Genes Are Mediated by Protein Kinase C - Ha ras Activated Alterations in Murine Keratinocyte Differentiation Induced by Updated

Primary mouse keratinocytes expressing the v.ras@I*oncogene 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@ras@I* induce similar phenotypic changes in cultured keratinocytes, and cellular diacylglycerol levels are constitutively elevated in ras-transformed kern tinocytes, we tested whether PKC is a downstream target for oncogenic ma in this cell type. Ca2'-dependent PKC activity was increased in lysates from cultured v@rasHa keratinocytes when compared to control cells; in contrast, Ca2@-independent activity decreased. Similar to PKC activators, v-ras5@blocked Ca2@-mediatedexpressionof the early epidermal differ entiation markers keratins Ki and K1O while Inducing aberrant expres slon of K8 Pretreatment of v-ms'@keratinocytes with bryostatin to block PKC function restored Ca2'-mediated expression of K! and K1O and blocked abnormal expression of KS, suggesting that these responses are mediated by the PKC pathway. Furthermore, expression of Kl is restored at bryostatin doses which specifically down-modulate PKC-a, the only Ca2@-dependent PKC isozyme detected in cultured keratinocytes. In con treat to the inhibition of K! and K1O, Ca2@-induced expression of the late epidermal differentiation markers loricrin, filaggrin, and keratinocyte transglutaminase was accelerated by v@ras@@M, @ previously reported in normal keratinocytes treated with PKC activators. Pretreatment of v ma'@ keratinocytes with bryostatin blocked expression oflate markers in these cells, and this response was correlated with down-regulation of PKC-a The results of this study suggest that oncogenic ras alters kern tinocyte differentiation by altering the function of the PKC signaling pathway, and that PKC-a Is the specific isozyme involved in down modulating expression of keratins K! and K1Oand up-regulating expres sion of loricrin, filaggrin, and keratinocyte transglutaminase.