Regulation of the Rat Follicle-Stimulating Hormone β-Subunit Promoter by Activin

Abstract

FSH is controlled by a variety of positive and negative stimuli, and the unique FSHbeta-subunit is a major target for this regulation. Activin is a key modulator of FSHbeta transcription and hormone secretion. The signal transduction pathway leading to FSH expression was previously unknown. Here, we show that the transcription factors Smad3 and Smad4 mediate activin-stimulated activity of the rat FSHbeta promoter in a pituitary-derived cell line, LbetaT2. Cells were transiently transfected with the rat FSHbeta promoter fused to a luciferase reporter gene (-338rFSHbeta-Luc), and a minimal activin-responsive region was identified. Transfection of Smad3, but not the highly related Smad2, led to a ligand-independent stimulation of the FSHbeta promoter activity. As expected, activin caused an additional increase of luciferase expression, which was blocked by cotreatment with follistatin. Although Smad4 alone had no effect on FSHbeta transcription, it significantly augmented Smad3 and activin-mediated stimulation of the promoter. A palindromic consensus Smad-binding element in the proximal promoter was found to bind Smad4, and elimination of the region resulted in a loss of activin-mediated FSHbeta transcription. The activin signaling pathway is conserved in a number of cells, but FSHbeta expression is restricted to gonadotropes. A pituitary-specific transcription factor necessary for activin-dependent induction of the FSHbeta promoter has been identified that permits FSHbeta expression in nongonadotrope cells. Pitx2 is a member of Pitx subfamily of bicoid-related homeodomain factors that is required for pituitary development and is present in the adult pituitary. This factor was transfected into LbetaT2 cells, where it caused up-regulation of basal and activin-mediated FSHbeta promoter activity. Furthermore, cotransfection of Pitx2c with Smad3 in kidney-derived TSA cells resulted in activin-regulated FSHbeta response, suggesting its important role in tissue-restricted regulation of FSHbeta by activin. A Pitx2c binding site was identified within the proximal promoter, and elimination of this region also resulted in a loss of activin-regulated FSHbeta promoter activity. Taken together, these studies suggest that the regulation of FSHbeta is dependent on activin-mediated signaling factors in concert with pituitary-derived nuclear regulatory proteins.

Activin and inhibin receptor gene expression in the ewe pituitary throughout the oestrous cycle

“…In good agreement with this hypothesis, in the sheep and the rat, the FSH subunit gene is a direct transcriptional target of activin signals (Dupont et al 2003, Suszko et al 2003. Moreover, in the mouse gonadotrope L T2 cell line, using the Northern blot technique and the transient transfection of a Smad2 dominant negative construct, our group has previously shown that the Smad2 signalling pathway is partly involved in the activin-induced FSH synthesis and release (Dupont et al 2003).…”

Urinary beta-luteinizing hormone and beta-follicle stimulating hormone immunoenzymometric assays for population research

“…Free excess alpha may be found in the blood, whereas the free β subunits of each, which confer specific biological actions, are rarely found in the plasma in healthy individuals (23,24). Regulation of bioactive LH and FSH is most likely mediated by production of the β-subunits; for example, changes in the frequency and amplitude of gonadotropin releasing hormone pulses differentially stimulate synthesis of the β-subunits of each (25,26). Production of the β-subunit is likely the limiting step in production of the bioactive heterodimer forms of both LH and FSH (23), and would thus be expected to be proportional to the amount of bioactive hormone in circulation.…”

Inhibin α-Subunit N Terminus Interacts with Activin Type IB Receptor to Disrupt Activin Signaling

“…/␤A, and ␣ Hwt /␤A were determined using L␤T2 gonadotrope cells stably transfected with the Ϫ338 region of the FSH␤ promoter conjugated to a luciferase reporter (L␤T2-F338) (39). Cells were maintained as reported previously and treated with ligands for 6 h at 37°C in serum-free, phenol redfree DMEM-F12 supplemented with 1% penicillin/streptomycin.…”