p53 is phosphorylated at the carboxyl terminus and promotes the differentiation of human HaCaT keratinocytes

Abstract

The p53 phosphoprotein acts as a tumor-suppressor gene product through the inhibition of cell growth and induction of apoptosis in a transcription-dependent manner. These functions require p53 activation through different biochemical postranslational modifications. Given the relevance of this protein in ultraviolet light-induced carcinogenesis, whose targets are primarily skin keratinocytes, we studied the functions of p53 in epidermal cell differentiation. We selected HaCaT cells, a human keratinocyte cell line bearing point-mutated, transcriptionally inactive, but highly stable p53, which facilitates immunochemical and biochemical analysis. In addition, a reliable in vitro differentiation system has been developed with these cells (Paramio et al. Oncogene 17:949, 1998). We report that during HaCaT differentiation there is a loss of immunoreactivity of p53 against antibodies that specifically recognize epitopes located at the carboxyl terminus of the protein. Because treatment with phosphatase restores this immunoreactivity, we conclude that p53 is phosphorylated at the carboxyl terminus during keratinocyte differentiation. This biochemical modification has been associated with the transcriptional activation of the molecule, and because p53 is involved in differentiation processes in other cell types, we investigated the potential functions of p53 during epidermal differentiation. To this end, we generated HaCaT clones expressing a murine temperature-sensitive p53 (Mp53ts) by transfection because the endogenous p53 is not functional even with phosphorylation. We characterized the expression and effects of the transfected protein in different selected clones. The ultraviolet-light response of these clones was restored, demonstrating the functionality of Mp53ts in these cells. We also observed that, with induction of differentiation, Mp53ts transfected cells differentiate faster than the parental or vector-transfected control cells, demonstrating that p53 promotes epidermal differentiation. The sustained expression of p53 in differentiating cells leads to massive cell death and detachment, a phenomenon that may be similar to epidermal terminal differentiation. In addition, we observed that the expression of p53-dependent genes such as p21waf/cip1 and mdm2 (which are known to participate in epidermal differentiation) increases during HaCaT differentiation, i.e., in a p53-independent manner.