Definition by specific antisense oligonucleotides of a role for protein kinase Cα in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated, stepwise process that requires protein kinase C (PKC) activation. Studies using cultured mouse keratinocytes induced to differentiate with Ca²⁺ have indirectly implicated the α isoform of PKC in upregulation of 'late' (granular cell) epidermal differentiation markers. Activation of this isoform is also implicated in the suppression of 'early' differentiation markers keratin (K) 1 and 10 that characterizes the neoplastic phenotype produced by the v-Ha-ras oncogene. We used antisense oligonucleotides (AS) to directly address the role of PKCα in regulating expression of these markers in normal and v-Ha-ras-transduced primary keratinocytes and a keratinocyte cell line (SP-1) containing an activating mutation of the c-Ha-ras gene. Transfection of PKCα AS reduced the PKCα protein level in a dose-dependent manner, with a maximum effect at doses of 100 nM or higher. Immunoblot analysis with antibodies against PKCα, PKCδ, PKCε, and PKCη confirmed that PKCα AS selectively reduced the level of PKCα but not the other isoforms. In vitro kinase assays also revealed suppression of Ca²⁺-dependent PKC activity, which is the PKCα activity in this cell type, after transfection of PKCα AS. When PKCα AS-treated normal keratinocytes were stimulated to terminally differentiate with Ca²⁺, induction of the late differentiation markers loricrin, filaggrin, and SPR-1, as well as transglutaminase K mRNA, was suppressed when compared with their induction in scrambled AS-treated controls. In neoplastic v-Ha-ras-transduced keratinocytes and SP-1 cells, transfection of PKCα AS, but not the scrambled AS control, selectively downregulated PKCα and restored differentiation-specific expression of K1. These findings directly confirm that PKCα is an important component of the signaling pathway regulating terminal differentiation of normal keratinocytes and that activation of PKCα contributes to the altered differentiation program of neoplastic murine keratinocytes.