Stravitz R T, Rao Y P, Vlahcevic Z R, Gurley E C, Jarvis W D, Hylemon P B
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23219, USA.
Am J Physiol. 1996 Aug;271(2 Pt 1):G293-303. doi: 10.1152/ajpgi.1996.271.2.G293.
We have recently shown that taurocholate (TCA) represses the transcriptional activity of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of the bile acid biosynthetic pathway, through a protein kinase C (PKC)-dependent mechanism in primary cultures of rat hepatocytes. The present studies sought to determine the mechanisms by which bile acids activate hepatic PKC activity and the consequences of this activation on isoform distribution and cholesterol 7 alpha-hydroxylase mRNA levels. TCA (12.5-100 microM for 15 min) increased membrane-associated "classic" isoenzyme cPKC-alpha and "novel" isoenzymes nPKC-delta, and nPKC by two- to sixfold. Membrane-associated PKC progressively increased, and cytosolic PKC decreased, for 1 h after the addition of TCA (50 microM); after 24 h whole cell cPKC-alpha, nPKC-delta, and nPKC were downregulated by 35-55% compared with untreated controls. In a reconstituted assay system, TCA or taurodeoxycholate (10-100 microM) increased calcium-dependent and -independent PKC activity by three- and fourfold, respectively. Taurine-conjugated bile acids stimulated PKC activity in proportion to their hydrophobicity index (r = 0.99). Finally, cholesterol 7 alpha-hydroxylase mRNA was repressed > 75% by phorbol 12-myristate 13-acetate (100 nM for 3 h), a nonselective activator of PKC isoforms. In contrast, selective cPKC-alpha activation with thymeleatoxin (100 nM for 3 h) had no significant effect on cholesterol 7 alpha-hydroxylase mRNA levels. We conclude that bile acids activate hepatocellular PKC, resulting in sequential redistribution and down-regulation of calcium-dependent and -independent isoforms. The calcium-independent PKC isoforms may mediate the repression of cholesterol 7 alpha-hydroxylase mRNA by TCA.
我们最近发现，牛磺胆酸盐（TCA）通过蛋白激酶C（PKC）依赖的机制，在大鼠原代肝细胞培养物中抑制胆汁酸生物合成途径的限速酶胆固醇7α-羟化酶的转录活性。本研究旨在确定胆汁酸激活肝PKC活性的机制，以及这种激活对同工型分布和胆固醇7α-羟化酶mRNA水平的影响。TCA（12.5 - 100 microM，作用15分钟）使膜相关的“经典”同工酶cPKC-α和“新型”同工酶nPKC-δ以及nPKC增加了2至6倍。添加TCA（50 microM）后1小时，膜相关PKC逐渐增加，胞质PKC减少；24小时后，与未处理的对照相比，全细胞cPKC-α、nPKC-δ和nPKC下调了35 - 55%。在重组测定系统中，TCA或牛磺去氧胆酸盐（10 - 100 microM）分别使钙依赖性和非钙依赖性PKC活性增加了3倍和4倍。牛磺酸结合的胆汁酸刺激PKC活性的程度与其疏水指数成正比（r = 0.99）。最后，佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（100 nM，作用3小时），一种PKC同工型的非选择性激活剂，使胆固醇7α-羟化酶mRNA被抑制> 75%。相比之下，百里酚毒素（100 nM，作用3小时）对cPKC-α的选择性激活对胆固醇7α-羟化酶mRNA水平没有显著影响。我们得出结论，胆汁酸激活肝细胞PKC，导致钙依赖性和非钙依赖性同工型的顺序重新分布和下调。非钙依赖性PKC同工型可能介导TCA对胆固醇7α-羟化酶mRNA的抑制作用。
