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FoxL2 Is required for activin induction of the mouse and human follicle-stimulating hormone beta-subunit genes.

ABSTRACT: Activin is a major physiological regulator of FSH. We identify FoxL2 as a critical component in activin induction of FSHbeta, both for the mouse gene, induction of which is Sma- and Mad-related protein (Smad) dependent, and for the human gene that is Smad independent. FoxL2 has been shown to regulate gonadotrope gene expression (GnRH receptor, alpha-glycoprotein subunit, porcine FSHbeta, and follistatin), but the mechanisms of action are not well understood. We identify novel sites required for activin action in both the mouse and human FSHbeta promoters, some of which bind FoxL2, and show that the FoxL2-binding element encompasses a larger region (12 bp) than the previously identified forkhead-binding consensus (7 bp). Remarkably, although required for activin induction, FoxL2 sites neither contribute to basal FSHbeta promoter activity nor confer activin response to a heterologous promoter; thus, they are neither classical activin-response elements nor is their role solely to recruit Smads to the promoter. FoxL2 overexpression can potentiate activin induction in gonadotropes and can confer activin responsiveness to FSHbeta in heterologous cells where this promoter is normally refractory to activin induction. Although Smad3 requires the presence of FoxL2 sites to induce mouse FSHbeta, even through its consensus Smad-binding element; the human promoter, which is induced by activin independently of Smad3, also requires FoxL2 sites for its induction by activin; thus the actions of FoxL2 are not exclusively through interactions with the Smad pathway. Thus, FoxL2 plays a key role in activin induction of the FSHbeta gene, by binding to sites conserved across multiple species.

SUBMITTER: Corpuz PS

PROVIDER: S-EPMC2870942 | biostudies-literature |

REPOSITORIES: biostudies-literature

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FOXL2 is involved in the synergy between activin and progestins on the follicle-stimulating hormone β-subunit promoter.

Project description:Differential regulation of gonadotropin hormone production in the pituitary is critical for fertility. Activin and progesterone signaling in gonadotrope cells is important for Fshb gene expression. Previously, we reported that synergy between activin and progestins required the binding of SMAD proteins and the progesterone receptor (PR) to the murine Fshb promoter. In this study, we demonstrate that the FOXL2 transcription factor is also necessary for the full synergistic response between activin and progestins. We show that this synergy occurs in a species-specific manner and that multiple elements in the Fshb promoter that bind forkhead box L2 (FOXL2), SMA/mothers against decapentaplegic homologs (SMAD), and PR are required. Furthermore, we demonstrate that FOXL2 can physically interact with PR and SMAD3. Thus, it is likely that protein-protein interactions among FOXL2, SMAD, and PR recruited to the Fshb promoter play a key role in facilitating Fshb transcription before the secondary FSH surge in rodents.

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Project description:Synthesis of the FSH beta-subunit (FSHbeta) is critical for normal reproduction in mammals, and its expression within the pituitary gonadotrope is tightly regulated by activin. Here we show that Runt-related (RUNX) proteins, transcriptional regulators known to interact with TGFbeta signaling pathways, suppress activin induction of FSHbeta gene expression. Runx2 is expressed within the murine pituitary gland and dramatically represses activin-induced FSHbeta promoter activity, without affecting basal expression in LbetaT2 cells, an immortalized mouse gonadotrope cell line. This repressive effect is specific, because RUNX2 induces LHbeta transcription (with or without activin) and does not interfere with GnRH induction of either gonadotropin beta-subunit gene. Analysis of the murine FSHbeta promoter by transfection and gel shift assays reveals that RUNX2 repression localizes to a Runx-binding element at -159/-153, which is adjacent to a previously recognized region critical for activin induction. Mutation of this -153 activin-response element or, indeed, any of the five activin-responsive regions prevents activin induction and, in fact, RUNX2 suppression, instead converting RUNX2 to an activator of the FSHbeta gene. Although the Runx-binding element is required for RUNX2-mediated repression of FSHbeta induction by either activin or Smad3, confirming a functional role of this novel site, protein interactions in addition to those between RUNX2 and Smads are necessary to account for full repression of activin induction. In summary, the present study provides evidence for Runx2-mediated repression of activin-induced FSHbeta gene expression and reveals the context dependence of Runx2 action in hormonal regulation of the gonadotropin genes.

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A novel role for the forkhead transcription factor FOXL2 in activin A-regulated follicle-stimulating hormone beta subunit transcription.

Project description: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.

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Project description:Follicle-stimulating hormone beta subunit (Fshb) expression is regulated by transforming growth factor beta superfamily ligands. Recently, we demonstrated that bone morphogenetic proteins (BMPs) stimulate Fshb transcription alone and in synergy with activins. Also, transfection of the BMP type II receptor (BMPR2) and constitutively active forms of the type I receptors (activin A receptor type I [ACVR1] or BMP receptor type IA [BMPR1A]) in immortalized gonadotroph cells (LbetaT2) stimulated murine Fshb promoter-reporter activity. A third type I receptor (BMP receptor type IB [BMPR1B]) is also expressed in LbetaT2 cells, but we did not previously assess its functional role. A point mutation in BMPR1B (Q249R) is associated with increased ovulation rates and elevated FSH levels in Booroola (FecB) sheep. Herein, we assessed whether BMPR1B can regulate Fshb transcription in LbetaT2 cells and whether its ability to do so is altered by the Q249R mutation. As with ACVR1 and BMPR1A, coexpression of BMPR1B with BMPR2 increased Fshb promoter-reporter activity in BMP2-dependent and BMP2-independent fashions. Unexpectedly, the BMPR1B-Q249R mutant was equivalent to the wild type in its ability to stimulate SMAD1/5 phosphorylation and Fshb transcription. Pharmacological inhibition of ACVR1, BMPR1A, and BMPR1B confirmed that one or more of these receptors are required for BMP2-stimulated SMAD1/5 phosphorylation and Fshb reporter activity. Knockdown of endogenous BMPR1A, but not ACVR1 or BMPR1B, significantly impaired the synergism of BMP2 with activin A. Collectively, these data suggest that BMPR1A is the preferred BMP2 type I receptor in LbetaT2 cells and that neither ACVR1 nor BMPR1B compensates for its loss. The specific mechanism(s) through which the Booroola FecB mutation alters BMPR1B function remains to be determined.

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Project description: Not available

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Project description:Follicle-stimulating hormone (FSH) is essential for human reproduction. The unique functions of this hormone are provided by the FSH receptor-binding beta-subunit encoded by the FSHB gene. Resequencing and genotyping of FSHB in three European, two Asian and one African population, as well as in the great apes (chimpanzee, gorilla, orangutan), revealed low diversity and significant excess of polymorphisms with intermediate frequency alleles. Statistical tests for FSHB showed deviations from neutrality in all populations suggesting a possible effect of balancing selection. Two core haplotypes were identified (carried by 76-96.6% of each population's sample), the sequences of which are clearly separated from each other. As fertility most directly affects an organism's fitness, the carriers of these haplotypes have apparently had more success in human history to contribute to the next generation. There is a preliminary observation suggesting that the second most frequent FSHB haplotype may be associated with rapid conception success in females. Interestingly, the same haplotype is related to an ancestral FSHB variant shared with the ancestor of the great apes. The determination of the functional consequence of the two core FSHB variants may have implications for understanding and regulating human fertility, as well as in assisting infertility treatments.

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