CROSS-TALK BETWEEN GONADOTROPIN-

RELEASING HORMONES AND PROGESTERONE

RECEPTOR IN NEUROENDOCRINE CELLS

by

BEUM-SOO AN

D.V.M., Chung-buk National University, 2000

M.Sc, Chung-buk National University, 2002

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

(Reproductive & Developmental Sciences)

THE UNIVERSITY OF BRITISH COLUMBIA

March 2007

© Beum-soo AN, 2007 ABSTRACT

Hypothalamic gonadotropin-releasing hormone (GnRH) is a decapeptide that plays a pivotal role in mammalian reproduction. It is hypothesized that progesterone (P4) may regulate

GnRH I, GnRH II (a second form of GnRH) and GnRH I receptor (GnRH I R) at the transcriptional level. Alternatively, GnRHs may stimulate transactivation of the progesterone receptor (PR), thereby, modulating gonadotropin subunit gene expression. Treatment of human neuronal cells with P4 suppressed GnRH I R promoter activity. This P4-stimulated inhibition was enhanced when PR A was over-expressed. With respect to the two GnRHs, P4 increased

GnRH I mRNA levels, but did not significantly affect GnRH II gene expression.

Regulation of gonadotropin production involves interplay between steroids and neuro• peptides, thus we have examined the effects of GnRHs on PR activation in pituitary cells.

Treatment with GnRHs increased a progesterone response element (PRE)-luciferase reporter gene activity. PR was phosphorylated at Ser294 and translocated into nucleus after GnRH treatment in the absence of P4. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR: Steroid Receptor Coactivator-3 (SRC-3) interaction. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 to the PRE reporter gene was also increased by GnRHs. The knockdown of GnRH I R and SRC-3 levels by siRNA treatment reduced GnRH-induced PR transactivation. Gonadotropin subunit gene expression was evaluated following treatment with GnRHs, and common a-subunit and FSHjS transcription were upregulated by GnRHs. We used siRNA for PR to examine the involvement of

PR in GnRH I-induced FSH/3 gene expression. The effect of GnRH I on FSH/3, but not a-subunit gene expression was reduced when siRNA targeting PR was introduced.

In summary, these results indicate that P4 is a potent regulator of GnRH I R and GnRH I at

ii the transcriptional level, and this distinct effect of P4 on the GnRH system may be derived from the differential action of PR A or PR B. Conversely, GnRHs can activate PR-mediated transcription in the absence of P4, and this ligand-independent mechanism of PR additionally regulates FSH/3 subunit gene expression.

in TABLE OF CONTENTS

ABSTRACT ii

TABLE OF CONTENTS iv

LIST OF TABLES viii

LIST OF FIGURES viii

LIST OF ABBREVIATIONS xi

1. INTRODUCTION 1

1.1 The hypothalamic-pituitary gonadal axis 1

1.2 GnRH I and its receptor 4

1.3 GnRH I R-induced signaling 7

1.4 GnRH II and its receptor 10

1.5 Regulation of the GnRH system 13

1.6 Classification and structure of nuclear receptor superfamily 14

1.7 Structure and mechanism of action of PR 18

1.8 Phosphorylation of PR 22

1.9 Interactions between PR and coregulators 25

1.10 Transactivation of PR in the absence of P4 27

1.11 Transactivation of PR by GnRH I in the absence of P4 30

1.12 Pituitary gonadotropin hormones 30

1.13 Regulation of gonadotropin subunit genes 31

1.13.1 GnRH regulation of common a-subunit (q-GSU) gene expression.... 31

iv 1.13.2 GnPvH regulation of LH/3-subunit gene expression 32

1.13.3 GnRH regulation of FSH /3-subunit gene expression 32

1.13.4 P4 regulation of j3-subunit gene transcription 33

1.14 Hypothesis 33

1.15 Specific Obj ectives 33

2. MATERIALS AND METHODS 35

2.1. Materials 35

2.2 Cell cultures 35

2.3 Plasmids 35

2.4 PPvE-luciferase reporter gene assays 36

2.5 Transient transfection of GnRH I R promoter and over-expressing vectors for PR

isoforms 37

2.6 In vitro transfection with small interference RNAs 38

2.7 Western blot analysis 39

2.8 Immunoprecipitation 40

2.9 Immunocytochemistry 41

2.10 RNA extraction and reverse transcriptase-PCR 41

2.11 Real-time RT-PCR 42

2.12 Chromatin immunoprecipitation (ChIP) Assay 45

2.13 Statistical analysis 46

3. RESULTS 47

3.1 Regulation of the GnRH system by P4 47

3.1.1 P4 regulates human GnRH I R promoter activity 47

v 3.1.2 PR A but not PR B mediates P4 induced repression of GnRH IR

promoter... ! 50

3.1.3 Over-expression of PR A or PR B has distinct effects on PRE promoter

activity 50

3.1.4 Effects of P4 on human GnRH I and GnRH II inRNA levels 54

3.1.5 Effects of PR A and PR B on human GnRH I and GnRH II rnRNA

levels 58

Ligand-independent activation of PR by GnRHs 61

3.2.1 Transactivation of PR by GnRH I and GnRH II in o/T3-l cells 61

3.2.2 Treatment with GnRHs affects PR phosphorylation 65

3.2.3 Treatment with GnRHs affects PR sub-cellular distribution 71

3.2.4 Interaction between SRC-3 and PR increases in aT3-l cell after treatment

with GnRHs or P4 73

3.2.5 Recruitment of PR and SRC-3 to PREs is promoted by GnRHs 76

3.2.6 GnRH I R and SRC-3 are required for GnRH-mediated PR

activation 78

3 GnRH-induced FSH/3 subunit gene transcription involves the ligand-independent

transactivation of PR 84

3.3.1 Transactivation of PR by GnRH I in oT3-l and L(8T2 cells 84

3.3.2 Transcriptional regulation of gonadotropin subunit genes by

GnRH I and GnRH II in pituitary cells 87

3.3.3 Effects of signaling pathway inhibitors on GnRH I-induced trans•

activation of PR and gene expression of gonadotropin subunits 93

3.3.4 PR mediates GnRH I-induced FSH{3 gene expression 99

vi 4. DISCUSSION 102

4.1 Regulation of the GnRH system by P4 102

4.2 Ligand-independent activation of PR by GnRHs 106

4.3 Ligand-independent transactivation of PR mediates GnRH-induced FSH(3

subunit gene transcription 109

4.4 Clinical implications Ill

5. SUMMARY AND FUTURE STUDIES 115

5.1 Summary 115

5.1.1 Different regulation of GnRH system by PR isoforms 115

5.1.2 Ligand-independent activation of the PR by GnRHs 116

5.1.3 PR mediates GnRH-induced FSH(3 gene transcription via a

ligand-independent transactivation 117

5.2 Future Studies 120

6. REFERENCES 122

7. APPENDICES 138

vii LIST OF TABLES

Table. 1. Primers for Real-time PCR genes

i

viii LIST OF FIGURES

Figure 1. The hypothalamic-pituitary-gonadal axis 3

Figure 2. Two-dimensional representation of the GnRH I R 6

Figure 3. Schematic representation of GnRH I signaling in oT3-l, COS7 and DU145 cells.... 9

Figure 4. Schematic representation of the human GnRH I and GnRH II genes 11

Figure 5. Shared functional domains of the nuclear receptor superfamily and SRC/pl60

family members 17

Figure 6. Structures of PR A and PR B 19

Figure 7. Model of the mechanism of action of PR in the presence of P4 21

Figure 8. Phosphorylation sites in human PR 24

Figure 9. Model of molecular mechanism of action of PR in the absence of P4 29

Figure 10. Dose- and time-dependent effects of P4 on GnRH I R promoter activity 48

Figure 11. Effects of RU486 on P4-induced GnRH I R promoter activity 49

Figure 12. Effects of PR A or PR B over-expression on GnRH I R or PRE

promoter activities.... 53

Figure 13. Time- and dose-dependent effects of P4 on GnRH I mRNA levels 56

Figure 14. RU486 reverses P4-induced GnRH I mRNA levels 57

Figure 15. Effects of P4 on GnRH I and GnRH II mRNA levels after PR over-expression.... 59 ix Figure 16. Effects of GnRH I and II on PR-mediated trans-activation of a PRE-reporter gene in aT3-l cells 62

Figure 17. PKC and PKA inhibitors reverse GnRH-induced PR-mediated transactivation of a PRE-luciferase reporter gene in o;T3-l cells, but RU486 does not 64

Figure 18. Alignment of the amino acid sequences and phosphorylation sites of PR in mouse, rat and human 68

Figure 19. Regulation of PR phosphorylation at Ser294 by GnRHs 69

Figure 20. Cytoplasmic to nuclear translocation of PR in rxT3-l cells following treatments with GnRHs 72

Figure 21. Interaction between SRC-3 and PR increases in

Figure 22. Recruitment of PR and SRC-3 on the PREs is promoted by GnRHs 77

Figure 23. GnRH I R mediates both GnRH I - and GnRH II-induced ligand-independent activation ol PR 79

Figure 24. SRC-3 is essential for the ligand-independent activation of PR by GnRH I and GnRH II, and the synergistic amplification of this effect by P4 80

Figure 25. Effects of GnRH I on PR-mediated transactivation of a PRE-reporter gene in

0.T3-1 and L{3T2 cells 86

Figure 26. The effects of GnRH I on a-GSU, FSH|3 and LHJ3 mRNA levels 88

Figure 27. P4 does not have synergistic effects with GnRH I and GnRH II at the level of gonadotropin subunit gene expression 91

Figure 28. PKC and PKA inhibitors, but not RU486, reduce GnRH-induced PR-mediatec transactivation of a PRE-luciferase reporter gene in

Figure 29. Effects of PKC and PKA inhibitors on GnRH-induced gonadotropin subunit

x gene expression 96

Figure 30. PR mediates GnRH I-induced FSH/3 gene expression 101

Figure 31. Proposed cross-talk between PR and the GnRH system in pituitary cells 119

XI LIST OF ABBREVIATIONS

AF Activation function domain ANOVA Analysis of variance AR Androgen receptor Bp Base pare C Celcius Ca2+ Calcium cAMP Cyclic adenosine monophosphate CBP Creb-binding protein cDNA Complementary deoxyribonucleic acid CDK Cyclin-dependent protein kinase ChIP Chromatin Immunoprecipitation DBD DNA-binding domain DMEM Dulbecco's modified eagle medium DNA Deoxyribonucleic acid DAG Diacylglycerol E2 Estradiol ECL Enhanced chemiluminescence EDTA Ethylene diaminetetraacetic acid EGF Epidermal growth factor ER Estrogen receptor ERK Extracellular signal-regulated kinase Fas L Fas ligand FBS Fetal bovine serum FSH Follicle-stimulating hormone GnRH Gonadotropin-releasing hormone GnRHa Gonadotropin-releasing hormone agonist GnRH II Gonadotropin-releasing hormone-II GnRH IR Gonadotropin-releasing hormone I receptor GnRH II R Gonadotropin-releasing hormone I receptor G-protein GTP-binding protein GPCR G-protein coupled receptors GR Glucocorticoid receptor GTFs General transcription factors

xii GTP Guanosine triphosphate H Hour Hsp Heat shock protein IP Inositol phosphate

IP3 Inositol 1, 4, 5-triphosphate JDP Jun dimerization protein-2 LBD Ligand-binding domain LH Luteinizing hormone M Micro MAPK Mitogen-activated protein kinase MAPKKs (=MEK) MAPK kinases MEK1/2 MAPK/ERK kinase 1/2 Min Minutes MMP Matrix metalloproteinases MR Mineralocorticoid receptor mRNA Messenger ribonucleic acid Mw Molecular weight n (as in nM) Nano NR Nuclear receptors NCOA Nuclear receptor coactivator P4 Progesterone p (as in pM) Pico PAGE Polyacrylamide gel electrophoresis PBS Phosphatase buffered saline PCR Polymerase chain reaction PI Phosphatidylinositol PKA Protein kinase A PKC Protein kinase C PLC Phospholipase C PMSF Phenylmethylsulfonyl fluoride PR Progesterone receptor PRE Progesterone-responsive element RAR AW-trans retinoic acid receptor RSV Rous sarcoma virus RXR 9-cis retinoic acid receptor rpm Revoultions per min sec Seconds

xiii SD Standard deviation SDS Sodium dodecyl sulphate. siRNA Small interference RNA SRC Steroid receptor coactivator TEMED N, N, N', N'-tetramethylethlenediamine TR Thyroid hormone receptor Tris Tris(hydroxy methyl) aminomethane VDR Vitamin D3 receptor

xiv 1. INTRODUCTION

1.1 The hypothalamic-pituitary gonadal axis

Gonadotropin releasing hormone (GnRH I) is secreted by specific neurons located in the

anterior and mediobasal hypothalamus, and stimulates pituitary gonadotroph cell action (Fig. 1).

GnRH I secretion is pulsatile and is correlated to the electrical activity of the GnRH secreting neurons (Knobil 1988). GnRH I is liberated in the median eminence in the perivascular space

and then enters the capillaries of the primary portal system. After entering portal capillaries,

GnRH I reaches the anterior lobe of the pituitary and the gonadotroph cells. In the gonadotroph

cells, GnRH I induces the synthesis of the a subunit, common to gonadotropins and of follicle-

stimulating hormone (FSH) /3 and luteinizing hormone (LH) f3 specific subunits (Chabbert-

Buffeta et al. 2000). GnRH I regulates the biosynthesis and secretion of LH and FSH from the pituitary gonadotropes. LH and FSH function mainly on the ovaries to regulate folliculogenesis, ovulation and steroidogenesis. Steroid hormones including estrogen (E2) and progesterone (P4) mediate the ovarian effects on hypothalamic-pituitary system. Menstrual cyclicity in women is greatly dependent on negative and positive ovarian feedback mechanisms. During the follicular phase, E2 plays a key role, while P4 (in low concentrations) contribute to the control of LH and

FSH secretion. It has been demonstrated that exogenous E2 is able to suppress FSH and LH levels in the follicular phase (Messinis and Templeton 1990). In this experiment, the two gonadotropins were equally sensitive to the negative feedback effect of E2.

During the luteal phase, both E2 and P4 regulate the maintenance of the low FSH and LH

levels. This negative feedback effect of steroids also controls GnRH secretion. The frequency of

1 GnRH pulses decreases, while the amplitude increases during the luteal phase. Although this could be due to the high P4 concentrations, it seems that both E2 and P4 are required to maintain this pattern (Nippoldt et al. 1989).

Steroid hormones conversely have positive feedback mechanisms to regulate hypogonadal-pituitary levels. The positive feedback effect has been known to play an important role in the GnRH self-priming effect (Lasley et al. 1975). The response to the second GnRH pulse is greater than the response to the first pulse and is called the self-priming effect of GnRH on the pituitary. It has been known for years that E2 is the main component of the positive feedback effect of the ovaries (Lasley et al. 1975). P4 in the follicular phase of the menstrual cycle, although in low concentrations, probably sensitizes the pituitary to GnRH and in that way facilitates the positive effect of E2. Even, in the absence of P4, GnRH self-potentiation requires a cross-talk with the progesterone receptor (PR) (Waring and Turgeon 1992). The interaction between steroid hormones and GnRH and the self-priming effect of GnRH are important for the expression of the endogenous gonadotrophin surge at midcycle (Messinis 2006); however, the mechanism of GnRH-induced PR activation is still unknown.

The level of the hormones is various depending on stages of the cycle. The level of

GnRH in hypophysial portal plasma is 2-50 pg/ml during ovine estrus cycle (Clarke and

Pompolo 2005). During the female menstrual cycle, the amount of plasma concentration of P4,

E2, FSH and LH is 2-40 nmol/L, 200-1300 pmol/L, 2-15 U/L and 2-40 U/L (Groome et al. 1996).

2 Hypothalamus^^)

GnRH

V (^Pita

Gonadotropins

Ovary

Steroid Hormones

Figure 1. The hypothalamic-pituitary-gonadal axis. Secretion of GnRH occurs in a pulsatile fashion and control synthesis and release of FSH and LH to regulate the function of ovary.

Steroid hormones have negative or positive feedback on the axis.

3 1. 2 GnRH I and its receptor

It is well documented that GnRH I plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins such as FSH and LH from the anterior pituitary.

The amino acid sequence of GnRH I was reported in the 1970s (Matsuo et al. 1971;

Burgus et al. 1972). The expression levels of GnRH I and GnRH receptors are considered to be important for gonadal steroidogenesis and maintenance of pregnancy, and efforts have focused on their molecular biology. Low doses of synthetic GnRH I delivered in a pulsatile fashion in the portal vessels restore fertility in hypogonadal men and women, and are also effective in the treatment of undescended testes and delayed puberty (Millar et al. 2004). However, high doses of

GnRH I or agonist analogs desensitize the gonadotrope resulting in a decrease in LH and FSH and reduced ovarian and testicular function (Millar et al. 2004). Initially the structure of the mammalian GnRH I receptor (GnRH I R) was determined from an immortalized murine gonadotrope cell line (oT3-l) (Reinhart et al. 1992; Tsutsumi et al. 1992). The GnRH I R is a G protein-coupled receptor (GPCR) (Kraus et al. 2001) and a member of the 7 transmembrane receptor superfamily that transduces an extracellular signal into an intracellular signal. The signal transduction pathway following the binding of GnRH I to its receptor has been extensively studied (Millar et al. 2004). Intracellular signalling of the mammalian GnRH I R is unique because it lacks the common carboxyl-terminal cytoplasmic domain and possesses a relatively short intracellular third loop in comparison to other GPCRs (Reinhart et al. 1992) (Fig. 2). The

GPCRs can activate protein kinase C (PKC) and various downstream signal transduction cascades, including the mitogen-activated protein kinase pathways (MAPK) (Harris et al. 1997).

The activation of the PKC pathway has been well documented in response to GnRH I

4 stimulation and this induces the phosphorylation of MAPK, which may participate in gonadotropin release or synthesis in pituitary cells (Shacham et al. 2001). In the pituitary, GnRH

I R have been identified in the animal and human (Clayton et al. 1979; Bourne et al. 1980;

Clayton and Catt 1980; Clayton et al. 1980; Naor et al. 1980; Wormald et al. 1985; Pal et al.

1992; Weil et al. 1992; Schulz et al. 1993). In extra-pituitary tissues, the gene of GnRH I and its receptors are expressed in gonads (Currie et al. 1981; Iwashita et al. 1986) and the placenta

(Miller et al. 1985; Emons et al. 1992), but not in the liver and spleen (Kakar et al. 1992).

5 GnRH I 328 aa Receptor

coo Intracellular

Figure 2. Two-dimensional representation of the GnRH I R. Note that the absence of a carboxy-terminal cytoplasmic tail in the mammalian GnRH I R is the most unique feature of this

GPCR.

6 1. 3 GnRH I R-induced signaling

After GnRH I binds to cell surface receptors, GnRH receptors become internalized and enter a degradation pathway involving lysosomes and/ or undergo receptor recycling (Hazum and Conn 1988). Binding of GnRH I to the GnRH I R leads to conformational changes in the receptor. GnRH I via GnRH I R transmits extracellular signals into the intracellular milieu via heterotrimeric (a, ft and 7 subunits) GTP-binding proteins (G-proteins) (Birnbaumer 1992).

