p63 is a recently identified homolog of p53 that is found in the basal layer of several stratified epithelial tissues such as the epidermis, oral mucosa, prostate, and urogenital tract. Studies with p63 ؊/؊ mice and analysis of several human autosomal-dominant disorders with germ line p63 mutations suggest p63 involvement in maintaining epidermal stem cell populations. The p63 gene encodes six splice variants with reported transactivating or dominant-negative activities. The goals of the current study were to determine the splice variants that are expressed in primary human epidermal keratinocytes (HEKs) and the biochemical activity p63 has in these epithelial cell populations. We found that the predominant splice variant expressed in HEKs was ⌬Np63␣, and it was present as a phosphorylated protein. During HEK differentiation, ⌬Np63␣ and p53 levels decreased, while expression of p53 target genes p21 and 14-3-3 increased. ⌬Np63␣ had transcriptional repressor activity in vitro, and this activity was reduced in ⌬Np63␣ proteins containing point mutations, corresponding to those found in patients with Hay-Wells syndrome. Further, we show that ⌬Np63␣ and p53 can bind the p21 and 14-3-3 promoters in vitro and in vivo, with decreased binding of p63 to these promoters during HEK differentiation. These data suggest that ⌬Np63␣ acts as a transcriptional repressor at select growth regulatory gene promoters in HEKs, and this repression likely plays an important role in the proliferative capacity of basal keratinocytes.

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Although a number of target genes for the tumor suppressor p53 have been described, the mechanism of p53-dependent apoptosis is incompletely understood. Thus, it is essential to identify and characterize additional target genes that could mediate apoptosis. In the study reported here, we isolated a p53-regulated gene named NDRG1 (N-Myc down-regulated gene 1). Its expression is induced by DNA damage in a p53-dependent fashion. The promoter region of the NDRG1 gene contains a p53 binding site that confers p53-dependent transcriptional activation via a heterologous reporter. RNA interference and inducible gene expression approaches suggest that NDRG1 is necessary but not sufficient for p53-mediated caspase activation and apoptosis. This report further supports the notion that p53 controls a network of genes that are required for its apoptotic function.

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DeltaNp63alpha, a homologue of the tumor suppressor p53, acts as a transcriptional repressor with dominant negative effects towards p53. Additionally, DeltaNp63alpha is overexpressed in a number of squamous cell carcinomas, suggesting a potential role in oncogenesis. However, the mechanisms regulating p63 have yet to be elucidated. The goal of the current study was to determine the effect of various genotoxic stresses on DeltaNp63alpha posttranslational modification and stability in normal and transformed squamous epithelial cells. We found that DeltaNp63alpha protein levels decreased after ultraviolet radiation and paclitaxel treatment of both normal and transformed cells. After UV and paclitaxel treatment, DeltaNp63alpha phosphorylation was significantly modulated. Additionally, DeltaNp63alpha protein levels were regulated in a proteasome-dependent manner in control and UV treated cells with increased DeltaNp63alpha ubiquitination after UV treatment or proteasome inhibition. Our studies provide insight to a mechanism for DeltaNp63alpha regulation during normal cell proliferation and, in particular, after stress. Further, the inverse regulation of p53 and DeltaNp63alpha protein levels after cell stress through opposing regulation of proteasome-mediated degradation may allow for rapid transcriptional changes of specific target genes that are consistent with the roles of these family members in tumor suppression and cell growth.