A functional genomics approach for elucidation of novel mechanisms involved in GnRH regulation of the gonadotropins

A FUNCTIONAL GENOMICS APPROACH FOR ELUCIDATION OF NOVEL MECHANISMS INVOLVED IN GnRH REGULATION OF THE GONADOTROPINS LUO MIN NATIONAL UNIVERSITY OF SINGAPORE 2007 A FUNCTIONAL GENOMICS APPROACH FOR ELUCIDATION OF NOVEL MECHANISMS INVOLVED IN GnRH REGULATION OF THE GONADOTROPINS By LUO MIN (B. SC.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 NOVEL MECHANISMS INVOLVED IN GnRH Luo Min REGULATION OF THE GONADOTROPINS 2007 ACKNOWLEDGEMENTS This thesis is the result of four years work whereby I have been accompanied and supported by many people. It is a pleasant aspect that I have now the opportunity to express my heartfelt thanks and gratitude to all of them. The first person I would like to thank is my supervisor Dr. Philippa Melamed for her encouragement, patience, guidance and advice throughout this project, without which this thesis could not have been possible. I would like to express my gratitude to my wonderful labmates, especially Ms Koh Mingshi, Ms Tan Siew Hoon, Ms Wang Sihui, Mr Feng Jiajun, Mr Lim Yi Wei Stefan and Mr Yang Meng for all their suggestions and help. I also would like to thank my friends: Ms Hu Zhehua, Ms Qin Yafeng, Ms Wang Xiaoxing, Mr Li Mo, Ms Qian Zhuolei, Mr Yu Hongbing and Mr Hu Yi, for their invaluable friendship and encouragement. I am really glad that I have come to know them in my life. The financial assistance in the form of a research scholarship provided by NUS is greatly acknowledged. Finally, I wish to thank my family, for their constant support and unconditional love. i ABSTRACT The pituitary gonadotropes synthesize and secrete luteinizing hormone (LH) and folliclestimulating hormone (FSH), which control reproductive development and function. In mature gonadotropes and in the LβT2 cell line, both hormones are regulated by GnRH, but the hormone-specific β subunits are not expressed in the αT3-1 cells, which represent an immature gonadotrope. In order to identify novel factors and mechanisms involved in basal and GnRH-induced gonadotropin gene transcription, subtractive hybridization was carried out to reveal genes expressed in mature LβT2 but not in immature αT3-1 cells, or those whose expression in LβT2 cells is induced following GnRH treatment. A number of candidate genes was identified, among them the ubiquitin-conjugating enzyme (ubc4), and calmodulin-dependent serine/threonine protein phosphatase calcineurin, both of which are up-regulated following GnRH treatment. Functional studies revealed that GnRH increases estrogen receptor α (ERα) degradation and transactivation of the LHβ gene in LβT2 cells, apparently through stimulation of ubc4 expression. It was further demonstrated that the stimulatory effect of ERα on LHβ expression is mediated through interactions with other regulatory transcription factors Pitx1 and Sf-1 on the proximal promoter, without necessarily requiring an ERE. Calcineurin is activated by GnRH and regulates both basal and GnRH stimulated human αGSU promoter activity, through its target NFAT proteins. NFAT4, which is not affected by GnRH treatment, is constitutively associated with the human αGSU promoter and mediates the promoter basal activity, while NFAT3, activated by GnRH through calcineurin, is associated with the human αGSU promoter only after GnRH treatment and may mediate the GnRH effect on the human αGSU promoter. Furthermore, calcineurin plays a role in the GnRHmediated derepression of the FSHβ gene in the immature gonadotrope αT3-1 cells, possibly by activating its targets MEF2D and Nur77. Nur77 expression is induced by GnRH, which is calcineurin-dependent. Both of the two factors are associated with the FSHβ gene promoter and activate FSHβ gene transcription or promoter activity when over-expressed. It was further demonstrated that GnRH-activated CaMKI is also required for GnRH to overcome the histone deacetylase (HDAC)-mediated repression of the FSHβ gene. ii Table of contents Page ACKNOWLEDGEMENTS……………………………………………………………… .I ABSTRACT……………………………………………………………… .II TABLE OF CONTENTS…………………………………………………………………… III LIST OF FIGURES……………………………………………………………………… .VII LIST OF TABLES………………………………………………………………… .