The GnRH I R can be coupled to the Gotyn protein that activates phospholipase Cft leading to the stimulation of PKC and various downstream signal transduction cascades, including the MAPK pathways (Harris et al. 1997). The activation of PKC is reported as one of the most important signaling pathways in the stimulation of MAPK by GnRH I in pituitary cells

(Andrews and Conn 1986; Zheng et al. 1994). The G-protein involved in the GnRH I signaling pathway in pituitary gland cells is not God but might be Gq/11, which activates phospholipase C

(PLC) to mediate inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) production (Hsieh and Martin 1992; Anderson et al. 1993). IP3 releases calcium from intracellular stores

(Stojilkovic et al. 1994; Tse et al. 1997) and DAG stimulates the PKC pathway in pituitary gonadotropes. Unlike other tissues, activated PKC might be involved in the Ca influx and up- regulate GnRH receptors in the pituitary (Naor 1990). The activation of PKC by an increase in cytoplasmic Ca2+ concentration is important for mediating GnRH I action such as gonadotropin secretion in the pituitary gland. In granulosa cells, PKC pathway has been known as a major component in activating MAPK signaling from GnRH I R (Kang et al. 2001a). Activation of

PKC is an important second messenger mediating GnRH I-induced MAPK stimulation in pituitary cells (Harris et al. 1997) and ovarian cancer cells (Chamson-Reig et al. 2003).

7 GnRH I R signaling activate diverse cytoplasmic proteins to transfer its signal into the nucleus, and MAPK is considered to be one of the important pathways in GnRH I signaling pathway (Naor et al. 2000; Kraus et al. 2001). MAPK cascades are activated via a variety of cell surface molecules including receptor tyrosine kinases and GPCRs. Signals transmitted through

GnRH I R induced-cascades induce the activation of diverse molecules which regulate cell growth, survival and differentiation (Naor et al. 2000).

GnRH I revealed distinct differences in signaling pathways in different cell types (Fig. 3).

The main G-protein that transmits the signals in the pituitary is the Goq/11, and it was shown that GnRH I R is able to couple to both Gaq and Gal 1 in mice gonadotropes (Kraus et al. 2006).

The signaling mechanisms mediating the activation of MAPKs by GnRH I also seem to be significantly different in various cell types. In pituitary-derived oT3-l and LpT2 cells, the GnRH

I R signals are mediated via all four major MAPK cascades including MAPK, INK, p38 MAPK and BMK1/ERK5 (Naor et al. 2000; Kraus et al. 2001) (Reiss et al. 1.997; Levi et al. 1998;

Roberson et al. 1999; Liu et al. 2002b). However, in GnRH I R-expressing COS7 cells, the Geo is the main intermediate in the GnRHR to MAPK signaling pathway, but the same MAPK activation in these cells can be obtained when the receptor is coupled to Goq (Kraus et al. 2006).

8 aT3 COS7 DU145

GnRH-I EGFR EGFR

i MMP^v__^ Gaq

i PI3K Ras PKC Ras ^PT3K) MLK3 Ras T ^Raf

MEK t INK ERK P38 ERK JNK ERK JNK

Gonadotropin Growth arrest Apoptosis synthesis and secretion

Figure 3. Schematic representation of GnRH I signaling in pituitary (aT3-l), extra-pituitary

(COS7) and prostate cancer (DU145) cells (Adapted from Kraus et al., 2006).

9 1. 4 GnRH II and its receptors

Three structural variants of GnRH exist in non-mammalian vertebrates. These GnRH variants have similar amino acid sequences but different functions in the regulation of reproduction (Sherwood et al. 1993; Sealfon et al. 1997). One of these GnRH variants is GnRH

II (also called chicken GnRH II), which is conserved in structure from fish to mammals (Fig. 4).

Recently, several groups have identified GnRH II in mammalian species including human (Millar et al. 2004). In contrast to GnRH I, human GnRH II mRNA is expressed at significantly higher levels outside the brain, particularly in the kidney, bone marrow, prostate and ovary (Cheng and

Leung 2005). The evolutionary conservation of GnRH II and its wide distribution in tissues suggest that this neuropeptide has vital biological functions. Although the normal physiologic function of GnRH II is poorly understood, it has been reported that GnRH II suppresses the proliferation of some reproductive tissue-derived tumors (Choi et al. 2001; Chen et al. 2002a), regulates human chorionic gonadotropin release from the placenta (Siler-Khodr and Grayson

2001), and inhibits ovarian steroidogenesis (Kang et al. 2001b). In addition, GnRH II has been shown to preferentially stimulate FSH release in the pituitary (Millar et al. 2004).

1 0 Human GnRH-l

mRNA: 5.1 kb

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

Human GnRH-ll

mRNA : 2.1 kb Exon 1 Exon 2 Exon 3 Exon 4

• 5'-Untranslated Signal sequence GnRH GAP 3'-Untranslated

region region

pTyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- NH2

100 bp GAP : Gonadotropin releasing hormone associated protein

Figure 4. Schematic representation of the human GnRH I and GnRH II genes. Only exonic regions are drawn to scale (Adapted from White et al., 1998).

1 1 In most vertebrates, several structural variants of GnRH exist, suggesting that additional receptors for GnRH in mammals may exist. Indeed, three distinct types of GnRH receptor were identified in the bullfrog (Wang et al. 2001b) and a novel chicken pituitary GnRH receptor has been cloned (Sun et al. 2001). Neill et al. observed that the GnRH II receptor (GnRH II R) gene is present in human (Neill et al. 2001). In mammals, GnRH II has been found to be more widely expressed than GnRH I (Millar et al. 1999; Kang et al. 2000; Millar et al. 2001), suggesting that

GnRH II R may have other functions.

Regarding the role of GnRH I and II receptors, there are discrepancies among previous reports. It has been suggested that the signal transduction pathways coupled to the GnRH II R may be different from those triggered by activation of the GnRH I R (Millar et al. 2001; Neill et al. 2001). Enomoto et al. showed that GnRH II R is necessary to mediate the effect of GnRH II

(Enomoto et al. 2004) and Grundker et al. reported that the anti-proliferative effect induced by

GnRH II is not mediated through GnRH I R (Grundker et al. 2004). However, a functional human GnRH II R protein has not been verified (Grundker et al. 2002), since the identified human GnRH II R transcript has a frame-shift resulting in a premature stop codon (Morgan et al.

2003). In some mammals including mice, the gene encoding this receptor is inactivated or deleted from the genome (Morgan et al. 2003). Thus, the issue of whether this transcript encodes a functional receptor protein in the tissues and the potential roles of the GnRH II R in mediating the effects of GnRH I and II remains obscure. In addition, a recent study showed that GnRH II R inhibits the expression of GnRH I R, indicating that GnRH I R may be a common receptor that mediates the effects of both GnRH I and GnRH II in ovarian cancer cell lines (Pawson et al.

2005).

1 2 1.5 Regulation of the GnRH system

It has been demonstrated that the expression levels of the GnRH I, GnRH II and GnRH I

R genes are regulated, at least in part, at the transcriptional levels (Duval et al. 1997; Ngan et al.

1999; Khosravi and Leung 2003). The change in GnRH I R mRNA levels in the pituitary

gonadotropes throughout the estrous cycle (Bauer-Dantoin et al. 1993; Funabashi et al. 1994)

and after gonadectomy (Funabashi et al. 1994; Sakurai et al. 1997) suggests a possible role of

gonadal steroids in the regulation of the GnRH I R gene. It is established that gonadal steroids

can influence GnRH I secretion. Estrogen (E2) acts in a classic feedback loop between the

gonads and the brain (Gore and Roberts 1997; Herbison 1998). It has both a positive and

negative effect on the secretion of GnRH I. For the greater part of the ovarian cycle, E2 restrains

the GnRH I and LH secretion by negative feedback action (Chongthammakun and Terasawa

1993) (Fig. 1).

P4, the other dominant ovarian steroid in the mammalian reproductive cycle also serves a number of important regulatory roles. P4 regulated the hypothalamic pituitary functions through

a feedback mechanism in animals (Sagrillo et al. 1996; Schumacher et al. 1999) and humans

(Poindexter et al. 1993; Alexandris et al. 1997) (Fig. 1). An elevation of P4 in the luteal phase

inhibits a pulsatile secretion of GnRH I and LH (Goodman and Karsch 1980; Karsch et al. 1987;

O'Byrne et al. 1991). This prevents the occurrence of GnRH I (Kasa-Vubu et al. 1992) and LH

(Scaramuzzi et al. 1971) surges in response to fluctuations in peripheral E2 levels that

accompany the waves of follicular growth occurring in the ovary (Souza et al. 1997). Even

though several studies demonstrated that P4 regulates GnRH I secretion through a negative

feedback mechanism, this scenario of P4-induced gene regulation for GnRH I and GnRH I R is

still controversial, and the regulation of GnRH II by P4 is virtually unknown. 1 3 Several studies have demonstrated that the expression of the human GnRH I and GnRH II genes are regulated in different ways. The transcription of the GnRH II gene is strongly up- regulated by cAMP, which only induces a modest stimulation of the GnRH I promoter activity in neuronal medulloblastoma cells. This cAMP-stimulated GnRH II expression is mediated via a putative cAMP-response element located between nucleotide (nt) -860 and-853 (relative to the translation start codon), which is also critical for the basal transcription of the gene (Chen et al.

2001a). In addition, the mRNA levels of GnRH II in human granulosa-luteal cells were up- regulated by gonadotropins (Kang et al. 2001b). The expression of these neuropeptides has also been shown to be regulated differentially by E2 in such a way that it increases the transcription and mRNA levels of the GnRH II but decreases GnRH I gene expression (Chen et al. 2002b).

Together with their distinct tissue expression patterns, these observations indicate that the two forms of GnRH play distinct biological roles in humans.

1. 6 Classification and structure of nuclear receptor superfamily

In these studies, the projects focused on PR and GnRH system. The PR is a member of the type I nuclear receptor families. In general, type I and II nuclear receptors are ligand- dependent transcription factors that control many biological functions through regulation of specific genes involved in metabolism, development, and reproduction. The primary function of these receptors is to mediate the response of hormones in target cells. Many nuclear receptors have been shown to exist, and these receptors comprise the largest family of transcription factors, the nuclear receptor superfamily.

1 4 Phylogenetic analysis has identified several subfamilies in this superfamily: type I receptors include PR, estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor

(GR), and mineralocorticoid receptor (MR), whereas type II receptors include thyroid hormone receptor (TR), all-trans retinoic acid receptor (RAR), 9-cis retinoic acid receptor (RXR), and vitamin D3 receptor (VDR). A third subclass contains orphan receptors. Although they have common structural features, divergence of subclasses is supported by differences in their functional characteristics, as well as by their different recognition of czs-acting hormone response elements. Type I receptors, in the absence of ligand, are typically sequestered in inactive complexes with heat shock proteins. However, type II receptors are able to bind DNA in the absence of ligand and some times have a repressive effect on target promoters (Tsai and

O'Malley 1994). Type I receptors usually bind to specific palindromic repeats generally in a homodimeric arrangement in the presence of ligand, whereas type II receptors generally bind to response elements that contain direct repeats. In addition, type II receptors exhibit promiscuous dimerization patterns.

A number of functional domains in the nuclear receptor superfamily have been identified. Broadly, the receptor structure is comprised of: an amino-terminal activation function

(AF), AF-1 (A/B domain); the DNA-binding domain (DBD) (C); a hinge region (D) (Fig. 5); and a carboxy-terminal ligand-binding domain (LBD) (E). Mutational analysis of the E domain led to the designation of a second activation function, AF-2, which is for ligand-dependent activation of nuclear receptors (Ham and Parker 1989). Other functions have been ascribed to the

E domain, including ligand binding (Dobson et al. 1989), heat shock protein interactions

(Housley et al. 1990), and nuclear localization (Picard and Yamamoto 1987).

These functional domains reflect an intricate, but well characterized, ligand-dependent receptor activation pathway. This multistep-process involves activation of receptor by binding to

1 5 the cognate hormone, a change in receptor structure and dissociation of heat shock proteins, nuclear translocation of the activated receptor (in the case of ER, GR, MR, AR, and PR), and dimerization and apposition of the nuclear translocated receptor to its DNA response elements.

While the role of general transcription factors (GTFs) in mediating basal transcription is well documented (see Section LB. below), it has recently been documented that nuclear receptors recruit coregulators that create a transcriptionally permissive state depending upon the activation of the receptor, or nonpermissive state at the promoter, and/or communicate with the GTFs

(McKenna et al. 1999).

1 6 NR superfamily N

g| A/B domain H Ligand-bmding (E) domain | DNAbmding (C) domain | Autonomous activation functions [ ] Hinge (D) domain

SRC/p160 family N~

BHLH • CBP interaction domain PAS A/B B CARM1 interaction domain LXXLL/NR Doxes H Acetyltransferase activity

Figure 5. Schematic representation of the functional domains of the nuclear receptor superfamily and SRC/pl60 family members. (Top) General structure of NRs; AF-1 is embedded in the N terminus of type I NRs and AF-2 is found in the C terminus of all NRs. (Bottom) General structure of the SRC/pl60 family. The CBP interaction domain and the CARM1 interaction

domain overlap with the transferable activation domains 1 and 2 of the SRC/pl60 family.

(Adapted from (McKenna and O'Malley 2002).

1 7 1. 7 Structure and mechanism of action of PR

In this section of the introduction, the structure and mechanism of action of PR are described in detail. P4 plays a pivotal role in female reproduction, it is involved in the control of ovulation, prepares the endometrium for implantation, regulates the implantation processes, and is responsible for the maintenance of pregnancy at later stages (Csapo 1956). P4 mediates its physiological effects through interaction with the PR that expressed as two isoforms, PR-A and

PR-B, in multiple tissues. These isoforms are derived from the same gene by the use of two different promoters (Richer et al. 2002). The full-length PR B and N-terminus truncated PR A have highly conserved DNA and ligand LBDs.

Both isoforms have a similar architecture composed of the ligand-dependent AF-2 present in the carboxyl terminus, and AF-1, a transcription domain present in the amino terminus

(Leonhardt and Edwards 2002) (Fig. 6). The constitutive AF-1 can function independently of

AF-2 or with AF-2 in a ligand-dependent manner. The AF-3 domain, is only located in the upstream sequence region of PR B isoform (Pratt and Toft 1997; Leonhardt and Edwards 2002) and composed of approximately 164 amino acids.

1 8 AF3 AF1 AF2

I 165 1 556 642 1 933 PR-B DBD B LBD

PR-A DBD B LBD

Figure 6. Structures of PR A and PR B. Domain organization of the human PR A and B isoforms, H, hinge region; LDB, ligand binding domain; DBD, DNA binding domain the numbers denote the positions of amino acids for isoform proteins and each domains. AF-1, -2 and -3 are transcription activation domains.

1 9 The DNA binding domain contains two asymmetric zinc fingers and two u-helices perpendicular to one another that facilitate interaction with the hormone response element present in PR-target genes. The domains present in the PR undergo conformational modifications to accommodate the ligand. The P4-induced changes in PR help to orchestrate the responses on PR-responsive genes (Lonard and O'Malley 2005; Wardell and Edwards 2005).

In the absence of P4, the transcriptionally inactive PR remains associated with a large complex of heat shock proteins in the nuclei or cytoplasm of target cells (Fig. 7). Upon hormone binding, the receptor dissociated from the heat shock protein complex, dimerizes, and binds to

P4-responsive elements (PREs) within the regulatory regions of target genes (Wardell and

Edwards 2005).

2 0 Figure 7. Model of PR action in the presence of P4. In the absence of P4, PRs are associated with a preformed heat shock protein (hsp) complex. P4 diffuses into the cell, binds to the PR

resulting in dissociation of associated proteins, dimerization of the PR and binding to PRE. P4

and other signaling pathways phosphorylate PR and enhance PR transactivation. Other proteins

including coactivators and GTFs bind to the DNA and to the receptor producing a

transcriptionally active complex.

2 1 1.8 Phosphorylation of PR

Phosphorylation-dephosphorylation events provide an additional level of complexity to

PR action. Like other nuclear receptor family members, PR isoforms are phosphorylated by multiple protein kinases on primarily serine residues (Takimoto et al. 1996). PRs contain 14 known phosphorylation sites (Zhang et al. 1994; Zhang et al. 1997; Knotts et al. 2001) (Fig. 8).

Serines at positions 81, 162, 190, and 400 are defined as "basal" sites (Zhang et al. 1997), constitutively phosphorylated in the absence of P4 (Fig. 8), while serines 102, 294, and 345 sites are hormone-dependent (Zhang et al. 1995). Several specific kinases responsible for phosphorylation have been identified. The serines at 81 and 294 have been demonstrated to be phosphorylated by casein kinase II (Zhang et al. 1994) and MAPK (Lange et al. 2000; Shen et al.

2001), respectively. Eight of the total 14 sites (Ser 25, 162, 190, 213, and 400; Thr 430, 554, and

676) have been demonstrated to be phosphorylated by cyclin A/cyclin-dependent protein kinase

(CDK) 2 complexes in vitro (Zhang et al. 1997; Knotts et al. 2001).

Although the role of PR phosphorylation is not fully understood, it may influence aspects of transcriptional regulation such as interaction with coregulators, as has been found for

ER (Font de Mora and Brown 2000) and recently for PR (Lange 2004). Regulation of ligand- dependent (Shen et al. 2001) and -independent (Labriola et al. 2003) PR transcriptional activities

(Lange et al. 2000) have also been shown to involve phosphorylation. Phosphorylation is generally accepted as a positive regulator of steroid receptor function and may serve to integrate additional signals. Epidermal growth factor (EGF) and P4 synergistically up-regulate mRNA or protein levels for a number of growth-regulatory genes (Richer et al. 1998) including cyclin Dl and cyclin E (Haslam et al. 1993); the regulation of cyclins by P4 is MAPK dependent. Cyclins, in turn, regulate progression of cells through the cell cycle by interaction with CDKs. P4

2 2 activates CDK2 (Groshong et al. 1997), and PRs are predominantly phosphorylated by CDK2 at proline-directed sites (Zhang et al. 1997; Knotts et al. 2001), perhaps allowing for the coordinated regulation of PR action during cell cycle progression.

The Ser294 site of PR is a ligand-inducible phosphorylation site, becoming rapidly phosphorylated upon exposure to hormone (Zhang et al. 1995). Recently, MAPK-dependent PR phosphorylation at Ser294 has been shown to be required for nuclear translocation of unliganded

PR, suggesting that MAPK signaling may regulate PR action by altering nucleo-cytoplasmic shuttling (Qiu et al. 2003). Phosphorylation of PR at Ser294 by MAPK increases transcriptional activity of liganded PR in PRE-containing promoters (Shen et al. 2001). Interestingly, liganded mutant Ser294A PR is a weak transcriptional activator when stably expressed in breast cancer cells, and does not undergo synergistic regulation in response to agents that activate MAPK

(Shen et al. 2001). Phosphorylation of Ser294 may also mediate some aspects of ligand- independent PR action.

