MECHANISM OF MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION BY GONADOTROPIN-RELEASING-HORMONE IN THE PITUITARY ALPHA-T3-1 CELL-LINE - DIFFERENTIAL ROLES OF CALCIUM AND PROTEIN-KINASE-C

The mechanism of nitogen-activated protein kinase (MAPK, ERR) stimulat ion by the GnRH analog [d-Trp(6)]GnRH (GnRH-a) was investigated in the gonadotroph-derived alpha T3-1 cell line. GnRH-a as well as the prote in kinase C(PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal g rowth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mon o-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine p hosphorylation of several proteins, and this effect as well as the sti mulation of MAPK activity were inhibited by the PKC inhibitor GF 10920 3X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein inhibited protei n tyrosine phosphorylation and reduced GnRH-a-stimulated MAPK ac activ ity by 50%, suggesting the participation of genistein-sensitive and in sensitive pathways in GnRH-a action. Although Ca2+ ionophores have onl y a marginal stimulatory effect, the removal of Ca2+ markedly reduced MAPK activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA stimulation of protein tyrosine phosphorylation. Interestingly, the r emoval of Ca2+ also partly inhibited the activation of MAPK by EGF and vanadate H2O2. Thus, a calcium-dependent component(s) downstream of P KC and PTK might also participate in MAPK activation. Elevation of cAM P by forskolin exerted partial inhibition on EGF, but not on TPA or Gn RH-a action, suggesting that MEK activators other than Raf-l might be involved in GnRH action. We conclude that Ca2+, PTK, and PKC participa te in the activation of MAPK by GnRH-a, with Ca2+ being necessary down stream to PKC and PTK.