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Cross-talk between gonadotropin-releasing hormones and progesterone receptor in neuroendocrine cells An, Beum-Soo 2007

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CROSS-TALK BETWEEN GONADOTROPIN-RELEASING HORMONES AND PROGESTERONE RECEPTOR IN NEUROENDOCRINE CELLS by B E U M - S O O A N D . V . M . , Chung-buk National University, 2000 M . S c , Chung-buk National University, 2002 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES (Reproductive & Developmental Sciences) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A 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 G n R H I, G n R H II (a second form of GnRH) and G n R H I receptor ( G n R H 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 G n R H I R promoter activity. This P4-stimulated inhibition was enhanced when P R A was over-expressed. With respect to the two GnRHs, P4 increased G n R H I m R N A levels, but did not significantly affect G n R H II gene expression. Regulation of gonadotropin production involves interplay between steroids and neuro-peptides, thus we have examined the effects of GnRHs on P R activation in pituitary cells. Treatment with GnRHs increased a progesterone response element (PRE)-luciferase reporter gene activity. P R was phosphorylated at Ser294 and translocated into nucleus after G n R H treatment in the absence of P4. Interactions between the P R 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 P R and SRC-3 to the P R E reporter gene was also increased by GnRHs. The knockdown of G n R H I R and SRC-3 levels by s i R N A treatment reduced GnRH-induced P R transactivation. Gonadotropin subunit gene expression was evaluated following treatment with GnRHs, and common a-subunit and FSHjS transcription were upregulated by GnRHs. We used s i R N A for P R to examine the involvement of P R in G n R H I-induced FSH/3 gene expression. The effect of G n R H I on FSH/3, but not a-subunit gene expression was reduced when s i R N A targeting P R was introduced. In summary, these results indicate that P4 is a potent regulator of G n R H I R and G n R H I at ii the transcriptional level, and this distinct effect of P4 on the G n R H system may be derived from the differential action of P R A or P R B . Conversely, GnRHs can activate PR-mediated transcription in the absence of P4, and this ligand-independent mechanism of P R additionally regulates FSH/3 subunit gene expression. in TABLE OF CONTENTS A B S T R A C T i i T A B L E O F C O N T E N T S iv LIST O F T A B L E S v i i i LIST O F FIGURES v i i i LIST O F ABBREVIATIONS x i 1. INTRODUCTION 1 1.1 The hypothalamic-pituitary gonadal axis 1 1.2 G n R H I and its receptor 4 1.3 G n R H I R-induced signaling 7 1.4 G n R H II and its receptor 10 1.5 Regulation of the G n R H system 13 1.6 Classification and structure of nuclear receptor superfamily 14 1.7 Structure and mechanism of action of P R 18 1.8 Phosphorylation of P R 22 1.9 Interactions between P R and coregulators 25 1.10 Transactivation of P R in the absence of P4 27 1.11 Transactivation of P R by G n R H I in the absence of P4 30 1.12 Pituitary gonadotropin hormones 30 1.13 Regulation of gonadotropin subunit genes 31 1.13.1 G n R H regulation of common a-subunit ( q - G S U ) gene expression.... 31 iv 1.13.2 GnPvH regulation of LH/3-subunit gene expression 32 1.13.3 G n R H regulation of F S H /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. M A T E R I A L S AND M E T H O D S 35 2.1. Materials 35 2.2 Ce l l cultures 35 2.3 Plasmids 35 2.4 PPvE-luciferase reporter gene assays 36 2.5 Transient transfection of G n R H I R promoter and over-expressing vectors for P R isoforms 37 2.6 In vitro transfection with small interference R N A s 38 2.7 Western blot analysis 39 2.8 Immunoprecipitation 40 2.9 Immunocytochemistry 41 2.10 R N A extraction and reverse transcriptase-PCR 41 2.11 Real-time R T - P C R 42 2.12 Chromatin immunoprecipitation (ChIP) Assay 45 2.13 Statistical analysis 46 3. RESULTS 47 3.1 Regulation of the G n R H system by P4 47 3.1.1 P4 regulates human G n R H I R promoter activity 47 v 3.1.2 P R A but not P R B mediates P4 induced repression of G n R H I R promoter... ! 50 3.1.3 Over-expression of P R A or P R B has distinct effects on P R E promoter activity 50 3.1.4 Effects of P4 on human G n R H I and G n R H II i n R N A levels 54 3.1.5 Effects of P R A and P R B on human G n R H I and G n R H II r n R N A levels 58 Ligand-independent activation of P R by GnRHs 61 3.2.1 Transactivation of P R by G n R H I and G n R H II in o/T3-l cells 61 3.2.2 Treatment with GnRHs affects P R phosphorylation 65 3.2.3 Treatment with GnRHs affects P R sub-cellular distribution 71 3.2.4 Interaction between SRC-3 and P R increases in a T 3 - l cell after treatment with GnRHs or P4 73 3.2.5 Recruitment of P R and SRC-3 to PREs is promoted by GnRHs 76 3.2.6 G n R H I R and SRC-3 are required for GnRH-mediated P R activation 78 3 GnRH-induced FSH/3 subunit gene transcription involves the ligand-independent transactivation of P R 84 3.3.1 Transactivation of P R by G n R H I in o T 3 - l and L(8T2 cells 84 3.3.2 Transcriptional regulation of gonadotropin subunit genes by G n R H I and G n R H II in pituitary cells 87 3.3.3 Effects of signaling pathway inhibitors on G n R H I-induced trans-activation of P R and gene expression of gonadotropin subunits 93 3.3.4 P R mediates G n R H I-induced FSH{3 gene expression 99 vi 4. DISCUSSION 102 4.1 Regulation of the G n R H system by P4 102 4.2 Ligand-independent activation of P R by GnRHs 106 4.3 Ligand-independent transactivation of P R mediates GnRH-induced FSH(3 subunit gene transcription 109 4.4 Clinical implications I l l 5. S U M M A R Y AND F U T U R E STUDIES 115 5.1 Summary 115 5.1.1 Different regulation of G n R H system by P R isoforms 115 5.1.2 Ligand-independent activation of the P R by GnRHs 116 5.1.3 P R mediates GnRH-induced FSH(3 gene transcription via a ligand-independent transactivation 117 5.2 Future Studies 120 6. R E F E R E N C E S 122 7. APPENDICES 138 vi i LIST OF TABLES Table. 1. Primers for Real-time P C R genes i viii LIST OF FIGURES Figure 1. The hypothalamic-pituitary-gonadal axis 3 Figure 2. Two-dimensional representation of the G n R H I R 6 Figure 3. Schematic representation of G n R H I signaling in o T 3 - l , C O S 7 and DU145 cells.... 9 Figure 4. Schematic representation of the human G n R H I and G n R H II genes 11 Figure 5. Shared functional domains of the nuclear receptor superfamily and S R C / p l 6 0 family members 17 Figure 6. Structures of P R A and P R B 19 Figure 7. Model of the mechanism of action of P R in the presence of P4 21 Figure 8. Phosphorylation sites in human P R 24 Figure 9. Model of molecular mechanism of action of P R in the absence of P4 29 Figure 10. Dose- and time-dependent effects of P4 on G n R H I R promoter activity 48 Figure 11. Effects of RU486 on P4-induced G n R H I R promoter activity 49 Figure 12. Effects of P R A or P R B over-expression on G n R H I R or P R E promoter activities.... 53 Figure 13. Time- and dose-dependent effects of P4 on G n R H I m R N A levels 56 Figure 14. RU486 reverses P4-induced G n R H I m R N A levels 57 Figure 15. Effects of P4 on G n R H I and G n R H II m R N A levels after P R over-expression.... 59 ix Figure 16. Effects of G n R H I and II on PR-mediated trans-activation of a PRE-reporter gene in a T 3 - l cells 62 Figure 17. P K C and P K A 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 P R in mouse, rat and human 68 Figure 19. Regulation of P R phosphorylation at Ser294 by GnRHs 69 Figure 20. Cytoplasmic to nuclear translocation of P R in rxT3-l cells following treatments with GnRHs 72 Figure 21. Interaction between SRC-3 and P R increases in <xT3-l cells after treatment with GnRHs or P4 74 Figure 22. Recruitment of P R and SRC-3 on the PREs is promoted by GnRHs 77 Figure 23. G n R H I R mediates both G n R H I - and G n R H II-induced ligand-independent activation ol P R 79 Figure 24. SRC-3 is essential for the ligand-independent activation of P R by G n R H I and G n R H II, and the synergistic amplification of this effect by P4 80 Figure 25. Effects of G n R H I on PR-mediated transactivation of a PRE-reporter gene in 0.T3-1 and L{3T2 cells 86 Figure 26. The effects of G n R H I on a - G S U , FSH|3 and LHJ3 m R N A levels 88 Figure 27. P4 does not have synergistic effects with G n R H I and G n R H II at the level of gonadotropin subunit gene expression 91 Figure 28. P K C and P K A inhibitors, but not RU486, reduce GnRH-induced PR-mediatec transactivation of a PRE-luciferase reporter gene in <xT3-l or L/3T2 cells 94 Figure 29. Effects of P K C and P K A inhibitors on GnRH-induced gonadotropin subunit x gene expression 96 Figure 30. P R mediates G n R H I-induced FSH/3 gene expression 101 Figure 31. Proposed cross-talk between P R and the G n R H system in pituitary cells 119 X I LIST OF ABBREVIATIONS A F Activation function domain A N O V A Analysis of variance A R Androgen receptor Bp Base pare C Celcius C a 2 + Calcium c A M P Cycl ic adenosine monophosphate C B P Creb-binding protein c D N A Complementary deoxyribonucleic acid C D K Cyclin-dependent protein kinase ChIP Chromatin Immunoprecipitation D B D DNA-binding domain D M E M Dulbecco's modified eagle medium D N A Deoxyribonucleic acid D A G Diacylglycerol E2 Estradiol E C L Enhanced chemiluminescence E D T A Ethylene diaminetetraacetic acid E G F Epidermal growth factor E R Estrogen receptor E R K Extracellular signal-regulated kinase Fas L Fas ligand F B S Fetal bovine serum F S H Follicle-stimulating hormone G n R H Gonadotropin-releasing hormone G n R H a Gonadotropin-releasing hormone agonist G n R H II Gonadotropin-releasing hormone-II G n R H I R Gonadotropin-releasing hormone I receptor G n R H II R Gonadotropin-releasing hormone I receptor G-protein GTP-binding protein G P C R G-protein coupled receptors G R Glucocorticoid receptor GTFs General transcription factors xi i G T P Guanosine triphosphate H Hour Hsp Heat shock protein IP Inositol phosphate IP3 Inositol 1, 4, 5-triphosphate JDP Jun dimerization protein-2 L B D Ligand-binding domain L H Luteinizing hormone M Micro M A P K Mitogen-activated protein kinase M A P K K s (=MEK) M A P K kinases M E K 1 / 2 M A P K / E R K kinase 1/2 M i n Minutes M M P Matrix metalloproteinases M R Mineralocorticoid receptor m R N A Messenger ribonucleic acid M w Molecular weight n (as in n M ) Nano N R Nuclear receptors N C O A Nuclear receptor coactivator P4 Progesterone p (as in pM) Pico P A G E Polyacrylamide gel electrophoresis P B S Phosphatase buffered saline P C R Polymerase chain reaction PI Phosphatidylinositol P K A Protein kinase A P K C Protein kinase C P L C Phospholipase C P M S F Phenylmethylsulfonyl fluoride P R Progesterone receptor P R E Progesterone-responsive element R A R AW-trans retinoic acid receptor R S V Rous sarcoma virus R X R 9-cis retinoic acid receptor rpm Revoultions per min sec Seconds xi i i SD Standard deviation SDS Sodium dodecyl sulphate. s i R N A Small interference R N A S R C Steroid receptor coactivator T E M E D N , N , N ' , N'-tetramethylethlenediamine T R Thyroid hormone receptor Tris Tris(hydroxy methyl) aminomethane V D R Vitamin D3 receptor xiv 1. INTRODUCTION 1.1 The hypothalamic-pituitary gonadal axis Gonadotropin releasing hormone ( G n R H I) is secreted by specific neurons located in the anterior and mediobasal hypothalamus, and stimulates pituitary gonadotroph cell action (Fig. 1). G n R H I secretion is pulsatile and is correlated to the electrical activity of the G n R H secreting neurons (Knobil 1988). G n R H 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, G n R H I reaches the anterior lobe of the pituitary and the gonadotroph cells. In the gonadotroph cells, G n R H 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). G n R H I regulates the biosynthesis and secretion of L H and F S H from the pituitary gonadotropes. L H and F S H 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 L H and F S H secretion. It has been demonstrated that exogenous E2 is able to suppress F S H and L H 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 F S H and L H levels. This negative feedback effect of steroids also controls G n R H secretion. The frequency of 1 G n R H 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 G n R H self-priming effect (Lasley et al. 1975). The response to the second G n R H pulse is greater than the response to the first pulse and is called the self-priming effect of G n R H 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 G n R H and in that way facilitates the positive effect of E2. Even, in the absence of P4, G n R H self-potentiation requires a cross-talk with the progesterone receptor (PR) (Waring and Turgeon 1992). The interaction between steroid hormones and G n R H and the self-priming effect of G n R H are important for the expression o f the endogenous gonadotrophin surge at midcycle (Messinis 2006); however, the mechanism of GnRH-induced P R activation is still unknown. The level of the hormones is various depending on stages of the cycle. The level of G n R H 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, F S H and L H is 2-40 nmol/L, 200-1300 pmol/L, 2-15 U / L and 2-40 U / L (Groome et al. 1996). 