Growth factors such as EGF lead to phosphorylation of the Ser294 site within 5 min in the absence of P4. When Ser294 becomes phosphorylated, rapid nuclear accumulation of PR occurs, as measured by both fluorescence microscopy of intact cells and cellular fractionation experiments (Qiu et al. 2003). Mutation of the consensus MAPK site, Ser294 to Ala (Ser294A), abolished EGF-mediated translocation; however, the ability of progestin (R5020) to induce nuclear localization of Ser294A PR was unaffected (Lange 2004). EGF-induced nuclear accumulation requires p42/p44 MAPK activation and phosphorylation of Ser294, and occurs independently of progestin, suggesting a mechanism for ligand-independent transcriptional activation of PR (Lange 2004).

2 3 Hormone-dependent 51103(102, 294, 345)

hPR HBD

AF1 H AF2

Basal sites (81,162,190, 400)

Figure 8. Phosphorylation sites in human PR. Fourteen residues in human PR have been shown to represent basal (constitutive) and hormone-induced phosphorylation sites and may contribute to PR regulation by MAPK, casein kinase II, and CDK2. Individual PR phosphorylation sites may be regulated by multiple protein kinases and/or in a sequential manner, illustrating the complexity of PR regulation by phosphorylation (Adapted from (Lange 2004)).

2 4 1. 9 Interactions between PR and coregulators

After binding of PR to PREs, the receptors modulate target gene transcription by recruiting components of the transcriptional machinery directly or indirectly via coregulators such as coactivators and corepressors either positively or negatively (Wu et al. 2005). The coregulators including members of the steroid receptor coactivator (SRC/NCoA) family (Smith and O'Malley 2004).

Nuclear receptor coregulators are coactivators or corepressors that are required for efficient transcriptional regulation. Coactivators are defined as molecules that interact with nuclear receptors and prompt their transactivation. Nuclear receptor corepressors are defined as factors that interact with nuclear receptors and lower the transcription rate at their target genes.

Most coregulators are rate limiting for nuclear receptor activation and repression, but do not significantly alter basal transcription.

Initial contact between the activated nuclear receptor and coactivators is mediated in large part by an amphipathic helix conserved on most coactivators, the LXXLL motif, or NR box in coactivators (Heery et al. 1997) (Fig. 5). These factors include the SRC/pl60/NCoA family, creb-binding protein (CBP)/P300 (Xu and O'Malley 2002), CIA, ASC-2/TRBP/AIB3/

RAP250/PRIP/NCR, and PBP/DRIP205/TRAP220 (Li and O'Malley 2003). The SRC family is composed of three distinct but structurally related members: SRC-1, SRC-2 (NCoA2/TIF-

2/GRIP1), and SRC-3 (NCoA3/p/CIP/RAC3/ACTR/TRAM-l/AIBl) (McKenna et al. 1999). A different group of coactivators is specialized for interaction with the DBD of receptors. The

DBD of PR is required for binding to specific PRE sequences, but much less is known about the function of nuclear cofactors that bind to the DBD. This includes small nuclear RING finger

2 5 protein, SNURF, GT198, a tissue-specific coactivator, and high mobility group proteins (HMG).

PR appears to utilize HMG-1 or -2 proteins for high affinity interaction with DNA in vitro and for full transcription activity in vivo (Li and O'Malley 2003).

Compared with AF-2 and DBD interacting coregulators, cofactor interactions with the

AF-1 are less well characterized. AF-ls are the least conserved regions among PRs from different species and are likely associated with the differential ability of PR A and PR B to recruit specific coregulator proteins (Giangrande et al. 2000). A recent study has identified several N-terminal domain interacting factors, including Jun dimerization protein-2 (JDP-2) and nuclear receptor coactivator- 62 (NCOA-62) (Edwards et al. 2002). JDP-2, initially defined as a repressor of Jun and other bZIP transcription factors, functions as an AF-1 coactivator of PR. It has been shown that endogenous JDP-2 and PR are recruited in a hormone-dependent manner to a progesterone-responsive promoter in the context of chromatin in vivo. AF-1 cofactors may prompt PR function by recruiting or stabilizing other coactivators independent of the AF-2 and

SRC coactivators (Edwards et al. 2002). The recent description of PR coactivators, which influence RNA splicing, has revealed another mechanism by which PR regulates gene expression.

Transcription and mRNA processing are coupled events in vivo, whereas the mechanisms that coordinate these processes were largely unknown until recently.

The precise mechanisms by cofactors are not fully understood. However, a number of studies have indicated multiple possible mechanisms of action of coregulators. First, the structural properties of coactivators allow for multiple interactions among receptor and coactivator complexes (McKenna et al. 1999). The coupling of interaction domains within coactivators determines the recruitment of distinct acetyltransferases (CBP/P300, pCAF), methyltransferases (CARM1, PRMT1) (Wang et al. 2001a), kinases (Rsk-2, Msk-1) (Chen et al.

2 6 2001b), ubiquitin ligases (E6-AP, p300) (Grossman et al. 2003), ATP-dependent chromatin remodeling complexes (SWI/SNF) (Ostlund Farrants et al. 1997), and RNA splicing factors.

These factors facilitate the formation of a transcriptional complex and contribute to downstream events of transcription machinery. In addition, preferential recruitment of specific cofactors to the promoters in a cellular environment leads to various distinct patterns of gene expression.

Although all of these coactivators are implicated in PR-mediated gene activation, not all are functionally equivalent in vivo or expressed in the same manner in all cells. A recent study demonstrates that PR preferentially recruits SRC-1, SRC-3, and CBP, but not much SRC-2 or pCAF(Ostlund Farrants et al. 1997).

Corepressors also play a role in the regulation of coactivator function, and coactivator/corepressor ratios have been reported to modulate PR-mediated transcription (Liu et al. 2002c). Accumulating evidence has documented the functional significance of covalent modifications of cofactors. Phosphorylation of SRC-1 and SRC-3 at specific sites potentiates

PR-mediated transcription, probably due to the enhanced interaction with other histone acetyltransferases such as CBP (Rowan et al. 2000b). Acetylation of SRC-3 by p300/CBP at lysine residues adjacent to NR boxes disrupts its association with the receptor (Chen et al. 1999).

These potential mechanisms provide effective means of enhancing the functional plasticity of coregulators that will eventually result in reorganization of protein-protein or protein-DNA contacts and receptor-mediated transcription.

1.10 Transactivation of PR in the absence of P4

The activation of PR and other nuclear hormone receptors was initially considered to be

2 7 entirely steroid-dependent (Matkovits and Christakos 1995). However, many steroid receptors including PR have been shown to be activated in the absence of their cognate ligands by modulation of protein kinase or phosphatase (Power et al. 1991; Mani et al. 1994) (Fig. 9). In the absence of P4, PR can be activated by signaling pathways including cAMP, phorbol esters, dopamine, EGF, and phosphatase inhibitors (Zhang et al. 1997; Pierson-Mullany and Lange

2004). Ligand-independent activation of steroid receptors may have important physiological and clinical implications for the study and treatment of hormone responsive organs. Very little is known about the molecular mechanisms of ligand-independent activation of the receptors. Since steroid receptors are phosphoproteins, it is possible that alteration of receptor phosphorylation in response to signals mediates the ligand-independent activation (Pierson-Mullany and Lange

2004). The AF-2 region within the hormone binding domain is known to be regulated by hormone, while AF-1 is located in the amino-terminal region. The fact that AF-1 is located outside the hormone-binding domain raises the possibility that AF-1 might be activated by means such as phosphorylation rather than by ligand binding.

Modulation of coactivators provides another possibility of mechanism for ligand- independent steroid receptor activation because coregulators are themselves targets of multiple signal transduction pathways (Li and O'Malley 2003). Phosphorylation of SRCs was shown to be induced in response to EGF, cytokines and increased intracellular cAMP (Wu et al. 2005). In the case of SRC-1, phosphorylation at Thrll79 and Serll85 induced by cAMP were shown to enhance both ligand-dependent and -independent activity of PR (Rowan et al. 2000b). Similary, phosphorylation of SRC-3 induced by EGF and cytokines also was shown to be important for its coactivator activity (Font de Mora and Brown 2000).

2 8 Growth factors PKC PKA

MAPK CDKs Cytokine (GnRHs?)

Figure 9. Model of PR action in the absence of P4. Growth factors, cytokines, stimulators of

PKC, PKA, MAPK and CDKs, and neurotransmitters such as dopamine phosphorylated PR in the absence of P4. This activated PR binds to PRE and recruits coregulators. Coactivators including SRC family also can be phosphorylated by these signaling pathways and resulted in an increase unliganded PR activation. GnRHs activate PR in the absence of P4 however, the mechanism of it is unclear.

2 9 1.11 Transactivation of PR by GnRH I in the absence of P4

Crosstalk between the PR and GnRH I has been implicated in a GnRH I self-priming mechanism in the pituitary (Turgeon and Waring 1986; Waring and Turgeon 1992), which is defined as an enhanced LH secretion by pituitary gonadotropes in response to a second GnRH I stimulation (Fink 1995). This response appears to depend upon the capacity of E2 to induce PR expression in gonadotropes (Fink 1995), but it is completely absent in PR knockout mice

(Chappell et al. 1999). It has therefore been suggested that activation of GnRH I R in gonadotropes prompts a signaling pathway, which ultimately activates the PR in a ligand- independent manner (Turgeon and Waring 1994). However, the mechanisms responsible for

GnRH I self-priming and the ligand-independent activation of the PR by GnRHs in gonadotophs are still unclear.

1.12 Pituitary gonadotropin hormones

The main function of GnRHs in the pituitary is to regulate production of gonadotropins such as LH and FSH. Dynamic regulation of the pituitary LH and FSH is essential for mammalian reproduction. LH and FSH are comprised of two glycoprotein subunits, common a, and LH/3 and FSH/3 (Gharib et al. 1990). LH and FSH secreted from the pituitary gonadotropes act on the ovaries and the testes to regulate folliculogenesis, ovulation, spermatogenesis and steroidogenesis (Marshall and Kelch 1986; Wu et al. 1990; Burger et al. 2004). The synthesis and secretion of the gonadotropins are primarily regulated by the hypothalamic GnRH in a pulsatile manner (Clarke and Cummins 1982). The control of LH and FSH synthesis and secretion is complex and involves interplay between the gonads, pituitary and hypothalamus. LH and FSH act on gonadal steroids, E2 and P4, and mediate positive and negative feedback 3 0 influences on LH and FSH in both the pituitary and the hypothalamus. Both hormones have been shown to contribute to positive or negative feedback effect of LH and FSH secretion, through regulation of hypothalamic GnRH neurosecretion, and/or modulation of pituitary responsiveness to the decapeptide (Chappell et al. 1997).

1.13 Regulation of gonadotropin subunit genes

The differential control of LH and FSH secretion can be dynamically regulated by hormones that alter their synthesis, storage and /or release. GnRH, E2, and P4 are involved in the sensitivity to gonadotropes. GnRH and combined effects with steroids are instrumental in generating the preovulatory LH surge.

1.13.1 GnRH regulation of common a-subunit (a-GSU) gene expression

GnRH-responsive regions have been mapped in the a-GSU promoter of several species.

In humans, cows and mice, the DNA elements that confer GnRH responsiveness all reside in the proximal promoter: human -346 to -244 (Kay and Jameson 1992), cow -315 (Hamernik et al.

1992) and mouse -406 to -399 and -337 to -330 bp (Schoderbek et al. 1993). The -337 to -330 bp site in the mouse a-GSU promoter binds a LIM homeodomain transcription factor that directs basal expression. The second identified site at -406 to -399 binds a transcription factor that is stimulated by GnRH via the MAPK (Roberson et al. 1995). Saunders et al. (Saunders et al.

1998) have shown that cAMP stimulates a-GSU transcription, and this is additive to the stimulation mediated by the PKC pathway. However, it is unclear whether this is mediated via a creb-response element (CRE). GnRH I R also increases a-GSU transcription by mobilizing

3 1 extracellular calcium, and the DNA elements responsible map are between —420 and -244 bp in the human a-GSU promoter (Holdstock et al. 1996). Differences in the intracellular signaling of the GnRH I R mediated by PKC- and cAMP-activated pathways, and by calcium influx, may be part of the mechanism that ensures adequate amounts of a-GSU in different physiological states, especially since a-GSU is always synthesized in excess over the /3-subunits.

1.13. 2 GnRH regulation of LH/3-subunit gene expression

LH/3-subunit transcriptional stimulation by GnRH I is mediated by stimulation of PKC

(Sartorius et al. 2003), MAPK (Week et al. 1998) and cAMP pathways (Saunders et al. 1998).

Two regions have been identified in the rat LH/3-subunit promoter that mediate GnRH I stimulation: at -490 to -352 and -207 to -82 bp (Kaiser et al. 1998b). The -490 to -352 region binds the ubiquitous transcription factor, Sp-1 (Kaiser et al. 1998a). SF-1 is also involved in the

GnRH regulation of LH/3-subunit gene expression. The SF-1 DNA binding site discovered in

LH/J-subunit promoters is highly conserved across species and occurs at approximately -130 bp.

A second SF-1 site in the rat promoter at -59 bp is also conserved across species (Halvorson et al.

1998). The stimulatory ligand for SF-1 was initially thought to be GnRH (Haisenleder et al.

1996).

1.13. 3 GnRH regulation of FSH /3-subunit gene expression

Regions responsible for activation of FSH/?-subunit gene expression have been defined as two activating protein 1 (AP-1) sites, which localized to -215 bp of the promoter at positions

-120 and -83 bp (Strahl et al. 1997). The AP-1 sites confer GnRH responsiveness (Strahl et al.

1998) and this is relayed by the PKC pathway (Saunders et al. 1998; Strahl et al. 1998). Calcium

3 2 influx appears to have no major role in the regulation of FSH/3-subunit gene expression

(Saunders etal. 1998).

1.13. 4 P4 regulation of /3-subunit gene transcription

P4 also alters the pattern and magnitude of GnRH-stimulated calcium signals in pituitary gonadotropes (Ortmann et al. 1994). In pituitary gonadotrophs, acute P4 pretreatment shifts

GnRH induced calcium oscillations towards a biphasic calcium signal, whereas in 0.T3-1 cells the amplitude of both phases of the biphasic calcium response increase. P4 can stimulate transcription of the FSH/3 gene, as over-expression of the PR increased rat FSH/3 promoter activity, and this action was mapped to three specific regions of the promoter, that contained PR- response element (PRE)-like sequences (Webster et al. 1995; O'Conner et al. 1999). These regions bind PR with high affinity and are sufficient for P4 responsiveness (O'Conner et al.

1999).

1.14 Hypotheses

1. There is a differential role of PR isoforms in the regulation of human GnRH I R,

GnRH I and GnRH II gene expression.

2. GnRHs induce transactivation of PRs in a ligand-independent manner, thereby

modulating FSH|3 subunit gene expression in the absence of P4.

1.15 Specific Objectives

Hypothesis 1

1. To investigate the regulation of GnRH I R promoter activity by P4, and to determine the 3 3 relative importance of specific isoforms of PR.

2. To examine the nature and mechanism of the transcriptional regulation of GnRH I and GnRH II by P4. Hypothesis 2

1. To elucidate the signaling pathways mediating GnRH I or GnRH II-induced ligand-

independent activation of PR.

2. To elucidate the involvement of coactivators and their recruitment to specific PRE

promoter regions by GnRHs.

3. To examine the involvement of the ligand-independent transactivation of PR in GnRH-

induced regulation of endogenous gonadotropin subunit gene expression. 2 MATERIALS AND METHODS

2.1 Materials

GnRH I agonist, (D-Trp6)-GnRH, H-89, Staurosporin (PKC inhibitor), E2, PR

antagonist RU486, and P4 were purchased from Sigma-Aldrich Corp (Oakville, Canada). A

GnRH II analogue, D-Arg(6)-Azagly(10)-NH2, was purchased from Peninsula Laboratories

(Belmont, CA). GF109203X, inhibitor of PKC, was purchased from EMD Biosciences, Inc.

Staurosporin and GnRH I R antagonist (Antide) were obtained from Sigma-Aldrich Corp.

2.2 Cell culture

The mouse gonadotrope-derived clonal oT3-l and L/3T2 cell lines were provided by Dr.

P. L. Mellon (Department of Reproductive Medicine, University of California, San Diego, CA).

The human cerebellar medulloblastoma (TE671) cells were obtained from American Type

Culture Collection (Manassas, VA). All cells were maintained in DMEM (Life Technologies,

Inc., Burlington, Canada) supplemented with 10 % fetal bovine serum (FBS; Hyclone, Logan,

USA). Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air. The cells were passaged when they reached about 90 % confluence using a trypsin/EDTA solution

(0.05 % trypsin, 0.5 mM EDTA).

2.3 Plasmids

A PRE-luciferase reporter plasmid, containing two copies of consensus PRE upstream

3 5 of the thymidine kinase promoter, was provided by Dr. D. P. McDonnell (Department of

Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC). Human

GnRH I R-luciferase construct (p2300-LucF) was prepared as previously described (Ngan et al.

1999; Cheng et al. 2001b). PR B construct (pSG5-PR B) was kindly provided by Dr. P.

Chambon (INSERM, University Louis Pasteur, Paris, France) and PR A construct (pOP13-PR

A) was a gift from Dr. Graham (University of Sydney Westmead Hospital, NSW, Australia).

Plasmid DNAs for transfection studies were prepared using QIAGEN Plasmid Maxi Kits

(QIAGEN, Chatsworth, CA) following the manufacturer's suggested procedure. The concentration and integrity of DNA were determined by measuring absorbance at 260 nm and agarose gel electrophoresis, respectively.

2.4 PRE-luciferase reporter gene assay

Transient transfections of PRE-lueciferase reporter gene were performed using

FuGENE 6.0 (Roche Diagnostics, Quebec, Canada) following the manufacturer's procedure.

Briefly, 4 x 105 cells were seeded into six-well tissue culture plates two day before transfection in 2 ml phenol red-free DMEM (Life Technologies, Inc., Burlington, Canada) containing 10 % charcoal-dextran-treated FBS (HyClone Laboratories, Inc. Logan, UT), which was used as standard culture medium in all experiments unless indicated. One microgram of the PRE- luciferase reporter plasmid and 0.5 pg RSV-/acZ were dissolved in 100 pl standard culture medium containing 3 pl FuGENE 6.0 without serum. The DNA mixture was incubated for 45 min at room temperature and then applied to the cells. Incubation of the cells with transfection medium continued for 24 h at 37 °C in 5 % CO2, and a further 48 h in culture medium with or without E2 (0.2 nM) prior to treatments with GnRHs (I or II) or P4. The cellular lysates were

3 6 collected with 150 pl reporter lysis buffer, and assayed for luciferase activity, and B- galactosidase activity to normalize transfection efficiencies, with commercially available reagents (Promega Corp., Nepean, Canada). Promoter activities were calculated as the luciferase activity/B-galactosidase activity.

2.5 Transient transfection of GnRH I R promoter and over-expressing vectors for PR isoforms

Transfections for GnRH I R promoter containing plasmids and over-expression vectors for PR isoforms were carried out using FuGENE 6.0 following the manufacturer's procedure.