………X LIST OF ABBREVIATIONS……………………………………………………………… .XII Chapter Introduction…………………………………………………………… …1 1.1 Gonadotropins . 1.1.1 Physiology of the gonadotropins 1.1.2 Genomic organization of the gonadotropins 1.1.3 Biological functions of the gonadotropins 1.1.4 Murine αT3-1 and LβT2 gonadotrope cell lines . 1.2 Molecular regulation of gonadotropin synthesis and secretion . 1.2.1 Transcriptional regulation of gonadotropin subunits 1.2.1.1 Transcriptional regulation of the αGSU subunit 1.2.1.2 Transcriptional regulation of the LHβ and FSHβ subunits 11 1.2.2 GnRH induced signaling pathways in stimulation of gonadotropins . 14 1.2.2.1 Calcium . 16 1.2.2.2 PKC/MAPK pathway . 17 1.2.2.3 cAMP/PKA pathway 18 1.2.3 Gonadal peptide mediated regulation of FSHβ gene expression 20 1.2.4 Estrogen (E2)-mediated regulation of LHβ gene expression 21 1.3 High throughput approaches for studying gene expression 26 1.4 Hypothesis and aims 31 Chapter Materials and Methods ………………………………………………….32 iii 2.1 Tissue culture . 32 2.1.1 Medium and culture conditions 32 2.1.2 Storing of cells 32 2.1.3 Recovery of cells 33 2.1.4 Transient transfection of cells . 33 2.1.5 Chemical treatment of cells 34 2.2 Plasmid construction . 35 2.2.1 Site-directed mutagenesis of promoters . 35 2.2.2 Contruction of expression vectors 38 2.2.3 Construction of siRNA constructs 39 2.2.3.1 Oligonucleotide design 39 2.2.3.2 Annealing of oligos . 40 2.2.3.3 Restriction digestion of vectors . 41 2.2.3.4 Extraction of DNA from gel 41 2.2.3.5 Ligation of annealed oligos and linearized pSUPER vector . 41 2.2.4 Constructs for mammalian two-hybrid assay 42 2.3 Isolation, verification and maxiprep of plasmids . 43 2.3.1 Transformation of plasmids into Escherichia coli (E.coli) cells 43 2.3.2 Plasmid isolation and verification 44 2.3.3 Large scale plasmid isolation and purification . 46 2.4 RT-PCR analysis 46 2.4.1 RNA isolation . 46 2.4.2 First strand cDNA synthesis . 46 2.4.3 PCR and gel electrophoresis analysis . 47 2.4.4 Real-time PCR quantification analysis . 50 2.5 Chloroamphenicol acetyl transferase (CAT) assay 52 2.6 Luciferase analysis . 53 2.6.1 Mammalian two-hybrid assay 53 2.6.2 Promoter activity study . 54 2.7 Statistical analysis 54 iv 2.8 Western blot . 54 2.9 Subtractive hybridization . 57 2.9.1 RNA extraction and mRNA isolation . 57 2.9.2 cDNA synthesis and digestion 57 2.9.3 Ligation of tester with two different adaptors 57 2.9.4 First hybridization . 58 2.9.5 Second hybridization 59 2.9.6 Primary PCR amplification 59 2.9.7 Secondary PCR amplification 60 2.9.8 Ligation and sequencing the clones 61 2.10 Chromatin Immunoprecipitation (ChIP) 61 2.11 Plasmid Immunoprecipitation (PIP) 64 Chapter Results . 66 3.1 Subtractive hybridization . 66 3.1.1 Subtractive hybridization of LβT2 and αT3-1 cells . 66 3.1.2 Subtractive hybridization of LβT2 cells with and without GnRH treatment . 68 3.2 GnRH induction of ubc4 expression promotes estrogen receptor ubiquitylation and trans-activation of the LHβ gene……………………………… …………… .72 3.2.1 GnRH induces ubc4 expression in LβT2 cells…………………………… .72 3.2.2 Over-expression of ubc4 reduces ERα protein levels, as does GnRH…… .74 3.2.3 GnRH reduction of ERα protein levels in gonadotropes is proteasome dependent………………………………………………………………………… 76 3.2.4 The liganded ERα transactivates LHβ directly in synergy with Sf-1 and Pitx1 without requiring a consensus ERE… ……………………………………… 77 3.2.5 GnRH-induced ubc4 enhances ERα transactivation of the LHβ gene…… .81 3.2.6 Ubc4 increases the synergistic effect of ERα with Sf-1 and Pitx1 on the LHβ promoter………… .……………………………………………………………….83 3.2.7 Ubc4 over-expression increases the interaction of ERα with Sf-1or Pitx1 .84 v 3.3 Calcineurin is involved in the GnRH activation of the αGSU gene promoter 86 3.3.1 Calcineurin catalytic subunit A expression levels increase in response to GnRH………………………………………………………………………………86 3.3.2 Calcineurin mediates the basal and GnRH stimulatory effect on the human αGSU promoter……….