Molecular oncology and endocrinology research group, Department of Chemistry-Biology, University of Quebec at Trois-Rivières, 3351 Blv Des Forges, Trois-Rivières, Québec, Canada G9A 5H7.
Exp Mol Pathol. 2010 Oct;89(2):117-25. doi: 10.1016/j.yexmp.2010.07.004. Epub 2010 Jul 17.
Keratins 8 and 18 (K8/18) intermediate filament proteins are believed to play an essential role in the protection of hepatocytes against mechanical and toxic stress. This assertion is mainly based on increased hepatocyte fragility observed in transgenic mice deficient in K8/18, or carrying mutations on K8/18. The molecular mechanism by which keratins accomplish their protective functions has not been totally elucidated. Liver diseases such as alcoholic hepatitis and copper metabolism diseases are associated with modifications, in hepatocytes, of intermediate filament organisation and the formation of K8/18 containing aggregates named Mallory-Denk bodies. Treatment of mice with a diet containing griseofulvin induces the formation of Mallory-Denk bodies in hepatocytes. This provides a reliable animal model for assessing the molecular mechanism by which keratins accomplish their protective role in the response of hepatocytes to chemical injuries. In this study, we found that griseofulvin intoxication induced changes in keratin solubility and that there was a 5% to 25% increase in the relative amounts of soluble keratin. Keratin phosphorylation on specific sites (K8 pS79, K8 pS436 and K18 pS33) was increased and prominent in the insoluble protein fractions. Since at least six K8 phosphoepitopes were detected after GF treatment, phosphorylation sites other than the ones studied need to be accounted for. Immunofluorescence staining showed that K8 pS79 epitope was present in clusters of hepatocytes that surrounded apoptotic cells. Activated p38 MAPK was associated with, but not present in K8 pS79-positive cells. These results indicate that griseofulvin intoxication mediates changes in the physicochemical properties of keratin, which result in the remodelling of keratin intermediate filaments which in turn could modulate the signalling pathways in which they are involved by modifying their binding to signalling proteins.
角蛋白 8 和 18（K8/18）中间丝蛋白被认为在保护肝细胞免受机械和毒性应激方面发挥着重要作用。这一断言主要基于 K8/18 缺失的转基因小鼠或携带 K8/18 突变的小鼠观察到的肝细胞脆性增加。角蛋白发挥其保护功能的分子机制尚未完全阐明。酒精性肝炎和铜代谢疾病等肝脏疾病与中间丝组织的改变以及形成称为 Mallory-Denk 体的含有 K8/18 的聚集体有关。用含有灰黄霉素的饮食治疗小鼠会在肝细胞中诱导 Mallory-Denk 体的形成。这为评估角蛋白在肝细胞对化学损伤的反应中发挥保护作用的分子机制提供了一个可靠的动物模型。在这项研究中，我们发现灰黄霉素中毒诱导角蛋白溶解度发生变化，并且可溶性角蛋白的相对量增加了 5%至 25%。特定部位（K8 pS79、K8 pS436 和 K18 pS33）的角蛋白磷酸化增加，并且在不溶性蛋白部分中明显增加。由于在 GF 处理后至少检测到六个 K8 磷酸化表位，因此需要考虑除研究的表位之外的其他磷酸化位点。免疫荧光染色显示，K8 pS79 表位存在于围绕凋亡细胞的肝细胞簇中。激活的 p38 MAPK 与 K8 pS79 阳性细胞相关，但不存在于其中。这些结果表明，灰黄霉素中毒介导角蛋白理化性质的变化，导致角蛋白中间丝的重塑，这反过来又可以通过改变它们与信号蛋白的结合来调节它们所涉及的信号通路。