Briefly, 4 x 105TE671 cells were seeded into six-well tissue culture plates before the day of transfection in 2 ml phenol red-free DMEM containing 10 % charcoal-dextran-treated FBS. One microgram of the GnRH I R promoter-luciferase construct, 0.5 pg RSV-/acZ and an indicated amount of expression plasmids (PR A or PR A/B) were dissolved in 100 pl phenol red-free

DMEM containing 3 pl FuGENE 6.0. The DNA mixture was incubated for 45 min at room temperature and then applied to the cells. Incubation of the cells with transfection medium continued for approximately 24 h at 37 °C in 5 % co2. After 24 h transfection, the cells were treated with various concentration of P4 or RU486 at different time periods before harvest.

Ethanol was added to the control media in the same final solvent concentration (typically 0.1 %).

The cellular lysates were collected with 150 pl reporter lysis buffer and cell lysis buffer, and assayed for luciferase activity and B-galactosidase activity immediately with the Luciferase

Assay system and B-Galactosidase Enzyme Assay System. Promoter activity was calculated as luciferase activity/B-galactosidase activity. A promoterless pGL2-Basic vector was included as a

3 7 control in the transfection experiments. To monitor the PRs over-expression, immunoblot analysis was performed using specific antibodies for PRs.

2.6 In Vitro transfection with small interference RNAs (siRNAs)

The shRNA (5'-TGACGGTTGCATTTGCCACTTCAAGAGAGTGGC AAATG

CAACCGTCA) for GnRH I R was produced using pSuper.gfp/neo vector. Two siRNAs for

SRC-3 (siSRC-3(a); 5'-UUACUGCUGCUUCUUGGCC and siSRC-3(b) (Liu et al. 2002c)) were obtained from QIAGEN (Chatsworth, CA). The siRNA (5'-

GUAUGGCUUUGAUUCCUUA) for PR was purchased from QIAGEN. In addition, a nonspecific siRNA was purchased from QIAGEN and used as a negative control. The siRNA transfection was performed according to the manufacturer's suggested procedure (QIAGEN). In brief, 2 days before transfection, 4 x 105 cells per well of a 6-well plate were seeded in 2 ml phenol red-free DMEM containing 10 % charcoal-dextran-treated FBS. The cells were transfected with 1 pg GnRH I R, or 12.5 pM (final concentration) of SRC-3 or PR siRNAs. The transfection was performed by 3 pl Lippfectamine 2000 reagent (Invitrogen, Burlington, ON), following the manufacturer's protocol. Then, the cells were challenged with GnRH I, GnRH II or P4. To monitor the siRNA transfection efficiency, immunoblot analysis was performed for PR,

SRC-3 or GnRH IR.

3 8 2.7 Western blot analysis

Immunoblot analysis was performed as previously described (Kang et al. 2001a; Choi et al. 2002; Kim et al. 2004). The cells were seeded at a density of 4 x 105 cells per well of a 6-well plate in 2 ml phenol red-free DMEM containing 10 % charcoal-dextran-treated FBS. The cells treated with GnRH I, II or P4, and then washed once with ice-cold PBS and lysed in 100 jrxl of in ice-cold lysis buffer (150 mM NaCl, 1 % Nondiet P-40, 0.5 % deoxycholate, 0.1 % SDS, 50 mM Tris (pH7.5) 1 mM PMSF, 10 /xg/ml leupeptin, 100 /ig/ml aprotinin). The cells were washed once with ice-cold PBS and lysed in 100 fi\ of in ice-cold lysis buffer (10 mM Tris pH

7.5, 150 mM NaCl, 1 % Triton X-100, 1 mM PMSF, 0.2 mM sodium orthovanadate, 0.5 % N-

40). The extracts were placed on ice for 10 min, collected into 1.5 ml tube and centrifuged for

10 min at 14,000 rpm. The supernatants were transfered to new tubes and the concentration of supernatants was determined using Bradford assay (Bio-rad Laboratories). Thirty five microgram of total protein was mixed with 6x sample buffer (75 mM Tri-HCl of pH 6.8, 15 %

SDS, 0.15 % bromophenol blue, 15 % glycerol, 37.2 % 2-mercapthoethanol) and boiled for 10 min. The sample mixture was run on 10 % SDS-PAGE gels (acrylamide: bisacrylamide =29:1) in lx gel running buffer (25 mM Tris/250 mM glycine, pH 8.3/0.1 % SDS) at 100 V for 2.5 h and electrotransferred to a nitrocellulose membrane (Hybond C, Amersham Pharmacia Biotech

Inc., Oakville, ON) at 100 V for 1.5 h. The resulting Western blots were blocked with Tris buffered saline (20 mM Tris-Cl, pH 7.4, 500 mM NaCl, 0.1 % Tween 20) containing 5 %

(wt/vol) nonfat milk for 2 h before addition of antibodies. The membrane was immunoblotted using a rabbit polyclonal antibody for PR (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), PR-

Ser294 (Neomarker, Fremont, CA) or PR-Ser400 with protein molecular marker (New England

Biolabs, Inc., Ontario). The anti-PR (phosopho-Ser400) antibody was provided Dr. C. A. Lange

(Department of Medicine, University of Minnesota, MN). Alternatively, the membrane was 3 9 immunoblotted with anti-/3-actin antibody (Santa Cruz Biotechnology, Inc). After washing three times with TBS-T (0.1 % Tween-20 in TBS) for 15 min, the signals were detected with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech Inc.) and visualized using the ECL chemiluminescent system (Amersham Pharmacia Biotech Inc.).

2.8 Immunoprecipitation

The cells were seeded at a density of 4 x 105 cells per well of a 6-well plate in 2 ml phenol red-free DMEM containing 10 % charcoal-dextran-treated FBS. The cells were treated with GnRH I, II or P4, and then washed once with ice-cold PBS and lysed in 100 fi\ of in ice- cold lysis buffer. The extracts were placed on ice for 10 min, collected into 1.5 ml tube and centrifuged for 10 min at 14,000 rpm. The supernatants were moved to new tubes and the concentration of the proteins was determined using Bradford assay. Endogenous PRs were immunoprecipitated from cell extracts with PR antibody (10 pg/ml) for 1 h at 4 °C, followed by incubation with protein A-Magnetic beads (BioLabs, Inc., Ipswich, MA) for 1 h at 4 °C. The beads were washed three times with lysis buffer. The PR-bound proteins were released by incubating the beads in SDS-PAGE sample buffer. The sample mixture was run on 10 % SDS-

PAGE gels (acrylamide: bisacrylamide =29:1) in lx gel running buffer (25 mM Tris/250 mM glycine, pH 8.3/0.1 % SDS) at 100 V for 2.5 h and electrotransferred to a nitrocellulose membrane at 100 V for 1.5 h.

The resulting Western blots were blocked with 5 % (wt/vol) nonfat milk for 2 h before addition of antibodies. Antibodies were obtained from Upstate, Lake Placid, NY (SRC-1, catalogue #05-522; GRIP-1, catalogue #06-986; SRC-3, catalogue #05-490), Neomarker,

4 0 Fremont, CA (GnRH I R, catalogue #MS-1139) or Santa Cruz Biotechnology, Inc, Santa Cruz,

CA (pCAF, catalogue #sc-13124). Incubation with primary antibodies and horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech Inc.), and washing of blots were performed three times for 15 min in Tris buffered saline with 0.1%Tween 20. The enhanced chemiluminescence system was used for detection, and signals were visualized by exposure to Kodak X-omat film.

2.9 Immunocytochemistry

Monolayer-cultured oT3-l cells were grown in standard culture medium without serum for 16 h, and subsequently incubated with serum or hormones (see figure legend for details).

After stimulation, the cells were washed in PBS and fixed in 4 % paraformaldehyde for 15 min, washed with PBS and permeabilised with methanol for 10 min at -20 °C. Endogenous peroxidase was blocked with 0.35 % H2O2 for 10 min, and fixed cells were incubated for 2 h at

RT with the'corresponding anti-PR antibody diluted 1:100 in PBS/1 % BSA. After three washes with PBS, detection of the primary antibody was performed with an ABC peroxidase staining kit

(DaKoCytomation, Corp, Carpinteria, CA).

2.10 RNA extraction and reverse transcriptase-PCR (RT-PCR)

Total RNA was isolated from cell cultures by RNeasy Mini Kit (QIAGEN, Chatsworth,

CA). One pg of extracted total RNA from each cell line was reverse transcribed using the

Superscript™ II Reverse Transcriptase (Invitrogen) according to the manufacturer's suggested

4 1 protocol. PCR amplifications were carried out in 20 pl reactions containing 1 pl cDNA, 2.5 U

Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mm MgC^, 2 mm deoxynucleotide triphosphate, and 50 pmol forward and reverse primers. Primers for GnRH I and GnRH II were designed based on the published sequence (Khosravi and Leung 2003), (Nathwani et al. 2000).

The forward-and reverse primers for GnRH I (accession number: M12578) were 5-

ATTCTACTGACTTGGTGCGTG-3 and 5-GGAATATGTGCAACTTGG TGT-3, respectively.

Forward and reverse primers for GnRH II (accession number: AF036329) were 5-

GCCCACCTTGGACCCTCAGAG-3 and 5-CCAATAAAGTGTGA GGTTCTCCG-3, respectively PCR amplification for GnRH I was carried out for 27 cycles with denaturing at 94

°C for 60 sec, annealing at 60 °C for 60 sec and extension at 72 °C for 90 sec, followed by a final extension at 72 °C for 15 min. The PCR for GnRH II was performed with denaturing for 1 min at 94 °C, annealing for 60 sec at 60 °C,. extension for 90 sec at 72 °C, and a final extension for 15 min at 72 °C for 29 cycles (Nathwani et al. 2000). Ten microliters of PCR products were fractionated on a 1.5 % agarose gel with ethidium bromide. The expected PCR product of GnRH

I and GnRH II were isolated from the gel and sequenced by the dideoxy nucleotide chain termination method. Sequence analysis revealed that GnRH I and GnRH II cDNAs have identical sequence to those from the published human GnRH I and GnRH II. The sizes for

GnRH II and GnRH I cDNAs were 327 and 380 bp, respectively.

2.11 Real time RT-PCR

Total RNA (2.5 pg) was reverse transcribed into first-strand cDNA. The primers used for SYBR Green real-time RT-PCR were designed using the Primer Express Software v 2.0

(Perkin-Elmer Applied Biosystems, Foster City, CA) and tested previously. The primers for the

4 2 real time PCR are described in Table. 1. Real-time PCR was performed using the ABI prism

7000 Sequence Detection System (Perkin-Elmer Applied Biosystems, CA, USA) equipped with a 96-well 10 optical reaction plate. The reactions were set up with 12.5 [i\ SYBR® Green PCR

Master Mix (Perkin-Elmer Applied Biosystems). All real-time experiments were run in triplicate and a mean value was used for the determination of mRNA levels. Negative controls, containing water instead of sample cDNA, were used in each real-time plate. The amount of transcript in each sample was calculated by interpolation using the following formula: (threshold cycle-y 15 intercept)/S. The steady-state concentrations of mRNA for a-GSU, FSH(3, and LH(3 in aT3-l and L[3T2 cells were normalized to the amount of GAPDH mRNA.

4 3 Genes Direction Sequences Gene ID FSHB sense CCC AGC TCG GCC CAA TA NM_008045.2 anti-sense GCA ATC TTA CGG TCT CGT ATA CCA LHp sense GGC CGC AGA GAA TGA GTT CT NM_008497.2 anti-sense CTC GGA CCA TGC TAG GAC AGT AG a-GSU sense TGT TGC TTC TCC AGG GCA TAT NM_009889.1 anti-sense TGG AAC CAG CAT TGT CTT CTT G GAPDH sense CAT GGC CTT CCG TGT TCC TA M32599.1 anti-sense GCG GCA CGT CAG ATC CA

Table. 1. Primers for Real-time PCR 2.12 Chromatin immunoprecipitation (ChIP) assay

Unless otherwise stated, all reagents, buffers and supplies were included in a ChIP-IT1M kit (Active Motif, Inc., Carlsbad, CA). Briefly, the oT3-l cells were cross-linked with 1 % formaldehyde for 10 min at room temperature. After washing and treatment with glycine Stop-

Fix solution, the cells were re-suspended in lysis buffer and incubated for 30 min on ice. The cells were homogenized and nuclei were re-suspended in shearing buffer, and subjected to optimized ultrasonic disruption conditions to yield 100-400 bp DNA fragments. The chromatin was pre-cleared with Protein G beads and incubated (overnight at 4 °C) with 1 pg of the following antibodies; negative control mouse IgG, anti-PR or anti-SRC-3. Protein G beads were then added to the antibody/chromatin incubation mixtures and incubated for 1.5 h at 4 °C.

After extensive washings, immuno-precipitated DNA was removed from the beads in an elution buffer. To reverse cross-links and remove RNA, 5 M NaCl and RNase was added to the samples and incubated for 4 h at 65 °C. The samples were then treated with proteinase K for 2 h at 42 °C and the DNA was purified using gel exclusion columns. The purified DNA was subjected to

PCR amplification (1 cycle of 94 °C for 3 min; 40 cycles of 94 °C for 20 sec; 64 °C for 30 sec and 72 °C for 30 sec) of the PRE-luciferase promoter using specific forward (5'-

AGAACTCTTGCTTGCTTTGC) and reverse (5'-AATAGCAGACACTCTATGCC) primers.

As an input control, 10 % of each chromatin preparation was used. The PCR products were resolved by electrophoresis in a 2.5 % acrylamide gel and visualized after ethidium bromide staining. 2.13 Statistical analysis

Data are shown as means of three individual experiments and presented as the mean ±

SD. Data were analyzed by ANOVA followed by Tukey's multiple comparison test. P< 0.05

was considered statistically significant.

4 6 3. RESULTS

3.1 Regulation of the GnRH system by P4

3.1.1 P4 regulates human GnRH IR promoter activity

To examine the transcriptional regulation of human GnRH I R gene by P4, a full-length human GnRH I R promoter-Iuciferase construct (p2300-LucF) was transiently transfected into human neuronal TE671 cells, and treated with P4 for 24 h. A significant decrease of promoter activity was observed following treatment with P4 at 10"6M and 10"5M doses (Fig. 10). This inhibitory effect on the transcriptional level of GnRH I R was shown at 12 and 24 h treatment with 10"5M P4 (Fig. 10B). To further confirm the specificity of PR of P4-mediated effect in the expression of human GnRH I R, the human neuronal cells were co- treated with RU486 (10"5M), an antagonist of PR. Although RU486 itself had no effect on expression of GnRH I R transcript, it blocked P4 effect on GnRH I R transcriptional levels when the cells were co-treated with P4

(Fig. 11). The antagonistic effect of RU486 suggested that P4 regulation on GnRH I R promoter activity might be mediated by specific receptors for PR.

4 7 Control 10-9 lO8 107 10"6 10s Concentration of Progesterone (Log M)

Figure 10. Dose- and time-dependent effects of P4 on GnRH I R promoter activity. The human GnRH I R promoter-luciferase construct p2300-LucF was transiently transfected into

TE671 cells by FuGENE 6.0 reagent. The RSV-/acZ vector was also cotransfected in order to normalize the transfection efficiency. After 24 h transfection, the cells were treated with P4 in a dose (A) or time-dependent manner with 10"5 M P4 (B). The relative promoter activity is represented as luciferase activity/p-galactosidase activity. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate, a, P <

0.05 vs control.

4 8 300

C3 T3 T 2 250 H T o b too T 5 200 a o «> 150 T

C3

100 J3 5 50

Control P4 P4+RU486 RU486

Figure 11. Effects of RU486 on P4-induced GnRH I R promoter activity. The TE671 cells were transiently transfected with p2300-LucF and treated with P4 (10"5 M), RU486 (10 "5 M) or

P4 plus RU486 for 24h. The RSV-lacZ vector was cotransfected to normalize varying transfection efficiencies. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate, a, P<0.05 compared with control; b,

P<0.05 compared with P4.

4 9 3.1.2 PR A but not PR B mediates P4 induced repression of GnRH I R promoter

Since RU486 reduced the P4 effect on GnRH I R transcription, roles of specific isoforms

of PR were further analyzed in this study. The presence of endogenous PR A and PR B in TE671

cells was observed by immunoblot analysis. As seen in Fig. 12A, PR A was highly expressed in

these cells, while PR B was weakly detected. Molecular weights of the detected human PR A

(95-kDa) and PR B (114 to 120-kDa) were similar to those that reported previously (Cheng et al.

2001a). To further evaluate the mechanism of P4 action in the promoter activity of GnRH I R,

the full-length versions of PR A or PR B were cotransfected into TE671 cells. The over-

expression of PR A or PR B was monitored by immunoblot analysis (Fig. 12A).

Over-expression of PR A enhanced a P4-mediated decrease in GnRH I R promoter

activity (Fig. 12B) in a dose-dependent manner. Interestingly, PR B over-expression reversed the

effect of P4 in the GnRH I R promoter activity. This result suggests distinct function of PR A and

PR B in the regulation of GnRH I R gene at the transcriptional level.

3.1.3 Over-expression of PR A or PR B has distinct effects on PRE promoter activity

Since P4-stimulated PR A and PR B showed distinct regulation of GnRH IR transcription,

transcriptional properties of PR-isoforms were further examined in this study. The cells were

cotransfected with the reporter plasmid 2 X PRE-tk-Luc and either PR A or PR B, then treated

with 10"5 M P4 (Fig. 12C). The reporter plasmid 2 X PRE-tk-Luc contains two copies of 5 0 consensus PRE upstream of the thymidine kinase promoter. Over-expression of PR A reduced the PRE reporter gene activity under P4-treated conditions, but over-expression of PR B increased it. However, without over-expression of PR A or PR B, P4 had no significant effect on

PRE promoter activity. These results indicate that isoforms of PR have different transcription properties in human neuronal cells. The PR B potentiates PRE Luciferase reporter gene activity, while PR A represses. The distinct promoter activities of PR A and B on the target gene promoter have been reported in other studies in a cell and promoter specific manner (Richer et al. 2002;

Jacobsen et al. 2005).

5 1 PR A PR B 1 2 3 > ^ PI c

I Control a 400 H ] P4

o 300 A

200 -j

'§ 100 J

OH

0 PRE-tk-Luc + + + PR A + PR B +

Figure 12. Effects of PR A or PR B over-expression on GnRH I R or PRE promoter activities. (A) Basal expression levels of PRs (lane 1), and over-expression of PR A (lane 2) and

PR B (lane 3) were investigated by Western blot analysis. (B) TE671 cells were cotransfected with GnRH I R luciferase construct and increasing amounts of the PR A or PR B plasmid DNA

(0.1, 0.5 and 1 pg). The RSV-lacZ vector was cotransfected to normalize varying transfection efficiencies. Two days after transfection, the cells were treated with P4 (10"5 M) for 24 h. (C)

The reporter plasmid 2XPRE-tk-Luc was cotransfected with 0.5 pg of either PR A or PR B into

TE671 cells. Following transfection, the cells were treated with P4 (10"5 M) for 24 h. The relative promoter activity of PRE is represented as the percentage of the respective control grou p, of which the activity is set as 100 % after being normalized by P-galactosidase activity.

Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate, a, P < 0.05 vs control.

5 3 3.1.4 Effects of P4 on human GnRH I and GnRH II mRNA levels

The PR A and PR B showed different transcription activities on the PRE Luciferase reporter gene and GnRH I R gene promoter. This dynamic action of PR isoforms in TE-671 cells led us to evaluate other possible P4 target genes, GnRH I and GnRH II. Semi-quantitative RT-

PCR was performed to examine the mRNA levels of GnRH I and GnRH II by P4 in TE671 cells.

The 380-bp product corresponding to GnRH I and, a 327-bp product for GnRH II were examined to evaluate P4 regulation of these genes. The 372-bp product for GAPDH was used as an internal control. A linear relationship was found between the cycle numbers and optical density for

GAPDH, GnRH I and GnRH II, respectively (Data not shown). As results, 29 cycles for GnRH I,

27 cycles for GnRH II and 20 cycles for GAPDH were employed for semi-quantification, and the PCR products were sequenced to assure authenticity. Treatment with P4 (10"6 and 10"5M) for

24h resulted in increases in mRNA levels of GnRH I (40 % and 100 %, respectively), compared with control (Fig. 13A). In a time-dependent experiment, treatment with P4 (10~6 M) increased the expression of GnRH I gene significantly at 12 and 24 h as shown in Fig. 13B. In contrast, it did not significantly affect mRNA levels of GnRH II (Fig. 15B). The P4-induced increases in

GnRH I mRNA levels were completely reversed by RU486 (10"5 M), whereas RU486 itself had no significant effect on it (Fig. 14).

5 4 A

5 5 B

Control 3h 6h 12h 24h

o -I 1—i—I 1—i—I 1—,—< 1—i—I 1—,—I Control 3h 6h 12h 24h

Treatments with P41106 M L Figure 13. Time- and dose-dependent effects of P4 on GnRH I mRNA levels. The TE671 cells were treated with P4 for 24 h in a dose (A) and time-dependent manner (B). Total RNA was extracted from TE671 cells, and 1 pg of total RNA was reverse transcribed. The expression levels of GnRH I mRNA was estimated by semi-quantitative RT-PCR and normalized by

GAPDH. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate, a, P < 0.05 vs control.

5 6 Figure 14. RU486 reverses P4-induced GnRH I mRNA levels. TE671 cells were treated with P4 (106 M), or P4 plus RU486 (10"6 M) for 24h. The levels of GnRH I mRNA was estimated by RT-PCR and normalized by GAPDH. Experiments were repeated three times independently. Bars represent mean + SD of representative experiments with triplicate, a, P <

0.05 vs control; b, PO.05 P4-treated.

5 7 3.1.5 Effects of PR A and PR B on human GnRH I and GnRH II mRNA levels

The plasmid encoding PR A or PR B mRNA was transiently transfected into TE671 cells to investigate roles of P4-stimulated PR isoforms in the modulation of GnRH I and GnRH II gene expression. Without PR expression vectors, treatment with P4 induced an increase in mRNA levels of GnRH I, while it did not affect GnRH II gene expression. In this context, the cells were introduced with expression vector for PR B, and this enhanced P4 effects on the

GnRH I gene expression (1.5-fold vs P4 only treated group) as shown in Fig. 15. However, the over-expression of PR A or PR B did not effectively alter the levels of GnRH II mRNA in the absence or presence of P4.

5 8 Figure 15. Effects of P4 on GnRH I and GnRH II mRNA levels after PR over-expression.

The over-expressing vector of PR A or PR B (0.5 pg) was transiently transfected into TE671 cells, and after 24 h of transfection, the cells were treated with P4 (10"6 M). The expression levels of GnRH I (A) and GnRH II (B) mRNAs were estimated by semi-quantitative RT-PCR and normalized by GAPDH. Experiments were repeated three times independently. Bars represent mean ± SD of Bars represent mean ± SD of representative experiments with triplicate, a, P < 0.05 vs. control; b, P<0.05 vs. P4-treated.

5 9

3.2 Ligand-independent activation of PR by GnRHs

3.2.1 Transactivation of PR by GnRH I and GnRH II in aT3-l cells

In the previous study, P4 regulated GnRH system in a PR-isoform specific manner in

TE671 neuronal cells. The results showed dynamic and complex interplay between the PR and

GnRH systems. In the following study, the effects of GnRHs on PR action were tested. The ability of GnRHs to activate PR-mediated transcription in mouse pituitary-derived o/T3-l cells was studied in the absence or presence of P4. In the initial experiments, o/T3-l cells were transfected with the PRE-luciferase reporter plasmid, and then treated with either GnRH I or II

7 7

(10" M) alone or with P4 (10" M) alone. Under these conditions, P4 increased the transcriptional activity of PR in a time-dependent manner with maximal activation at 24 h (3.5-fold vs control), while GnRH I and II showed maximal effects (5.5-fold vs control) on PR activation at 8 h (Fig.

16A). When these effects of GnRH I and II were studied in the presence of 10"7M P4, this resulted in a synergistic increase (2-fold vs GnRH-treated group without P4) in PR transactivation of the reporter plasmid after an 8 h treatment (Fig. 16B).

These initial experiments led us to suspect that the temporal difference in stimulation of

PR by GnRHs and P4 could be attributed to PR acting through ligand-independent and ligand- dependent pathways, respectively. To explore this, cells were co-treated with 10"5M PKA (H89),

10"6M PKC inhibitors (Staurosporin and GF109203X), 10"5M PR antagonist (RU486) or 10"7M

GnRH I R antagonist (Antide). This showed that co-treatments with Staurosporin, GF109203X,

H89 and Antide completely blocked the trans-activitation of the PR that was mediated by

GnRHs, while RU486 did not (Fig. 17). By contrast, activation of the PR by P4 was blocked completely by RU486 under the same conditions (data not shown).

6 1 Figure 16. Effects of GnRH I and II on PR-mediated transactivation of a PRE-reporter gene in aT3-l cells. (A) The PRE-luciferase reporter gene was transiently transfected into aT3-l cells by FuGENE 6.0 reagent. After 2 days in standard culture medium + 0.2 nM E2, the cells were treated with 10"7M GnRH I, GnRH II or P4 over a 24 h time course. (B) Ligand-dependent and ligand-independent transactivation of PR was tested after treatment with 10~7M GnRHs in the absence or presence of 10"7M P4. The cells were transiently trasfected with the reporter gene and treated with GnRH I or GnRH II with or without P4 for 8 h. In both experiments, a RSV- lacZ reporter plasmid was also co-transfected to control for transfection efficiency, and PRE- reporter gene activities were expressed in terms of luciferase activity/p-galactosidase activity. In both experiments, Experiments were repeated three times independently. Bars represent mean ±

SD of representative experiments with triplicate.

6 2 A

P4 600 rMllGnRH I i Ell] GnRH II ro 400

1 300

= 200 1 1 1 I 4h 8h 16h 24h

6 3 I I Control 1250 T^m GnRH I II GnRH II

+ + + + + No Stauro GF H89 RU486 Antide Cotreatment

Figure 17. PKC and PKA inhibitors reverse GnRH-induced PR-mediated transactivation of a PRE-luciferase reporter gene, but a PR antagonist (RU486) does not. The PRE- luciferase reporter gene was transiently transfected into oT3-l cells. After 2 d in standard culture medium + 0.2 nM E2, the cells were treated with 10"7M GnRH I or GnRH II alone (no co- treatment) or together with Stauro (staurosporin, PKC inhibitor), GF (GF109203X, PKC inhibitor), H89 (PKA inhibitor), RU486 (PR antagonist) or Antide (GnRH I R antagonist). After incubation for 8 h, cell lysates were analyzed for luciferase activity. A RSV-lacZ vector was co• transfected to control for transfection efficiency, and PRE-reporter gene activities were expressed in terms of luciferase activity/B-galactosidase activity. Control cells were not treated with

GnRHs but inhibitors alone. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate.

6 4 3.2.2 Treatment with GnRHs affects PR phosphorylation

The majority of PR phosphorylation sites contain a Ser-Pro consensus sequence for proline-directed kinases (Zhang et al. 1995). Since PKC and PKA inhibitors reduced the transcriptional activity of the PR, it was investigated whether PR is phosphorylated by GnRHs or P4. Amino acid sequences of murine, rat and human PR were compared with major phosphorylation sites (Fig. 18). The amino acid identity of human PR was 78 % to that of mouse.

Most of the murine PR phosphorylation sites were conserved with other species including

Ser294 and Ser400. Ser294 and Ser400 sites have known to be hyperphosphorylated in response to ligand and mitogen (Zhang et al. 1995; Zhang et al. 1997). Moreover, Ser400 phosphorylation mediates ligand-independent transactivation of the CDK-2 gene by the human PR (Pierson-

Mullany and Lange 2004).

To investigate the regulation of PR phosphorylation in oT3-l cells, antibodies that recognize both isoforms of PR (PR A and PR B) as well as antibodies against phosporylated-PR at Ser294 or Ser400 were used in Western blotting experiments (Fig. 19). This demonstrated that both PR A and PR B isoforms are present in oT3-l cells, but it was not able to detect PR phosphorylated at Ser400 in these cells. Low levels of Ser294-phosphorylated PR B were detected, while phosphorylation of PR A at this site was undetectable. Phosphorylation of mouse

PR B at Ser294 in GT3-1 cells tended to increase at 1-4 h following treatment with GnRH I or

GnRH II (Fig. 19). By contrast, there was no increase in PR B phosphorylation at this site after

P4 treatment within this time frame.

6 5 Ser20 Ser25 mPR MTELQAKDPQVLHTSGASfStPHlfislPLLARLDSGPFQGSQHSDVSSVVSPIPISLDGLL 60 rPR MTELQAKDPRTLHTSGAAPS PTHVGS PLLARLDPDPFQGSQHSDASS WS PI PI SLDRLL 60 hPR MTELKAKGPRAPHVAGGPPS ?-EV(3SPLLCRPAAGPFPGSQTSDTLPEVSAIPISLDGLL 59

Ser81 mPR FPRSCRGPELPDGKTGDQQSlCsbVEGAFSGVEATHREGGRNSRPP--EKDSRLLDSVLDS 118 rPR FSRSCQAQELPDEKTQNQQSliSDVEGAFSGVEASRRRS-RNPRAP--EKDSRLLDSVLDT 117 hPR FPRPCQGQDPSDEKTQDQQSLS3VEGAYSRAEATRGAGGSSSSPP--EKDSGLLDSVLDT 117

Serl62 mPR LLTPSGPEQSHASPPACEAITSWCLFGPELPEDPRSVPATKGLljSPLMSRPEIKVGDQSG 178 rPR LLAPSGPEQSQTSPPACEAITSWCLFGPELPEDPRSVPATKGLLSPLMSRPESKAGDSSG 177 hPR LLAPSGPGQSQPSPPACEVTSSWCLFGPELPEDPPAAPATQRviaPLMSRSGCKVGDSSG 177

Serl90 Ser213 mPR TGRGQKVLPKG: SJPPRQLLLPTSGSAHWPGAGVKPSlPQPAAGEVEEDSGLETEGSASPLL 23 8

3 rPR TGAGQKVLPKA' PPRQLLLPTSGSAHWPGAGVK: S 2QPATVEVEEDGGLETEGSAGPLL 23 7 hPR TAAAHKVLPRG: SPARQLLLPASESPHWSGAPVKPSPQAAAVEVEEEDGSESEESAGPLL 23 7

Ser294 mPR KSKPRALEGTGQGGGVAANAPSAAPGGVTLVPKEDSRFSAPRVS-LEQDSPIAPGfSJPLA 2 97 rPR. KSKPRALEGMCSGGGVTANAPGAAPGGVTLVPKEDSRFSAPRVS-LEQDAPVAPGI ISPLA 2 96 hPR KGKPRALGGAAAGGGAAAVPPGAAAGGVALVPKEDSRFSAPRVALVEQDAPMAPG] ISPLA 2 97

Ser345 mPR TTWDFIHVPILPLNHALLAARTRQLLEGESYDGGATAG-PFCPPRFslPSAPSTPVPRGD 3 55 rPR TTWDFIHVPILPLNHALLAARTRQLLEGDSYDGGAAAQVPFAPPRI3SPSAPSPPVPCGD 356 hPR TTVMDFIHVPILPLNHALLAARTRQLLEDESYDGGAGAASAFAPPRoSPCASSTPVAVGD 357

Ser400 mPR FPDCTYPLEGDPKEDVFPLYGDFQTPGLKIKEEEEGADAAVfSJPRPYLSAGASSSTFPDF 415 rPR FPDCTYPPEGDPKEDGFPVYGEFQPPGLKIKEEEEGTEAASRSPRPYLLAGASAATFPDF 416 hPR FPDCAYPPDAEPKDDAYPLYSDFQPPALKIKEEEEGAEASARSPRSYLVAGANPAAFPDF 417

6 6 mPR PLAPAP QAAPSSRPGEAAVAGGPSSAAVSPASSSGSALECILYKAE-APPTQGSFAP 471 rPR PLPPRP PRAPPSRPGEAAVAAP--SAAVSPVSSSGSALECILYKAEGAPPTQGSFAP 471 hPR PLGPPPPLPPRATPSRPGEAAVTAAPASASVSSASSSGSTLECILYKAEGAPPQQGPFAP 4 77

mPR LPCKPPAAASCLLPRDSLP AAPGTAAAPAIYQPLGLNG-LPQLGYQAAVLKDSLPQ 526 rPR LPCKPPAASSCLLPRDSLP AAPTSSAAPAIYPPLGLNG-LPQLGYQAAVLKDSLPQ 526 hPR PPCKAPGASGCLLPRDGLPSTSASAAAAGAAPALYPALGLNG-LPQLGYQAAVLKEGLPQ 536

Ser554 mPR VYPPYLNYLRPDSEAS(5lPQYGFDSLPQKICLICGDEASGCHYGVLTCGSCKVFFKRAME 586 rPR. VYPPYLNYLRPDSEAS()SPQYGFDSLPQKICLICGDEASGCHYGVLTCGSCKVFFKRAME 586 hPR VYPPYLNYLRPDSEASC13PQYSFESLPQKICLICGDEASGCHYGVLTCGSCKVFFKRAME 596

mPR GQHNYLCAGRNDCIVDKIRRKNCPACRLRKCCQAGMVLGGRKFKKFNKVRVMRTLDGVAL 646 rPR GQHNYLCAGRNDCIVDKIRRKNCPACRLRKCCQAGMVLGGRKFKKFNKVRVMRALDGVAL 646 hPR GQHNYLCAGRNDCIVDKIRRKNCPACRLRKCCQAGMVLGGRKFKKFNKVRWRALDAVAL 656

Ser676 mPR PQSVGLPNESQALSQRITi^SJ'NQEIQLVPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLT 706 rPR PQSVAFPNESQTLGQRIT7SPNQEIQLVPPLINLLMSIEPDWYAGHDNTKPDTSSSLLT 706 hPR PQPVGVPNESQALSQRFTFSpGQDIQLIPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLT 716

mPR SLNQLGERQLLSWKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQML 766 rPR SLNQLGERQLLSWKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQML 766 hPR SLNQLGERQLLSWKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQML 776

mPR YFAPDLILNEQRMKELSFYSLCLTMWQIPQEFVKLQVTHEEFLCMKVLLLLNTIPLEGLR 826 rPR YFAPDLILNEQRMKELSFYSLCLTMWQIPQEFVKLQVTHEEFLCMKVLLLLNTIPLEGLR 826

6 7 hPR YFAPDLILNEQRMKESSFYSLCLTMWQIPQEFVKLQVSQEEFLCMKVLLLLNTIPLEGLR 836

mPR SQSQFEEMRSSYIRELIKAIGLRQKGWPTSQRFYQLTKLLDSLHDLVKQLHLYCLNTFI 88 6 rPR SQSQFEEMRSSYIRELIKAIGLRQKGWPSSQRFYQLTKLLDSLHDLVKQLHLYCLNTFI 88 6 hPR SQTQFEEMRSSYIRELIKAIGLRQKGWSSSQRFYQLTKLLDNLHDLVKQLHLYCLNTFI 8 96

mPR QSRTLAVEFPEMMSEVIAAQLPKILAGMVKPLLFHKK 92 3 rPR QSRALAVEFPEMMSEVIAAQLPKILAGMVKPLLFHKK 923 hPR QSRALSVEFPEMMSEVIAAQLPKILAGMVKPLLFHKK 93 3

Figure 18. Alignment of the amino acid sequences and phosphorylation sites of PR in mouse, rat and human. The derived amino acid sequences and phosphorylation sites of the PR for mouse, human and rat were aligned. Murine, rat and human PR are highly conserved, especially in regions encompassing the phosphorylation sites. The PR at Ser294 and Ser400 are conserved in mouse, rat and human. The amino acid sequence identity of human PR was 78 % with mouse and 79 % with rat PR.

6 8 Figure 19. Regulation of PR phosphorylation at Ser294 by GnRHs. The oT3-l cells expressing endogenous PR A and PR B isoforms were treated with 10"7M GnRH I, GnRH II or

P4 for 1-8 h. Equal amounts of cell lysates (100 pg) were electrophoresed on SDS-7 % PAGE gels, transferred to nitrocellulose, and Western blotted using antibodies specific to either PR A and PR B (upper panel), phospho-Ser294 PR (middle panel) or actin as a control (lower panel).

Control (C) represents untreated cells at time zero. Experiments were repeated three times independently. Bars represent mean ± SD of three independent experiments.

6 9 GnRH I GnRH II P4 C 8 1 4 8 14 8 PR-B IMP HHp M

PR-A *** mm vmf^mm-

Phospho Ser294 PR-B

Actin

500 n u < 400 a: S 300 CO CM 200

& 100 o 0 C 1 8 1 4 8 1 4 8 GnRH I GnRH II P4

7 0 3.2.3 Treatment with GnRHs affects PR sub-cellular distribution

Since phosphorylation has been reported to influence the cellular distribution of the PR

(Qiu et al. 2003), the sub-cellular localization of PR was examined after treatment with GnRHs over a period of 24 h (Fig. 20). When o/T3-l cells were cultured for 16 h in serum free medium, the PR was predominantly cytoplasmic (Fig. 20A), while immunoreactive PR is located predominantly in the nucleus of ctT3-l cells cultured in the presence of serum (Fig. 20B).

Importantly, when the cells in serum free medium were treated with 10"7 M GnRH I or GnRH II, the PR accumulated in the nucleus within 1 h (Fig. 20C and 4D) and this persisted up to 24 h

(not shown).

7 1 Figure 20. Cytoplasmic to nuclear translocation of PR in aT3-l cells following treatments with GnRHs. (A) Immunocytochemical localization of PR in aT3-l cells grown in standard culture medium + 0.2 nM E2 but without serum (B) standard culture medium + 0.2 nM E2 (i.e, containing 10 % charcoal dextran-treated FBS), or (C) standard culture medium + 0.2 nM E2 without serum and containing GnRH I or (D) GnRH II. After 16 h in culture, 10"7M GnRH I or

GnRH II was added to the culture media for 1 h. After fixation, the cells were subjected to immuno-cytochemistry with anti-PR antibody followed by DAB staining. Experiments were repeated three times independently.