2 Hypothalamus^^) G n R H V ( ^ P i t a Gonadotropins Ovary Steroid Hormones Figure 1. The hypothalamic-pituitary-gonadal axis. Secretion of G n R H occurs in a pulsatile fashion and control synthesis and release of F S H and L H 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 G n R H I plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins such as F S H and L H from the anterior pituitary. The amino acid sequence of G n R H I was reported in the 1970s (Matsuo et al. 1971; Burgus et al. 1972). The expression levels of G n R H I and G n R H receptors are considered to be important for gonadal steroidogenesis and maintenance of pregnancy, and efforts have focused on their molecular biology. L o w doses of synthetic G n R H 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 (Mil lar et al. 2004). However, high doses of G n R H I or agonist analogs desensitize the gonadotrope resulting in a decrease in L H and F S H and reduced ovarian and testicular function (Mil lar et al. 2004). Initially the structure of the mammalian G n R H I receptor ( G n R H I R) was determined from an immortalized murine gonadotrope cell line (oT3-l) (Reinhart et al. 1992; Tsutsumi et al. 1992). The G n R H 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 G n R H I to its receptor has been extensively studied (Mil lar et al. 2004). Intracellular signalling of the mammalian G n R H 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 G P C R s (Reinhart et al. 1992) (Fig. 2). The G P C R s can activate protein kinase C (PKC) and various downstream signal transduction cascades, including the mitogen-activated protein kinase pathways ( M A P K ) (Harris et al. 1997). The activation of the P K C pathway has been well documented in response to G n R H I 4 stimulation and this induces the phosphorylation of M A P K , which may participate in gonadotropin release or synthesis in pituitary cells (Shacham et al. 2001). In the pituitary, G n R H 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 G n R H 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 Receptor coo Intracellular 328 aa Figure 2. Two-dimensional representation of the G n R H I R. Note that the absence of a carboxy-terminal cytoplasmic tail in the mammalian G n R H I R is the most unique feature of this G P C R . 6 1. 3 GnRH I R-induced signaling After G n R H I binds to cell surface receptors, G n R H receptors become internalized and enter a degradation pathway involving lysosomes and/ or undergo receptor recycling (Hazum and Conn 1988). Binding of G n R H I to the G n R H I R leads to conformational changes in the receptor. G n R H I via G n R H I R transmits extracellular signals into the intracellular milieu via heterotrimeric (a, ft and 7 subunits) GTP-binding proteins (G-proteins) (Birnbaumer 1992). The G n R H I R can be coupled to the Gotyn protein that activates phospholipase Cft leading to the stimulation of P K C and various downstream signal transduction cascades, including the M A P K pathways (Harris et al. 1997). The activation of P K C is reported as one of the most important signaling pathways in the stimulation of M A P K by G n R H I in pituitary cells (Andrews and Conn 1986; Zheng et al. 1994). The G-protein involved in the G n R H 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 ( D A G ) production (Hsieh and Martin 1992; Anderson et al. 1993). IP3 releases calcium from intracellular stores (Stojilkovic et al. 1994; Tse et al. 1997) and D A G stimulates the P K C pathway in pituitary gonadotropes. Unlike other tissues, activated P K C might be involved in the Ca influx and up-regulate G n R H receptors in the pituitary (Naor 1990). The activation of P K C by an increase in cytoplasmic C a 2 + concentration is important for mediating G n R H I action such as gonadotropin secretion in the pituitary gland. In granulosa cells, P K C pathway has been known as a major component in activating M A P K signaling from G n R H I R (Kang et al. 2001a). Activation of P K C is an important second messenger mediating G n R H I-induced M A P K stimulation in pituitary cells (Harris et al. 1997) and ovarian cancer cells (Chamson-Reig et al. 2003). 7 G n R H I R signaling activate diverse cytoplasmic proteins to transfer its signal into the nucleus, and M A P K is considered to be one of the important pathways in G n R H I signaling pathway (Naor et al. 2000; Kraus et al. 2001). M A P K cascades are activated via a variety of cell surface molecules including receptor tyrosine kinases and G P C R s . Signals transmitted through G n R H I R induced-cascades induce the activation of diverse molecules which regulate cell growth, survival and differentiation (Naor et al. 2000). G n R H 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 G n R H I R is able to couple to both G a q and G a l 1 in mice gonadotropes (Kraus et al. 2006). The signaling mechanisms mediating the activation o f M A P K s by G n R H I also seem to be significantly different in various cell types. In pituitary-derived o T 3 - l and L p T 2 cells, the G n R H I R signals are mediated via all four major M A P K cascades including M A P K , I N K , p38 M A P K and B M K 1 / E R K 5 (Naor et al. 2000; Kraus et al. 2001) (Reiss et al. 1.997; Lev i et al. 1998; Roberson et al . 1999; L i u et al . 2002b). However, in G n R H I R-expressing COS7 cells, the Geo is the main intermediate in the G n R H R to M A P K signaling pathway, but the same M A P K activation in these cells can be obtained when the receptor is coupled to G o q (Kraus et al. 2006). 8 aT3 G n R H - I i Gaq i P K C Ras T I N K ^ R a f M E K E R K P38 Gonadotropin synthesis and secretion C O S 7 E G F R Ras ^ P T 3 K ) E R K t J N K Growth arrest DU145 E G F R M M P ^ v _ _ ^ Ras P I 3 K M L K 3 E R K J N K Apoptosis Figure 3. Schematic representation of G n R H 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 G n R H exist in non-mammalian vertebrates. These G n R H 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 G n R H variants is G n R H II (also called chicken G n R H II), which is conserved in structure from fish to mammals (Fig. 4). Recently, several groups have identified G n R H II in mammalian species including human (Millar et al. 2004). In contrast to G n R H I, human G n R H II m R N A 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 G n R H II and its wide distribution in tissues suggest that this neuropeptide has vital biological functions. Although the normal physiologic function of G n R H II is poorly understood, it has been reported that G n R H 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, G n R H II has been shown to preferentially stimulate F S H release in the pituitary (Mil lar et al. 2004). 1 0 Human GnRH-l m R N A : 5.1 kb pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 Human GnRH-ll mRNA : 2.1 kb • 5'-Untranslated region 100 bp Exon 1 Exon 2 Exon 3 Exon 4 Signal sequence GnRH G A P 3'-Untranslated region pTyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- N H 2 G A P : Gonadotropin releasing hormone associated protein Figure 4. Schematic representation of the human G n R H I and G n R H II genes. Only exonic regions are drawn to scale (Adapted from White et al., 1998). 1 1 In most vertebrates, several structural variants of G n R H exist, suggesting that additional receptors for G n R H in mammals may exist. Indeed, three distinct types of G n R H receptor were identified in the bullfrog (Wang et al. 2001b) and a novel chicken pituitary G n R H receptor has been cloned (Sun et al. 2001). Ne i l l et al. observed that the G n R H II receptor ( G n R H II R) gene is present in human (Neil l et al. 2001). In mammals, G n R H II has been found to be more widely expressed than G n R H I (Mil lar et al. 1999; Kang et al. 2000; M i l l a r et al. 2001), suggesting that G n R H II R may have other functions. Regarding the role of G n R H I and II receptors, there are discrepancies among previous reports. It has been suggested that the signal transduction pathways coupled to the G n R H II R may be different from those triggered by activation of the G n R H I R (Mil lar et al. 2001; Ne i l l et al. 2001). Enomoto et al. showed that G n R H II R is necessary to mediate the effect of G n R H II (Enomoto et al. 2004) and Grundker et al. reported that the anti-proliferative effect induced by G n R H II is not mediated through G n R H I R (Grundker et al. 2004). However, a functional human G n R H II R protein has not been verified (Grundker et al. 2002), since the identified human G n R H 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 G n R H II R in mediating the effects of G n R H I and II remains obscure. In addition, a recent study showed that G n R H II R inhibits the expression of G n R H I R, indicating that G n R H I R may be a common receptor that mediates the effects of both G n R H I and G n R H 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 G n R H I, G n R H II and G n R H 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 G n R H I R m R N A 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 G n R H I R gene. It is established that gonadal steroids can influence G n R H 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 G n R H I. For the greater part of the ovarian cycle, E2 restrains the G n R H I and L H 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). A n elevation of P4 in the luteal phase inhibits a pulsatile secretion of G n R H I and L H (Goodman and Karsch 1980; Karsch et al. 1987; O'Byrne et al. 1991). This prevents the occurrence of G n R H I (Kasa-Vubu et al. 1992) and L H (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 G n R H I secretion through a negative feedback mechanism, this scenario of P4-induced gene regulation for G n R H I and G n R H I R is still controversial, and the regulation of G n R H II by P4 is virtually unknown. 1 3 Several studies have demonstrated that the expression of the human G n R H I and G n R H II genes are regulated in different ways. The transcription of the G n R H II gene is strongly up-regulated by c A M P , which only induces a modest stimulation of the G n R H I promoter activity in neuronal medulloblastoma cells. This cAMP-stimulated G n R H 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 m R N A levels of G n R H 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 m R N A levels of the G n R H II but decreases G n R H I gene expression (Chen et al. 2002b). Together with their distinct tissue expression patterns, these observations indicate that the two forms of G n R H play distinct biological roles in humans. 1. 6 Classification and structure of nuclear receptor superfamily In these studies, the projects focused on P R and G n R H system. The P R 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 ( A R ) , glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) , whereas type II receptors include thyroid hormone receptor (TR), all-trans retinoic acid receptor ( R A R ) , 9-cis retinoic acid receptor ( R X R ) , and vitamin D3 receptor ( V D R ) . 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 D N A 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-bind ing domain ( D B D ) (C); a hinge region (D) (Fig. 5); and a carboxy-terminal ligand-binding domain ( L B D ) (E). Mutational analysis of the E domain led to the designation of a second activation function, A F - 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 E R , G R , M R , A R , and PR), and dimerization and apposition of the nuclear translocated receptor to its D N A 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 S R C / p l 6 0 family members. (Top) General structure of NRs ; A F - 1 is embedded in the N terminus of type I N R s and A F - 2 is found in the C terminus of all N R s . (Bottom) General structure of the S R C / p l 6 0 family. The C B P interaction domain and the C A R M 1 interaction domain overlap with the transferable activation domains 1 and 2 of the S R C / p l 6 0 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 P R 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 P R that expressed as two isoforms, P R - A and P R - 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 P R B and N-terminus truncated P R A have highly conserved D N A and ligand L B D s . Both isoforms have a similar architecture composed of the ligand-dependent A F - 2 present in the carboxyl terminus, and A F - 1 , a transcription domain present in the amino terminus (Leonhardt and Edwards 2002) (Fig. 6). The constitutive A F - 1 can function independently of A F - 2 or with A F - 2 in a ligand-dependent manner. The A F - 3 domain, is only located in the upstream sequence region of P R B isoform (Pratt and Toft 1997; Leonhardt and Edwards 2002) and composed of approximately 164 amino acids. 