Transforming growth factor-beta modulates inhibin A bioactivity in the LbetaT2 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 decreased 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 [(125)I]inhibin for the binding to LbetaT2 cells (IC(50) = 280 pM and 72 pM, respectively). Immunoprecipitation studies of [(125)I]inhibin A cross-linked 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.
Ethier
Jean-François
JF
Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia. jfethier@uottawa.ca
Farnworth
Paul G
PG
Findlay
Jock K
JK
Ooi
Guck T
GT
D028341:Activins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D037201:Follicle Stimulating Hormone, beta Subunit / Q000235:genetics; D006801:Humans; D028322:Inhibin-beta Subunits / Q000037:antagonists & inhibitors / Q000494:pharmacology; D007265:Inhibins / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008156:Luciferases / Q000235:genetics; D051379:Mice; D010902:Pituitary Gland / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011509:Proteoglycans / Q000378:metabolism*; D011966:Receptors, LHRH / Q000235:genetics; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D001616:beta-Galactosidase / Q000235:genetics
D037201:Follicle Stimulating Hormone, beta Subunit; D007457:Iodine Radioisotopes; D011509:Proteoglycans; D011966:Receptors, LHRH; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; C508960:TGFB1 protein, human; C509041:TGFB2 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; C422405:activin A; C422362:inhibin A; D028341:Activins; C071736:betaglycan; D007265:Inhibins; D028322:Inhibin-beta Subunits; D008156:Luciferases; D001616:beta-Galactosidase