7 2 3.2.4 Interaction between SRC-3 and PR increases in aT3-l cell after treatment with GnRHs or P4

To examine whether the PR associates with specific coactivators in oT3-l cells after treatment with GnRHs, cell lysates were immunoprecipitated with anti-PR antibody and then immunoblotted with antibodies to various coactivators. As shown in Fig. 21A, there was no increase in the co-immunopreciptation of pCAF, SRC-1, or SRC-2 with the PR after cells were stimulated with GnRH I or GnRH II. In contrast, both GnRHs increased interaction of the PR with SRC-3, and this again was most apparent at 4-8 h after treatment with GnRH I.

Since P4 binding to the PR promotes its interaction with SRC-3 (Torchia et al. 1997), a

P4-dependent and ligand-independent (i.e., GnRH-mediated) recruitment of SRC-3 by PR was further compared in aT3-l cells after 8h of treatment. Under these conditions, none of the hormones influence the total levels of either PR A or PR B.

Although the amount of SRC-3 that immunoprecipitates with PR after GnRH I treatment was increased to about the same extent as that observed after P4 treatment, there was a more modest increase in the ligand-independent interactions between SRC-3 and the PR after GnRH

II treatment (Fig. 21B).

7 3 Figure 21. Interaction between SRC-3 and PR increases in aT3-l cells after treatment with

GnRHs or P4. (A) After 2 d in standard culture medium + 0.2 nM E2, the cells were treated with GnRH I or II for increasing lengths of time, and lysates were immunoprecipitated (IP) using anti-PR antibody. The immunoprecipitates were then probed with anti-SRC-1, anti-SRC-2, anti- pCAF or anti-SRC-3 antibody. (B) After treating the cells with 10"7M GnRH I, GnRH II or P4 for 8 h, cell lysates were prepared and immunoprecipitated with anti-PR antibody.

Immunoprecipitates were then analyzed by Western blotting with anti-SRC-3 antibody.

Experiments were repeated three times independently.

7 4 GnRH I GnRH

0 2 4 8 24h 0 2 4 8 24h

SRC-1

SRC-2

pCAF

SRC-3 •

IP with anti-PR

B

Control P4 GnRH I GnRH

SRC-3 IP with anti-PR 3.2.5 Recruitment of PR and SRC-3 to PREs is promoted by GnRHs

Since SRC-3 possesses HAT activity, which affects chromatin remodeling and transcription (Chen et al. 1997), it was explored whether or not GnRH I or II treatments influence PR-mediated assembly of SRC-3 at PREs within target genes by ChIP assays. For this purpose, the same synthetic PRE containing reporter gene construct was introduced by transient transfection into aT3-l cells. In this context, it was found that GnRHs promote very similar levels of PR recruitment to the PRE as that observed after treatment with P4, and this occurred within 4 h of treatment (Fig. 22A). This also showed that treatment with GnRH I induced a robust recruitment of SRC-3 to the same site within the same time frame, which was much greater than the recruitment of SRC-3 to this site after an 8 h treatment with P4. Interestingly, although treatment with either GnRHs caused a similar level of PR recruitment to this PRE, the increase in SRC-3 recruitment to this site after GnRH II treatment was not as effective as after

GnRH I treatment, but it was still greater than that observed after P4 treatment (Fig. 22A).

These results are consistent with the changes in PRE reporter gene activity after treatment with GnRHs (Fig. 16), and the observations that GnRH I consistently enhances ligand- independent transactivation of the gene by PR to a greater extent than GnRH II.

7 6 Figure 22. Recruitment of PR and SRC-3 on the PREs is promoted by GnRHs. The PRE-tk- luciferase reporter gene was transiently transfected into GT3-1 cells. Nuclear proteins bound to

PREs in aT3-l cells treated with GnRH I, GnRH II or P4 for 1, 4 or 8 h were cross-linked and subjected to ChIP assay using antibodies against SRC-3 and PR. The oligonucleotide primers for

PCR amplify the region containing the PRE in the PRE-tk luciferase reporter gene promoter. A non-specific mouse IgG was used in all ChIP reactions as a control for non-specific immuno- precipitation. Positive PCR controls of sheared genomic DNA templates indicated the integrity of the input DNA used in the ChIP reactions, while PCR reactions performed in the absence of template were used as a negative control. Experiments were repeated two times independently. 3.2.6 GnRH I R and SRC-3 are required for GnRH-mediated PR activation

Since mouse o/D-l cells only possess the GnRH I R, siRNA was used to decrease its expression in order to determine whether it mediates the ligand-independent transactivation of the PR by GnRH I or GnRH II in these cells (Fig. 23). A Western blot demonstrated that the siRNA treatment effectively decreased GnRH I R levels prior to the introduction of the PRE- luciferase reporter gene (Fig. 23). When these cells were then treated with GnRH I or GnRH II, the PRE-reporter gene was reduced substantially (66 % and 48 % for GnRH I and GnRH II) over that observed in cells that contain normal levels of the GnRH I R (Fig. 23). In this context, it should also be noted that the siRNA-induced loss of GnRH I R had no influence on the ligand

(P4)-dependent transactivation of the PRE-reporter gene (Fig. 23). These data confirm that the

GnRH I R mediates the ligand-independent activation of the PRE-reporter gene by GnRH I and

GnRH II.

7 8 Control siGnRH I R

GnRH I R M

Actin

Control SiGnRH I R 500 -t

>400 4 o re g|300 H 2 S=200 H o ujioo H Q.

Control P4 GnRH I GnRH II

Figure 23. GnRH I R mediates both GnRH I- and GnRH II-induced ligand-independent activation of PR. The oT3-l cells were co-transfected with a PRE-luciferase reporter gene and siRNA for GnRH I R. After 2 d in standard culture medium + 0.2 nM E2, the cells were treated with 10"7 M GnRH I, GnRH II or P4 for 8 h. The efficiency of the siRNA was tested by immunoblotting for GnRH I R (upper panel), and the cell lysates were assayed for luciferase activity. In these experiments, a RSV-/acZ reporter plasmid was co-transfected to normalize for transfection efficiency, and PRE-reporter gene activities were expressed in terms of luciferase activity/p-galactosidase activity. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with duplicate.

7 9 The siRNAs for SRC-3 also used to explore whether it is essential for the GnRH- induced transactivation of the PRE-luciferase reporter gene. Transfection of oT3-l cells with two siRNAs from different target regions of the gene resulted in substantial decreases in the cellular content of SRC-3, as shown by Western blotting, with a greater decrease being observed with the siSRC-3 (b) treatment (73 % and 67 % for GnRH I and GnRH II) (Fig. 24A). The results of this experiment are particularly important because they demonstrate that loss of SRC-3 has a much greater impact on the rapid (within 8 h), ligand-independent effects of the GnRHs on PRE- luciferase reporter gene activation, as compared to the ligand (P4)-dependent transactivation of the PR (not significant) within this same time frame. In fact treatment with siSRC-3 (b) completely blocked the ligand-independent transactivation of the PRE-luciferase reporter by both GnRHs acting either alone (Fig. 24A) or in synergy with P4 (Fig. 24B).

8 0 Figure 24. SRC-3 is essential for the ligand-independent activation of PR by GnRH I and

GnRH II (A), and the synergistic amplification of this effect by P4 (B). The aT3-l cells were co-transfected with PRE-luciferase reporter gene alone (no siRNA) or together with siRNAs for

SRC-3 (siSRC-3 (a) and siSRC-3 (b)). After 2 d in standard culture medium + 0.2 nM E2, the cells were treated with 10"7M GnRH I, GnRH II or P4 alone or in combination with each other for 8 h. The efficiency of the siRNA was tested by immunoblotting for SRC-3 (upper panel), and the cell lysates were assayed for luciferase activity. In these experiments, a RSV-/acZ reporter plasmid was co-transfected to normalize for transfection efficiency, and PRE-reporter gene activities were expressed in terms of luciferase activity/B-galactosidase activity. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate

8 1 * ST*?

SRC-3 Act in

O Control 5001 > cm GnRH I u 4001 ro C3 GnRH II d) c/> ro 300 i S_ CD o 200 I 5 3 _l I LU 100 CC CL nil! nlin No siRNA siSRC-3(a) siSRC-3(b)

8 2 B

3500 i No siRNA siSRC-3 (b) > 3000 o 2500 (0 Q> (A 2000 2 1500 U 3 _l 1000 UJ OC 500 CL 0 P4 + + + + + + GnRH I + + + + GnRH II + + + +

8 3 3.3 GnRH-induced FSHjft subunit gene transcription involves the ligand- independent transactivation of PR

3.3.1 Transactivation of PR by GnRH I in oT3-l and L0T2 cells

In the previous studies, GnRHs activated PR-mediated transcription in the absence of P4, and these data led us to examine the regulation of endogenous GnRH-target genes, gonadotropin subunit genes, by GnRH-induced PR transactivation in a ligand-independent fashion. In initial experiment, the ability of GnRHs to activate PR-mediated transcription in o/T3-l and L/3T2 cells was studied in the absence or presence of P4. The cells were cultured in the absence or presence of 0.2 nM E2, transfected with the PRE-luciferase reporter plasmid and then treated with GnRH I

(10"7M). Under these conditions, GnRH I increased the transcriptional activity of PR in a time- dependent manner with maximal activation at 8h in GT3-1 (1.7-fold vs control) or 24 h in L/3T2 cells (20-fold vs control) (Fig. 25). When the effects of GnRH I were studied in the presence of

0.2 nM E2, a synergistic effect was observed in oT3-l cells (4.3-fold vs GnRH I-treated alone), but not in L/3T2 cells (Fig. 25). It was reasoned that this differential synergistic effect might result from E2-induced PR expression. Indeed, the presence of E2 for 2 days increased PRs expression in oT3-l cells (Fig. 25A), however, it was not able to induce PRs in L/3T2 cells (Fig.

25B). In a previous study GnRHs with P4 showed synergistic effect on PR mediated transactivation in o/T3-l cells (Fig. 16). Thus, it was tested whether P4 alone or combination with GnRHs had effects on PRE-luciferase activities or not. Interestingly PRE luciferase gene activity was not regulated by P4 and no synergistic effect was detected with GnRH I (Fig. 25C).

8 4 E2 +E2

PR B PR A

1000n >» E2- ;> 33 E2+ o ra 75

B

-E2 +E2

3000-1 PR B PR A E2- > E2+ re i cu 2000- CO ro o 3 1000« -J 111 CC CL I-I^ r-ir-i nn i 8 24

8 5 c

Figure 25. Effects of GnRH I on PR-mediated transactivation of a PRE-reporter gene in a

T3-1 and LJ3T2 cells. A PRE-luciferase reporter gene was transiently transfected into QT3-1

(A) and LPT2 (B) cells with FuGENE 6.0 reagent. After 2 d in culture medium with or without

0.2 nM E2, the cells were treated with 10~7M GnRH I (A and B) over a 24 h time course. The expression of E2-induced PRs was tested by immunoblot assay (upper panel). To examine ligand-dependent and -independent transactivation of PR, L (3T2 cells were treated for 24 h with

10"7M GnRH I in the absence or presence of 10"7M P4 (C). The cells were transiently trasfected with the reporter gene before treatments. In both experiments, a RSV-/acZ reporter plasmid was also co-transfected to control for transfection efficiency, and PRE-reporter gene activities were expressed in terms of luciferase activity/B-galactosidase activity. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate.

8 6 3.3.2 Transcriptional regulation of gonadotropin subunit genes by GnRH I and GnRH II in pituitary cells

To investigate GnRH I regulation of gonadotropin subunit gene transcription, mRNA levels of a-GSU, FSH/3 and LH/3 were analyzed by real time PCR. It was shown that L/3T2 cells express a-GSU, FSH/3 and LH/3, while oT3-l cells only express a-GSU. In both cell lines, a-

GSU mRNA levels were significantly increased by GnRH I in a time-dependent manner with maximum at 8 h in oT3-l cells (2.4-fold vs control) (Fig. 26A) or 24 h in L/3T2 cells (8.5-fold vs control) (Fig. 26B). Interestingly GnRH I induced an increase in FSH/3 subunit mRNA expression at 8 h (4-fold vs control), this suddenly returned to control levels by 24 h (Fig. 26C).

LH/3 subunit gene expression was not significantly regulated by GnRH I at any time point (Fig.

26D). In addition, the presence of E2 did not result in any effect (Fig. 26) on GnRH I-induced modulation of the subunits gene even in oT3-l cells that have a robust increase in PR transactivation by GnRH I with the presence of E2 (Fig. 25A). To compare the effects of GnRH I and GnRH II on the regulation of gonadotropin subunit gene expression, the cells were incubated with GnRH I or GnRH II in the absence or presence of P4 (Fig. 27). Treatment with GnRH II resulted in the induction of a-GSU transcripts with a pattern similar that of GnRH I, while it had relatively less effect on FSH/3 mRNA content. For both a- and /3-subunits, P4 did not have any effects alone or in combination with GnRHs (Fig. 27).

8 7 Figure 26. The effects of GnRH I on a-GSU, FSHB and LH|3 mRNA levels. Changes in a-

GSU (A and B), FSHfi (C) and LHB (D) mRNA content as determined by real-time PCR in a

T3-1 (A) or LJ3T2 (B, C and D). After 2 days in culture medium with or without 0.2 nM E2, the cells were treated with 10"7M GnRH I over a 24 h time course. The mRNA levels of a-GSU and FSH{3 were significantly increased (*, P<0.05) after treatment with GnRH I, whereas changes in LH{3 mRNA during this time course were not remarkable. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate.

8 8 a-GSU mRNA expression a-GSU mRNA expression cn b cn -I l • o-l • o.

£-1

224 CO.

CO CO

10 , ro

Dm Dm IS+) I•O Dm Dm to ro + • c

5 4-5-1 O E2- w 4.0- E2+ % 3.5- 6 3.0- ° 25- Z 2.0- E 1-5-

CQ. 1.0- V) 0.5- LL 0.0-ID D 4h 8h 24h

1.25-1 E2- 1.00H E2+

0.75-

0.50H

0.25H

0.00- 4h 8h 24h

9 0 Figure 27. P4 does not have synergistic effects with GnRH I and GnRH II at the level of gonadotropin subunit gene expression. oT3-l (A) and L/3T2 (B and C) cells were treated with 10"7 M GnRH I or GnRH II in the absence or presence of 10"7 M P4 for 8 h (A and C) or 24 h (B). Following treatment the expression levels of a-GSU (A and B) and FSH/3 (C) were analyzed by real time PCR. Control cells were not treated with GnRHs or treated with P4 alone at each time point. Experiments were repeated three times independently. Bars represent mean ±

SD of representative experiments with triplicate.

9 1 FSHB mRNA expression n Q-GSU mRNA expression a-GSU mRNA expression

DD + • 3.3.3 Effects of signaling pathway inhibitors on GnRH I-induced transactivation of PR and gene expression of gonadotropin subunits

To examine the signaling pathways mediating GnRH I-induced PR transactivation (Fig.

28) and gonadotropin gene expression (Fig. 29), the cells were co-treated with GnRH I and 10"5

M PKA (H89), 10"6M PKC inhibitors (GF109203X), or a 10~7M PR antagonist (RU486). Co- treatment with GF109203X or H89 completely blocked the GnRH I-induced transactivation of

PR in 0.T3-1 cells (Fig. 28A) but MAPK inhibitor did not (data not shown), whereas PR transactivation was reduced half in LBT2 cells (Fig. 28B). In contrast, GF109203X reduced the effect of GnRH I on a-GSU mRNA levels in both cell lines (Fig. 29A and B) while H89 reduced only GnRH I induced FSHJ3 transcription by 40 % (Fig. 29C). The inhibitory effects of

GF109203X and H89 on GnRH I-stimulated FSH|3 transcripts were similar to those of PRE- luciferase activation by GnRH I in L{3T2 cells (Fig. 28B). This suggests the possibility that

GnRH I-mediated transactivation of the PR could influence the regulation of FSHJ3 gene expression in the pituitary. For both u-and 13-subunit genes, RU486 did not affect GnRH I- induced transcriptional regulation (Fig. 28 and 29).

9 3 Figure 28. PKC and PKA inhibitors, but not RU486, reduce GnRH-induced PR-mediated transactivation of a PRE-luciferase reporter gene in aT3-l or L/8T2 cells. The PRE- luciferase reporter gene was transiently transfected into oT3-l (A) or L/3T2 (B) cells. After 2 d in standard culture medium supplemented with 0.2 nM E2, the cells were treated with 10"7M

GnRH I alone or together with, GF (GF109203X, PKC inhibitor), H89 (PKA inhibitor) or

RU486 (PR antagonist). After incubation for 8 h cell lysates were analyzed for luciferase activity.

A RSV-/acZ vector was co-transfected to control for transfection efficiency and PRE-reporter gene activities are expressed in terms of luciferase activity/B-galactosidase activity. Control cells were not treated with GnRHs. Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate.

9 4 A

400i

300-

200-

100- II n C H-89 GF RU H-89 GF RU B GnRH I

nnnn T 1 1 T" t i i i C H-89 GF RU H-89 GF RU GnRH I

9 5 Figure 29. Effects of PKC and PKA inhibitors on GnRH-induced gonadotropin subunit gene expression. The o/T3-l (A) and L/3T2 (B and C) cells were treated with 10"7 M GnRH I alone or together with GF (GF109203X, PKC inhibitor), H89 (PKA inhibitor) or RU486 (PR antagonist). Following treatment for 8 h (A and C) or 24 h (B), the mRNA levels of a-GSU or

FSH/3 were analyzed by real time PCR. Control cells were not treated with GnRHs. Experiments were repeated three times independently. Bars represent mean + SD of representative experiments with triplicate. A

c 2n o

GF H89 RU GF H89 RU B GnRH I

c .2 5n (/> X a. 4H x 3H z E 2H z> w o• a Hnnn GF H-89 RU GF H-89 RU GnRH

9 7 c

c 7.5n o '35 co i0_ Q. 5.0H X CD a: E 2.5H ca. X if) 0.0- a GF H89 RU GF H89 RU GnRH I 3.3.4 PR mediates GnRH I-induced FSH]3 gene expression

The siRNA targeting PR was used to determine whether PR mediates GnRH I-induced gonadotropin subunit gene expression in L|3T2 cells (Fig. 30). When the cells were then treated with GnRH I, siRNAs for PR resulted in a decrease in FSH/3 gene expression by 32 %

(Fig. 30C), while it did not affect a-GSU transcripts (Fig. 30A and B). These data suggest that

PR mediates the transcriptional regulation of FSH|3 in a ligand-independent manner by GnRH I.

A Western blot demonstrated that the siRNA treatment effectively decreased PR levels prior to

GnRH I treatment (D).

