Differential regulation of follicle stimulating hormone by activin A and TGFB1 in murine gonadotropes

• Main Authors: Miller William L, Philips Daniel P, Gore A Jesse, Bernard Daniel J
• Format: Article
• Language: English
• Published: BMC 2005-12-01
• Series: Reproductive Biology and Endocrinology
• Online Access: http://www.rbej.com/content/3/1/73

<p>Abstract</p> <p>Background</p> <p>Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (<it>Fshb</it>). Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1) in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of <it>Fshb </it>in LbetaT2 cells as well as in purified primary murine gonadotropes.</p> <p>Methods</p> <p>Murine <it>Fshb </it>promoter-reporter (-1990/+1 m<it>Fshb</it>-luc) activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine <it>Fshb </it>mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR.</p> <p>Results</p> <p>Activin A dose-dependently stimulated -1990/+1 m<it>Fshb</it>-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D) and TGFB (TGFBR1-T204D) type I receptors strongly stimulated -1990/+1 m<it>Fshb</it>-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed <it>Tgfbr1 </it>mRNA, but that <it>Tgfbr2 </it>was not detected in LbetaT2 cells. When cells were transfected with a human TGFBR2 expression construct, TGFB1 acquired the ability to significantly stimulate -1990/+1 m<it>Fshb</it>-luc activity. In contrast to LbetaT2 cells, primary murine gonadotropes from young mice (8–10 weeks) contained low, but detectable levels of <it>Tgfbr2 </it>mRNA and these levels increased in older mice (1 yr). A second surprise was the finding that treatment of purified primary gonadotropes with TGFB1 decreased murine <it>Fshb </it>mRNA expression by 95% whereas activin A stimulated expression by 31-fold.</p> <p>Conclusion</p> <p>These data indicate that TGFB1-insensitivity in LbetaT2 cells results from a deficiency in <it>Tgfbr2 </it>expression. In primary gonadotropes, however, expression of <it>Tgfbr2 </it>does occur, and its presence permits TGFB1 to inhibit <it>Fshb </it>transcription, whereas activin A stimulates it. These divergent actions of activin A and TGFB1 were unexpected and show that the two ligands may act through distinct pathways to cause opposing biological effects in primary murine gonadotropes.</p>