1 8 AF3 I 165 AF1 1 AF2 556 642 1 933 P R - B DBD B LBD P R - A DBD B LBD Figure 6. Structures of P R A and P R B . Domain organization of the human PR A and B isoforms, H , hinge region; L D B , ligand binding domain; D B D , D N A 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 D N A 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 P R undergo conformational modifications to accommodate the ligand. The P4-induced changes in P R 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 P R 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 P R action in the presence of P4. In the absence o f 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 P R and binding to P R E . P4 and other signaling pathways phosphorylate PR and enhance P R transactivation. Other proteins including coactivators and GTFs bind to the D N A 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 P R action. L ike other nuclear receptor family members, P R 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 o f 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 M A P K (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 ( C D K ) 2 complexes in vitro (Zhang et al. 1997; Knotts et al. 2001). Although the role of P R phosphorylation is not fully understood, it may influence aspects of transcriptional regulation such as interaction with coregulators, as has been found for E R (Font de Mora and Brown 2000) and recently for P R (Lange 2004). Regulation of ligand-dependent (Shen et al. 2001) and -independent (Labriola et al. 2003) P R 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 m R N A or protein levels for a number of growth-regulatory genes (Richer et al. 1998) including cyclin D l and cyclin E (Haslam et al. 1993); the regulation o f cyclins by P4 is M A P K dependent. Cyclins, in turn, regulate progression of cells through the cell cycle by interaction with C D K s . P4 2 2 activates C D K 2 (Groshong et al. 1997), and PRs are predominantly phosphorylated by C D K 2 at proline-directed sites (Zhang et al. 1997; Knotts et al. 2001), perhaps allowing for the coordinated regulation of P R action during cell cycle progression. The Ser294 site of P R is a ligand-inducible phosphorylation site, becoming rapidly phosphorylated upon exposure to hormone (Zhang et al. 1995). Recently, MAPK-dependent P R phosphorylation at Ser294 has been shown to be required for nuclear translocation of unliganded PR, suggesting that M A P K signaling may regulate P R action by altering nucleo-cytoplasmic shuttling (Qiu et al. 2003). Phosphorylation of P R at Ser294 by M A P K increases transcriptional activity o f liganded P R in PRE-containing promoters (Shen et al. 2001). Interestingly, liganded mutant Ser294A P R is a weak transcriptional activator when stably expressed in breast cancer cells, and does not undergo synergistic regulation in response to agents that activate M A P K (Shen et al. 2001). Phosphorylation of Ser294 may also mediate some aspects of ligand-independent P R action. Growth factors such as E G F lead to phosphorylation of the Ser294 site within 5 min in the absence of P4. When Ser294 becomes phosphorylated, rapid nuclear accumulation of P R occurs, as measured by both fluorescence microscopy of intact cells and cellular fractionation experiments (Qiu et al. 2003). Mutation of the consensus M A P K site, Ser294 to A l a (Ser294A), abolished EGF-mediated translocation; however, the ability of progestin (R5020) to induce nuclear localization of Ser294A P R was unaffected (Lange 2004). EGF-induced nuclear accumulation requires p42/p44 M A P K activation and phosphorylation of Ser294, and occurs independently of progestin, suggesting a mechanism for ligand-independent transcriptional activation of P R (Lange 2004). 2 3 Hormone-dependent 51103(102, 294, 345) hPR AF1 H Basal sites (81,162,190, 400) HBD AF2 <D MAPK consensus sites (20, 294, 345) Casein kinase II site (81) if CDK2 sites (25, 162, 190, 213, 400, 430, 554, 676) * Unknown kinases (102, 130) Figure 8. Phosphorylation sites in human PR. Fourteen residues in human P R have been shown to represent basal (constitutive) and hormone-induced phosphorylation sites and may contribute to P R regulation by M A P K , casein kinase II, and C D K 2 . Individual P R phosphorylation sites may be regulated by multiple protein kinases and/or in a sequential manner, illustrating the complexity of P R regulation by phosphorylation (Adapted from (Lange 2004)). 2 4 1. 9 Interactions between PR and coregulators After binding of P R 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 ( S R C / N C o A ) 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 N R box in coactivators (Heery et al. 1997) (Fig. 5). These factors include the S R C / p l 6 0 / N C o A family, creb-binding protein (CBP)/P300 (Xu and O'Malley 2002), C I A , A S C - 2 / T R B P / A I B 3 / RAP250 /PRIP /NCR, and PBP/DRIP205/TRAP220 ( L i and O'Malley 2003). The S R C family is composed of three distinct but structurally related members: S R C - 1 , SRC-2 (NCoA2/TIF-2/GRIP1), and SRC-3 ( N C o A 3 / p / C I P / R A C 3 / A C T R / T R A M - l / A I B l ) (McKenna et al. 1999). A different group of coactivators is specialized for interaction with the D B D of receptors. The D B D of P R is required for binding to specific P R E sequences, but much less is known about the function of nuclear cofactors that bind to the D B D . This includes small nuclear R I N G finger 2 5 protein, S N U R F , GT198, a tissue-specific coactivator, and high mobility group proteins ( H M G ) . P R appears to utilize H M G - 1 or -2 proteins for high affinity interaction with D N A in vitro and for full transcription activity in vivo ( L i and O'Malley 2003). Compared with A F - 2 and D B D interacting coregulators, cofactor interactions with the AF-1 are less well characterized. A F - l s are the least conserved regions among PRs from different species and are likely associated with the differential ability of P R A and P R 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 P R are recruited in a hormone-dependent manner to a progesterone-responsive promoter in the context of chromatin in vivo. AF-1 cofactors may prompt P R function by recruiting or stabilizing other coactivators independent of the A F - 2 and S R C coactivators (Edwards et al. 2002). The recent description of P R coactivators, which influence R N A splicing, has revealed another mechanism by which P R regulates gene expression. Transcription and m R N A 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, p C A F ) , methyltransferases ( C A R M 1 , P R M T 1 ) (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 R N A 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 P R preferentially recruits S R C - 1 , SRC-3 , and C B P , 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 C B P (Rowan et al. 2000b). Acetylation of SRC-3 by p300/CBP at lysine residues adjacent to N R 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 wi l l 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 P R and other nuclear hormone receptors was initially considered to be 2 7 entirely steroid-dependent (Matkovits and Christakos 1995). However, many steroid receptors including P R have been shown to be activated in the absence of their cognate ligands by modulation of protein kinase or phosphatase (Power et al. 1991; M a n i et al. 1994) (Fig. 9). In the absence of P4, P R can be activated by signaling pathways including c A M P , 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 o f 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 A F - 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 A F - 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 (L i and O'Malley 2003). Phosphorylation of SRCs was shown to be induced in response to E G F , cytokines and increased intracellular c A M P (Wu et al. 2005). In the case of S R C - 1 , phosphorylation at T h r l l 7 9 and Se r l l85 induced by c A M P were shown to enhance both ligand-dependent and -independent activity of P R (Rowan et al. 2000b). Similary, phosphorylation of SRC-3 induced by E G F 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 P R action in the absence of P4. Growth factors, cytokines, stimulators of P K C , P K A , M A P K and C D K s , and neurotransmitters such as dopamine phosphorylated P R in the absence of P4. This activated PR binds to P R E and recruits coregulators. Coactivators including S R C family also can be phosphorylated by these signaling pathways and resulted in an increase unliganded P R activation. GnRHs activate P R 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 P R and G n R H I has been implicated in a G n R H I self-priming mechanism in the pituitary (Turgeon and Waring 1986; Waring and Turgeon 1992), which is defined as an enhanced L H secretion by pituitary gonadotropes in response to a second G n R H I stimulation (Fink 1995). This response appears to depend upon the capacity of E2 to induce P R expression in gonadotropes (Fink 1995), but it is completely absent in P R knockout mice (Chappell et al. 1999). It has therefore been suggested that activation of G n R H I R in gonadotropes prompts a signaling pathway, which ultimately activates the P R in a ligand-independent manner (Turgeon and Waring 1994). However, the mechanisms responsible for G n R H I self-priming and the ligand-independent activation of the P R 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 L H and F S H . Dynamic regulation of the pituitary L H and F S H is essential for mammalian reproduction. L H and F S H are comprised of two glycoprotein subunits, common a, and LH/3 and FSH/3 (Gharib et al. 1990). L H and F S H secreted from the pituitary gonadotropes act on the ovaries and the testes to regulate folliculogenesis, ovulation, spermatogenesis and steroidogenesis (Marshall and Kelch 1986; W u et al. 1990; Burger et al. 2004). The synthesis and secretion of the gonadotropins are primarily regulated by the hypothalamic G n R H in a pulsatile manner (Clarke and Cummins 1982). The control of L H and F S H synthesis and secretion is complex and involves interplay between the gonads, pituitary and hypothalamus. L H and F S H act on gonadal steroids, E2 and P4, and mediate positive and negative feedback 3 0 influences on L H and F S H in both the pituitary and the hypothalamus. Both hormones have been shown to contribute to positive or negative feedback effect of L H and F S H secretion, through regulation of hypothalamic G n R H 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 L H and F S H secretion can be dynamically regulated by hormones that alter their synthesis, storage and /or release. G n R H , E2, and P4 are involved in the sensitivity to gonadotropes. G n R H and combined effects with steroids are instrumental in generating the preovulatory L H surge. 1.13.1 GnRH regulation of common a-subunit (a-GSU) gene expression GnRH-responsive regions have been mapped in the a - G S U promoter of several species. In humans, cows and mice, the D N A elements that confer G n R H 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 - G S U promoter binds a L I M homeodomain transcription factor that directs basal expression. The second identified site at -406 to -399 binds a transcription factor that is stimulated by G n R H via the M A P K (Roberson et al. 1995). Saunders et al. (Saunders et al. 1998) have shown that c A M P stimulates a - G S U transcription, and this is additive to the stimulation mediated by the P K C pathway. However, it is unclear whether this is mediated via a creb-response element (CRE) . G n R H I R also increases a - G S U transcription by mobilizing 3 1 extracellular calcium, and the D N A elements responsible map are between —420 and -244 bp in the human a - G S U promoter (Holdstock et al. 1996). Differences in the intracellular signaling of the G n R H I R mediated by P K C - and cAMP-activated pathways, and by calcium influx, may be part of the mechanism that ensures adequate amounts of a - G S U in different physiological states, especially since a - G S U 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 G n R H I is mediated by stimulation of P K C (Sartorius et al. 2003), M A P K (Week et al. 1998) and c A M P pathways (Saunders et al. 1998). Two regions have been identified in the rat LH/3-subunit promoter that mediate G n R H 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 G n R H regulation of LH/3-subunit gene expression. The SF-1 D N A 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 G n R H (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 G n R H responsiveness (Strahl et al. 1998) and this is relayed by the P K C 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 G n R H 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 P R 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 P R with high affinity and are sufficient for P4 responsiveness (O'Conner et al. 1999). 1.14 Hypotheses 1. There is a differential role of P R isoforms in the regulation of human G n R H I R, G n R H I and G n R H 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 G n R H 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 G n R H I and G n R H II by P4. Hypothesis 2 1. To elucidate the signaling pathways mediating G n R H I or G n R H II-induced ligand-independent activation of PR. 2. To elucidate the involvement of coactivators and their recruitment to specific P R E promoter regions by GnRHs. 3. To examine the involvement of the ligand-independent transactivation of P R in G n R H -induced regulation of endogenous gonadotropin subunit gene expression. 2 MATERIALS AND METHODS 2.1 Materials G n R H I agonist, (D-Trp6)-GnRH, H-89, Staurosporin ( P K C inhibitor), E2, P R antagonist RU486, and P4 were purchased from Sigma-Aldrich Corp (Oakville, Canada). A G n R H II analogue, D-Arg(6)-Azagly(10)-NH2, was purchased from Peninsula Laboratories (Belmont, C A ) . GF109203X, inhibitor of P K C , was purchased from E M D Biosciences, Inc. Staurosporin and G n R H I R antagonist (Antide) were obtained from Sigma-Aldrich Corp. 2.2 Cell culture The mouse gonadotrope-derived clonal o T 3 - l and L/3T2 cell lines were provided by Dr. P. L . Mel lon (Department of Reproductive Medicine, University of California, San Diego, C A ) . The human cerebellar medulloblastoma (TE671) cells were obtained from American Type Culture Collection (Manassas, V A ) . A l l cells were maintained in D M E M (Life Technologies, Inc., Burlington, Canada) supplemented with 10 % fetal bovine serum (FBS; Hyclone, Logan, U S A ) . 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 m M E D T A ) . 