Activin A induction of murine and ovine follicle-stimulating hormone β transcription is SMAD-dependent and TAK1 (MAP3K7)/p38 MAPK-independent in gonadotrope-like cells

Abstract

Activins stimulate follicle-stimulating hormone (FSH) β subunit (Fshb) gene transcription in pituitary gonadotrope cells. Previous studies suggest that activins signal via homolog of Drosophila mothers against decapentaplegic (SMAD) proteins to stimulate murine or porcine Fshb promoter activity in the gonadotrope-like cell line, LβT2. In contrast, activins were suggested to regulate the ovine Fshb promoter via a SMAD-independent pathway involving TGFβ associated kinase 1 (TAK1, MAP3K7) and p38 mitogen activated protein kinase (MAPK). Here, we examined roles for TAK1 and p38 in activin A-stimulated murine and ovine Fshb transcription. The TAK1 inhibitor 5Z-7-Oxozeanol (Oxo) significantly impaired fold activin A induction of murine and ovine Fshb promoter-reporters (Fshb-luc) in LβT2 cells, but only at concentrations 50-100 fold greater than its IC(50) for TAK1. Moreover, Oxo failed to inhibit activin A induction of endogenous Fshb mRNA levels or fold induction of Fshb-luc activity by a constitutively active form of the activin type I receptor (ALK4). Oxo, at a concentration 5-10 fold greater than its IC(50) for TAK1, attenuated TAK1/TAB2 stimulation of a p38-dependent reporter in the same cells. A Map3k7 siRNA impaired TAK1/TAB2-stimulated p38-dependent reporter activity, but failed to antagonize activin A-stimulated Fshb-luc. Though TAK1 was previously suggested to act via p38 to stimulate the ovine Fshb promoter, activin A failed to stimulate p38 phosphorylation in LβT2 cells. In apparent contrast, however, the p38 inhibitors SB203580 and SB202190 concentration-dependently attenuated activin A-induced Fshb-luc activity. Given the lack of p38 activation, we postulated that the inhibitors might non-selectively antagonize ALK4 activity. Indeed, both attenuated activin A-stimulated SMAD2 phosphorylation, consistent with direct antagonism of ALK4 kinase activity. Finally, we observed that RNA-mediated suppression of Smad4, and to a lesser extent Smad3, attenuated activin A induction of both murine and ovine Fshb promoter-reporters. Collectively, these data suggest that activin A signals via SMAD proteins, but not TAK1 or p38, to regulate murine and ovine Fshb transcription in gonadotrope-like cells.