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 β-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 TGFβ bind betaglycan, we examined whether TGFβ 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 FSHβ promoter (oFSHβluc), 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 LβT2. The oFSHβluc and 3XGRAS-PRL-lux activities stimulated by 0.5 nM activin A were decreased by up to 50% in a dose-dependent manner by inhibin A. TGFβ₁ and TGF₂ (0-4 nM), alone or in the presence of activin A, did not significantly affect the promoter elements. However, with increasing doses of TGFβ₁ or TGFβ₂, inhibin A antagonism of activin A activity was partly or completely reversed. Competition studies with radiolabeled inhibin A showed that TGFβ₁ and TGFβ₂ competed with [^125I]inhibin for the binding to LβT2 cells (IC₅₀ = 280 pM and 72 pM, respectively). Immunoprecipitation studies of [¹²⁵I]inhibin A cross-linked receptor complexes confirmed that TGFβ₁ and TGFβ₂ competed with inhibin A for the binding of betaglycan. These results suggest that TGFβ competition with inhibin for binding to betaglycan interferes with inhibin's suppression of activin-induced FSHβ and GnRHR promoters in LβT2 cells. We propose that under certain circumstances, TGFβ may facilitate activin biological activity by hindering the access of inhibin to its coreceptor betaglycan.