determines whether a specific genomic region interacts with the protein of interest
Transcription factors on promoters or other DNA binding sites
If promoter known, can determine if ERbeta and BMAL1 binding site close enough for direct contact where on the Kiss1R promoter ERbeta and BMAL1 are binding Domain(s) of each transcription factor is/are required for this
interaction
Kiss-1R PromoterE2 response elements (ERE) and E-boxes within the
GPR54 promoter sequenceCLOCK/BMAL1 suspected binding sites
Infertility“Circadian health” essential
to fertilityProper rhythmic cycling
necessary for ovulationClock disregulation could
cause missed surgesLess ovulation
Decreasing fertility rate since Industrial Revolution“Abnormal” sleep wake
cyclesSwing shift workers
Nurses, flight attendents
• Increase of hormone dependent cancers• Breast and Prostate cancers combine to roughly 30% of total new
cancer cases each year-American Cancer Association