9 9 1 0 0 c

11 siControl (A IsiPR aif)> 5- &«• o < 3- Z " a: E 2- ca 1 1- LL

GnRH I

PRB

PR A

Actin

Figure 30. PR mediates GnRH I-induced FSHjS gene expression. o/F3-l (A) and L/3T2 (B and C) cells were transfected with control siRNA or siRNA for PR. Two days after transfection, the cells were treated with 10"7 M GnRH I for 8 (A and C) or 24 h (B) and a-GSU or FSH/3 mRNA expression was analyzed by real-time PCR. The efficiency of the siRNA was tested by immunoblotting for PR (D) in LbT2 cells, Controls were not treated with GnRH. In the experiments, Experiments were repeated three times independently. Bars represent mean ± SD of representative experiments with triplicate.

1 0 1 4. DISCUSSION

4.1 Regulation of the GnRH system by P4

Although it is now well established that the feedback actions of gonadal steroid hormones play an important role in regulating the function of the GnRH neurons, the mechanism of this influence is not fully understood (Levine 1997; Herbison 1998). P4, one of the principal ovarian steroid hormones, is known to exert inhibitory and stimulatory effects on gonadotropin secretion in several species, including human, and these actions involve the modulation of pulsatile GnRH

I secretion (Leipheimer et al. 1984; Skinner et al. 1998). The precise mechanism through which

P4 modulates the activity of the GnRH neurons is presently unknown. To date, only a few studies have been done in the regulation of GnRH in the human, because of the lack of appropriate cell models.

Previously, it has been shown that P4 treatment resulted in a decrease in GnRH I R promoter activity in the mouse pituitary cells, while P4 increased its activity in human placenta cells (Cheng et al. 2001a). A putative PRE was identified between -535 and -521 related to translation start site at P2300-LucF, GnRH I R promoter construct, which is responsible for the

P4 action. Recently, the human medulloblastoma cell line, TE671, has been demonstrated to express and secrete GnRH I and GnRH II (Chen et al. 2002b). To further elucidate the molecular mechanism of P4 in the regulation of different components of the human GnRH system, the transcriptional regulation of GnRH I, GnRH II, and GnRH I R was investigated after P4 treatment in TE671 cells. In the present study, the results indicate that P4 had an inhibitory role in human GnRH I R promoter activity, and this effect was reversed by RU486 which is a known

PR and GR antagonist. This suggests that the P4 effect on GnRH I R promoter activity is mainly

10 2 mediated by PRs.

In order to further investigate the mechanism of P4 function on the expression of GnRH I

R gene, the cells were co-transfected with P2300-LucF construct and PR A or PR B. The over- expression of PR A in TE671 cells enhanced P4 effects in the repression of GnRH I R promoter activity. Interestingly, over-expression of PR B reversed the PR A-mediated inhibition of GnRH

I R transcription in a dose-dependent manner. This suggests that the P4-induced inhibitory effect on the GnRH I R gene expression is mediated by PR A, not PR B. PRs are ligand-inducible transcriptional regulators that control the gene expression upon binding to the PRE in the vicinity of target promoters or influence the gene expression by interaction with other transcriptional factors, i.e., NF-kB and STAT, and these interactions result in the repression of transcriptional activities (Truss and Beato 1993).

To investigate transcriptional activity of PR A and PR B, the cells were co-transfected with 2 X PRE-tk-Luc vector and PR A or PR B. In TE-671 cells, PR A was shown to be a transcriptional repressor, whereas PR B acted as an activator in the presence of P4. These results indicate that PR A and PR B have distinct transcriptional properties in a gene-specific manner on the GnRH I R promoter. Although PR B acts as a strong transcriptional activator of the PRE promoter, it did not induce transcriptional activity on GnRH I R promoter. It might be because the PR B expression vector also could over-express PR A gene, and the PR A probably blocks the function of PR B on the GnRH I R promoter. Taken together, PR A is more functional on the

GnRH I R gene expression in this promoter context, and PR B could play a role as a transdominant regulator of PR A action. Due to their different transcriptional properties, the relative expression levels of PR A and PR B within target cells may direct the overall functional responses to P4. The expression of PR A and PR B isoforms was examined by immunoblot

1 0 3 analysis in this study. Both of PR isoforms exist in TE671 cells. The expression level of PR A was high, while PR B was relatively low in these cells. These results support the scenario that an inhibitory role of P4 on the GnRH I R gene expression is mediated by PR A because of much higher endogenous levels of PR A than PR B in this cell line. Thereby, PR A mediates P4- mediated down-regulation of the human GnRH I R at the transcriptional levels

In the present study, the mRNA levels of GnRH I and GnRH II were distinctly regulated by P4. Treatment with P4 in TE671 cells resulted in an increase of GnRH I mRNA levels, whereas it had no significant effect on GnRH II gene transcription. Since the transcriptional activities of PR A and PR B on the GnRH I R and PRE luciferase promoter were distinct in this cell line, the biological pathway of P4 action was also examined in the regulation of GnRH I gene. Surprisingly, the transfection of these cells with PR B-expressing plasmid enhanced a P4- induced increase in the GnRH I gene expression. This finding suggests that the effect of P4 on

GnRH I mRNA levels is mediated by PR B not PR A even though the expression level of PR B is relatively low.

The differential regulation of two forms of GnRHs has been recently observed. In gold salmon fish, the ratio between GnRH I and GnRH II changed with sexual maturation

(Rosenblum et al. 1994). A smaller increase in the level of GnRH II than in salmon GnRH I was observed in the pituitary (Rosenblum et al. 1994). In the chicken, castration led to a change in the level of only cGnRH I but not cGnRH II (Sharp et al. 1990). In addition, the differential regulation of GnRH I and GnRH II by E2 has been recently demonstrated in TE671 cells, indicating that E2 increased endogenous GnRH II mRNA levels and decreased endogenous

GnRH I mRNA levels (Chen et al. 2002b). The differential regulation of two forms of GnRHs by sex steroid hormones suggests that GnRH I and GnRH II may be temporally regulated by

1 0 4 steroids during different phases of the menstrual cycle.

To date, the effect of P4 on the expression of the GnRH I gene is still controversial, as it has both stimulatory and inhibitory actions. Even though P4 has a negative or no effect on the expression of GnRH I, co-treatment of P4 with E2 can induce secretion of GnRH I, and the reason for this difference may be due to the induction of PRs by E2 (Barni et al. 1999; Robinson et al. 2000). The differential expression and gene-specific action of PR A or PR B could be one of the clues for explaining various P4 effects on the regulation of GnRH system. Taken together, these results indicate that P4 is a potent regulator of GnRH I R and GnRH I gene expression.

This distinct effect of P4 on GnRH system may be derived from different pathways through PR

A or PR B.

Two PR isoforms have been known to show distinct transcriptional properties in a cell- and promoter-specific manner. In general, PR B acts as a potent transcriptional activator, whereas the transcriptional activity of PR A is cell- or gene-specific dependent manner (Vegeto et al. 1993; McDonnell and Goldman 1994; Sartorius et al. 1994; Wen et al. 1994). Where PR A is inactive as a transcription factor, it has the ability to repress PR B and other steroid receptors such as ER in vitro (Smith and O'Malley 2004). Functional evaluation studies of the AF-3 (exists in only PR B not PR A) (Fig. 6) suggest that it mediates PR B transactivational activity, through inhibition of the inhibitory domain common to PR A and PR B (Pratt and Toft 1997; Leonhardt and Edwards 2002). Moreover, distinct interaction of PR isoforms with cofactors possibly mediates different transcriptional properties of PR A and PR B. Inhibitory domain of PR A recruits corepressor SMRT with greater affinity than PR B, while PR B preferentially interacts with SRC-1 and SRC-2 (Giangrande et al. 2000). This differential recruitment of cofactors to PR

10 5 leads to specific histone modification of the promoter. Competition between cofactors binding to receptors may influence the interaction with other complexes (Yang et al. 2000).

The functions of PR isoforms have recently been characterized in an in vivo system

(Mulac-Jericevic and Conneely 2004). Selective ablation of PR A produced a phenotype characterized by infertility, severe endometrial hyperplasia, anovulation, and ovarian abnormalities in the presence of normal mammary gland in the response to P4. In contrast, mice with ablation of PR B were fertile, did not show altered responses to P4 in the uterus, but exhibited severely disrupted pregnancy- induced mammary gland morphogenesis.

4.2 Ligand-independent activation of PR by GnRHs

The main function of GnRH I in the pituitary is to promote gonadotropin secretion

(Kaiser et al. 1997; Stanislaus et al. 1998). In female rats, sequential treatments of GnRH I enhance substantially the production of gonadotropins (Turgeon and Waring 1994), and this self priming effect is thought to involve the PR because it is absent in PRA/B KO mice (Chappell et al. 1999). Moreover, this effect has been reported to be due to the ligand- independent activation of the PR by GnRH I in primary pituitary cell cultures (Turgeon and Waring 1994).

This latter observation was confirmed in the present study by using an established mouse pituitary cell line (o/T3-l cells), and it has also been shown that GnRH II promotes the ligand- independent activation of the PR in these cells. Although both GnRHs function rapidly in this context, i.e., within 8h, GnRH I consistently evoked a more robust response than GnRH II.

However these data indicate that the ligand-independent activation of the PR by both GnRHs is mediated via the GnRH I R, and involves the PKA and PKC pathways.

1 0 6 Numerous studies have indicated that the ligand-independent activation of nuclear hormone receptors, including the PR, involve an alteration in the phosphorylation of the receptors themselves (Zhang et al. 1997; Labriola et al. 2003) or their various co-regulatory proteins (Rowan et al. 2000a), and these are likely to vary depending on the cell-type and hormone stimulus. Although treatment with GnRHs did not result in a significant increase in the phosphorylation of PR B at Ser294 within 1-4 h in o/T3-l cells, this may still contribute (Qiu et al. 2003) in part to the rapid (within 1 h) relocation of the PR from cytoplasm to the nucleus of serum starved cells after treatment with either GnRH I or GnRH II.

The present study has not explored the mechanisms responsible for the cellular redistribution of the PR, but receptor coactivators, such as SRC-3, also undergo rapid cytoplasmic to nuclear translocations under similar conditions (Qutob et al. 2002). We therefore set out to examine the interaction between PR and various coactivator proteins within aT3-l cells after stimulation with GnRHs in the absence of P4. These studies showed that a substantial and specific increase in PR interaction with SRC-3 occurs 8h after treatment with

GnRHs, and this again was most evident after GnRH I treatment. Thus, PR phosphorylation and its translocation to the nucleus appear to occur prior to its increased association with SRC-3.

To explore the relevance of GnRH-induced interactions between PR and SRC-3 in relation to the ligand-independent activation of PR responsive genes, ChIP assays were performed to examine the loading of PR and SRC-3 onto PREs within the promoter of a transiently transfected reporter gene. In these assays, a rapid and robust recruitment of both PR and SRC-3 to the multiple PREs within the PRE-luciferase reporter gene was observed after

GnRH I treatment. Although GnRH II increased recruitment of PR to this PRE, it occurred in concert with much less SRC-3 than that observed after GnRH I treatment. However, in both

1 0 7 cases, treatments with GnRHs elicited a more robust response than that observed after the ligand-dependent recruitment of PR to the PRE.

In these ChiP assays, substantially more SRC-3 appeared to be recruited to PREs by

GnRHs than that observed after P4 treatment, while the PR: SRC-3 interactions observed in co- immunoprecipitation assays showed a similar pattern after treatments with both GnRHs and P4.

As suggested by recent studies (Wu et al. 2004; Yi et al. 2005), multiple cellular signaling pathways phosphorylate SRC-3 and regulate the activities of steroid receptors. It is therefore possible that GnRHs increase phosphorylation of SRC-3 and this induces recruitment of SRC-3 to PREs in a ligand-independent manner.

To demonstrate that SRC-3 plays a pivotal role in the GnRH-induced ligand-independent activation of the PR in ctT3-l cells, the SRC-3 levels were substantially reduced using a siRNA approach. These studies clearly indicated that loss of SRC-3 from the cells essentially eliminates the ability of GnRH I and GnRH II to activate the PRE-luciferase reporter gene in the absence or presence of P4. In this context, it also appears that loss of SRC-3 affects the ligand- independent activation of the reporter gene by GnRHs more effectively than that observed after

P4 treatment. This suggests a qualitative difference in the transcriptional complexes that assemble at the PREs in response to the ligand-dependent vs ligand-independent activation of the PR.

Taken together, these studies indicate that the self-priming of gonadotropin gene expression in pituitary cells by GnRH is mediated via the GnRH I R. More importantly, it was shown that treatment of aT3-l cells with GnRHs, and GnRH I, in particular, results in rapid changes in PR phosphorylation and its translocation to the nucleus, where it interacts with PREs

1 0 8 followed by the recruitment of SRC-3 (Fig. 31). This study also provides evidence that the interaction between the PR and SRC-3 is essential for the ligand-independent transactivation of the PR in response to GnRH I and GnRH II treatment.

4.3 Ligand-independent transactivation of PR mediates GnRH-induced FSH0 subunit gene transcription

The previous study confirmed GnRH-induced transcativation of PR and suggested phosphorylation followed by translocation of the PR. It was also found that recruitment of coactivator SRC-3 is involved in GnRH-induced ligand-independent PR transactivation.

However, it is still unclear whether or not PR mediates GnRH-induced regulation of endogenous gonadotropin gene expression in the absence of P4. To investigate this, involvement of PR in

GnRH regulation of gonadotropin gene expression was explored in pituitary cells.

The synthesis and secretion of the gonadotropins is regulated primarily by the pulsatile release of hypothalamic GnRH I. LH and FSH are comprised of two glycoprotein subunits, a common osubunit linked to a specific /5-subunit, LH/3 and FSH/3. The differential regulation of

LH and FSH production is complex and involves the interplay of gonadal, hypothalamic and pituitary factors. It has been documented that PR is involved in the GnRH-self priming effect, in vivo and in vitro, in a ligand-independent manner (Turgeon and Waring 1986; Waring and

Turgeon 1992).

Analysis of the regulation of gonadotropin gene expression in the pituitary has been hampered by the dearth of cell lines. Much of work was performed on primary cultures of

1 0 9 pituitary cells. Although the primary cultures contain about 5-10 % mature gonadotropes, they represent a heterogeneous population of cells and are difficult to manipulate in vitro (Liu et al.

2002a). The development of immortalized pituitary cell lines, aT3-l, by targeted expression of

SV40 large T antigen driven by the common glycoprotein hormone a-GSU promoter has greatly furthered our understanding of GnRH signaling (Mellon et al. 1991). The aT3-l cells express both a-GSU and GnRH I R, but are considered immature, because they do not express the FSH

(3 and LHP genes (Alarid et al. 1996). More recently other immortalized pituitary cell lines have been developed by utilizing the LHJ3 promoter for targeted expression of SV40 T-antigen in transgenic mice (Turgeon et al. 1996). These cells, LJ3T2 cells, express a-GSU, GnRH I R,

FSHP and LHP genes. Moreover, both FSH13 and LHP genes are regulated by GnRH, thus, representative of mature pituitary gonadotropes (Liu et al. 2002a).

In aT3-l and LPT2 cells, PR was activated by GnRH I in the absence of P4. When the cells were precultured with E2 for 2 days, GnRH I showed a synergistic increase in PR transactivation in aT3-l cells, whereas E2 priming did not affect the GnRH response in LPT2 cells. Interestingly, a low dose of E2 (0.2 nM) for 2 days induced PR expression in aT3-l cells, but not in LPT2 cells, and this induction of PR may underlie the synergistic PR transactivation observed in aT3-l cells. To compare a ligand -dependent and -independent PR activation, the cells were co-treated with GnRH I and P4. Co-treatment with P4 had a synergistic effect on

PRE-luciferase activity in aT3-l cells, whereas it did not in LPT2 cells. This suggests that, in

LPT2 cells which are more differentiated cells than aT3-l, PR does not undergo a ligand- dependent activation its activation is ligand-independent.

1 1 0 Despite of altered PR activation mechanisms, GnRH I prompted a-GSU gene expression in both cells, and it increased FSHP, but not LH(3 mRNA levels in LPT2 cells. The regulation of a-GSU mRNA levels by GnRH I was time-dependent and maximum at 24 h, while

GnRH I regulation of FSHG resulted in a maximum increase at 8 h and a return to control level at 24 h. GnRH II treatment enhanced a-GSU and FSHp mRNA levels. There was no synergistic response when GnRH I and II were combined with E2 or P4. Even though PRE- luciferase gene activity was reduced by PKC and PKA pathway inhibitors, only GnRH I- mediated transcription of the FSHP gene was reduced by both PKC and PKA inhibitors.

Interestingly the PKC, but not the PKA inhibitor effectively reduced GnRH I-induced a-GSU gene expression. The similarity of the signaling pathways between GnRH I-mediated transactivation of the PR and induction of FSHP gene expression led us to test the direct involvement of PR in GnRH I-induced FSH(3 gene expression. Although P4 did not augment

FSH|3 gene expression in this study, it has been reported that P4 response elements are present within the FSHP promoter, moreover, P4 stimulated FSHP promoter activity when PR was over-expressed in rat pituitary cells (O'Conner et al. 1999). In this context, siRNA for PR was used to examine the role of PR in GnRH I-induced gonadotropin subunit gene expression. The siRNA for PR reduced the effects of GnRH I on FSHp mRNA levels but did not affect GnRH I- stimulated a-GSU mRNA levels.

1 1 1 Taken together, these data indicate that GnRH I stimulates a-GSU and FSHB gene expression in pituitary cells. GnRH I activates PR in a ligand-independent manner, and GnRH I- induced FSHB gene regulation requires PR transactivation in the absence of P4.

4.4 Clinical implications

During the menstrual cycle, E2 and P4 exert negative and positive feedbacks at the hypothalamus and pituitary. These steroid hormones inhibit the release of GnRH and gonadotropins and maintain normal cyclicity during follicular phase of the cycle. At midcycle, these steroids induce positive feedback at the pituitary level, this results in the preovulatory LH and FSH surges. In women, disturbances in the negative and positive feedback may occur and cause menstrual irregularities. In terms of the negative feedback, a possible abnormality is a reduced suppression of gonadotrophin secretion thereby leading to premature ovarian failure and ovulatory cycles with higher FSH (Messinis 2006). In cases of follicle arrest including hyperandrogenic condition, hyperprolactinaemia, hypogonadotrophic-hypogonadism and premature ovarian failure, there is no regular expression of a positive feedback effect of steroid hormones (Messinis 2006). To change the abnormal feedback system, treatment with various pharmaceutical compounds is available containing ethinylestradiol, progestagen and GnRH agonist / antagonist. However, it has not been clarified what the specific role and mechanism is each of the steroids and GnRH.

The primary action of P4 is to maintain menstrual cycle, and it also initiates and maintains pregnancy. P4 maintains the uterus in a quiescent state by inhibiting myometrial contractility. It also facilitates the LH surge, transforms the endometrium to a secretory from a proliferative state and maintains endometrial integrity with E2 (Spitz 2003). Therefore, it is not

1 1 2 surprising that P4 or PR modulators including RU486 have clinical application in medical termination of pregnancy, in producing cervical softening, and in menstrual regulation. PR and

ER modulators together with GnRH analogues are considered for clinical application in premenopausal women. PR modulators may display antiproliferative effects in the endometrium.

They may suppress E2-dependent endometrial proliferation and mitotic activity, secretory activity and reduce endometrial thickness and wet weight (Slayden et al. 1998; Baird et al. 2003).

