Up-regulation of p21WAF1 by phorbol ester and calcium in human keratinocytes through a protein kinase C dependent pathway

Terminal differentiation in a variety of cell types has been associated with p53-independent up-regulation of p21(WAF1). P21(WAF1) mRNA and protein are expressed at low levels in normal human skin, but overexpression of p21(WAF1) has been observed in differentiating keratinocytes in involved psoriatic epidermis and in human squamous cell carcinoma. In this study we investigated by immunohistochemistry and Western blotting whether calcium and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, well characterized differentiation signals, induce p21(WAF1) in cultured normal human keratinocytes and whether induction of p21(WAF1) in this system depends on protein kinase C activation or functional p53. Phorbol ester induced p21(WAF1) expression, which was maximal at 4 to 8 h with reduction back to baseline by 24 to 48 h. In contrast, increasing the extracellular Ca2+ concentration from 70 mu mol/L to 1.5 mmol/L resulted in upregulation of p21(WAF1) expression with a slower time course, with peak induction at 18 to 24 h. No parallel increase in p53 expression was observed in normal human keratinocytes. Up-regulation of p21(WAF1) was also observed in response to phorbol ester in HaCaT cells, which carry homozygous and inactivating mutations for p53. Induction of p21(WAF1) by phorbol ester and Ca2+ was inhibited by the specific protein kinase C inhibitor Ro 31-8220, The results demonstrate a differential time course of p21(WAF1) protein up-regulation in response to phorbol ester and Ca2+, signals that result in keratinocyte differentiation, and suggest that induction of p21(WAF1) in differentiating human keratinocytes occurs through protein kinase C-dependent and p53-independent mechanisms.