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Transforming Growth Factor-ß Modulates Inhibin A Bioactivity in the LßT2 Gonadotrope Cell Line by Competing for Binding to Betaglycan

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Jean-François Ethier, Ph.D., Ottawa Regional Cancer Centre, Centre for Cancer Therapeutics, Third Floor, 503 Smyth Road, Ottawa, Ontario, K1H 1C4, Canada. E-mail: jfethier{at}uottawa.ca.

Activin stimulates expression of GnRH receptor (GnRHR) and FSH ß-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 TGFß bind betaglycan, we examined whether TGFß 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 FSHß promoter (oFSHßluc), 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 LßT2. The oFSHßluc 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. TGFß₁ and TGFß₂ (0–4 nM), alone or in the presence of activin A, did not significantly affect the promoter elements. However, with increasing doses of TGFß₁ or TGFß₂, inhibin A antagonism of activin A activity was partly or completely reversed. Competition studies with radiolabeled inhibin A showed that TGFß₁ and TGFß₂ competed with [¹²⁵I]inhibin for the binding to LßT2 cells (IC₅₀ = 280 pM and 72 pM, respectively). Immunoprecipitation studies of [¹²⁵I]inhibin A cross-linked receptor complexes confirmed that TGFß₁ and TGFß₂ competed with inhibin A for the binding of betaglycan. These results suggest that TGFß competition with inhibin for binding to betaglycan interferes with inhibin’s suppression of activin-induced FSHß and GnRHR promoters in LßT2 cells. We propose that under certain circumstances, TGFß may facilitate activin biological activity by hindering the access of inhibin to its coreceptor betaglycan.

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