title: Activator protein-1 and smad proteins synergistically regulate human follicle-stimulating hormone beta-promoter activity.
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.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada. daniel.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Jérôme
authors.initials: J
authors.lastname: Fortin
authors.forename: Pankaj
authors.initials: P
authors.lastname: Lamba
authors.forename: Marco
authors.initials: M
authors.lastname: Bonomi
authors.forename: Luca
authors.initials: L
authors.lastname: Persani
authors.forename: Mark S
authors.initials: MS
authors.lastname: Roberson
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Bernard
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D037201:Follicle Stimulating Hormone, beta Subunit / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011485:Protein Binding; D051785:Smad Proteins / Q000378:metabolism / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
chemical_list: D037201:Follicle Stimulating Hormone, beta Subunit; D051785:Smad Proteins; D018808:Transcription Factor AP-1; D007987:Gonadotropin-Releasing Hormone