Stimulation of Luteinizing Hormone Gene Promoter Activity by the Orphan Nuclear Receptor, Steroidogenic Factor-1

Abstract Ovarian inhibin production is stimulated by FSH and several TGFβ family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFβ/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-α content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-α and inhibin-β subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-α gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-α promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-α gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-α promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.

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Pituitary homeobox 1 (Pitx1) stimulates rat LHβ gene expression via two functional DNA-regulatory regions

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01754 ◽

2005 ◽

Vol 35 (1) ◽

pp. 145-158 ◽

Cited By ~ 14

Author(s):

Qiaorong Jiang ◽

Kyeong-Hoon Jeong ◽

Cheryl D Horton ◽

Lisa M Halvorson

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Gene Promoter ◽

Orphan Nuclear Receptor ◽

Mobility Shift ◽

Regulatory Regions ◽

Steroidogenic Factor 1 ◽

Reproductive Axis ◽

Functional Dna ◽

Mobility Shift Assay

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH β-subunit gene (LHβ). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHβ gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHβ gene promoter. While investigating the role of Pitx1 in regulating rat LHβ gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions −73 to −52 in the rat LHβ gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5′Pitx1 site as well as this putative 3′Pitx1 region. In transient transfection analysis, mutation of the LHβ-3′Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5′Pitx1 site with further loss following mutation of the 3′Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHβ gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

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Activation of the thyroid hormone receptor α gene promoter by the orphan nuclear receptor ERRα

Oncogene ◽

10.1038/sj.onc.1202167 ◽

1998 ◽

Vol 17 (19) ◽

pp. 2429-2435 ◽

Cited By ~ 48

Author(s):

Jean-Marc Vanacker ◽

Edith Bonnelye ◽

Cateline Delmarre ◽

Vincent Laudet

Keyword(s):

Thyroid Hormone ◽

Nuclear Receptor ◽

Hormone Receptor ◽

Thyroid Hormone Receptor ◽

Gene Promoter ◽

Orphan Nuclear Receptor ◽

Thyroid Hormone Receptor Α

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Dax1 Up-Regulates Oct4 Expression in Mouse Embryonic Stem Cells via LRH-1 and SRA

Molecular Endocrinology ◽

10.1210/me.2010-0133 ◽

2010 ◽

Vol 24 (12) ◽

pp. 2281-2291 ◽

Cited By ~ 40

Author(s):

Victoria R. Kelly ◽

Bin Xu ◽

Rork Kuick ◽

Ronald J. Koenig ◽

Gary D. Hammer

Keyword(s):

Nuclear Receptor ◽

Transcriptional Activation ◽

Chromatin Immunoprecipitation ◽

Target Genes ◽

Embryonic Stem ◽

Steroid Receptor ◽

Orphan Nuclear Receptor ◽

Steroidogenic Factor 1 ◽

Oct4 Expression ◽

Mes Cells

Abstract Dax1 (Nr0b1) is an atypical orphan nuclear receptor that has recently been shown to play a role in mouse embryonic stem (mES) cell pluripotency. Here we describe a mechanism by which Dax1 maintains pluripotency. In steroidogenic cells, Dax1 protein interacts with the NR5A nuclear receptor steroidogenic factor 1 (Nr5a1) to inhibit transcription of target genes. In mES cells, liver receptor homolog 1 (LRH-1, Nr5a2), the other NR5A family member, is expressed, and LRH-1 has been shown to interact with Dax1. We demonstrate by coimmunoprecipitation that Dax1 is, indeed, able to form a complex with LRH-1 in mES cells. Because Dax1 was historically characterized as an inhibitor of steroidogenic factor 1-mediated transcriptional activation, we hypothesized that Dax1 would inhibit LRH-1 action in mES cells. Therefore, we examined the effect of Dax1 on the LRH-1-mediated activation of the critical ES cell factor Oct4 (Pou5f1). Chromatin immunoprecipitation localized Dax1 to the Oct4 promoter at the LRH-1 binding site, and luciferase assays together with Dax1 overexpression and knockdown experiments revealed that, rather than repress, Dax1 accentuated LRH-1-mediated activation of the Oct4 gene. Similar to our previously published studies that defined the RNA coactivator steroid receptor RNA activator as the critical mediator of Dax1 coactivation function, Dax1 augmentation of LRH-1-mediated Oct4 activation is dependent upon steroid receptor RNA activator. Finally, utilizing published chromatin immunoprecipitation data of whole-genome binding sites of LRH-1 and Dax1, we show that LRH-1 and Dax1 commonly colocalize at 288 genes (43% of LRH-1 target genes), many of which are involved in mES cell pluripotency. Thus, our results indicate that Dax1 plays an important role in the maintenance of pluripotency in mES cells through interaction with LRH-1 and transcriptional activation of Oct4 and other genes.

