ALTERATIONS IN MURINE KERATINOCYTE DIFFERENTIATION-INDUCED BY ACTIVATED RAS(HA) GENES ARE MEDIATED BY PROTEIN-KINASE C-ALPHA

Primary mouse keratinocytes expressing the v-ras(Ha) oncogene (v-ras(H a) keratinocytes) produce squamous papillomas when grafted onto nude m ice and respond abnormally to signals for terminal differentiation bot h in vivo and ill 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 targe t for oncogenic ras in this cell type, Ca2+-dependent PKC activity was increased in lysates from cultured v-ras(Ha) keratinocytes when compa red to control cells; in contrast, Ca2+-independent activity decreased . Similar to PKC activators, v-ras(Ha) blocked Ca2+-mediated expressio n of the early epidermal differentiation markers keratins K1 and K10 w hile inducing aberrant expression of K8. Pretreatment of v-ras(Ha) ker atinocytes with bryostatin to block PKC function restored Ca2+ mediate d expression of K1 and K10 and blocked abnormal expression of K8, sugg esting that these responses are mediated by the PKC pathway. Furthermo re, expression of K1 is restored at bryostatin doses which specificall y down-modulate PKC-alpha, the only Ca2+-dependent PKC isozyme detecte d in cultured keratinocytes. In contrast to the inhibition of K1 and K 10, Ca2+-induced expression of the late epidermal differentiation mark ers loricrin, filaggrin, and keratinocyte transglutaminase was acceler ated by v-ras(Ha), as previously reported in normal keratinocytes trea ted with PKC activators, Pretreatment of vras(Ha) keratinocytes with b ryostatin 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 differen tiation by altering the function of the PKC signaling pathway, and tha t PKC-alpha is the specific isozyme involved in down-modulating expres sion of keratins K1 and K10 and up-regulating expression of loricrin, filaggrin, and keratinocyte transglutaminase.