Differential activation of protein kinase C δ and ε gene expression by gonadotropin-releasing hormone in αT3-1 cells: Autoregulation by protein kinase C

Abstract

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) δ and PKCε gene expression was investigated in the gonadotroph- derived αT3-1 cell line. Stimulation of the cells with a stable analog [D- Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCε mRNA levels (1 h), while PKCδ mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCε mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O- tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCε mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCδ mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCδ mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of αT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCδ mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCδ and PKCε. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCδ and PKCε mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCδ but not PKCε mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCδ mRNA by GnRH-A which can be memorized for 24 h. PKCδ and PKCε gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.