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Steroidogenic Factor-1 and The Gonadotrope-Specific Element Enhance Basal and Pituitary Adenylate Cyclase-Activating Polypeptide-Stimulated Transcription of the Human Glycoprotein Hormone α-Subunit Gene in Gonadotropes

Molecular Endocrinology ◽

10.1210/me.2002-0393 ◽

2003 ◽

Vol 17 (11) ◽

pp. 2177-2188 ◽

Cited By ~ 21

Author(s):

Robert C. Fowkes ◽

Marion Desclozeaux ◽

Mayur V. Patel ◽

Simon J. B. Aylwin ◽

Peter King ◽

...

Keyword(s):

Adenylate Cyclase ◽

Promoter Activity ◽

Kinase Inhibitor ◽

Mutant Form ◽

Orphan Nuclear Receptor ◽

Glycoprotein Hormone ◽

Steroidogenic Factor 1 ◽

Specific Element ◽

Α Subunit ◽

Pituitary Adenylate Cyclase

Abstract In the anterior pituitary, expression of the common glycoprotein hormone α-subunit (αGSU) is mediated in part by multiple response elements residing in the distal promoter (−435 bp). One such site is the gonadotrope-specific element (GSE), which is bound by the orphan nuclear receptor steroidogenic factor-1 (SF-1) and confers pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated αGSU expression. Here we investigated the functional importance of the GSE and SF-1 phosphorylation in both basal and stimulated αGSU transcription. Mutation of the GSE reduced basal and PACAP-stimulated αGSU promoter activity in the αT3-1 gonadotrope cell line. Overexpression of wild-type SF-1, but not an S203A mutant form of SF-1, enhanced basal and PACAP-stimulated αGSU promoter activity. The effect of PACAP on αGSU promoter activity was inhibited after overexpression of MAPK phosphatase. Helix assembly of the SF-1 ligand-binding domain was stimulated by PACAP in vitro via a MAPK-dependent pathway, as determined using a mammalian two-hybrid assay. PACAP quickly activated MAPK (within 5 min) and also resulted in elevated levels of phospho-cAMP response element-binding protein and phospho-SF-1, as judged by a specific antiphospho-S203 antibody; this effect was blocked by the MAPK kinase inhibitor, UO126. Collectively, these data demonstrate that SF-1 binds to the GSE and activates both basal and PACAP-stimulated αGSU transcription, which is further increased by phosphorylation at Ser203 via MAPK. These data suggest strongly that the induction of αGSU gene expression by peptide hormone signaling is coupled directly to the posttranslational status of SF-1.

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Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1811 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1811-1814 ◽

Cited By ~ 26

Author(s):

F P Lemaigre ◽

D A Lafontaine ◽

S J Courtois ◽

S M Durviaux ◽

G G Rousseau

Keyword(s):

Growth Hormone ◽

Promoter Activity ◽

Gene Promoter ◽

Human Growth ◽

Growth Hormone Gene ◽

Dnase I Footprinting ◽

Kinetics Of ◽

Distal Site ◽

Human Growth Hormone Gene ◽

Stimulation Of

DNase I footprinting experiments showed that binding activities of Sp1 and of GHF-1 to its distal site on the human growth hormone gene promoter are mutually exclusive. The kinetics of GHF-1 binding were indicative of positive cooperativity. The Sp1 site did not affect promoter activity in cell-free transcription. Still, Sp1 could compensate partially for the decreased stimulation of transcription seen at low GHF-1 concentrations.

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