… ………………………………………………… ……88 3.3.3 The calcineurin target, NFAT, is necessary for the human αGSU promoter activity…………………………………………………………………………… .93 3.4 Calcineurin plays a role in the GnRH-mediated derepression of the FSHβ gene in the immature gonadotrope…………………………….…………………… .…102 3.4.1 Inhibition of calcineurin abolishes the GnRH derepression effects on the FSHβ gene……………………………………….………………….…………….102 3.4.2 Nur77 and MEF2D activate the FSHβ gene.……… .… .………….…… 103 3.4.3 The mechanism for Nur77 and MEF2D activation of the FSHβ gene….….107 3.4.4 CaMKs roles in mediating of GnRH effects on the FSHβ gene……… … 108 Chapter Discussion ………………….………………………… ……………… 110 4.1 Differential gene expression in gonadotropes…………………………………110 4.1.1 Differential gene expression in the differentiating gonadotrope 110 4.1.2 Genes up-regulated following GnRH treatment in mature gonadotropes 112 4.2 Ubc4 regulation of LHβ gene expression through increasing ERα transactivation . 115 4.3 Calcineurin is involved in GnRH-stimulated human αGSU promoter activity . 122 4.4 The role of calcineurin in GnRH-mediated derepression of the FSHβ gene in the immature gonadotrope . 132 4.5 General conclusion and future work . 140 Chapter References 142 vi LIST OF FIGURES Page Figure 1.1: Anatomical and functional connections of the hypothalamic-pituitary axis Figure 1.2: A diagrammatic representation of the gonadotrope cell lineage development in the mouse. Figure 1.3: Overview of the regulation of gonadotropins in the hypothalamic-pituitary-gonadal axis Figure 1.4: Several elements define the αGSU gene expression 10 Figure 1.5: Signal transduction pathways activated by GnRH. . .15 Figure 1.6: Schematic model of basal and GnRH-stimulated gonadotropin subunit gene expression. 19 Figure 1.7: Disparity between the binding sites on the LHβ gene proximal promoters of teleosts and mammals. . 22 Figure 1.8: Genomic organization and functional domains of murine ERα. 23 Figure 1.9: The ubiquitin-proteasome pathway 25 Figure 1.10: Overview of the BD PCR-Select subtractive hybridization method. . 30 Figure 3.1: The subtracted PCR products for the control skeletal muscle cDNA. . 69 Figure 3.2: The subtracted PCR products for the LβT2 cDNA following GnRH treatment. 70 Figure 3.3: Subtractive efficiency was confirmed by reduction of GAPDH abundance after PCR-select subtraction 70 Figure 3.4: The mRNA levels of ubc4 increase following GnRH treatment in LβT2 cells 73 Figure 3.5: GnRH treatment increases of the protein levels of ubc4 73 Figure 3.6: Transfection of siRNA to knockdown ubc4 increases ERα protein levels in cells exposed to GnRH . 75 Figure 3.7: GnRH exposure of gonadotropes causes a reduction in ERα protein levels. . 75 Figure 3.8: Proteasome inhibitor MG132 abates the GnRH effect on ERα protein levels. 76 Figure 3.9: The liganded ERα transactivates two vertebrate LHβ gene promoters in synergy with Sf-1 and Pitx1. 78 Figure 3.10: The response elements required for the activation of the LHβ gene promoters by ERα 80 Figure 3.11: Ubc4 is involved in mediating the effect of GnRH on the LHβ gene and increases ERα transactivation. . 82 Figure 3.12: Ubc4 increases ERα transactivation, and the synergistic effect of ERα with Sf-1 and Pitx-1 . 83 Figure 3.13: Ubc4 increases ERα interaction with Sf-1 and Pitx1 . 85 Figure 3.14: GnRH exposure of gonadotropes is followed by an increase the mRNA levels of CnA…… .87 vii Kaiser, U. 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Proc Natl Acad Sci U S A 98: 1057210577. 167 168 [...]