Ila Richard, Solem Michele
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Alcohol. 2006 Jul;39(3):169-78. doi: 10.1016/j.alcohol.2006.08.006.
Previously, we have demonstrated that chronic-alcohol exposure alters insulin-like growth factor 1 (IGF1) signaling in adult rat heart cells. This report examines the effects of alcohol in vitro on the expression of protein kinase C (PKC) alpha, delta, and epsilon using the embryonic heart cell line, H9c2, and how this may be linked to changes in IGF1 signal transduction. Western blot analyses of H9c2 protein preparations demonstrate that there are significant increases in the total protein levels of PKC delta and epsilon after 4 days exposure to alcohol, and similar increases were found after 2 and 6 days exposure. In addition, there was a significant increase in PKC delta and epsilon in the membranal fractions and a decrease in the cytosolic fractions. No change was found in the expression or activity levels for PKC alpha. Chronic-alcohol exposure (100 mM, 4 days) increased the basal tyrosine kinase activity of the IGF1 receptor (IGF1R), and altered its rate of activation. Chronic-alcohol exposure also reduced the rate of Erk1/Erk2 activation by IGF1. Chronic alcohol blocked the proliferative effects of IGF1 on cell growth and reduced cell viability both in the presence and absence of IGF1, and this alcohol-induced reduction in cell viability was blocked using siRNA to inhibit PKC delta. In addition, a reduction in the amount of myosin light chain 2 was found in the alcohol-exposed cells. In conclusion, chronic alcohol alters PKC delta and epsilon expression and activity, and suppresses the IGF1 signaling pathway in embryonic heart cell culture. Blockage of PKC delta expression using siRNA inhibits the suppressive effects of alcohol on cell viability.
此前，我们已经证明，长期酒精暴露会改变成年大鼠心脏细胞中的胰岛素样生长因子1（IGF1）信号传导。本报告使用胚胎心脏细胞系H9c2研究了酒精在体外对蛋白激酶C（PKC）α、δ和ε表达的影响，以及这可能如何与IGF1信号转导的变化相关联。对H9c2蛋白质制剂的蛋白质印迹分析表明，暴露于酒精4天后，PKCδ和ε的总蛋白水平显著增加，在暴露2天和6天后也发现了类似的增加。此外，膜组分中的PKCδ和ε显著增加，而胞质组分中的则减少。未发现PKCα的表达或活性水平有变化。长期酒精暴露（100 mM，4天）增加了IGF1受体（IGF1R）的基础酪氨酸激酶活性，并改变了其激活速率。长期酒精暴露还降低了IGF1对Erk1/Erk2的激活速率。长期酒精暴露在有和没有IGF1的情况下均阻断了IGF1对细胞生长的增殖作用并降低了细胞活力，并且使用小干扰RNA（siRNA）抑制PKCδ可阻断这种酒精诱导的细胞活力降低。此外，在酒精暴露的细胞中发现肌球蛋白轻链2的量减少。总之，长期酒精会改变PKCδ和ε的表达及活性，并抑制胚胎心脏细胞培养中的IGF1信号通路。使用siRNA阻断PKCδ表达可抑制酒精对细胞活力的抑制作用。
