No abstract

In rodents, activins stimulate immediate-early increases in pituitary follicle-stimulating hormone (Fshb) subunit transcription. Here, we investigated the underlying signaling mechanisms using the mouse gonadotrope cell line, L T2. Activin A increased mouse Fshb-luciferase reporter activity within 4 h through a Smad-dependent signaling pathway. The ligand rapidly stimulated formation of SMAD2/3/4 complexes that could interact with a consensus palindromic Smad binding element (SBE) in the proximal Fshb promoter. SMAD over-expression potently stimulated transcription, with the combination of SMADs 2, 3 and 4 producing the greatest synergistic activation. A mutation in the SBE that abolished Smad binding greatly impaired the effects of acute (4 h) activin A treatment and SMAD over-expression on promoter activity, but did not abolish the effects of chronic (24 h) activin A exposure. Within activated SMAD complexes, SMADs 3 and 4 appeared to bind the SBE simultaneously and the binding of both was required for maximal transcriptional activation. Interestingly, the human FSHB promoter, which lacks the consensus SBE, was neither rapidly stimulated by activin A nor by over-expressed SMADs, but was activated by 24 h activin A. Addition of the SBE to the human promoter increased both SMAD2/3/4-sensitivity and acute regulation by activin A, though not to levels observed in mouse. We postulate that short reproductive cycles in female rodents, particularly the brief interval between the primary and secondary FSH surges of the estrous cycle, require the Fshb promoter in these animals to be particularly sensitive to the rapid, Smad-dependent actions of activins on transcription. The human FSHB promoter, in contrast, is chronically regulated by activins seemingly through a SMAD-independent pathway.

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.

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.

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.