Transforming growth factor-β modulates inhibin a bioactivity in the LβT2 gonadotrope cell line by competing for binding to betaglycan

Activin stimulates expression of GnRH receptor (GnRHR) and FSH beta-subunit in gonadotropes. Inhibin antagonizes activin actions on the gonadotropes, but its molecular mechanism of action remains poorly understood. It has been suggested that inhibin exerts its antagonistic effects by competing with activin for the binding of the activin receptor complex. Betaglycan has recently been identified as an inhibin-binding accessory protein in this process. Because both inhibin and TGFbeta bind betaglycan, we examined whether TGFbeta can modify inhibin's antagonism of activin-induced transcription in gonadotrope cells. Two activin-responsive reporter constructs were used, the first containing 5.5 kb of the ovine FSHbeta promoter (oFSHbetaluc), and the second containing three copies of the activin-responsive sequence of the GnRHR promoter (3XGRAS-PRL-lux). These constructs were transfected into the gonadotrope cell line LbetaT2. The oFSHbetaluc and 3XGRAS-PRL-lux activities stimulated by 0.5 nm activin A were de-creased by up to 50% in a dose-dependent manner by inhibin A. TGFbeta(1) and TGFbeta(2) (0-4 nm), alone or in the presence of activin A, did not significantly affect the promoter elements. However, with increasing doses of TGFbeta(1) or TGFbeta(2), inhibin A antagonism of activin A activity was partly or completely reversed. Competition studies with radiolabeled inhibin A showed that TGFbeta(1) and TGFbeta(2) competed with [1251]inhibin for the binding to LbetaT2 cells (IC50 = 280 pm and 72 pm, respectively). Immunoprecipitation studies of [1151]inhibin A crosslinked receptor complexes confirmed that TGFbeta(1) and TGFbeta(2) competed with inhibin A for the binding of betaglycan. These results suggest that TGFbeta competition with inhibin for binding to betaglycan interferes with inhibin's suppression of activin-induced FSHbeta and GnRHR promoters in LbetaT2 cells. We propose that under certain circumstances, TGFbeta may facilitate activin biological activity by hindering the access of inhibin to its coreceptor betaglycan.