Differential activation of protein kinase C delta and epsilon gene expression by gonadotropin-releasing hormone in alphaT3-1 cells. Autoregulation by protein kinase C.
The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta and PKCepsilon gene expression was investigated in the gonadotroph-derived alphaT3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon 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 PKCepsilon 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 PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta 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 alphaT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta 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 PKCdelta and PKCepsilon. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon 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 PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.
Harris
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D
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel.
Reiss
N
N
Naor
Z
Z
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D007987:Gonadotropin-Releasing Hormone / Q000502:physiology*; D006706:Homeostasis; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000502:physiology; D010902:Pituitary Gland / Q000166:cytology / Q000201:enzymology; D011493:Protein Kinase C / Q000235:genetics* / Q000502:physiology; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002273:Carcinogens; D007476:Ionophores; D007527:Isoenzymes; D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D015759:Ionomycin; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium