Transcripts encoding protein kinase C‐α, ‐ϵ, ‐ζ, and ‐ζ are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells

Abstract

The protein kinase C (PKC) family of phospholipid‐dependent serine‐threonine kinases has been implicated in keratinocyte differentiation and neoplastic transformation. To determine if Ca²⁺‐mediated keratinocyte differentiation is associated with changes in PKC isozyme gene expression, RNA was isolated from primary mouse keratinocytes grown in medium with 0.05, 0.12, or 1.4 mM Ca²⁺. Based on northern blot analysis, primary keratinocytes expressed mRNA encoding PKC‐α, ‐ϵ, ‐ζ, and ‐neta but not PKC‐p or ‐y. Relatively little change was detected in the level of these transcripts in cells induced to differentiate by exposure to elevated extracellular Ca²⁺. Interestingly, the PKC‐ζ transcripts detected in RNA isolated from keratinocytes were approximately 200 nucleotides longer than those from mouse brain, suggesting the existence of an alternative form of this isozyme. An early change in benign neoplastic transformation of keratinocytes is the inability to differentiate in response to Ca²⁺ or the PKC activator 12‐O‐tetradecanoylphorbol‐13‐acetate, which is consistent with altered PKC function in these cells. The PKC isozyme mRNA profile was examined in two benign neoplastic keratinocyte cell lines, 308 and SP‐1, which contain an activating mutation of the c‐Ha‐ras gene. Like normal keratinocytes, 308 and SP‐1 cells expressed mRNA encoding PKC‐α, ‐ϵ, ‐ζ, and ‐η However, the abundance of PKC‐η transcripts in both cell lines was reduced by 74–89% when compared with normal keratinocytes at similar Ca²⁺ levels. In addition, SP‐1 but not 308 cells exhibited a sevenfold increase in PKC‐η mRNA when cultured in medium with 1.4 mM Ca²⁺. To address whether these changes were related to the presence of an activated ras gene, RNA was isolated from primary keratinocytes transduced to a benign neoplastic phenotype with the v‐Ha‐ras oncogene. As with normal, 308, and SP‐1 cells, v‐Ha‐ras keratinocytes expressed mRNA encoding PKC‐α, ‐ϵ, ‐ζ, and ‐neta. The level of PKC‐η transcripts was similar in normal and v‐Ha‐ras keratinocytes, indicating that reduction of this mRNA in both 308 and SP‐1 cells was not a direct result of ras activation. As in SP‐1 cells, PKC‐η in v‐Ha‐ras keratinocytes was responsive to extracellular Ca²⁺, with a four‐fold increase in transcript abundance in 0.12 mM Ca²⁺ medium relative to 0.05 mM Ca²⁺ medium. These findings illustrate the complexity of the PKC‐signal transduction pathway in cultured mouse keratinocytes and indicate that the level of certain PKC isozyme transcripts is modulated during the neoplastic process.