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ABSTRACT: The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play essential roles in the regulation of vertebrate reproduction. Activins and inhibins have opposing actions on FSH (but not LH) synthesis, either inducing or inhibiting transcription of the FSHβ subunit gene (Fshb). The translational inhibitor cycloheximide (CHX) produces inhibin-like effects in cultured pituitary cells, selectively suppressing FSH production. Using the murine gonadotrope-like cell line, LβT2, as a model, we tested the hypothesis that a component of the activin pathway is highly labile in gonadotrope cells and that its rapid loss following CHX treatment impairs activin-stimulated Fshb transcription. Treatment of cells with CHX for 6hours, but not 1hour, blocked activin A-stimulated Fshb transcription. Pre-treatment of LβT2 cells with CHX for as few as 2-3hours inhibited activin A-stimulated SMAD2/3 phosphorylation without altering total SMAD2/3 protein levels. These data indicated that CHX affects activin signalling upstream of SMAD proteins, most likely at the receptor level. Indeed, CHX rapidly reduced activin A binding to LβT2 cells. We went on to show that activin A signals via the type II receptor ACVR2, rather than ACVR2B, to regulate Fshb transcription and that the receptor has a half life of ~2hours in LβT2 cells. The mechanism of ACVR2 turnover remains undefined, but appears to be ligand-, proteasome-, and lysosome-independent. Collectively, these data indicate that CHX produces inhibin-like effects in gonadotropes by preventing de novo synthesis of the highly labile ACVR2, thereby blocking activin signaling to the Fshb promoter.
Cellular signalling 03/2013; · 4.09 Impact Factor
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ABSTRACT: Successful mammalian reproduction depends on proper synthesis of the pituitary-derived glycoprotein hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several transcription factors cooperate to activate cell-specific and hormone-regulated expression of the gonadotropin beta subunits (Lhb and Fshb). Among these, NR5A1 (steroidogenic factor 1; SF1) has been shown to directly bind to the Lhb promoter, mediate basal and gonadotropin-releasing hormone (GnRH)-stimulated Lhb transcription, and possibly directly regulate Fshb expression. Recently, the closely-related NR5A2 was shown to activate the rat Lhb promoter in vitro. Here, we further characterized the role of NR5A2 in regulating gonadotropin synthesis. Ectopically expressed NR5A2 directly activated the murine Lhb promoter in a manner identical to that of NR5A1, whereas neither factor activated the murine Fshb promoter. In LβT2 gonadotrope-like cells, depletion of endogenous NR5A1 or NR5A2 impaired basal and GnRH-stimulated Lhb and Fshb transcription. To analyze the physiological role of NR5A2 in gonadotropes in vivo, we generated mice with a gonadotrope-specific deletion of Nr5a2. In contrast with our in vitro data, these mice had normal pituitary Lhb and Fshb expression and intact fertility. Together, our data establish that NR5A2 can act in a non-redundant manner to regulate Lhb and Fshb transcription in vitro, but is dispensable in vivo.
PLoS ONE 01/2013; 8(3):e59058. · 4.09 Impact Factor
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ABSTRACT: Activins stimulate follicle-stimulating hormone (FSH) β subunit (Fshb) gene transcription in pituitary gonadotrope cells. Previous studies suggest that activins signal via homolog of Drosophila mothers against decapentaplegic (SMAD) proteins to stimulate murine or porcine Fshb promoter activity in the gonadotrope-like cell line, LβT2. In contrast, activins were suggested to regulate the ovine Fshb promoter via a SMAD-independent pathway involving TGFβ associated kinase 1 (TAK1, MAP3K7) and p38 mitogen activated protein kinase (MAPK). Here, we examined roles for TAK1 and p38 in activin A-stimulated murine and ovine Fshb transcription. The TAK1 inhibitor 5Z-7-Oxozeanol (Oxo) significantly impaired fold activin A induction of murine and ovine Fshb promoter-reporters (Fshb-luc) in LβT2 cells, but only at concentrations 50-100 fold greater than its IC(50) for TAK1. Moreover, Oxo failed to inhibit activin A induction of endogenous Fshb mRNA levels or fold induction of Fshb-luc activity by a constitutively active form of the activin type I receptor (ALK4). Oxo, at a concentration 5-10 fold greater than its IC(50) for TAK1, attenuated TAK1/TAB2 stimulation of a p38-dependent reporter in the same cells. A Map3k7 siRNA impaired TAK1/TAB2-stimulated p38-dependent reporter activity, but failed to antagonize activin A-stimulated Fshb-luc. Though TAK1 was previously suggested to act via p38 to stimulate the ovine Fshb promoter, activin A failed to stimulate p38 phosphorylation in LβT2 cells. In apparent contrast, however, the p38 inhibitors SB203580 and SB202190 concentration-dependently attenuated activin A-induced Fshb-luc activity. Given the lack of p38 activation, we postulated that the inhibitors might non-selectively antagonize ALK4 activity. Indeed, both attenuated activin A-stimulated SMAD2 phosphorylation, consistent with direct antagonism of ALK4 kinase activity. Finally, we observed that RNA-mediated suppression of Smad4, and to a lesser extent Smad3, attenuated activin A induction of both murine and ovine Fshb promoter-reporters. Collectively, these data suggest that activin A signals via SMAD proteins, but not TAK1 or p38, to regulate murine and ovine Fshb transcription in gonadotrope-like cells.
