DIFFERENCES IN THE REGULATION OF PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C IN NORMAL AND NEOPLASTIC KERATINOCYTES

The induction of epidermal differentiation by Ca2+ in vitro is associa ted with enhanced activity of phosphatidylinositol-specific phospholip ase C (PLC). Neoplastic keratinocyte cell lines expressing a mutant c- Ha-ras gene and normal keratinocytes transformed to the neoplastic phe notype by transduction with the v-Ha-ras gene (v-Ha-ras keratinocytes) have elevated constitutive activity of PLC that increases further in response to Ca2+, but the cells do not differentiate normally. PLC-gam ma 1 (145 kDa) is the major isoform detected by immunoblotting of extr acts from control, v-Ha-ras, and neoplastic keratinocyte cell lines cu ltured in 0.05 mM Ca2+ medium. The amount of PLC-gamma 1 protein was h igher in neoplastic cell lines than in normal and v-Ha-ras keratinocyt es that had similar PLC-gamma 1 protein levels. Thus, higher PLC-gamma 1 protein levels cannot account for the elevated constitutive activit y PLC in v-Ha-ras keratinocytes. After induction of differentiation by Ca2+, the amount of PLC-gamma 1 protein increased in all cell types, and PLC-delta 1 (85 kDa), barely detectable in 0.05 mM Ca2+, increased . PLC-beta 1 was not detected at any Ca2+ concentration. PLC-gamma 1 a nd PLC-delta 1 mRNA did not increase after elevation of extracellular Ca2+, suggesting that posttranscriptional mechanisms can regulate PLC- gamma 1 and PLC-delta 1 protein levels in normal and neoplastic kerati nocytes. Activation of protein kinase C by treatment with 12-O-tetrade canoylphorbol-13-acetate (TPA) inhibited the stimulation of inositol p hosphate (InsP) formation by Ca2+ but did not alter basal InsP levels in normal keratinocytes. In contrast, TPA treatment reduced both Ca2+- stimulated and basal InsP formation in neoplastic cells lines and v-Ha -ras keratinocytes. In both normal and v-Ha-ras keratinocytes labeled with [P-32]orthophosphate, antibodies against PLC-gamma 1 immunoprecip itated a complex of P-32-labeled proteins. The relative labeling of th e PLC-gamma 1 band was greater in normal than in v-Ha-ras keratinocyte s. Furthermore, treatment with TPA specifically increased the relative phosphorylation of PLC-gamma 1 in v-Ha-ras keratinocytes but not in n ormal keratinocytes. These results suggest that the negative regulatio n of constitutive activity of PLC by protein kinase C differs in norma l and neoplastic keratinocytes and that this could be the mechanism of increased PLC activity produced by an oncogenic ras gene in keratinoc ytes. (C) 1994 Wiley-Liss. Inc.