2.3 Plasmids A PRE-luciferase reporter plasmid, containing two copies of consensus P R E upstream 3 5 of the thymidine kinase promoter, was provided by Dr. D . P. McDonnel l (Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, N C ) . Human G n R H I R-luciferase construct (p2300-LucF) was prepared as previously described (Ngan et al. 1999; Cheng et al. 2001b). P R B construct (pSG5-PR B) was kindly provided by Dr. P. Chambon ( I N S E R M , University Louis Pasteur, Paris, France) and P R A construct (pOP13-PR A ) was a gift from Dr. Graham (University of Sydney Westmead Hospital, N S W , Australia). Plasmid D N A s for transfection studies were prepared using Q I A G E N Plasmid M a x i Kits ( Q I A G E N , Chatsworth, C A ) following the manufacturer's suggested procedure. The concentration and integrity of D N A 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 F u G E N E 6.0 (Roche Diagnostics, Quebec, Canada) following the manufacturer's procedure. Briefly, 4 x 10 5 cells were seeded into six-well tissue culture plates two day before transfection in 2 ml phenol red-free D M E M (Life Technologies, Inc., Burlington, Canada) containing 10 % charcoal-dextran-treated F B S (HyClone Laboratories, Inc. Logan, U T ) , which was used as standard culture medium in all experiments unless indicated. One microgram of the P R E -luciferase reporter plasmid and 0.5 pg R S V - / a c Z were dissolved in 100 pl standard culture medium containing 3 pl F u G E N E 6.0 without serum. The D N A 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 G n R H I R promoter containing plasmids and over-expression vectors for P R isoforms were carried out using F u G E N E 6.0 following the manufacturer's procedure. Briefly, 4 x 10 5 TE671 cells were seeded into six-well tissue culture plates before the day of transfection in 2 ml phenol red-free D M E M containing 10 % charcoal-dextran-treated F B S . One microgram of the G n R H I R promoter-luciferase construct, 0.5 pg R S V - / a c Z and an indicated amount of expression plasmids (PR A or P R A / B ) were dissolved in 100 p l phenol red-free D M E M containing 3 p l F u G E N E 6.0. The D N A 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 % c o 2 . 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 s h R N A ( 5 ' - T G A C G G T T G C A T T T G C C A C T T C A A G A G A G T G G C A A A T G C A A C C G T C A ) for G n R H I R was produced using pSuper.gfp/neo vector. Two s iRNAs for SRC-3 (siSRC-3(a); 5 ' - U U A C U G C U G C U U C U U G G C C and siSRC-3(b) (L iu et al. 2002c)) were obtained from Q I A G E N (Chatsworth, C A ) . The s i R N A (5'-G U A U G G C U U U G A U U C C U U A ) for P R was purchased from Q I A G E N . In addition, a nonspecific s i R N A was purchased from Q I A G E N and used as a negative control. The s i R N A transfection was performed according to the manufacturer's suggested procedure ( Q I A G E N ) . In brief, 2 days before transfection, 4 x 10 5 cells per well of a 6-well plate were seeded in 2 ml phenol red-free D M E M containing 10 % charcoal-dextran-treated F B S . The cells were transfected with 1 pg G n R H I R, or 12.5 p M (final concentration) of SRC-3 or P R s iRNAs . The transfection was performed by 3 p l Lippfectamine 2000 reagent (Invitrogen, Burlington, ON) , following the manufacturer's protocol. Then, the cells were challenged with G n R H I, G n R H II or P4. To monitor the s i R N A transfection efficiency, immunoblot analysis was performed for PR, SRC-3 or G n R H I R . 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 D M E M 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 T B S - 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 E C L chemiluminescent system (Amersham Pharmacia Biotech Inc.). 2.8 Immunoprecipitation The cells were seeded at a density of 4 x 10 5 cells per well o f a 6-well plate in 2 ml phenol red-free D M E M containing 10 % charcoal-dextran-treated F B S . The cells were treated with G n R H I, II or P4, and then washed once with ice-cold P B S 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 P R antibody (10 pg/ml) for 1 h at 4 °C, followed by incubation with protein A-Magnetic beads (BioLabs, Inc., Ipswich, M A ) 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 S D S - P A G E sample buffer. The sample mixture was run on 10 % SDS-P A G E gels (acrylamide: bisacrylamide =29:1) in l x gel running buffer (25 m M Tris/250 m M glycine, p H 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, N Y (SRC-1, catalogue #05-522; GRIP-1 , catalogue #06-986; SRC-3 , catalogue #05-490), Neomarker, 4 0 Fremont, C A ( G n R H I R, catalogue #MS-1139) or Santa Cruz Biotechnology, Inc, Santa Cruz, C A ( p C A F , 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 o T 3 - 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 P B S and fixed in 4 % paraformaldehyde for 15 min, washed with P B S 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 R T with the'corresponding anti-PR antibody diluted 1:100 in PBS/1 % B S A . After three washes with P B S , detection of the primary antibody was performed with an A B C peroxidase staining kit (DaKoCytomation, Corp, Carpinteria, C A ) . 2.10 RNA extraction and reverse transcriptase-PCR (RT-PCR) Total R N A was isolated from cell cultures by RNeasy M i n i K i t ( Q I A G E N , Chatsworth, C A ) . One pg of extracted total R N A from each cell line was reverse transcribed using the Superscript™ II Reverse Transcriptase (Invitrogen) according to the manufacturer's suggested 4 1 protocol. P C R amplifications were carried out in 20 pl reactions containing 1 p l c D N A , 2.5 U Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mm M g C ^ , 2 mm deoxynucleotide triphosphate, and 50 pmol forward and reverse primers. Primers for G n R H I and G n R H II were designed based on the published sequence (Khosravi and Leung 2003), (Nathwani et al. 2000). The forward-and reverse primers for G n R H I (accession number: M12578) were 5-A T T C T A C T G A C T T G G T G C G T G - 3 and 5 - G G A A T A T G T G C A A C T T G G T G T - 3 , respectively. Forward and reverse primers for G n R H II (accession number: AF036329) were 5-G C C C A C C T T G G A C C C T C A G A G - 3 and 5 - C C A A T A A A G T G T G A G G T T C T C C G - 3 , respectively P C R amplification for G n R H 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 P C R for G n R H 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 P C R products were fractionated on a 1.5 % agarose gel with ethidium bromide. The expected P C R product of G n R H I and G n R H II were isolated from the gel and sequenced by the dideoxy nucleotide chain termination method. Sequence analysis revealed that G n R H I and G n R H II c D N A s have identical sequence to those from the published human G n R H I and G n R H II. The sizes for G n R H II and G n R H I c D N A s were 327 and 380 bp, respectively. 2.11 Real time RT-PCR Total R N A (2.5 pg) was reverse transcribed into first-strand c D N A . The primers used for S Y B R Green real-time R T - P C R were designed using the Primer Express Software v 2.0 (Perkin-Elmer Applied Biosystems, Foster City, C A ) and tested previously. The primers for the 4 2 real time P C R are described in Table. 1. Real-time P C R was performed using the A B I prism 7000 Sequence Detection System (Perkin-Elmer Applied Biosystems, C A , U S A ) equipped with a 96-well 10 optical reaction plate. The reactions were set up with 12.5 [i\ SYBR® Green P C R Master M i x (Perkin-Elmer Applied Biosystems). A l l real-time experiments were run in triplicate and a mean value was used for the determination of m R N A levels. Negative controls, containing water instead of sample c D N A , 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 m R N A for a - G S U , FSH(3, and LH(3 in a T 3 - l and L[3T2 cells were normalized to the amount of G A P D H m R N A . 4 3 Genes Direction Sequences Gene ID FSHB sense C C C A G C T C G G C C C A A T A NM_008045.2 anti-sense G C A A T C T T A C G G T C T C G T A T A C C A L H p sense G G C C G C A G A G A A T G A G T T C T NM_008497.2 anti-sense C T C G G A C C A T G C T A G G A C A G T A G a - G S U sense T G T T G C T T C T C C A G G G C A TAT NM_009889.1 anti-sense T G G A A C C A G C A T T G T C T T C T T G G A P D H sense C A T G G C C T T C C G T G T T C C T A M32599.1 anti-sense G C G G C A C G T C A G A T C C A Table. 1. Primers for Real-time P C R 2.12 Chromatin immunoprecipitation (ChIP) assay Unless otherwise stated, all reagents, buffers and supplies were included in a C h I P - I T 1 M kit (Active Motif, Inc., Carlsbad, C A ) . Briefly, the o T 3 - 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 D N A 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 D N A was removed from the beads in an elution buffer. To reverse cross-links and remove R N A , 5 M N a C l 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 D N A was purified using gel exclusion columns. The purified D N A was subjected to P C R 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'-A G A A C T C T T G C T T G C T T T G C ) and reverse ( 5 ' - A A T A G C A G A C A C T C T A T G C C ) primers. A s an input control, 10 % o f each chromatin preparation was used. The P C R 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 A N O V A 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 G n R H I R gene by P4, a full-length human G n R H 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 1 0 " 6 M and 1 0 " 5 M doses (Fig. 10). This inhibitory effect on the transcriptional level of G n R H I R was shown at 12 and 24 h treatment with 1 0 " 5 M P4 (Fig. 10B). To further confirm the specificity of P R of P4-mediated effect in the expression of human G n R H I R, the human neuronal cells were co- treated with RU486 (10" 5 M), an antagonist of PR. Although RU486 itself had no effect on expression of G n R H I R transcript, it blocked P4 effect on G n R H I R transcriptional levels when the cells were co-treated with P4 (Fig. 11). The antagonistic effect of RU486 suggested that P4 regulation on G n R H I R promoter activity might be mediated by specific receptors for PR. 4 7 Control 10-9 lO 8 107 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 G n R H I R promoter-luciferase construct p2300-LucF was transiently transfected into TE671 cells by F u G E N E 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 C 3 T 3 2 250 H o too 5 200 o «> 150 C3 J 3 5 100 50 T T b T a T Control P4 P4+RU486 RU486 Figure 11. Effects of RU486 on P4-induced G n R H 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 G n R H I R transcription, roles of specific isoforms of P R were further analyzed in this study. The presence of endogenous P R A and P R B in TE671 cells was observed by immunoblot analysis. A s seen in Fig. 12A, P R A was highly expressed in these cells, while P R B was weakly detected. Molecular weights of the detected human P R A (95-kDa) and P R 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 G n R H I R, the full-length versions of P R A or P R B were cotransfected into TE671 cells. The over-expression of P R A or P R B was monitored by immunoblot analysis (Fig. 12A). Over-expression of P R A enhanced a P4-mediated decrease in G n R H I R promoter activity (Fig. 12B) in a dose-dependent manner. Interestingly, P R B over-expression reversed the effect of P4 in the G n R H I R promoter activity. This result suggests distinct function of P R A and P R B in the regulation of G n R H 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 P R A and P R B showed distinct regulation of G n R H I R 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 P R A or P R 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 P R E upstream of the thymidine kinase promoter. Over-expression of P R A reduced the P R E reporter gene activity under P4-treated conditions, but over-expression of P R B increased it. However, without over-expression of P R A or P R B , P4 had no significant effect on P R E promoter activity. These results indicate that isoforms of P R have different transcription properties in human neuronal cells. The P R B potentiates P R E Luciferase reporter gene activity, while P R A represses. The distinct promoter activities of P R 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 P R A P R B 1 2 3 > ^ PI c 400 H I Control ] P4 a o 300 A 200 -j OH '§ 100 J 0 PRE-tk-Luc PR A PR B + + + + + Figure 12. Effects of PR A or PR B over-expression on G n R H I R or P R E promoter activities. (A) Basal expression levels of PRs (lane 1), and over-expression of P R A (lane 2) and P R B (lane 3) were investigated by Western blot analysis. (B) TE671 cells were cotransfected with G n R H I R luciferase construct and increasing amounts of the P R A or P R B plasmid D N A (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 P R A or P R B into TE671 cells. Following transfection, the cells were treated with P4 (10"5 M ) for 24 h. The relative promoter activity of P R E 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 P R A and P R B showed different transcription activities on the P R E Luciferase reporter gene and G n R H I R gene promoter. This dynamic action of P R isoforms in TE-671 cells led us to evaluate other possible P4 target genes, G n R H I and G n R H II. Semi-quantitative RT-P C R was performed to examine the m R N A levels of G n R H I and G n R H II by P4 in TE671 cells. The 380-bp product corresponding to G n R H I and, a 327-bp product for G n R H II were examined to evaluate P4 regulation of these genes. The 372-bp product for G A P D H was used as an internal control. A linear relationship was found between the cycle numbers and optical density for G A P D H , G n R H I and G n R H II, respectively (Data not shown). A s results, 29 cycles for G n R H I, 27 cycles for G n R H II and 20 cycles for G A P D H were employed for semi-quantification, and the P C R products were sequenced to assure authenticity. Treatment with P4 (10"6 and 10" 5 M) for 24h resulted in increases in m R N A levels of G n R H 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 G n R H I gene significantly at 12 and 24 h as shown in Fig. 13B. In contrast, it did not significantly affect m R N A levels of G n R H II (Fig. 15B). The P4-induced increases in G n R H I m R N A 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 G n R H I mRNA levels. The TE671 cells were treated with P4 for 24 h in a dose (A) and time-dependent manner (B). Total R N A was extracted from TE671 cells, and 1 pg of total R N A was reverse transcribed. The expression levels of G n R H I m R N A was estimated by semi-quantitative R T - P C R and normalized by G A P D H . 