Cellular signalling 04/2012; 24(8):1632-40. · 4.09 Impact Factor
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ABSTRACT: Pituitary FSH regulates ovarian and testicular function. Activins stimulate FSHβ subunit (Fshb) gene transcription in gonadotrope cells, the rate-limiting step in mature FSH synthesis. Activin A-induced murine Fshb gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor FOXL2 (FOXL2). Here, we demonstrate that FOXL2 synergizes with SMAD2, SMAD3, and SMAD4 to stimulate murine Fshb promoter-reporter activity in heterologous cells. Moreover, SMAD3-induction of Fshb promoter activity or endogenous mRNA expression is dependent upon endogenous FOXL2 in homologous cells. FOXL2/SMAD synergy requires binding of both FOXL2 and SMAD3 or SMAD4 to DNA. Of three putative forkhead-binding elements identified in the murine Fshb promoter, only the most proximal is absolutely required for activin A induction of reporter activity in homologous cells. Additionally, mutations to the minimal SMAD-binding element adjacent to the proximal forkhead-binding element abrogate activin A or FOXL2/SMAD3 induction of reporter activity. In contrast, a mutation that impairs an adjacent PBX1/PREP1 (pre-B cell leukemia transcription factor 1-PBX/knotted-1 homeobox-1) binding site does not alter activin A-stimulated promoter activity in homologous cells. Collectively, these and previous data suggest a model in which activins stimulate formation of FOXL2-SMAD2/3/4 complexes, which bind to the proximal murine Fshb promoter to stimulate its transcription. Within these complexes, FOXL2 and SMAD3 or SMAD4 bind to adjacent cis-elements, with SMAD3 brokering the physical interaction with FOXL2. Because this composite response element is highly conserved, this suggests a general mechanism whereby activins may regulate and/or modulate Fshb transcription in mammals.
Molecular Endocrinology 05/2011; 25(7):1170-83. · 4.54 Impact Factor
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ABSTRACT: Activins stimulate FSH synthesis and secretion by pituitary gonadotrope cells. Activin A induction of porcine and murine FSHβ (Fshb) gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor L2 (FOXL2). Using both heterologous and homologous cell models, we demonstrate that FOXL2 functionally synergizes with SMAD3/4 to stimulate porcine Fshb promoter-reporter activity. We further show that endogenous FOXL2 and SMAD2/3 physically interact in homologous cells. We identify two composite cis-elements of adjacent FOXL2 and SMAD binding sites in the proximal porcine Fshb promoter that mediate activin A, FOXL2, and SMAD3 actions. FOXL2 can bind these elements independently of SMADs, whereas SMAD3/4 binding requires high-affinity FOXL2 binding. Conversely, FOXL2 alone is insufficient to regulate Fshb transcription and requires SMADs to induce promoter activity. Collectively, our data suggest a model in which activins stimulate formation and nuclear accumulation of SMAD3/4 complexes, which interact with FOXL2 bound to at least two proximal promoter elements. This association stabilizes SMAD3/4 binding to adjacent SMAD binding elements. SMAD-FOXL2 complexes then mediate activation of transcription through a currently unknown mechanism. Conservation of one of the two composite cis-elements suggests that this may form part of a general mechanism whereby activins regulate Fshb subunit transcription and FSH synthesis.