... cAMP/PKA pathway GnRH activates the cAMP/PKA pathway, which has been shown to activate the mouse, rat and human αGSU promoter activity (Attardi and Winters, 1998; Maurer et al., 1999) In rat pituitary cells, a cAMP analogue increased αGSU mRNA levels, but not those of LHβ and FSHβ (Haisenleder et al., 1992) The crosstalk between the cAMP/PKA pathway and other intracellular signaling pathways (PKC and... Signal transduction pathways activated by GnRH GnRH activates a number of signaling pathways, including MEK, JNK, ERK1/2, cAMP/PKA, PKC, Ca2+ and CaM-dependent pathways A number of transcription factors are activated through phosphorylation by these kinases The abbreviations are given in the text (Ruf et al., 2003) 15 1.2.2.1 Calcium The roles for GnRH- induced Ca2+ signals in mediating gonadotropin... biologically active LH and FSH requires the coordinated transcription and noncovalent assembly of the two subunits 1 A B Fig 1.1: Anatomical and functional connections of the hypothalamic-pituitary axis (A) The pituitary is a small, bean-shaped gland that sits below the brain in a well-protected position (B) Embryologically, anatomically and functionally, the pituitary gland is divided into posterior and anterior... regulation of gonadotropin synthesis and secretion The differential synthesis and secretion of gonadotropins are regulated by a number of factors along the hypothalamus-pituitary-gonadal axis, including gonadotropin-releasing hormone (GnRH) , steroid hormones (estrogen, androgen and progesterone) and gonadal peptides (activin and inhibin; Gharib et al., 1990; Landefeld et al., 1983; Ling et al., 1986; Papavasiliou... secretion The gonadal peptides: inhibin, activin and follistatin (FS) also have roles in the regulation of gonadotropin gene expression by exerting positive or negative feedback Adapted from Brown and McNeilly, 1999 6 1.2.1 Transcriptional regulation of gonadotropin subunits Transcriptional regulation of gonadotropin subunit genes is mainly achieved by a series of temporally and spatially expressed transcription... Physiology of the gonadotropins The pituitary, a small gland located beneath the hypothalamus, rests in a depression of the skull base called the sella turcica It synthesizes and secretes polypeptide hormones essential for growth, reproduction, metabolic regulation, environmental adaptations and other biological activities The pituitary consists of three sections: the anterior lobe, the intermediate lobe and... signaling pathways in stimulation of gonadotropin gene expression The action of GnRH is mediated through binding a G-protein coupled seventransmembrane receptor, which is bound to two specific GTP-binding proteins (Gq, G11) (Hsieh and Martin, 1992; Liu et al., 2002b; Reinhart et al., 1992) GnRH receptor (GnRHr) activation induces activation of phospholipase C (PLC) and an increase in intracellular cAMP... Transcriptional regulation of the LHβ and FSHβ subunits In a fashion similar to the αGSU promoter, the LHβ gene is also regulated by a combinatorial array of transcription factors and regulatory elements on the promoter The proximal 140 bp region in the mammalian LHβ promoter containing Pitx1, Sf-1 and Egr-1 binding site is highly conserved across all species studied so far The three binding sites are... Hsieh and Martin, 1992) The elevated cAMP levels activate the downstream cAMP dependant kinase (PKA; Lippmann, 1975; Yoshida et al., 1975), while PLC accelerates the cleavage of phosphatidylinositol 4,5bisphosphate (PIP2), thereby stimulating production of 1, 4, 5-triphosphate (IP3) and diacylglycerol (DAG; Andrews and Conn, 1986) IP3 induces the calcium release from the intracellular stores, and also... also extracellular calcium influx through voltage-sensitive Ltype calcium channels (Naor, 1990) DAG activates protein kinase C (PKC), which activates the downstream mitogen-activated protein kinase (MAPK) pathways, including extracellular signal regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 (Liu et al., 200 2a; Mitchell et al., 1994; Roberson et al., 1999; Fig 1.5) 14 Gonadotropins (LHβ, . A FUNCTIONAL GENOMICS APPROACH FOR ELUCIDATION OF NOVEL MECHANISMS INVOLVED IN GnRH REGULATION OF THE GONADOTROPINS LUO MIN NATIONAL UNIVERSITY OF SINGAPORE. SINGAPORE 2007 A FUNCTIONAL GENOMICS APPROACH FOR ELUCIDATION OF NOVEL MECHANISMS INVOLVED IN GnRH REGULATION OF THE GONADOTROPINS By LUO MIN (B. SC.) A THESIS. Glycoprotein α subunit AP-1 Activating protein 1 ATF Activating transcription factor CA-CnA Constitutively activate CnA CaM Calmodulin CnA Calcineurin catalytic subunit A ChIP Chromatin immunoprecipitation