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 G n R H I mRNA levels. TE671 cells were treated with P4 ( 1 0 6 M ) , or P4 plus RU486 (10"6 M ) for 24h. The levels of G n R H I m R N A was estimated by R T - P C R and normalized by G A P D H . Experiments were repeated three times independently. Bars represent mean + SD of representative experiments with triplicate, a, P < 0.05 vs control; b, P O . 0 5 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 P R A or P R B m R N A was transiently transfected into TE671 cells to investigate roles of P4-stimulated P R isoforms in the modulation of G n R H I and G n R H II gene expression. Without P R expression vectors, treatment with P4 induced an increase in m R N A levels of G n R H I, while it did not affect G n R H II gene expression. In this context, the cells were introduced with expression vector for P R B , and this enhanced P4 effects on the G n R H I gene expression (1.5-fold vs P4 only treated group) as shown in Fig. 15. However, the over-expression of P R A or P R B did not effectively alter the levels of G n R H II m R N A in the absence or presence of P4. 5 8 Figure 15. Effects of P4 on G n R H I and G n R H II mRNA levels after PR over-expression. The over-expressing vector of P R A or P R 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 o f G n R H I (A) and G n R H II (B) m R N A s were estimated by semi-quantitative RT-PCR and normalized by G A P D H . 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 G n R H system in a PR-isoform specific manner in TE671 neuronal cells. The results showed dynamic and complex interplay between the P R and G n R H systems. In the following study, the effects of GnRHs on P R 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 G n R H I or II 7 7 (10" M ) alone or with P4 (10" M ) alone. Under these conditions, P4 increased the transcriptional activity of P R in a time-dependent manner with maximal activation at 24 h (3.5-fold vs control), while G n R H I and II showed maximal effects (5.5-fold vs control) on P R activation at 8 h (Fig. 16A). When these effects of G n R H I and II were studied in the presence of 1 0 " 7 M P4, this resulted in a synergistic increase (2-fold vs GnRH-treated group without P4) in P R 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 P R by GnRHs and P4 could be attributed to P R acting through ligand-independent and ligand-dependent pathways, respectively. To explore this, cells were co-treated with 1 0 " 5 M P K A (H89), 1 0 " 6 M P K C inhibitors (Staurosporin and GF109203X), 1 0 " 5 M P R antagonist (RU486) or 10" 7 M G n R H I R antagonist (Antide). This showed that co-treatments with Staurosporin, GF109203X, H89 and Antide completely blocked the trans-activitation of the P R that was mediated by GnRHs, while RU486 did not (Fig. 17). B y contrast, activation of the P R by P4 was blocked completely by RU486 under the same conditions (data not shown). 6 1 Figure 16. Effects of G n R H 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 F u G E N E 6.0 reagent. After 2 days in standard culture medium + 0.2 n M E2, the cells were treated with 1 0 " 7 M G n R H I, G n R H II or P4 over a 24 h time course. (B) Ligand-dependent and ligand-independent transactivation of P R was tested after treatment with 10~ 7 M GnRHs in the absence or presence of 1 0 " 7 M P4. The cells were transiently trasfected with the reporter gene and treated with G n R H I or G n R H II with or without P4 for 8 h. In both experiments, a R S V -lacZ reporter plasmid was also co-transfected to control for transfection efficiency, and P R E -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 600 ro 400 </> 1 300 = 200 1 i 4h 1 8h P4 rMllGnRH I Ell] GnRH II 1 I 16h 24h 6 3 1250 I I Control T^m G n R H I I I G n R H II + + + + + No Stauro GF H89 RU486 Antide Cotreatment Figure 17. P K C and P K A inhibitors reverse GnRH-induced PR-mediated transactivation of a PRE-luciferase reporter gene, but a PR antagonist (RU486) does not. The P R E -luciferase reporter gene was transiently transfected into o T 3 - l cells. After 2 d in standard culture medium + 0.2 n M E2, the cells were treated with 1 0 " 7 M G n R H I or G n R H II alone (no co-treatment) or together with Stauro (staurosporin, P K C inhibitor), G F (GF109203X, P K C inhibitor), H89 ( P K A inhibitor), RU486 (PR antagonist) or Antide ( G n R H 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 P R phosphorylation sites contain a Ser-Pro consensus sequence for proline-directed kinases (Zhang et al. 1995). Since P K C and P K A inhibitors reduced the transcriptional activity of the PR, it was investigated whether P R is phosphorylated by GnRHs or P4. Amino acid sequences of murine, rat and human P R were compared with major phosphorylation sites (Fig. 18). The amino acid identity of human P R was 78 % to that of mouse. Most of the murine P R 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 C D K - 2 gene by the human P R (Pierson-Mullany and Lange 2004). To investigate the regulation of P R phosphorylation in o T 3 - l cells, antibodies that recognize both isoforms of P R (PR A and P R B) as well as antibodies against phosporylated-PR at Ser294 or Ser400 were used in Western blotting experiments (Fig. 19). This demonstrated that both P R A and P R B isoforms are present in o T 3 - l cells, but it was not able to detect P R phosphorylated at Ser400 in these cells. L o w levels of Ser294-phosphorylated P R B were detected, while phosphorylation of P R A at this site was undetectable. Phosphorylation of mouse P R B at Ser294 in GT3-1 cells tended to increase at 1-4 h following treatment with G n R H I or G n R H II (Fig. 19). B y contrast, there was no increase in P R B phosphorylation at this site after P4 treatment within this time frame. 6 5 mPR rPR hPR Ser20 Ser25 MTELQAKDPQVLHTSGASfStPHlfislPLLARLDSGPFQGSQHSDVSSVVSPIPISLDGLL 60 MTELQAKDPRTLHTSGAAPS PTHVGS PLLARLDPDPFQGSQHSDASS W S PI PI SLDRLL 60 MTELKAKGPRAPHVAGGPPS ?-EV(3SPLLCRPAAGPFPGSQTSDTLPEVSAIPISLDGLL 59 mPR rPR hPR Ser81 FPRSCRGPELPDGKTGDQQSlCsbVEGAFSGVEATHREGGRNSRPP--EKDSRLLDSVLDS 118 FSRSCQAQELPDEKTQNQQSliSDVEGAFSGVEASRRRS-RNPRAP--EKDSRLLDSVLDT 117 FPRPCQGQDPSDEKTQDQQSLS3VEGAYSRAEATRGAGGSSSSPP--EKDSGLLDSVLDT 117 mPR rPR hPR Serl62 LLTPSGPEQSHASPPACEAITSWCLFGPELPEDPRSVPATKGLljSPLMSRPEIKVGDQSG 178 LLAPSGPEQSQTSPPACEAITSWCLFGPELPEDPRSVPATKGLLSPLMSRPESKAGDSSG 177 L L A P S G P G Q S Q P S P P A C E V T S S W C L F G P E L P E D P P A A P A T Q R v i a P L M S R S G C K V G D S S G 177 Serl90 Ser213 mPR rPR hPR TGRGQKVLPKG: TGAGQKVLPKA' TAAAHKVLPRG: SJPPRQLLLPTSGSAHWPGAGVKPSlPQPAAGEVEEDSGLETEGSASPLL 23 8 PPRQLLLPTSGSAHWPGAGVK: 3S 2QPATVEVEEDGGLETEGSAGPLL 23 7 SPARQLLLPASESPHWSGAPVKPSPQAAAVEVEEEDGSESEESAGPLL 23 7 mPR rPR. hPR Ser294 KSKPRALEGTGQGGGVAANAPSAAPGGVTLVPKEDSRFSAPRVS-LEQDSPIAPGfSJPLA 2 97 KSKPRALEGMCSGGGVTANAPGAAPGGVTLVPKEDSRFSAPRVS-LEQDAPVAPGI ISPLA 2 96 KGKPRALGGAAAGGGAAAVPPGAAAGGVALVPKEDSRFSAPRVALVEQDAPMAPG] ISPLA 2 97 mPR rPR hPR Ser345 TTWDFIHVPILPLNHALLAARTRQLLEGESYDGGATAG-PFCPPRFslPSAPSTPVPRGD 3 55 TTWDFIHVPILPLNHALLAARTRQLLEGDSYDGGAAAQVPFAPPRI3SPSAPSPPVPCGD 356 TTVMDFIHVPILPLNHALLAARTRQLLEDESYDGGAGAASAFAPPRoSPCASSTPVAVGD 357 mPR rPR hPR Ser400 FPDCTYPLEGDPKEDVFPLYGDFQTPGLKIKEEEEGADAAVfSJPRPYLSAGASSSTFPDF 415 FPDCTYPPEGDPKEDGFPVYGEFQPPGLKIKEEEEGTEAASRSPRPYLLAGASAATFPDF 416 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 mPR rPR. hPR Ser554 VYPPYLNYLRPDSEAS(5lPQYGFDSLPQKICLICGDEASGCHYGVLTCGSCKVFFKRAME 586 VYPPYLNYLRPDSEAS()SPQYGFDSLPQKICLICGDEASGCHYGVLTCGSCKVFFKRAME 586 VYPPYLNYLRPDSEASC13PQYSFESLPQKICLICGDEASGCHYGVLTCGSCKVFFKRAME 596 mPR GQHNYLCAGRNDCIVDKIRRKNCPACRLRKCCQAGMVLGGRKFKKFNKVRVMRTLDGVAL 646 rPR GQHNYLCAGRNDCIVDKIRRKNCPACRLRKCCQAGMVLGGRKFKKFNKVRVMRALDGVAL 646 hPR GQHNYLCAGRNDCIVDKIRRKNCPACRLRKCCQAGMVLGGRKFKKFNKVRWRALDAVAL 656 mPR rPR hPR Ser676 PQSVGLPNESQALSQRITi^SJ'NQEIQLVPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLT 706 PQSVAFPNESQTLGQRIT7SPNQEIQLVPPLINLLMSIEPDWYAGHDNTKPDTSSSLLT 706 PQPVGVPNESQALSQRFTFSpGQDIQLIPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLT 716 mPR SLNQLGERQLLSWKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQML 766 rPR SLNQLGERQLLSWKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQML 766 hPR SLNQLGERQLLSWKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQML 776 mPR rPR YFAPDLILNEQRMKELSFYSLCLTMWQIPQEFVKLQVTHEEFLCMKVLLLLNTIPLEGLR 826 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 P R for mouse, human and rat were aligned. Murine, rat and human P R are highly conserved, especially in regions encompassing the phosphorylation sites. The P R at Ser294 and Ser400 are conserved in mouse, rat and human. The amino acid sequence identity of human P R 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 P R A and P R B isoforms were treated with 1 0 " 7 M G n R H I, G n R H II or P4 for 1-8 h. Equal amounts of cell lysates (100 pg) were electrophoresed on SDS-7 % P A G E gels, transferred to nitrocellulose, and Western blotted using antibodies specific to either P R A and P R B (upper panel), phospho-Ser294 P R (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 PR-B PR-A Phospho Ser294 PR-B C GnRH I GnRH II P4 8 1 4 8 1 4 8 IMP HHp M *** mm vmf^mm-Actin 500 n 400 u < 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 P R (Qiu et al. 2003), the sub-cellular localization of P R 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 P R was predominantly cytoplasmic (Fig. 20A), while immunoreactive P R 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 G n R H I or G n R H II, the P R 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 P R in a T 3 - l cells grown in standard culture medium + 0.2 n M E2 but without serum (B) standard culture medium + 0.2 n M E2 (i.e, containing 10 % charcoal dextran-treated FBS) , or (C) standard culture medium + 0.2 n M E2 without serum and containing G n R H I or (D) G n R H II. After 16 h in culture, 10" 7 M G n R H I or G n R H 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 D A B 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 P R associates with specific coactivators in o T 3 - 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 p C A F , SRC-1 , or SRC-2 with the P R after cells were stimulated with G n R H I or G n R H II. In contrast, both GnRHs increased interaction of the P R with SRC-3 , and this again was most apparent at 4-8 h after treatment with G n R H I. Since P4 binding to the P R 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 P R was further compared in a T 3 - l cells after 8h of treatment. Under these conditions, none of the hormones influence the total levels of either P R A or P R B . Although the amount of SRC-3 that immunoprecipitates with P R after G n R H 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 P R after G n R H 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 n M E2, the cells were treated with G n R H I or II for increasing lengths o f time, and lysates were immunoprecipitated (IP) using anti-PR antibody. The immunoprecipitates were then probed with anti-SRC-1, anti-SRC-2, anti-p C A F or anti-SRC-3 antibody. (B) After treating the cells with 1 0 " 7 M G n R H I, G n R H 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 G n R H I 0 2 4 8 24h G n R H 0 2 4 8 24h SRC-1 S R C - 2 p C A F SRC-3 B • IP with anti-PR Control P4 G n R H I G n R H 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 H A T activity, which affects chromatin remodeling and transcription (Chen et al. 1997), it was explored whether or not G n R H 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 P R E containing reporter gene construct was introduced by transient transfection into a T 3 - l cells. In this context, it was found that GnRHs promote very similar levels of P R recruitment to the P R E as that observed after treatment with P4, and this occurred within 4 h of treatment (Fig. 22A). This also showed that treatment with G n R H 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 P R recruitment to this P R E , the increase in SRC-3 recruitment to this site after G n R H II treatment was not as effective as after G n R H I treatment, but it was still greater than that observed after P4 treatment (Fig. 22A). These results are consistent with the changes in P R E reporter gene activity after treatment with GnRHs (Fig. 16), and the observations that G n R H I consistently enhances ligand-independent transactivation of the gene by P R to a greater extent than G n R H 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 a T 3 - l cells treated with G n R H I, G n R H 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 P C R amplify the region containing the P R E 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 P C R controls of sheared genomic D N A templates indicated the