Abstract
BackgroundActivins stimulate pituitary FSH synthesis via transcriptional regulation of the FSHbeta subunit gene (Fshb). Like other members of the TGFbeta superfamily, these ligands signal through complexes of type I and type II receptor serine/threonine kinases. The type I receptors, or activin receptor-like kinases (ALKs), propagate intracellular signals upon ligand binding and phosphorylation by associated type II receptors. ALK4 is generally regarded as the type I receptor for activins; however, recent data suggested that activin B and AB might also signal through ALK7. Here, we examined a role for ALK7 in activin B-regulated Fshb transcription.MethodsWe analyzed ALK7 mRNA expression in immortalized gonadotrope cells, LbetaT2, and adult murine pituitary by RT-PCR. We next transfected LbetaT2 cells with wild-type and kinase-deficient (Lys to Arg, KR) forms of ALK4 and ALK7 and examined the effects of these receptors on activin A and B stimulated Fshb promoter-reporter activity. Cells were also transfected with constitutively active (Thr to Asp, TD) forms of the receptors and their effects on endogenous Fshb mRNA levels and phosphorylation of transfected Smad2/3 were measured by RT-PCR and Western blot, respectively. Finally, we measured ALK4(TD) and ALK7(TD) stimulation of Fshb transcription when endogenous Smad3 levels were depleted using short hairpin RNAs.ResultsALK7 mRNA was expressed in LbetaT2 cells and pituitary gland. Transfection of ALK4 cDNA potentiated the effects of both activin A and activin B on Fshb promoter-reporter activity in LbetaT2 cells. In contrast, ALK7 transfection selectively potentiated activin B's effects. Transfection of ALK4(KR) and ALK7(KR) partly inhibited basal and activin B-stimulated reporter activity, whereas ALK4(TD) and ALK7(TD) potently stimulated the Fshb promoter and endogenous mRNA levels. Transfection of both ALK4(TD) and ALK7(TD) stimulated Smad2/3 phosphorylation, and the effects of both receptors on Fshb promoter activity were inhibited by depletion of endogenous Smad3 protein levels.ConclusionThese data suggest that immortalized gonadotropes express ALK7 and that activin B can signal through this receptor to stimulate Fshb transcription. The relative roles of endogenous ALK4 and ALK7 receptors in mediating activin B's effects in these cells have yet to be determined.
Highlights
Activins stimulate pituitary FSH synthesis via transcriptional regulation of the FSHbeta subunit gene (Fshb)
ALK7 potentiates activin B signaling in LβT2 cells we transfected LβT2 cells with a murine Fshb promoter-reporter construct [3] along with expression vectors encoding wild-type ALK2, activin receptor-like kinase 4 (ALK4) or ALK7, followed by treatment with 25 ng/ml activin A or activin B for 24 h (Fig. 2A)
ALK7 transfection had no effect on the activin A response, but enhanced activin B activity (Fig. 2A, arrowhead); the potentiation with exogenous ALK7 was less than that observed with ALK4
Summary
Activins stimulate pituitary FSH synthesis via transcriptional regulation of the FSHbeta subunit gene (Fshb). Activins were first isolated from porcine follicular fluid during the purification of the inhibins and were shown to have potent and selective stimulatory effects on pituitary FSH secretion [1,2] Their effects are largely mediated via up-regulation of FSHβ (Fshb) subunit gene transcription [3,4,5]. The activin βA subunit (INHBA) is expressed throughout the pituitary, though not in LβT2 cells, and INHBA subunit bio-neutralizing antibodies have no effect on FSH secretion [8,9,10] These data suggest that activin B (a homodimer of INHBB subunits) is the physiologically relevant activin family member in the pituitary
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