118. Regulation of inhibin binding and action via betaglycan expression in mouse Leydig-like TM3 cells

Abstract

The actions of inhibin and transforming growth factor (TGF)-β2 are enhanced when their respective target cells express the TGF-β/inhibin co-receptor, betaglycan. In the present studies, we investigated the effects of multiple members of the TGF-β superfamily on betaglycan expression, and examined the consequences of such regulation for inhibin binding, and inhibin and TGF-β actions in mouse Leydig-like TM3 cells. Isoforms of activin (A and B), TGF-β (1 and 2) and BMP (2, 6 and 7) each suppressed the level of betaglycan mRNA in TM3 cells to 43–46%, 26–39% and 50–71% of control, respectively, during overnight treatment. Subsequent inhibin A binding was suppressed to 72–77%, 35–36% and 66–70% of control, respectively, with IC50s of 0.07–0.7, 0.05–0.5 and 0.4–0.6 nM, respectively. The effects of inhibiting betaglycan expression by TM3 cells on their responses to inhibin and TGF-β2 were examined by transfecting cells with a promoter construct that contains three copies of the activin-responsive sequence of the GnRHR promoter (3XpGRAS-PRL-lux) either alone or in the presence of small (21 bp) duplex siRNAs corresponding to the betaglycan gene. Activin A (0.5 nM) stimulated 3XpGRAS-PRL-lux expression 3–4 fold over control in TM3 cells, and inhibin dose-dependently abolished this stimulation, with no interference from the control siRNA (against BF-1 forkhead-like protein). However, inhibin suppression of activin-stimulated activity was antagonized in cells co-transfected with betaglycan siRNA. TGF-β (1 and 2) stimulated 3XpGRAS-PRL-lux expression 5–8 fold over control, and the action of TGF-β2, but not TGF-β1, was attenuated by the betaglycan siRNA. In summary, activin, TGF-β and BMP isoforms inhibit betaglycan expression by Leydig-like TM3 cells, and inhibin A binding is commensurately reduced. The ‘knock-down’ of betaglycan expression by specific siRNA inhibits TM3 responses to inhibin and TGF-β2. Whether the inhibition of betaglycan expression by activin, TGF-β and BMP has similar consequences for inhibin and/or TGF-β2 action is yet to be determined. These studies raise the possibility that multiple members of the TGF-β superfamily participate in cross-talk via the inhibin/TGF-β co-receptor, betaglycan. Funded by the NH&MRC of Australia (RegKey 241000 & 198705).