integrity of the input D N A used in the ChIP reactions, while P C R 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 G n R H I R, s i R N A was used to decrease its expression in order to determine whether it mediates the ligand-independent transactivation of the P R by G n R H I or G n R H II in these cells (Fig. 23). A Western blot demonstrated that the s i R N A treatment effectively decreased G n R H I R levels prior to the introduction of the P R E -luciferase reporter gene (Fig. 23). When these cells were then treated with G n R H I or G n R H II, the PRE-reporter gene was reduced substantially (66 % and 48 % for G n R H I and G n R H II) over that observed in cells that contain normal levels of the G n R H I R (Fig. 23). In this context, it should also be noted that the siRNA-induced loss of G n R H I R had no influence on the ligand (P4)-dependent transactivation of the PRE-reporter gene (Fig. 23). These data confirm that the G n R H I R mediates the ligand-independent activation of the PRE-reporter gene by G n R H I and G n R H II. 7 8 GnRH I Actin Control s iGnRH I R R M 500 -t >400 4 o re g|300 H 2 S=200 H o ujioo H Q. Control SiGnRH I R Control P4 GnRH I GnRH II Figure 23. G n R H I R mediates both G n R H I- and G n R H II-induced ligand-independent activation of PR. The o T 3 - l cells were co-transfected with a PRE-luciferase reporter gene and s i R N A for G n R H I R. After 2 d in standard culture medium + 0.2 n M E2 , the cells were treated with 10"7 M G n R H I, G n R H II or P4 for 8 h. The efficiency o f the s i R N A was tested by immunoblotting for G n R H 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 s i R N A s for SRC-3 also used to explore whether it is essential for the G n R H -induced transactivation of the PRE-luciferase reporter gene. Transfection of o T 3 - l cells with two s iRNAs 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 G n R H I and G n R H II) (Fig. 24A). The results of this experiment are particularly important because they demonstrate that loss o f SRC-3 has a much greater impact on the rapid (within 8 h), ligand-independent effects of the GnRHs on P R E -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 G n R H I and G n R H 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 s i R N A ) or together with s iRNAs for SRC-3 (siSRC-3 (a) and siSRC-3 (b)). After 2 d in standard culture medium + 0.2 n M E2, the cells were treated with 10" 7M G n R H I, G n R H II or P4 alone or in combination with each other for 8 h. The efficiency of the s i R N A 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 SRC-3 Act in * ST*? > u ro d) c/> ro S _ CD 5001 4001 300 i 5 200 o 3 _ l LU CC CL 100 I I O Control c m G n R H I C 3 G n R H II nil! nlin No siRNA siSRC-3(a) siSRC-3(b) 8 2 B > o (0 Q> (A 2 U 3 _l UJ OC CL 3500 i 3000 2500 2000 1500 1000 500 0 P4 GnRH I GnRH II No siRNA siSRC-3 (b) + + + + + + + + + + + + + + 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 P R 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 n M E2 , transfected with the PRE-luciferase reporter plasmid and then treated with G n R H I (10" 7 M). Under these conditions, G n R H I increased the transcriptional activity of P R 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 G n R H I were studied in the presence of 0.2 n M E2, a synergistic effect was observed in o T 3 - l cells (4.3-fold vs G n R H I-treated alone), but not in L/3T2 cells (Fig. 25). It was reasoned that this differential synergistic effect might result from E2-induced P R expression. Indeed, the presence of E2 for 2 days increased PRs expression in o T 3 - 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 P R 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 P R E luciferase gene activity was not regulated by P4 and no synergistic effect was detected with G n R H I (Fig. 25C). 8 4 E2 +E2 >» ;> 33 o ra d) to CO o 3 _ l UJ CC CL 1000n 75<H 500H PR B PR A 250-Jn/l nfl nfl fl C 2h 4h 8h E2-E2+ fl 24h B > re cu CO ro o 3 - J 111 CC CL 3000-1 2000-1000« PR B PR A -E2 +E2 I - I ^ r - i r - i n n i E2-E2+ 8 i 24 8 5 c Figure 25. Effects of G n R H 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 L P T 2 (B) cells with F u G E N E 6.0 reagent. After 2 d in culture medium with or without 0.2 n M E2, the cells were treated with 10~ 7 M G n R H 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 1 0 " 7 M G n R H I in the absence or presence of 1 0 " 7 M 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 G n R H I regulation of gonadotropin subunit gene transcription, m R N A levels of a - G S U , FSH/3 and LH/3 were analyzed by real time P C R . It was shown that L/3T2 cells express a - G S U , FSH/3 and LH/3, while o T 3 - l cells only express a - G S U . In both cell lines, a-G S U m R N A levels were significantly increased by G n R H I in a time-dependent manner with maximum at 8 h in o T 3 - l cells (2.4-fold vs control) (Fig. 26A) or 24 h in L/3T2 cells (8.5-fold vs control) (Fig. 26B). Interestingly G n R H I induced an increase in FSH/3 subunit m R N A 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 G n R H I at any time point (Fig. 26D). In addition, the presence of E2 did not result in any effect (Fig. 26) on G n R H I-induced modulation of the subunits gene even in oT3- l cells that have a robust increase in P R transactivation by G n R H I with the presence of E2 (Fig. 25A). To compare the effects of G n R H I and G n R H II on the regulation of gonadotropin subunit gene expression, the cells were incubated with G n R H I or G n R H II in the absence or presence of P4 (Fig. 27). Treatment with G n R H II resulted in the induction of a - G S U transcripts with a pattern similar that of G n R H I, while it had relatively less effect on FSH/3 m R N A 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 G n R H I on a-GSU, F S H B and LH|3 mRNA levels. Changes in a-G S U (A and B) , F S H f i (C) and L H B (D) m R N A content as determined by real-time P C R in a T3-1 (A) or LJ3T2 (B, C and D). After 2 days in culture medium with or without 0.2 n M E2, the cells were treated with 1 0 " 7 M G n R H I over a 24 h time course. The m R N A levels of a - G S U and FSH{3 were significantly increased (*, P<0.05) after treatment with G n R H I, whereas changes in LH{3 m R N A 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 o-l • • £-1 224 CO CO 1 0 , cn b cn D D m m IS) IO + • a-GSU mRNA expression o. C O . r o -I l D D m m to ro + • c 5 4-5-1 O w 4.0-% 3.5-6 3.0-° 25-Z 2.0-E 1-5-CQ. 1.0-V) 0.5-LL 0.0-ID E2-E2+ D 4h 8h 24h 1.25-1 1.00H E2-E2+ 0.75-0.50H 0.25H 0.00-4h 8h 24h 9 0 Figure 27. P4 does not have synergistic effects with G n R H I and G n R H II at the level of gonadotropin subunit gene expression. o T 3 - l (A) and L/3T2 (B and C) cells were treated with 10"7 M G n R H I or G n R H 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 - G S U (A and B) and FSH/3 (C) were analyzed by real time P C R . 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 - G S U 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 G n R H I-induced P R transactivation (Fig. 28) and gonadotropin gene expression (Fig. 29), the cells were co-treated with G n R H I and 10"5 M P K A (H89), 1 0 " 6 M P K C inhibitors (GF109203X), or a 10~ 7 M P R antagonist (RU486). Co-treatment with GF109203X or H89 completely blocked the G n R H I-induced transactivation of P R in 0.T3-1 cells (Fig. 28A) but M A P K inhibitor did not (data not shown), whereas P R transactivation was reduced half in L B T 2 cells (Fig. 28B). In contrast, GF109203X reduced the effect o f G n R H I on a - G S U m R N A levels in both cell lines (Fig. 29A and B) while H89 reduced only G n R H I induced FSHJ3 transcription by 40 % (Fig. 29C). The inhibitory effects of GF109203X and H89 on G n R H I-stimulated FSH|3 transcripts were similar to those of P R E -luciferase activation by G n R H I in L{3T2 cells (Fig. 28B). This suggests the possibility that G n R H I-mediated transactivation of the P R could influence the regulation of FSHJ3 gene expression in the pituitary. For both u-and 13-subunit genes, RU486 did not affect G n R H I-induced transcriptional regulation (Fig. 28 and 29). 9 3 Figure 28. P K C and P K A inhibitors, but not RU486, reduce GnRH-induced PR-mediated transactivation of a PRE-luciferase reporter gene in aT3-l or L/8T2 cells. The P R E -luciferase reporter gene was transiently transfected into o T 3 - l (A) or L/3T2 (B) cells. After 2 d in standard culture medium supplemented with 0.2 n M E2, the cells were treated with 1 0 " 7 M G n R H I alone or together with, G F (GF109203X, P K C inhibitor), H89 ( P K A 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 C H-89 GF RU B n H-89 GF RU GnRH I n n n n T 1 1 T " H-89 GF RU t i i i C H-89 GF RU GnRH I 9 5 Figure 29. Effects of P K C and P K A 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 G n R H I alone or together with G F (GF109203X, P K C inhibitor), H89 ( P K A inhibitor) or RU486 (PR antagonist). Following treatment for 8 h (A and C) or 24 h (B), the m R N A levels of a - G S U or FSH/3 were analyzed by real time P C R . 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 (/> </> a. 4H x a> X < z E z> w o • a 3H 2H Hnnn GF H-89 RU GF H-89 RU GnRH 9 7 c c o '35 co 0 i_ Q. X CD 7.5n 5.0H a: E ca. X if) 2.5H 0.0-a GF H89 RU GF H89 RU GnRH I 3.3.4 PR mediates GnRH I-induced FSH]3 gene expression The s i R N A targeting P R was used to determine whether P R mediates G n R H I-induced gonadotropin subunit gene expression in L|3T2 cells (Fig. 30). When the cells were then treated with G n R H I, s iRNAs for P R resulted in a decrease in FSH/3 gene expression by 32 % (Fig. 30C), while it did not affect a - G S U transcripts (Fig. 30A and B). These data suggest that P R mediates the transcriptional regulation of FSH|3 in a ligand-independent manner by G n R H I. A Western blot demonstrated that the s i R N A treatment effectively decreased P R levels prior to G n R H I treatment (D). 9 9 1 0 0 c 11 (A if) 5-a> &«• o < 3-Z " a: E 2-ca 1 1-LL siControl IsiPR G n R H I P R B PR A Actin Figure 30. PR mediates G n R H I-induced FSHjS gene expression. o/F3-l (A) and L/3T2 (B and C) cells were transfected with control s i R N A or s i R N A for PR. Two days after transfection, the cells were treated with 10"7 M G n R H I for 8 (A and C) or 24 h (B) and a - G S U or FSH/3 m R N A expression was analyzed by real-time PCR. The efficiency of the s i R N A was tested by immunoblotting for P R (D) in LbT2 cells, Controls were not treated with G n R H . 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 G n R H 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 G n R H I secretion (Leipheimer et al. 1984; Skinner et al. 1998). The precise mechanism through which P4 modulates the activity of the G n R H neurons is presently unknown. To date, only a few studies have been done in the regulation of G n R H in the human, because of the lack of appropriate cell models. Previously, it has been shown that P4 treatment resulted in a decrease in G n R H I R promoter activity in the mouse pituitary cells, while P4 increased its activity in human placenta cells (Cheng et al. 2001a). A putative P R E was identified between -535 and -521 related to translation start site at P2300-LucF, G n R H 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 G n R H I and G n R H II (Chen et al. 2002b). To further elucidate the molecular mechanism of P4 in the regulation of different components of the human G n R H system, the transcriptional regulation of G n R H I, G n R H II, and G n R H 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 G n R H I R promoter activity, and this effect was reversed by RU486 which is a known P R and G R antagonist. This suggests that the P4 effect on G n R H I R promoter activity is mainly 1 0 2 mediated by PRs. In order to further investigate the mechanism of P4 function on the expression of G n R H I R gene, the cells were co-transfected with P2300-LucF construct and P R A or P R B . The over-expression of P R A in TE671 cells enhanced P4 effects in the repression of G n R H I R promoter activity. Interestingly, over-expression of P R B reversed the P R A-mediated inhibition of G n R H I R transcription in a dose-dependent manner. This suggests that the P4-induced inhibitory effect on the G n R H I R gene expression is mediated by P R A , not P R B . PRs are ligand-inducible transcriptional regulators that control the gene expression upon binding to the P R E in the vicinity of target promoters or influence the gene expression by interaction with other transcriptional factors, i.e., N F - k B and S T A T , and these interactions result in the repression of transcriptional activities (Truss and Beato 1993). To investigate transcriptional activity of P R A and P R B , the cells were co-transfected with 2 X PRE-tk-Luc vector and P R A or P R B . In TE-671 cells, P R A was shown to be a transcriptional repressor, whereas P R B acted as an activator in the presence of P4. These results indicate that P R A and P R B have distinct transcriptional properties in a gene-specific manner on the G n R H I R promoter. Although P R B acts as a strong transcriptional activator of the P R E promoter, it did not induce transcriptional activity on G n R H I R promoter. It might be because the P R B expression vector also could over-express P R A gene, and the P R A probably blocks the function of P R B on the G n R H I R promoter. Taken together, P R A is more functional on the G n R H I R gene expression in this promoter context, and P R B could play a role as a transdominant regulator of P R A action. Due to