Endocrinology 11/2010; 151(11):5456-67. · 4.46 Impact Factor
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Mélanie Robitaille,
Sarah Gora, Ying Wang,
Eugénie Goupil,
Darlaine Pétrin,
Danny Del Duca,
Louis R Villeneuve,
Bruce G Allen,
Stéphane A Laporte,
Daniel J Bernard,
Terence E Hébert
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ABSTRACT: Following stimulation of G protein-coupled receptors (GPCRs) at the cell surface, heterotrimeric G proteins are activated. Both Galpha and Gbetagamma subunits regulate specific effectors to transmit signals received by the receptor. Recent data suggest potential nuclear localization or translocation of the Gbetagamma subunit. Here, we show that co-expression of the Gbetagamma dimer decreased phorbol 12-myristate 13-acetate (PMA)-stimulated AP-1 gene reporter activity in HEK293 cells as well as the AP-1 dependent gonadotropin-releasing hormone-stimulated human follicle-stimulating hormone beta reporter activity in LbetaT2 gonadotrope cells. Further, we identify Fos transcription factors as novel interactors of the Gbeta1 subunit, using protein fragment complementation assays, as well as co-immunoprecipitation in vivo and in vitro. Fos proteins dimerize with Jun proteins to form activator protein-1 (AP-1) transcription factor complexes, which regulate target gene expression. Gbetagamma did not interfere with the dimerization of Fos and Jun or their ability to bind DNA. Rather, Gbetagamma co-localized with the AP-1 complex in the nucleus and recruited histone deacetylases (HDACs) to inhibit AP-1 transcriptional activity. Our data indicate a novel role for Gbetagamma subunits as transcriptional regulators.
Cellular signalling 08/2010; 22(8):1254-66. · 4.09 Impact Factor
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ABSTRACT: The pituitary gonadotropin hormones, FSH and LH, are key regulators of reproductive physiology. Though the two hormones are produced by the same cell type, often in response to the same endocrine and paracrine regulators, they sub-serve different biological functions and their synthesis and secretion are differentially regulated. This stems largely from differences in transcriptional, post-transcriptional, and post-translational regulation of their unique beta subunits. That is, both hormones are dimeric glycoproteins and share a common alpha subunit. Their unique beta subunits, however, derive from different genes encoding distinct proteins. Past and recent research indicates synthesis and release of the two hormones are subject to extensive and independent regulation. LH appears to be secreted predominantly via the regulated secretory pathway, whereas FSH release is largely constitutive. As such, investigations of FSH-beta subunit synthesis may lend direct insight into mechanisms underlying patterns of secreted FSH, more so than investigations of the LHbeta subunit. Here, we review recent investigations of transcriptional regulation of the FSH-beta subunit gene from different mammalian species, including humans. The results reveal both conserved and species-specific regulatory mechanisms that might contribute to inter-species variation in FSH release.
Fertility and sterility 05/2010; 93(8):2465-85. · 3.97 Impact Factor
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ABSTRACT: Activins regulate FSH synthesis by stimulating the phosphorylation and nuclear accumulation of SMAD2 and SMAD3, which bind to a consensus SMAD-binding element in the proximal murine FSHbeta (Fshb) subunit gene to drive transcription. Previous over-expression and in vitro DNA binding analyses suggested that SMAD4 participates in complexes with SMAD2 and SMAD3 to regulate Fshb expression. Here, we have characterized the role of endogenous SMAD4 in activin A induction of Fshb transcription in immortalized murine gonadotropes (LbetaT2). We identified five murine Smad4 mRNA isoforms, of which, four are newly described; however, the canonical full-length form predominated at both the mRNA and protein levels. Depletion of endogenous SMAD4 by RNA interference (RNAi) abolished activin A-induced Fshb promoter-reporter activity and greatly attenuated constitutively active activin type IB receptor-stimulated Fshb mRNA levels. The activin A response was rescued with an RNAi-resistant form of wild-type SMAD4, but not with a DNA-binding-deficient (Lys88Arg) SMAD4, suggesting that DNA binding by SMAD4 is necessary for activin induction of the Fshb gene. Though SMAD2 and SMAD3 are generally thought to partner with SMAD4 prior to accumulation in the nucleus, treatment with leptomycin B, an inhibitor of SMAD4 nuclear export, reduced but did not prevent activin A induction of Fshb mRNA levels or promoter activity. In addition, a constitutively nuclear form of SMAD4 rescued the effect of endogenous SMAD4 depletion. Collectively, these data demonstrate a necessary role for SMAD4 in activin A induction of the murine Fshb gene in immortalized gonadotropes.
Journal of Molecular Endocrinology 04/2010; 44(6):349-62. · 3.48 Impact Factor
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ABSTRACT: Selective synthesis and release of FSH from pituitary gonadotropes is regulated by activins. Activins directly stimulate murine FSHbeta (Fshb) subunit gene transcription through a consensus 8-bp Sma- and Mad-related protein-binding element (SBE) in the proximal promoter. In contrast, the human FSHB promoter is relatively insensitive to the direct effects of activins and lacks this SBE. The proximal porcine Fshb promoter, which is highly conserved with human, similarly lacks the 8-bp SBE, but is nonetheless highly sensitive to activins. We used a comparative approach to determine mechanisms mediating differential activin induction of human, porcine, and murine Fshb/FSHB promoters. We mapped an activin response element in the proximal porcine promoter and identified interspecies variation in a single base pair in close proximity that conferred strong binding of the forkhead transcription factor FOXL2 to the porcine, but not human or murine, promoters. Introduction of the human base pair into the porcine promoter abolished FOXL2 binding and activin A induction. FOXL2 conferred activin A induction to the porcine promoter in heterologous cells, whereas knockdown of the endogenous protein in gonadotropes inhibited the activin A response. The murine Fshb promoter lacks the high-affinity FOXL2-binding site, but its activin induction is FOXL2 sensitive. We identified a more proximal FOXL2-binding element in the murine promoter, which is conserved across species. Mutation of this site attenuated activin A induction of both the porcine and murine promoters. Collectively, the data indicate a novel role for FOXL2 in activin A-regulated Fshb transcription.
Molecular Endocrinology 04/2009; 23(7):1001-13. · 4.54 Impact Factor
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ABSTRACT: Activins are pleiotropic members of the TGFbeta superfamily and were initially characterized based on their abilities to stimulate FSH synthesis and secretion by gonadotrope cells of the anterior pituitary gland. Here, we identified the gene encoding the steroidogenic enzyme, 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD1; Hsd17b1), as an activin-responsive gene in immortalized gonadotrope cells, LbetaT2. 17beta-HSD1 catalyzes the conversion of estrone to the more active 17beta-estradiol, and activin A stimulated an increase in this enzymatic activity in these cells. We demonstrated that activins signaled via the type I receptor, activin receptor-like kinase (ALK4), and the intracellular signaling protein, SMAD2, to regulate Hsd17b1 transcription in immediate-early fashion. Critical cis-elements, including a minimal SMAD-binding element, were mapped to within 100 bp of the start of transcription. Activin/ALK4 signaling also regulated Hsd17b1 transcription in both immortalized and primary cultured murine granulosa cells. The promoter regions mediating basal and activin/ALK4-regulated promoter activity were generally conserved across the different cell types. The data show that activin A rapidly regulates Hsd17b1 transcription in gonadotrope and granulosa cells and may thereby regulate local 17beta-estradiol synthesis.
Journal of Endocrinology 02/2009; 201(1):89-104. · 3.55 Impact Factor
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ABSTRACT: Gonadotrophin-releasing hormone (GNRH1) regulates pituitary luteinizing hormone (LH). Previous studies have delineated a mechanism for GNRH1-induced LHbeta subunit gene (Lhb) transcription, the rate-limiting step in LH production. GNRH1 induces expression of early growth response 1 (EGR1), which interacts with steroidogenic factor 1 (SF1) and paired-like homeodomain transcription factor 1 (PITX1) to regulate Lhb promoter activity. Though the cis-elements for these factors are conserved across species, regulation of human LHB transcription has not been thoroughly investigated. We therefore characterized LHB transcriptional regulation by GNRH1 using promoter-reporter analyses in LbetaT2 cells. GNRH1 stimulated LHB transcription via an extracellular signal-regulated kinase 1/2 pathway. EGR1 bound to two binding sites on the LHB promoter and this binding was increased by GNRH1. Mutation of either site or knockdown of endogenous EGR1 decreased basal and/or GNRH1-regulated promoter activity. The human LHB promoter also contains low and high affinity SF1 binding sites. Mutation of these elements or depletion of endogenous SF1 impaired basal and ligand-induced transcription. Knockdown of PITX1 or PITX2 isoforms impaired GNRH1 induction, and endogenous PITX1 bound to the candidate PITX binding site on the LHB promoter. Thus, the mechanism described for GNRH1 regulation of Lhb in other species is largely conserved for human LHB. We also uncover a previously unappreciated role for PITX2 isoforms in this process.
