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

Acknowledgements The Chappell Lab

Dr. Patrick ChappellCheri GoodallIan Hilgart, Briana Knight, Robinson Taylor, Alex Koosman,

Kristen TonsfeldWanda Crannell Funding

ER Jackman Grant Honors Experience Award