Acute regulation of murine follicle-stimulating hormone β subunit transcription by activin A

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.

Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone β subunit transcription

“…A greater concentration of BMP2 was used here to facilitate detection of changes occurring at earlier time points. As we reported previously (Lamba et al 2006), activin A time-dependently increased transcription, reaching 14-fold by 24 h (Fig. 3A).…”

“…The truncated Smad8 construct was produced by introduction of a premature stop codon just N-terminal to the C-terminal serines by site-directed mutagenesis. The K1990/C 1mFshb-luc reporter construct and 5 0 deletions thereof were described previously (Bernard 2004, Lamba et al 2006. The BREX4-luc BMP-responsive reporter (Hata et al 2000) and human BMPR2 expression construct (Liu et al 1995) were provided by Dr Joan Massague (Memorial Sloan Kettering Cancer Center, New York, NY, USA).…”

“…Cells were plated and transfected in 6-or 24-well plates with Lipofectamine/Plus or Lipofectamine 2000 (Invitrogen) as previously described (Bernard 2004, Lamba et al 2006. Specific treatment conditions are described in the text or figure legends where appropriate.…”

“…In many cases, singular changes in FSH synthesis and secretion are attributable to actions of transforming growth factor (TGF)b superfamily ligands, the activins and inhibins, in particular. Recent data show that activins stimulate FSH synthesis by upregulating FSHb subunit gene (Fshb) transcription, at least in part, through the activation of the receptor-regulated signaling proteins, Smads 2 and 3 (Dupont et al 2003, Suszko et al 2003, Bernard 2004, Gregory et al 2005, Suszko et al 2005, Lamba et al 2006. In the case of the rat and mouse genes, these Smads, in concert with Smad4, bind to a consensus Smad-binding element in the proximal promoter to exert their actions.…”

“…In the case of the rat and mouse genes, these Smads, in concert with Smad4, bind to a consensus Smad-binding element in the proximal promoter to exert their actions. The promoters in sheep, pig, and human lack this element and therefore mechanisms of activin/Smad action appear to differ in these species (Bailey et al 2004, West et al 2004, Safwat et al 2005, Lamba et al 2006.…”