DEFINITION BY SPECIFIC ANTISENSE OLIGONUCLEOTIDES OF A ROLE FOR PROTEIN-KINASE C-ALPHA IN EXPRESSION OF DIFFERENTIATION MARKERS IN NORMAL AND NEOPLASTIC MOUSE EPIDERMAL-KERATINOCYTES

Epidermal keratinocyte differentiation is a tightly regulated, stepwis e process that requires protein kinase C (PKC) activation. Studies usi ng cultured mouse keratinocytes induced to differentiate with Ca2+ hav e indirectly implicated the alpha isoform of PKC in upregulation of '' late'' (granular cell) epidermal differentiation markers. Activation o f this isoform is also implicated in the suppression of ''early'' diff erentiation markers keratin (K) 1 and 10 that characterizes the neopla stic phenotype produced by the v-Ha-ras oncogene. We used antisense ol igonucleotides (AS) to directly address the role of PKC alpha in regul ating expression of these markers in normal and v-Ha-ras-transduced pr imary keratinocytes and a keratinocyte cell line (SP-1) containing an activating mutation of the c-Ha-ras gene. Transfection of PKC alpha AS reduced the PKC alpha protein level in a dose-dependent manner, with a maximum effect at doses of 100 nM or higher. Immunoblot analysis wit h antibodies against PKC alpha, PKC delta, PKC epsilon, and PKC eta co nfirmed that PKC alpha AS selectively reduced the level of PKC alpha b ut not the other isoforms. In vitro kinase assays also revealed suppre ssion of Ca2+-dependent PKC activity, which is the PKC alpha activity in this cell type, after transfection of PKC alpha AS. When PKC alpha AS-treated normal keratinocytes were stimulated to terminally differen tiate with Ca2+, induction of the late differentiation markers loricri n, filaggrin, and SPR-1, as well as transglutaminase K mRNA, was suppr essed when compared with their induction in scrambled AS-treated contr ols. In neoplastic v-Ha-ras-transduced keratinocytes and SP-1 cells, t ransfection of PKC alpha AS, but not the scrambled AS control, selecti vely downregulated PKC alpha and restored differentiation-specific exp ression of K1. These findings directly confirm that PKC alpha is an im portant component of the signaling pathway regulating terminal differe ntiation of normal keratinocytes and that activation of PKC alpha cont ributes to the altered differentiation program of neoplastic murine ke ratinocytes. (C) 1997 Wiley-Liss, Inc.dagger