their different transcriptional properties, the relative expression levels of P R A and P R B within target cells may direct the overall functional responses to P4. The expression of P R A and P R B isoforms was examined by immunoblot 1 0 3 analysis in this study. Both of P R isoforms exist in TE671 cells. The expression level of P R A was high, while P R B was relatively low in these cells. These results support the scenario that an inhibitory role of P4 on the G n R H I R gene expression is mediated by P R A because of much higher endogenous levels of P R A than P R B in this cell line. Thereby, P R A mediates P4-mediated down-regulation of the human G n R H I R at the transcriptional levels In the present study, the m R N A levels of G n R H I and G n R H II were distinctly regulated by P4. Treatment with P4 in TE671 cells resulted in an increase of G n R H I m R N A levels, whereas it had no significant effect on G n R H II gene transcription. Since the transcriptional activities of P R A and P R B on the G n R H I R and P R E luciferase promoter were distinct in this cell line, the biological pathway of P4 action was also examined in the regulation of G n R H I gene. Surprisingly, the transfection of these cells with P R B-expressing plasmid enhanced a P4-induced increase in the G n R H I gene expression. This finding suggests that the effect of P4 on G n R H I m R N A levels is mediated by P R B not P R A even though the expression level of P R B is relatively low. The differential regulation of two forms of GnRHs has been recently observed. In gold salmon fish, the ratio between G n R H I and G n R H II changed with sexual maturation (Rosenblum et al. 1994). A smaller increase in the level of G n R H II than in salmon G n R H I was observed in the pituitary (Rosenblum et al. 1994). In the chicken, castration led to a change in the level of only c G n R H I but not c G n R H II (Sharp et al. 1990). In addition, the differential regulation of G n R H I and G n R H II by E2 has been recently demonstrated in TE671 cells, indicating that E2 increased endogenous G n R H II m R N A levels and decreased endogenous G n R H I m R N A levels (Chen et al. 2002b). The differential regulation of two forms of GnRHs by sex steroid hormones suggests that G n R H I and G n R H 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 G n R H 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 G n R H I, co-treatment of P4 with E2 can induce secretion of G n R H 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 P R A or P R B could be one of the clues for explaining various P4 effects on the regulation of G n R H system. Taken together, these results indicate that P4 is a potent regulator o f G n R H I R and G n R H I gene expression. This distinct effect of P4 on G n R H system may be derived from different pathways through P R A or P R B . Two P R isoforms have been known to show distinct transcriptional properties in a cell-and promoter-specific manner. In general, P R B acts as a potent transcriptional activator, whereas the transcriptional activity of P R A is cell- or gene-specific dependent manner (Vegeto et al. 1993; McDonnel l and Goldman 1994; Sartorius et al. 1994; Wen et al. 1994). Where P R A is inactive as a transcription factor, it has the ability to repress P R B and other steroid receptors such as E R in vitro (Smith and O'Malley 2004). Functional evaluation studies of the A F - 3 (exists in only P R B not P R A ) (Fig. 6) suggest that it mediates P R B transactivational activity, through inhibition of the inhibitory domain common to P R A and P R B (Pratt and Toft 1997; Leonhardt and Edwards 2002). Moreover, distinct interaction of P R isoforms with cofactors possibly mediates different transcriptional properties of P R A and P R B . Inhibitory domain of P R A recruits corepressor S M R T with greater affinity than P R B , while P R B preferentially interacts with SRC-1 and SRC-2 (Giangrande et al. 2000). This differential recruitment of cofactors to P R 1 0 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 P R isoforms have recently been characterized in an in vivo system (Mulac-Jericevic and Conneely 2004). Selective ablation of P R 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 P R 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 G n R H I in the pituitary is to promote gonadotropin secretion (Kaiser et al. 1997; Stanislaus et al. 1998). In female rats, sequential treatments of G n R H I enhance substantially the production of gonadotropins (Turgeon and Waring 1994), and this self priming effect is thought to involve the P R because it is absent in P R A / B K O mice (Chappell et al. 1999). Moreover, this effect has been reported to be due to the ligand- independent activation of the P R by G n R H 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 G n R H II promotes the ligand-independent activation of the P R in these cells. Although both GnRHs function rapidly in this context, i.e., within 8h, G n R H I consistently evoked a more robust response than G n R H II. However these data indicate that the ligand-independent activation of the P R by both GnRHs is mediated via the G n R H I R, and involves the P K A and P K C 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 P R 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 P R from cytoplasm to the nucleus of serum starved cells after treatment with either G n R H I or G n R H 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 P R and various coactivator proteins within a T 3 - l cells after stimulation with GnRHs in the absence of P4. These studies showed that a substantial and specific increase in P R interaction with SRC-3 occurs 8h after treatment with GnRHs, and this again was most evident after G n R H I treatment. Thus, P R 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 P R and SRC-3 in relation to the ligand-independent activation of P R responsive genes, ChIP assays were performed to examine the loading of P R and SRC-3 onto PREs within the promoter of a transiently transfected reporter gene. In these assays, a rapid and robust recruitment of both P R and SRC-3 to the multiple PREs within the PRE-luciferase reporter gene was observed after G n R H I treatment. Although G n R H II increased recruitment of P R to this P R E , it occurred in concert with much less SRC-3 than that observed after G n R H 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 P R to the P R E . 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. A s suggested by recent studies (Wu et al. 2004; Y i 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 P R in ctT3-l cells, the SRC-3 levels were substantially reduced using a s i R N A approach. These studies clearly indicated that loss of SRC-3 from the cells essentially eliminates the ability of G n R H I and G n R H I I 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 G n R H is mediated via the G n R H I R. More importantly, it was shown that treatment of a T 3 - l cells with GnRHs, and G n R H I, in particular, results in rapid changes in P R 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 P R and SRC-3 is essential for the ligand-independent transactivation of the P R in response to G n R H I and G n R H II treatment. 4.3 Ligand-independent transactivation of PR mediates GnRH-induced FSH0 subunit gene transcription The previous study confirmed GnRH-induced transcativation of P R 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 P R transactivation. However, it is still unclear whether or not P R mediates GnRH-induced regulation of endogenous gonadotropin gene expression in the absence of P4. To investigate this, involvement of P R in G n R H 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 G n R H I. L H and F S H are comprised of two glycoprotein subunits, a common osubunit linked to a specific /5-subunit, LH/3 and FSH/3. The differential regulation of L H and F S H production is complex and involves the interplay of gonadal, hypothalamic and pituitary factors. It has been documented that P R is involved in the GnRH-se l f 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 (L iu et al. 2002a). The development of immortalized pituitary cell lines, a T 3 - l , by targeted expression of SV40 large T antigen driven by the common glycoprotein hormone a - G S U promoter has greatly furthered our understanding of G n R H signaling (Mellon et al. 1991). The a T 3 - l cells express both a - G S U and G n R H I R, but are considered immature, because they do not express the F S H (3 and L H P 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 - G S U , G n R H I R, F S H P and L H P genes. Moreover, both FSH13 and L H P genes are regulated by G n R H , thus, representative of mature pituitary gonadotropes (Liu et al. 2002a). In a T 3 - l and L P T 2 cells, P R was activated by G n R H I in the absence of P4. When the cells were precultured with E2 for 2 days, G n R H I showed a synergistic increase in P R transactivation in aT3- l cells, whereas E2 priming did not affect the G n R H response in L P T 2 cells. Interestingly, a low dose of E2 (0.2 nM) for 2 days induced P R expression in aT3- l cells, but not in L P T 2 cells, and this induction of P R may underlie the synergistic P R transactivation observed in a T 3 - l cells. To compare a ligand -dependent and -independent P R activation, the cells were co-treated with G n R H I and P4. Co-treatment with P4 had a synergistic effect on PRE-luciferase activity in aT3- l cells, whereas it did not in L P T 2 cells. This suggests that, in L P T 2 cells which are more differentiated cells than a T 3 - l , P R does not undergo a ligand-dependent activation its activation is ligand-independent. 1 1 0 Despite of altered P R activation mechanisms, G n R H I prompted a-GSU gene expression in both cells, and it increased F S H P , but not LH(3 m R N A levels in L P T 2 cells. The regulation of a-GSU m R N A levels by G n R H I was time-dependent and maximum at 24 h, while G n R H I regulation of F S H G resulted in a maximum increase at 8 h and a return to control level at 24 h. G n R H II treatment enhanced a - G S U and F S H p m R N A levels. There was no synergistic response when G n R H I and II were combined with E2 or P4. Even though P R E -luciferase gene activity was reduced by P K C and P K A pathway inhibitors, only G n R H I-mediated transcription of the F S H P gene was reduced by both P K C and P K A inhibitors. Interestingly the P K C , but not the P K A inhibitor effectively reduced G n R H I-induced a-GSU gene expression. The similarity of the signaling pathways between G n R H I-mediated transactivation of the P R and induction of F S H P gene expression led us to test the direct involvement of P R in G n R H 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 F S H P promoter, moreover, P4 stimulated F S H P promoter activity when P R was over-expressed in rat pituitary cells (O'Conner et al. 1999). In this context, s i R N A for P R was used to examine the role of P R in G n R H I-induced gonadotropin subunit gene expression. The s i R N A for P R reduced the effects of G n R H I on F S H p m R N A levels but did not affect G n R H I-stimulated a-GSU m R N A levels. 1 1 1 Taken together, these data indicate that G n R H I stimulates a -GSU and F S H B gene expression in pituitary cells. G n R H I activates P R in a ligand-independent manner, and G n R H I-induced F S H B gene regulation requires P R transactivation in the absence of P4. 4.4 Clinical implications During the menstrual cycle, E 2 and P4 exert negative and positive feedbacks at the hypothalamus and pituitary. These steroid hormones inhibit the release of G n R H and gonadotropins and maintain normal cyclicity during follicular phase of the cycle. A t midcycle, these steroids induce positive feedback at the pituitary level, this results in the preovulatory L H and F S H 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 F S H (Messinis 2 0 0 6 ) . 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 2 0 0 6 ) . To change the abnormal feedback system, treatment with various pharmaceutical compounds is available containing ethinylestradiol, progestagen and G n R H agonist / antagonist. However, it has not been clarified what the specific role and mechanism is each of the steroids and G n R H . 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 L H surge, transforms the endometrium to a secretory from a proliferative state and maintains endometrial integrity with E 2 (Spitz 2 0 0 3 ) . Therefore, it is not 1 1 2 surprising that P4 or P R modulators including RU486 have clinical application in medical termination of pregnancy, in producing cervical softening, and in menstrual regulation. P R and E R modulators together with G n R H analogues are considered for clinical application in premenopausal women. P R 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, P R modulators have application in the treatment of uterine myoma and endometriosis. Long-acting G n R H 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 E R and P R (Pritchard 2005). The expression and ratios of P R A and P R B isoforms vary between normal and malignant tissues. In breast cancer cells with P R A or P R B , P4 stimulated gene regulation in a P R isoform-specific manner. The majority of the genes were regulated by P R B , with smaller subsets regulated by P R A or both isoforms (Richer et al. 2002; Jacobsen et al. 2005). It has recently been demonstrated (Jacobsen et al. 2005) that P R 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 P R A in the unliganded state. Because the P R A / P R 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 G n R H 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 E R and P R may predict response to endocrine therapy, and the P R have cross-talk with E R and growth factors (Pritchard 2005). However, the mechanisms between P R and other signaling pathways including 1 1 3 G n R H 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 P R isoforms in the absence or presence of P4 in neuroendocrine cells and ligand-independent P R transactivation by GnRHs. The suggested mechanisms of GnRH-induced P R 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 P R activation suggests a noble mechanism for interactions between P R and other signaling pathways in pituitary and extrapituitary tissues. 