FOXL2 Regulation of Murine Follicle-Stimulating Hormone Beta Transcription in Primary and Immortalized Murine Gonadotrophs.

Abstract

Follicle-stimulating hormone (FSH) regulates ovarian and testicular function. Impairments in FSH synthesis or action have profound effects on fertility. FSH is a heterodimeric glycoprotein composed of a common α and unique β subunit. The transforming growth factor β superfamily ligands, activins and inhibins, were identified based on their respective abilities to selectively stimulate and suppress FSH release from pituitary gonadotroph cells. Activins stimulate FSHβ subunit (Fshb) transcription, the rate-limiting step in mature FSH synthesis. We and others demonstrated that activin A regulates murine Fshb transcription in immortalized gonadotroph cells (LβT2), at least in part, via the actions of SMAD proteins and the forkhead transcription factor FOXL2. Here, we investigated FOXL2's role in endogenous Fshb expression in primary murine pituitary cells and defined a mechanism for SMAD/FOXL2 synergistic activation of the murine Fshb promoter. Pituitaries from adult "floxed" Foxl2 mice were dissociated and plated in primary culture. Cells were then incubated with control or Cre recombinase (Ad-Cre) encoding adenoviruses. Cells were efficiently transduced and the single exon Foxl2 locus successfully recombined. Activin A stimulated Fshb mRNA levels in control, but not Ad-Cre cultures. Basal Fshb expression, which is dependent on autocrine/paracrine activins, was also significantly impaired in Ad-Cre relative to control cultures. To define mechanisms of FOXL2 action, we used a combination of heterologous (HepG2) and homologous (LβT2) immortalized cell-based assays. In HepG2 cells, over-expressed FOXL2 synergized with SMADs 2, 3, and 4 to stimulate murine Fshb promoter-reporter activity. Moreover, the combination of FOXL2 and SMAD3 conferred activin-induction, which was otherwise absent in these (and other) heterologous cells. In LβT2 cells, SMAD3-induction of Fshb promoter activity or endogenous mRNA expression was dependent on endogenous FOXL2. FOXL2/SMAD synergy required binding of both FOXL2 and SMAD4 to DNA. Three putative forkhead binding elements (FBE) have been identified in the murine Fshb promoter. Of these, only the most proximal was absolutely required for activin A induction of promoter activity in homologous cells. Additionally, mutations in a SMAD binding element (SBE) adjacent to the proximal FBE abrogated activin A or FOXL2/SMAD3 induction of reporter activity. 5' to the composite SBE/FBE is a PBX1/PREP1 binding site previously thought to mediate activin A-induction of murine and ovine Fshb promoter-reporters. However, introduction of a 2 base pair mutation that impaired PBX1/PREP1 (but neither SMAD nor FOXL2) binding did not alter activin A-stimulated promoter activity in homologous cells. Taken together, 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 regulate its transcription. Within these complexes, FOXL2 and SMAD4 bind to adjacent cis-elements and SMAD3 brokers the physical interaction between the two. As this composite response element is highly conserved, our data suggest a general mechanism whereby activins may regulate and/or modulate Fshb transcription in mammals. This research was supported by operating funds from the Canadian Institutes of Health Research (MOP 89991) to D.J.B. D.J.B. and S.T. hold a Chercheur-boursier (Senior) and a doctoral fellowship, respectively, from the Fonds de la recherche en sante du Quebec. (poster)