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

Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892

Primary mouse keratinocytes expressing the v-ras^Ha oncogene (v-ras^Ha 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^Ha 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. Ca²⁺-dependent PKC activity was increased in lysates from cultured v-ras^Ha keratinocytes when compared to control cells; in contrast, Ca²⁺-independent activity decreased. Similar to PKC activators, v-ras^Ha blocked Ca²⁺-mediated expression of the early epidermal differentiation markers keratins K1 and K10 while inducing aberrant expression of K8. Pretreatment of v-ras^Ha keratinocytes with bryostatin to block PKC function restored Ca²⁺-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-, the only Ca²⁺-dependent PKC isozyme detected in cultured keratinocytes. In contrast to the inhibition of K1 and K10, Ca²⁺-induced expression of the late epidermal differentiation markers loricrin, filaggrin, and keratinocyte transglutaminase was accelerated by v-ras^Ha, as previously reported in normal keratinocytes treated with PKC activators. Pretreatment of v-ras^Ha keratinocytes with bryostatin blocked expression of late markers in these cells, and this response was correlated with down-regulation of PKC-. The results of this study suggest that oncogenic ras alters keratinocyte differentiation by altering the function of the PKC signaling pathway, and that PKC- is the specific isozyme involved in down-modulating expression of keratins K1 and K10 and up-regulating expression of loricrin, filaggrin, and keratinocyte transglutaminase.

¹ To whom requests for reprints should be addressed, at Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Building 37, Room 3B08, Bethesda, MD 20892. Recipient of a Thomas B. Fitzpatrick-Kao Corporation Research Award.

Received 9/ 1/94. Accepted 11/ 4/94.

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