For these reasons, PR modulators have application in the treatment of uterine myoma and endometriosis. Long-acting GnRH analogs are also generally used in the medical treatment of endometriosis and uterine myoma (Spitz 2003).

Many tumors, both benign and malignant, are steroid-dependent, and endocrine treatments work best in women whose tumors are positive for ER and PR (Pritchard 2005). The expression and ratios of PR A and PR B isoforms vary between normal and malignant tissues. In breast cancer cells with PR A or PR B, P4 stimulated gene regulation in a PR isoform-specific manner. The majority of the genes were regulated by PR B, with smaller subsets regulated by PR

A or both isoforms (Richer et al. 2002; Jacobsen et al. 2005). It has recently been demonstrated

(Jacobsen et al. 2005) that PR gene regulation in vitro is further differentiated by whether the receptor is bound by the ligand or not. Most of the genes are regulated by PR A in the unliganded state. Because the PR A/PR B ratio can vary in different physiological and pathological situations, the ultimate response to the ligand may be determined by concentration of the specific isoform (Mote et al. 2002; Sartorius et al. 2003). Recently, the development of

GnRH analogues has re-awakened interest and they have been shown to be effective in the treatment of metastatic breast cancer (Taylor et al. 1998). In many tumors ER and PR may predict response to endocrine therapy, and the PR have cross-talk with ER and growth factors

(Pritchard 2005). However, the mechanisms between PR and other signaling pathways including 1 1 3 GnRH and growth factors have not been studied in pituitary and extrapituitary tissues and need to be addressed to understand more effective endocrine therapy.

In these studies, we investigated the distinct mechanisms of PR isoforms in the absence or presence of P4 in neuroendocrine cells and ligand-independent PR transactivation by GnRHs.

The suggested mechanisms of GnRH-induced PR transactivation in these studies are important to understand negative and positive feedback effects of P4 in the reproductive system. Moreover, the involvement of SRC-3 coactivator for the ligand-independent PR activation suggests a noble mechanism for interactions between PR and other signaling pathways in pituitary and extrapituitary tissues.

114 5. Summary and Future Studies

5.1 Summary

5.1.1 Different regulation of GnRH system by PR isoforms

In this study, functional roles of PR A and B on GnRH I, GnRH II, and GnRH I R gene regulation were investigated in human neuronal cells.

1. P4 reduced promoter activity of GnRH I R. The effect of P4 on GnRH I R promoter

activity was blocked by RU486. This demonstrated that P4 regulates GnRH I R

transcription by PR. Dominant expressing isoform of PRs in these cells was PR A and it

mediated the repression of GnRH I R transcription following treatment with P4. In

contrast, transfection with PR B expressing vector reversed PR A-mediated effects of P4

on GnRH I R gene expression.

2. Treatment with P4 induced GnRH I mRNA levels, and this was reduced by RU486. P4

treatment did not have significant effect on GnRH II gene. Induction of GnRH I gene

expression was mediated by PR B, while PR A did not functionally affect the gene

regulation in this system.

3. P4 did not significantly affect PRE-luciferase gene activity. Under induction of each PR

isoforms by transfection with over-expression vectors, PR B increased transcriptional

activity of the reporter gene, while PR A decreased it.

1 1 5 5.1.2 Ligand-independent activation of the PR by GnRHs

In this study, transactivation of PR by GnRH I and GnRH II was evaluated. The mechanism of GnRHs in the modification of PR and recruitment of coactivators, in turn, PR transactivation on the promoter level was examined.

1. GnRH I and II activated PR-mediated transcription in aT3-l cells in the absence of or

presence of P4. PKC, PKA and GnRH I R inhibitors completely blocked GnRHs-induced

PR transactivation, while RU486 did not.

2. GnRH I and GnRH II phosphorylated PR, mainly B, at Ser294. Treatment with GnRHs in

these cells rapidly translocated PRs into nucleus from cytoplasm. Phosphorylation and

subcellular redistribution of PR may suggest that GnRHs phosphorylate PR at Ser294 and

this accumulate PR into the nucleus.

3. PR interaction with SRC-3 coactivator was enhanced by GnRHs and P4. At the

chromatin level, GnRHs promoted PR recruitment to the PRE luciferase reporter

promoter similar with that observed after treatment with P4. GnRH I and GnRH II also

recruited SRC-3 to the PRE promoter, but P4 did not.

4. Co-transfection of PRE-luciferase gene with siRNA for GnRH I R substantially

decreased GnRHs-induced PR transactivation, which did not affect P4 action. The loss of

SRC-3 by siRNA transfection has much more impact on the ligand-independent effects of

the GnRHs, as compared to the P4 dependent transactivation of the PR.

1 1 6 5.1.3 PR mediates GnRH-induced FSHP gene transcription via a ligand- independent transactivation

In this study, transcriptional regulation of gonadotropin subunit genes by GnRHs was examined in mouse pituitary cell lines. The involvement of PR activation in GnRH-induced gene expression was investigated in the absence and presence of ligand.

1. GnRH I activated PR-mediated transcription in pituitary cells. In E2-priming conditions,

GnRH I-induced PR transactivation was much greater in 0.T3-1, but not in LBT2 cells.

Expression of the PR was substantially induced in E2-priming conditions only in aT3-l

cells.

2. Treatment with GnRH I and GnRH II resulted in increases in a-GSU and FSHB mRNA

levels, which was not affected by co-treatment with P4 or E2.

3. PKC and PKA inhibitors blocked GnRH I-induced PR transactivation, whereas RU486

did not. PKC and PKA inhibitors reduced the increases in FSHP mRNA levels induced

by GnRH I, while only the PKC inhibitor decreased GnRH I-induced a-GSU gene

expression.

4. GnRH I-mediated increases in FSHP gene expression were reduced when siRNA for PR

was transfected. However, knock-down of PR did not affect GnRH I-mediated a-GSU

1 1 7 transcription.

Taken together, these results demonstrate that PR A and PR B have distinct mechanisms of transactivation in the absence or presence of P4, therefore regulate target genes by ligand- dependent or ligand-independent manner in the neuroendocrine system. P4-mediated PR A activation reduces GnRH I R promoter activity, while PR B results in an increase on GnRH I mRNA levels. Alternatively, in the absence of P4, PR is phosphorylated at Ser294 by GnRHs and translocated to the nucleus. In the nucleus, GnRH-activated PR interacts with coactivators including SRC-3 and then binds PRE. GnRHs-induced PR transactivation thereby regulate

FSH{3 gene in a ligand-independent manner (Fig. 31).

1 1 8 Figure 31. Proposed cross-talk between PR and the GnRH system in pituitary cells.

1 1 9 5.2 Future studies

1. Interactions of PR A or PR B with various coactivators after GnRH I treatment.

Future experiments need to examine whether or not PR A and PR B have differing

preferences to interact with various coactivators, and how treatment with GnRH I might

modulate these interactions. The precise mechanism of GnRH I-induced PRs

modification also need to be elucidated.

2. Recruitment of PR A or PR B to GnRH I target gene promoters including FSHJB, in vivo

and in vitro system

Recruitment of PR isoforms to the PRE-reporter and FSHP promoter should be

examined. In these studies, the mechanism of GnRH I-induced FSHB gene expression

and recruitment of PR A or PR B to the promoter should be investigated. Functional

GnRH I-stimulated PR response elements within the FSH|3 promoter should be

identified and examined for the response in the absence or presence of P4.

3. Ligand-independent PR transactivation in a cell and tissue specific fashion

Since ligand-independent activation of PR is controversial, especially in humans,

future studies need to be done to evaluate the activation of PR in the absence of P4.

1 2 0 Signaling pathways in PR post-translational modifications and its subcellular localization, and recruitment of coactivators or corepressors should be considered. Chromatin remodeling associated with unliganded PR and coregulators should also be studied.

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1 3 7 7. APPENDICES

7.1 Identification of PRE-like sequence in murine FSHj3 promoter

Murine FSH/3 promoter has been analyzed to identify PRE-like sequence. It has been reported that there are five PRE-like regions in rat FSH/3 promoter (Fig. 32). In electrophoretic mobility-shift assay, they demonstrated that PRE-like sequences 1, 2 and 3 were binding to the

PR. When it was analyzed, one PRE-like region is highly conserved between rat and murine

FSH/3 promoter (Fig. 33). Sequence homology has revealed that the PRE-like sequence is 92 % homologous to the consensus PRE

1 3 8 ! >I563 -866 CACATTCCCC TCACATTTTT MOGOtGAT CCl'ltSmtC AAACCACACT AVTAA1TAAT -806 GGATATTACA AAITAATAAA TtJAAITAAAC AACCTCMTC TCTATGGAGT TCATAQAATA -746 CTTAACA.AAT CGGTATATGA ATAACTACAA TCAGAAAGTA GAGATCATAA CrCTCClTAA -686 CTTTCGCCCT AAATTACACG 1GAAACAGCT CTG'J'36'l

-446 AAAGACACAO CCCATAGUAA TAAGCnfiCAC AAGTGTFTOC TATCTGTTCA TCCACTTCGA ; _>289

-386 (ymTTCAOTC TPTTCTltjCA CCAGTCAATT AGACATATTT TCGTGTCCTT TCTCAGTGGA

i-27-1 -326 GCCAAAGCAA TGCTCAGAAA GGATTCCGAG TTCTICAAGT TAAAGATGAC AkAGAACAGT

*-260 PRE #2 -266 CTAGACBZCA GAOGCACATC TAATITAGCA AGGTCMCGA OTRCCaW5C 'IT5CCATATCA

*-i7S *-16l ME #3 -206 GATrocwrrr GEVCAOAAAC ovnovimcr hAc^trxtrr TCTPCTCTGT orovnw;«

-HG CTCCnTGGC GAGGCTTGAT C1XXCTCTCT Cnt'l'AAACAA TGATCCCCIT TOViCCSOCi* 289 < — : y(51< ; TATA box - 86 TKiTBTrGGT ATTCCTCACC TTAACAOCGA CTAAATCCAT GGCGTGTTAG GATltTTAT.AA ) tituiacriptioii start site I - 26 AAGATCAGGT GTOACTTOAT TGAGTCTTCA GCriTDOCCA GOAGAdA'Mr rXAACKKMC + 35 AGGTGAGrrrr CAGCACITCKJ TOCAGCAAGO ACAGCTACTC 'IW1AGATTGC AGA1CTTTGG + 95 (JAtTTAAAGA AGGGATCAGA CGTCTCTGTC AGTGATTCTC TOTCACTGTA TAGAAACTJ'A + 1 r.f. CltrPTGCTAA CTTGGCT7CT TGTATGTGGT CAATOTOCTC TrATGAAGAA MCiXJAC-l'IAC +215 AGAGCAGlTr GOCTAGCTCC TCAGAnVifiAC TTTFCATCAA CAOATGTAGA CAAAG'AGAAA +275 CAGACGTTTA AATTGTGATA ACAAAGAOGC AA1TACAACG TGTAAA'mT AAACAAACTA +335 CTCTTTAGAA AACTGTTAAT GAATTOCAAA A'IYITAAGGAT CAC/UVPPATC TGATTAGAGT

M438 PHI! +395 AATGACTTTA AGATAGTAAT AA'nXXATCT (ffAAATACTG C77TOGTTAA ATGGTCATAC

+455 TGGAGTTrro ATCTATTTGA AVCTACTITA CTCTACCATA GTOGCATCCC ACAO2ACA0C +510 ACACCAAACA ATGAGGATTA TTGGTCCATO AAGCAAAAAG TAAAAAAAAA AAiAAAAAAT +575 CAGTXXrnGA GGAGAATCJ1T TOMSAAAGAT GAflTCGGTTA ACTOCTTCTC AACTCGTCAG

M-635 1+649 PRE *5 +635 TTTFCACrGT Cr?77TA'ITI' GATITOICTA TrGCTAGCAG GAGATAGCTG TITJACTTACC

+695 TGGCGATGAT GAAGTCCATC CWQCWtCCA fCCTACTCIO (rhXTKiAGA GCAftTCTGCT lS63< J +700 txunu. 8 Carl; site

+755 GCCATAGCTC IXiAACTCACC AACOTCACCA TCTCAGTAGA GAAGGAAGAG TCCCG

Figure 32. The promoter sequence of the rat FSH-/3 gene. Positions indicated are in reference to the transcription start site (position +1). PRE-like sequence 1 is 86 % homologous to the consensus GRE. PRE-like sequences 2 and 3 are 80 % homologous to the consensus PRE, and

PRE-like sequences 4 and 5 are 80 % homologous to the PRE

1 3 9 -611 AGGGCATTGG TGACAGAGAG GACATCACAT GCAGAGATCT GGAGGAACCC -511 ATCAGTATCA TAATTAGGGA ATATTTAGGG AATTACAATT TCTGATGCTC -461 TTCACAAAGC ATCAGAAAAA GGGGGGTTGA GATCAGGAGA ACTGAATGTG -411 GTCATAAAGA AAGACACAGC CCATAGGAAC AAGATGCAGA AGTACTTCCT

*PRE-like sequence -361 ATTTGTTCAT ACACTTGGAG TGTTCAGTCT GTTCTTGGAT CAATTAAGAC -311 ATATTTTGGT TTACCTTCGC AATGGAGCCA AAGCAATGTT CAGAAAGGAT -261 TCTGAGTTCG CCAAGTTAAA GATCAGAAAG AATAGTCTAG ACTCTAGAGT -211 CACATTTAAT TTACAAGGTG AGGGAGTGGG TGTGCTGCCA TATCAGATTC -161 GGTTTGTACA GAAACCATCA TCACTGATAG CATTTTCTGC TCTGTGGCAT -111 TTAGACTGCT TTGGCGAGGC TTGATCTCCC TGTCCGTCTA AACAATGATT - 61 CCCTTTCAGC AGGCTTTATG TTGGTATTGG TCATGTTAAC ACCCAGTAAA

+1 (transcription start site) - 11 TCCACAGGGT GTTCAGCTTT CCCCAGAAGA GACAGCTGAC TGCACAGGTG + 48 AGTAGCAGCA CTTGATGCAA CAAGGACAGC CACTTTGAAA ATTGCAGACC + 98 ATTAAGGATT TAAAGAAGGG ATAGGAGTTTT CTGCCGCTG CTGTGTAGAA +148 ACTTATTCTT GTTAACTTGG CTTCTTGAAT ATGGTCAATG TACAGTTATA +198 AGGAATCTGA CTTATAAAGC AGTTTGCCTA GCTTCTGACA TAGACTCTTT

Figure 33. Identification of PRE-like sequences in murine FSH-/3 promoter The 858 bp sequence spanned positions -611 to +247 of sequence of the mouse FSHP gene. Position of PRE-like sequence is indicated in reference to the transcription start site (position +1). Sequence homology has revealed that PRE-like sequence is 92 % homologous to the consensus PRE.

6.2 The effects of gonadotropins on upstream of GnRH II gene promoter

1 4 0 6.2.1 cAMP-induced promoter activity of GnRH II gene

To examine the transcriptional regulation of human GnRH II gene in different cells, human placenta JEG-3, human neuronal TE-671, human ovarian OVCAR-3, and mouse pituitary

QT3-1 cells were transiently transfected with a full-length human GnRH II promoter-luciferase construct (p2300-LucF). Since GnRH II gene has been reported to be regulated by cAMP in TE-

671 cells, transcriptional regulation of GnRH II was first tested by cAMP in the cells including

TE-671 cells (Fig. 34). After transfection, the cells were treated with cAMP in a time dependent manner, and the promoter activity of GnRH II was examined. In JEG-3, TE-671 and 0.T3-1 cells, the promoter activity of GnRH II gene was increased and maximized at 24 h; however, it showed maximal effect at 2 h and decreased in OVCAR-3 cells.

Figure 34. cAMP induces GnRH II promoter activity. The full-length human GnRH promoter-luciferase construct (p2300-LucF) was transiently transfected into JEG3 (A), TE-671

(B), OVCAR-3 (C) and aT3 (D) cells by FuGENE 6.0 reagent. After 2 d in DMEM, the cells were treated with cAMP (10~5 M) in a time dependent manner. The RSV-/acZ vector was also cotransfected in order to normalize the transfection efficiency. The relative promoter activity is

14 1 represented as luciferase activity/13-galactosidase activity.

1 4 2 B

>,600 ^200 n =o 500 '> CD Ti 05400 CO o o §300 100 oi_ . iI = 200 CL X X c 100 or

Control 2h 4h 8h 16h 24h Control 2h 4h 8h 16h 24h

D

300 i 350 >» ;> > 300 Tj ro CO 250 200 H £o o E E 200 o o

-100 Hr= Q.150 X CC | 100 c CD O 50 ^ T- Control 2h 4h r- 16h 24h Control 2h 16h 24h

1 4 3 6.2.2 LH increased transcriptional activity of GnRH II gene in OVCAR-3

and QT3-1 cells within 8h.

Since gonadotropins activate PKA pathway, the effects of LH and FSH on GnRH II transcription levels were further tested (Fig. 34). All four cell lines were transiently cotransfected a full length GnRH II promoter plasmid with LH or FSH receptor, and the cells were treated with

LH or FSH for 8 and 24 h. In JEG-3 and TE-671 cells, LH and FSH did not affect GnRH II promoter activity, while LH induced it in OVCAR-3 and 0.T3-1 cells within 8 h.

Figure 35. Effects of LH and FSH on the promoter activity of GnRH II gene. The full-length human GnRH promoter-luciferase construct (p2300-LucF) was transiently transfected into JEG3

1 4 4 (A), TE-671 (B), OVCAR-3 (C) and o;T3 (D) cells by FuGENE 6.0 reagent. After 2 d in DMEM the cells were treated with LH or FSH (100 nM) (8 or 24 h). The RSV-/acZ vector was also cotransfected in order to normalize the transfection efficiency. The relative promoter activity is represented as luciferase activity/p-galactosidase activity.

14 5 GnRH II promoter activity g g g v ' GnRH II promoter activity 6.2.3 cAMP phosphorylated CREB at Ser 133

To study biological pathway of LH on GnRH II promoter, phosphorylation was next

tested in CREB protein that has been known to response to cAMP (Fig. 35). The expression of

CREB was examined. All of the cells expressed CREB but the levels of it were not changed by

cAMP in any time points. However, cAMP induced phosphorylation of CREB. In JEG-3,

OVCAR-3 and 0.T3-1 cells it phosphorylated the CREB from 2 h and maintained up to 24 h. In

the case of TE-671 cells, it increased phosphorylation of the CREB from 2 h and showed

maximum effects at 4 h.

Figure 36. Regulation of CREB phosphorylation at Ser 133 by cAMP. The JEG3 (A), TE-

671 (B), OVCAR-3 (C) and aT3 (D) cells expressing total CREB were treated with LH for 2 to

24 h. Equal amounts of cell lysates (100 pg) were electrophoresed on SDS-7 % PAGE gels,

1 4 7 transferred to nitrocellulose, and blotted with antibodies specific for CREB phospho-Ser 133.

Western blotting was performed to detect phospho-Ser 133 CREB (upper panel) or total CREB

(lower panel)

1 4 8 A

p-CREB

CREB

c

p-CREB

CREB

1 4 9