Activation of protein kinase C beta gene expression by gonadotropin-releasing hormone in alpha T3-1 cell line. Role of Ca2+ and autoregulation by protein kinase C.
The gonadotroph-derived alpha T3-1 cell line was used to investigate the effect of gonadotropin-releasing hormone (GnRH) upon conventional protein kinase C sub-types (cPKCs) gene expression. Addition of the stable analog [D-Trp6]GnRH (GnRH-A, 0.1 nM) resulted in a rapid increase (30 min) of the steady state levels of PKC beta, but not PKC alpha, mRNA levels, while PKC gamma is not expressed in the cells. The rapid stimulatory effect of GnRH-A was blocked by pretreatment with actinomycin D or with the GnRH antagonist (D-pGlu1, pC1Phe2,D-Trp3,6)GnRH and was not mimicked by thyrotropin-releasing hormone. Addition of the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted also in a rapid (30 min) and selective increase in PKC beta, but not PKC alpha, mRNA levels. In contrast, the calcium ionophore, ionomycin, increased rapidly (30 min) both PKC alpha and PKC beta mRNA levels, and its stimulatory effect on PKC beta was not additive with that of TPA. The rapid stimulatory effect of GnRH-A was blocked by the PKC inhibitor bisindolylmaleimide (GF 109203X) or by down-regulation of endogenous PKC. Similarly, the rapid effect of GnRH-A was abolished by the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by removal of extracellular Ca2+. Stimulation of PKC beta mRNA levels by ionomycin was only reduced by GF 109203X and was not affected by down-regulation of PKC. In contrast the effect of TPA on PKC beta mRNA levels was reduced by BAPTA and abolished by removal of Ca2+. We conclude that Ca2+ and PKC act sequentially during GnRH-A-induced PKC beta gene expression and that PKC beta gene expression induced by GnRH-A is autoregulated by PKC.
Shraga-Levine
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Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Ben-Menahem
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Naor
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D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D015536:Down-Regulation; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000502:physiology*; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D064546:Protein Kinase C beta; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium