Stravitz R T, Vlahcevic Z R, Gurley E C, Hylemon P B
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
J Lipid Res. 1995 Jun;36(6):1359-69.
Inhibitors of protein kinases were screened for the ability to prevent the repression of cholesterol 7 alpha-hydroxylase mRNA by taurocholate in primary cultures of adult rat hepatocytes. The addition of taurocholate (25 microM) for 6 h decreased cholesterol 7 alpha-hydroxylase mRNA by 64 +/- 3%. However, after a preincubation with the protein kinase C inhibitors calphostin C or chelerythrine, taurocholate had no significant effect on cholesterol 7 alpha-hydroxylase mRNA, or decreased levels by only 23 +/- 8%, respectively. Protein kinase C activation with phorbol 12-myristate, 13-acetate (100 nM) decreased cholesterol 7 alpha-hydroxylase mRNA and transcriptional activity by 71 +/- 5% and 60 +/- 16%, respectively, within 3 h. mRNA levels recovered to control levels by 18-24 h, however, consistent with downregulation of protein kinase C. Furthermore, after depletion of protein kinase C with a 24-h preincubation with phorbol diesters, taurocholate (25 microM) repressed cholesterol 7 alpha-hydroxylase mRNA by only 14 +/- 17%. The addition of taurocholate (50 microM) to the culture medium transiently increased membrane-associated protein kinase C activity by approximately twofold, while causing a concomitant decrease in cytosolic activity. Other bile acids increased membrane-associated protein kinase C activity in approximate proportion to their relative hydrophobicity. Finally, immunoblotting experiments revealed translocation of the alpha isoform of protein kinase C to hepatocyte membranes in response to taurocholate. These data suggest that hydrophobic bile acids repress cholesterol 7 alpha-hydroxylase transcription through a protein kinase C-dependent mechanism.
在成年大鼠肝细胞原代培养物中，筛选蛋白激酶抑制剂，以检测其阻止牛磺胆酸盐对胆固醇7α-羟化酶mRNA的抑制作用的能力。添加牛磺胆酸盐（25μM）6小时后，胆固醇7α-羟化酶mRNA降低了64±3%。然而，在用蛋白激酶C抑制剂钙泊三醇C或白屈菜红碱预孵育后，牛磺胆酸盐对胆固醇7α-羟化酶mRNA没有显著影响，或分别仅使水平降低23±8%。用佛波酯12-肉豆蔻酸酯13-乙酸酯（100 nM）激活蛋白激酶C，在3小时内分别使胆固醇7α-羟化酶mRNA和转录活性降低71±5%和60±16%。然而，mRNA水平在18 - 24小时恢复到对照水平，这与蛋白激酶C的下调一致。此外，在用佛波酯预孵育24小时耗尽蛋白激酶C后，牛磺胆酸盐（25μM）仅使胆固醇7α-羟化酶mRNA抑制14±17%。向培养基中添加牛磺胆酸盐（50μM）可使膜相关蛋白激酶C活性瞬时增加约两倍，同时导致胞质活性相应降低。其他胆汁酸增加膜相关蛋白激酶C活性的程度与其相对疏水性大致成比例。最后，免疫印迹实验显示，牛磺胆酸盐可使蛋白激酶C的α亚型转位至肝细胞膜。这些数据表明，疏水性胆汁酸通过蛋白激酶C依赖性机制抑制胆固醇7α-羟化酶转录。