Molecular Human Reproduction 01/2009; 15(2):77-87. · 3.85 Impact Factor
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ABSTRACT: GnRH1 stimulates the synthesis and secretion of FSH and LH from the anterior pituitary gland. The molecular mechanisms through which GnRH1 produces these effects in humans have not been determined. Here, we examined transcriptional regulation of the human FSHbeta (FSHB) subunit using reporter assays in immortalized murine gonadotrope cells. GnRH1 dose and time dependently stimulated FSHB promoter activity, with peak stimulation occurring at 8 h. GnRH1 rapidly stimulated various MAPK cascades, though the ERK1/2 and p38 pathways appeared to be most critical for FSHB induction. Indeed, constitutively active forms of both Raf1 kinase and MAP2K6 (MKK6) were sufficient to stimulate reporter activity. GnRH1 stimulated activator protein-1 (AP-1) (FosB, c-fos, JunB, and cJun) synthesis and complex formation, the latter of which bound to a conserved cis-element within -120 bp of the transcription start site. A second, lower affinity, site was mapped more proximally. Mutations of both cis-elements diminished GnRH1-stimulated promoter activity, though disruption of the higher affinity site had a more dramatic effect. A dominant-negative Fos protein dose dependently inhibited GnRH1-stimulated FSHB transcription, confirming a role for endogenous AP-1 proteins. MAPK kinase 1 (MEK1) and p38 inhibitors significantly attenuated GnRH1-stimulated c-fos, FosB, and JunB synthesis, suggesting a mechanism whereby the ERK1/2 and p38 signaling pathways regulate FSHB transcription. Activins and inhibins potently regulate FSH synthesis in rodents, but their roles in FSH regulation in humans are less clear. Activin A, though weak on its own, synergized with GnRH1 to stimulate human FSHB promoter activity. In contrast, activin A partially inhibited GnRH1-stimulated LHbeta subunit (LHB) transcription. The GnRH1 and activin A signaling pathways appear to converge at the level of the high-affinity AP-1 site. Fos and Jun proteins synergistically regulate reporter activity through this element, and their effects are potentiated by coexpression of either Smad2 or Smad3, effectors in the activin signaling cascade. In summary, GnRH1 and activin A synergistically regulate human FSHB subunit transcription. The combined actions of AP-1 and Smad proteins acting through a conserved AP-1 element provide a candidate mechanism for this effect. The ability of activins to potentiate selectively the effects of GnRH1 on FSHB expression suggests a model for preferential increases in FSH secretion at the luteal-follicular transition of the menstrual cycle.
Endocrinology 11/2008; 149(11):5577-91. · 4.46 Impact Factor
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Mélanie Robitaille,
Sarah Gora, Ying Wang,
Eugénie Goupil,
Darlaine Pétrin,
Danny Del Duca,
Louis R. Villeneuve,
Bruce G. Allen,
Stéphane A. Laporte,
Daniel J. Bernard,
Terence E. Hébert
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ABSTRACT: Following stimulation of G protein-coupled receptors (GPCRs) at the cell surface, heterotrimeric G proteins are activated. Both Gα and Gβγ subunits regulate specific effectors to transmit signals received by the receptor. Recent data suggest potential nuclear localization or translocation of the Gβγ subunit. Here, we show that co-expression of the Gβγ dimer decreased phorbol 12-myristate 13-acetate (PMA)-stimulated AP-1 gene reporter activity in HEK293 cells as well as the AP-1 dependent gonadotropin-releasing hormone-stimulated human follicle-stimulating hormone β reporter activity in LβT2 gonadotrope cells. Further, we identify Fos transcription factors as novel interactors of the Gβ1 subunit, using protein fragment complementation assays, as well as co-immunoprecipitation in vivo and in vitro. Fos proteins dimerize with Jun proteins to form activator protein-1 (AP-1) transcription factor complexes, which regulate target gene expression. Gβγ did not interfere with the dimerization of Fos and Jun or their ability to bind DNA. Rather, Gβγ co-localized with the AP-1 complex in the nucleus and recruited histone deacetylases (HDACs) to inhibit AP-1 transcriptional activity. Our data indicate a novel role for Gβγ subunits as transcriptional regulators.
Cellular Signalling. 22(8):1254-1266.
