Dlugosz A A, Cheng C, Williams E K, Dharia A G, Denning M F, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
Cancer Res. 1994 Dec 15;54(24):6413-20.
Primary mouse keratinocytes expressing the v-rasHa oncogene (v-rasHa keratinocytes) produce squamous papillomas when grafted onto nude mice and respond abnormally to signals for terminal differentiation both in vivo and in vitro. Since protein kinase C (PKC) activators and v-rasHa induce similar phenotypic changes in cultured keratinocytes, and cellular diacylglycerol levels are constitutively elevated in ras-transformed keratinocytes, we tested whether PKC is a downstream target for oncogenic ras in this cell type. Ca(2+)-dependent PKC activity was increased in lysates from cultured v-rasHa keratinocytes when compared to control cells; in contrast, Ca(2+)-independent activity decreased. Similar to PKC activators, v-rasHa blocked Ca(2+)-mediated expression of the early epidermal differentiation markers keratins K1 and K10 while inducing aberrant expression of K8. Pretreatment of v-rasHa keratinocytes with bryostatin to block PKC function restored Ca(2+)-mediated expression of K1 and K10 and blocked abnormal expression of K8, suggesting that these responses are mediated by the PKC pathway. Furthermore, expression of K1 is restored at bryostatin doses which specifically down-modulate PKC-alpha, the only Ca(2+)-dependent PKC isozyme detected in cultured keratinocytes. In contrast to the inhibition of K1 and K10, Ca(2+)-induced expression of the late epidermal differentiation markers loricrin, filaggrin, and keratinocyte transglutaminase was accelerated by v-rasHa, as previously reported in normal keratinocytes treated with PKC activators. Pretreatment of v-rasHa keratinocytes with bryostatin blocked expression of late markers in these cells, and this response was correlated with down-regulation of PKC-alpha. The results of this study suggest that oncogenic ras alters keratinocyte differentiation by altering the function of the PKC signaling pathway, and that PKC-alpha is the specific isozyme involved in down-modulating expression of keratins K1 and K10 and up-regulating expression of loricrin, filaggrin, and keratinocyte transglutaminase.
表达v-rasHa癌基因的原代小鼠角质形成细胞（v-rasHa角质形成细胞）移植到裸鼠身上时会产生鳞状乳头状瘤，并且在体内和体外对终末分化信号均有异常反应。由于蛋白激酶C（PKC）激活剂和v-rasHa在培养的角质形成细胞中诱导相似的表型变化，并且在ras转化的角质形成细胞中细胞二酰甘油水平持续升高，我们测试了PKC是否是这种细胞类型中致癌性ras的下游靶点。与对照细胞相比，培养的v-rasHa角质形成细胞裂解物中的钙依赖性PKC活性增加；相反，非钙依赖性活性降低。与PKC激活剂相似，v-rasHa阻断了钙介导的早期表皮分化标志物角蛋白K1和K10的表达，同时诱导了K8的异常表达。用苔藓抑素预处理v-rasHa角质形成细胞以阻断PKC功能，恢复了钙介导的K1和K10的表达，并阻断了K8的异常表达，表明这些反应是由PKC途径介导的。此外，在特异性下调PKC-α（在培养的角质形成细胞中检测到的唯一钙依赖性PKC同工酶）的苔藓抑素剂量下，K1的表达得以恢复。与对K1和K10的抑制相反，如先前在用PKC激活剂处理的正常角质形成细胞中所报道的，v-rasHa加速了钙诱导的晚期表皮分化标志物兜甲蛋白、丝聚蛋白和角质形成细胞转谷氨酰胺酶的表达。用苔藓抑素预处理v-rasHa角质形成细胞可阻断这些细胞中晚期标志物的表达，并且这种反应与PKC-α的下调相关。本研究结果表明，致癌性ras通过改变PKC信号通路的功能来改变角质形成细胞的分化，并且PKC-α是参与下调角蛋白K1和K10表达以及上调兜甲蛋白、丝聚蛋白和角质形成细胞转谷氨酰胺酶表达的特异性同工酶。