1 1 4 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 P R A and B on G n R H I, G n R H II, and G n R H I R gene regulation were investigated in human neuronal cells. 1. P4 reduced promoter activity of G n R H I R. The effect of P4 on G n R H I R promoter activity was blocked by RU486. This demonstrated that P4 regulates G n R H I R transcription by PR. Dominant expressing isoform of PRs in these cells was P R A and it mediated the repression of G n R H I R transcription following treatment with P4. In contrast, transfection with P R B expressing vector reversed P R A-mediated effects of P4 on G n R H I R gene expression. 2. Treatment with P4 induced G n R H I m R N A levels, and this was reduced by RU486. P4 treatment did not have significant effect on G n R H II gene. Induction of G n R H I gene expression was mediated by P R B , while P R 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 P R isoforms by transfection with over-expression vectors, P R B increased transcriptional activity of the reporter gene, while P R A decreased it. 1 1 5 5.1.2 Ligand-independent activation of the PR by GnRHs In this study, transactivation of P R by G n R H I and G n R H II was evaluated. The mechanism of GnRHs in the modification of P R and recruitment of coactivators, in turn, P R transactivation on the promoter level was examined. 1. G n R H I and II activated PR-mediated transcription in a T 3 - l cells in the absence of or presence of P4. P K C , P K A and G n R H I R inhibitors completely blocked GnRHs-induced P R transactivation, while RU486 did not. 2. G n R H I and G n R H 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 P R may suggest that GnRHs phosphorylate P R at Ser294 and this accumulate P R into the nucleus. 3. P R interaction with SRC-3 coactivator was enhanced by GnRHs and P4. At the chromatin level, GnRHs promoted P R recruitment to the P R E luciferase reporter promoter similar with that observed after treatment with P4. G n R H I and G n R H II also recruited SRC-3 to the P R E promoter, but P4 did not. 4. Co-transfection of PRE-luciferase gene with s i R N A for G n R H I R substantially decreased GnRHs-induced P R transactivation, which did not affect P4 action. The loss of SRC-3 by s i R N A 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 P R activation in GnRH-induced gene expression was investigated in the absence and presence of ligand. 1. G n R H I activated PR-mediated transcription in pituitary cells. In E2-priming conditions, G n R H I-induced P R transactivation was much greater in 0.T3-1, but not in L B T 2 cells. Expression of the P R was substantially induced in E2-priming conditions only in aT3- l cells. 2. Treatment with G n R H I and G n R H II resulted in increases in a - G S U and F S H B m R N A levels, which was not affected by co-treatment with P4 or E2 . 3. P K C and P K A inhibitors blocked G n R H I-induced P R transactivation, whereas RU486 did not. P K C and P K A inhibitors reduced the increases in F S H P m R N A levels induced by G n R H I, while only the P K C inhibitor decreased G n R H I-induced a - G S U gene expression. 4. G n R H I-mediated increases in F S H P gene expression were reduced when s i R N A for P R was transfected. However, knock-down of P R did not affect G n R H I-mediated a-GSU 1 1 7 transcription. Taken together, these results demonstrate that P R A and P R 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 P R A activation reduces G n R H I R promoter activity, while P R B results in an increase on G n R H I m R N A levels. Alternatively, in the absence of P4, P R is phosphorylated at Ser294 by GnRHs and translocated to the nucleus. In the nucleus, GnRH-activated P R interacts with coactivators including SRC-3 and then binds P R E . GnRHs-induced P R transactivation thereby regulate FSH{3 gene in a ligand-independent manner (Fig. 31). 1 1 8 Figure 31. Proposed cross-talk between P R and the G n R H system in pituitary cells. 1 1 9 5.2 Future studies 1. Interactions of P R A or P R B with various coactivators after G n R H I treatment. Future experiments need to examine whether or not P R A and P R B have differing preferences to interact with various coactivators, and how treatment with G n R H I might modulate these interactions. The precise mechanism of G n R H I-induced PRs modification also need to be elucidated. 2. Recruitment of P R A or P R B to G n R H I target gene promoters including FSHJB, in vivo and in vitro system Recruitment of P R isoforms to the PRE-reporter and F S H P promoter should be examined. In these studies, the mechanism of G n R H I-induced F S H B gene expression and recruitment of P R A or P R B to the promoter should be investigated. Functional G n R H I-stimulated P R response elements within the F S H | 3 promoter should be identified and examined for the response in the absence or presence of P 4 . 3. Ligand-independent P R transactivation in a cell and tissue specific fashion Since ligand-independent activation of P R is controversial, especially in humans, future studies need to be done to evaluate the activation of P R in the absence of P 4 . 1 2 0 Signaling pathways in P R post-translational modifications and its subcellular localization, and recruitment of coactivators or corepressors should be considered. Chromatin remodeling associated with unliganded P R and coregulators should also be studied. 1 2 1 6. 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Sequence homology has revealed that the PRE- l ike sequence is 92 % homologous to the consensus P R E 1 3 8 ! >I563 -866 CACATTCCCC TCACATTTTT MOGOtGAT CCl ' l tSmtC AAACCACACT AVTAA1TAAT -806 GGATATTACA AAITAATAAA TtJAAITAAAC AACCTCMTC TCTATGGAGT TCATAQAATA -746 CTTAACA.AAT CGGTATATGA ATAACTACAA TCAGAAAGTA GAGATCATAA CrCTCClTAA -686 CTTTCGCCCT AAATTACACG 1GAAACAGCT CTG'J'<JKXrTA AAGOTTCGAG AAOTTGGGG -626 CCAGACGGAG AACAGACAOC AAACAAGCAA OGACATKiGT OrtCAGAGAGS ACATCACATC -566 CXGAGATCTG GTGGGATTCA TCAGTGTFAT AATTCOGCAA TAACAAACAA CGTTCACCTf -506 CTTCATAAAG GATAAGAAAA GCKKMCSATrG AIGACCAGGAC AGCI'CAATGT C-OTCATAAGO I >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 M E #3 - 2 0 6 GATrocwrrr GEVCAOAAAC ovnovimcr hAc^trxtrr TCTPCTCTGT o r o v n w ; « - H G CTCCnTGGC GAGGCTTGAT C1XXCTCTCT Cnt'l'AAACAA TGATCCCCIT TOViCCSOCi* 289 < — : y(51< ; TATA box - 86 TKiTBTrGGT ATTCCTCACC TTAACAOCGA CTAAATCCAT GGCGTGTTAG GATltTTAT.AA ) t i t u i a c r i p t i o i i start s i t e 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 ' m T AAACAAACTA +335 CTCTTTAGAA AACTGTTAAT GAATTOCAAA A'IYITAAGGAT CAC/UVPPATC TGATTAGAGT M438 PHI! +395 AATGACTTTA AGATAGTAAT AA'nXXATCT (ffAAATACTG C77TOGTTAA ATGGTCATAC +455 TGGAGTTrro ATCTATTTGA AVCTACTITA C T C T A C C A T A 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-l ike sequence 1 is 86 % homologous to the consensus G R E . PRE- l ike sequences 2 and 3 are 80 % homologous to the consensus P R E , and PRE-l ike sequences 4 and 5 are 80 % homologous to the P R E 1 3 9 -611 A G G G C A T T G G T G A C A G A G A G G A C A T C A C A T G C A G A G A T C T G G A G G A A C C C -511 A T C A G T A T C A T A A T T A G G G A A T A T T T A G G G A A T T A C A A T T T C T G A T G C T C -461 T T C A C A A A G C A T C A G A A A A A G G G G G G T T G A G A T C A G G A G A A C T G A A T G T G -411 G T C A T A A A G A A A G A C A C A G C C C A T A G G A A C A A G A T G C A G A A G T A C T T C C T *PRE-l ike sequence -361 A T T T G T T C A T A C A C T T G G A G T G T T C A G T C T G T T C T T G G A T C A A T T A A G A C -311 A T A T T T T G G T T T A C C T T C G C A A T G G A G C C A A A G C A A T G T T C A G A A A G G A T -261 T C T G A G T T C G C C A A G T T A A A G A T C A G A A A G A A T A G T C T A G A C T C T A G A G T -211 C A C A T T T A A T T T A C A A G G T G A G G G A G T G G G T G T G C T G C C A T A T C A G A T T C -161 G G T T T G T A C A G A A A C C A T C A T C A C T G A T A G C A T T T T C T G C T C T G T G G C A T -111 T T A G A C T G C T T T G G C G A G G C T T G A T C T C C C T G T C C G T C T A A A C A A T G A T T - 61 C C C T T T C A G C A G G C T T T A T G T T G G T A T T G G T C A T G T T A A C A C C C A G T A A A +1 (transcription start site) - 11 T C C A C A G G G T G T T C A G C T T T C C C C A G A A G A G A C A G C T G A C T G C A C A G G T G + 48 A G T A G C A G C A C T T G A T G C A A C A A G G A C A G C C A C T T T G A A A A T T G C A G A C C + 98 A T T A A G G A T T T A A A G A A G G G A T A G G A G T T T T C T G C C G C T G C T G T G T A G A A +148 A C T T A T T C T T G T T A A C T T G G C T T C T T G A A T A T G G T C A A T G T A C A G T T A T A +198 A G G A A T C T G A C T T A T A A A G C A G T T T G C C T A G C T T C T G A C A T A G A C T C T T T Figure 33. Identification of P R E - l i k e sequences in murine FSH-/3 promoter The 858 bp sequence spanned positions -611 to +247 of sequence of the mouse F S H P gene. Position of PRE- l ike sequence is indicated in reference to the transcription start site (position +1). Sequence homology has revealed that PRE- l ike sequence is 92 % homologous to the consensus P R E . 6.2 The effects of gonadotropins on upstream of GnRH II gene promoter 1 4 0 6.2.1 cAMP-induced promoter activity of G n R H II gene To examine the transcriptional regulation of human G n R H II gene in different cells, human placenta JEG-3 , human neuronal TE-671, human ovarian O V C A R - 3 , and mouse pituitary QT3-1 cells were transiently transfected with a full-length human G n R H II promoter-luciferase construct (p2300-LucF). Since G n R H II gene has been reported to be regulated by c A M P in T E -671 cells, transcriptional regulation of G n R H II was first tested by c A M P in the cells including TE-671 cells (Fig. 34). After transfection, the cells were treated with c A M P in a time dependent manner, and the promoter activity of G n R H II was examined. In JEG-3 , TE-671 and 0.T3-1 cells, the promoter activity of G n R H II gene was increased and maximized at 24 h; however, it showed maximal effect at 2 h and decreased in O V C A R - 3 cells. Figure 34. cAMP induces G n R H II promoter activity. The full-length human G n R H promoter-luciferase construct (p2300-LucF) was transiently transfected into JEG3 (A), TE-671 (B), O V C A R - 3 (C) and aT3 (D) cells by F u G E N E 6.0 reagent. After 2 d in D M E M , the cells were treated with c A M P (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 1 4 1 represented as luciferase activity/13-galactosidase activity. 1 4 2 B > , 6 0 0 = 500 o CD 05400 o §300 i_ o. = 200 X 100 c Control 2h 4h 8h 16h 24h ^200 n '> Ti CO o I 100 i CL X or Control 2h 4h 8h 16h 24h D 300 i >» ;> CO £ o E o - 1 0 0 Hr= 200 H X CC c CD Control 2h 4h r-16h T -24h 350 250 200 > 300 Tj ro o E o Q.150 | 100 O 50 ^ 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 P K A pathway, the effects of L H and F S H on G n R H II transcription levels were further tested (Fig. 34). A l l four cell lines were transiently cotransfected a full length G n R H II promoter plasmid with L H or F S H receptor, and the cells were treated with L H or F S H for 8 and 24 h. In JEG-3 and TE-671 cells, L H and F S H did not affect G n R H II promoter activity, while L H induced it in O V C A R - 3 and 0.T3-1 cells within 8 h. Figure 35. Effects of L H and F S H on the promoter activity of G n R H II gene. The full-length human G n R H promoter-luciferase construct (p2300-LucF) was transiently transfected into JEG3 1 4 4 (A), TE-671 (B), O V C A R - 3 (C) and o;T3 (D) cells by F u G E N E 6.0 reagent. After 2 d in D M E M the cells were treated with L H or F S H (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. 1 4 5 GnRH II promoter activity g g g v ' GnRH II promoter activity 6.2.3 cAMP phosphorylated C R E B at Ser 133 To study biological pathway of L H on G n R H II promoter, phosphorylation was next tested in C R E B protein that has been known to response to c A M P (Fig. 35). The expression o f C R E B was examined. A l l of the cells expressed C R E B but the levels of it were not changed by c A M P in any time points. However, c A M P induced phosphorylation of C R E B . In JEG-3 , O V C A R - 3 and 0.T3-1 cells it phosphorylated the C R E B from 2 h and maintained up to 24 h. In the case o f TE-671 cells, it increased phosphorylation o f the C R E B from 2 h and showed maximum effects at 4 h. Figure 36. Regulation of C R E B phosphorylation at Ser 133 by cAMP. The JEG3 (A), T E -671 (B), O V C A R - 3 (C) and aT3 (D) cells expressing total C R E B were treated with L H for 2 to 24 h. Equal amounts of cell lysates (100 pg) were electrophoresed on SDS-7 % P A G E gels, 1 4 7 transferred to nitrocellulose, and blotted with antibodies specific for C R E B phospho-Ser 133. Western blotting was performed to detect phospho-Ser 133 C R E B (upper panel) or total C R E B (lower panel) 1 4 8 A p-CREB CREB c p-CREB CREB 1 4 9 

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