Multiple protein kinase C (PKC) isoforms have been associated with the epidermal keratinocyte (KC) granular layer differentiation program. Here we show PKCalpha membrane localization and substrate phosphorylation in the first suprabasal KCs of normal human epidermis, suggesting activation in vivo in the lower spinous layers where terminal differentiation-associated growth arrest occurs. To determine if PKCalpha is sufficient for KC growth arrest, we expressed a constitutively active PKCalpha (PKCalpha Delta22-28) in normal human KCs and observed growth arrest and accumulation of cells in G1. PKCalpha Delta22-28 inhibited DNA synthesis through the induction of the cyclin-dependent kinase inhibitor p21. Furthermore, downregulation of PKCalpha in an in vitro organotypic epidermis resulted in increased basal and suprabasal proliferation marker expression, decreased differentiation, and reduced epidermal stratification. Together these results indicate that PKCalpha activation is both necessary and sufficient to trigger irreversible growth arrest during human KC differentiation.

SummaryProtein kinase C (PKC) is a heterogeneous family of serine ⁄ ⁄ threonine protein kinases that have different biological effects in normal and neoplastic melanocytes (MCs). To explore the mechanism behind their differential response to PKC activation, we analyzed the expression profile of all nine PKC isoforms in normal human MCs, HPV16 E6 ⁄ ⁄ E7 immortalized MCs, and a panel of melanoma cell lines. We found reduced PKCb and increased PKCf and PKCi expression at both the protein and mRNA levels in immortalized MCs and melanoma lines. We focused on PKCb as it has been functionally linked to melanin production and oxidative stress response. Re-expression of PKCb in melanoma cells inhibited colony formation in soft agar, indicating that PKCb loss in melanoma is important for melanoma growth. PKCbII, but not PKCbI, was localized to the mitochondria, and inhibition of PKCb significantly reduced UV-induced reactive oxygen species (ROS) in MCs with high PKCb expression. Thus alterations in PKCb expression in melanoma contribute to their neoplastic phenotype, possibly by reducing oxidative stress, and may constitute a selective therapeutic target.

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