Since the identification of the p53 homologue p63, several studies have investigated its function in epithelial cell growth and development. Using HEKs as a model system, we sought to further analyze the biochemical role p63 plays in keratinocyte growth and differentiation. We demonstrated that the primary splice variant of p63 expressed in HEKs is ΔNp63α, and its expression decreases as cells differentiate. Further, the ΔNp63α protein was present in differentiating HEKs as several phosphoforms. In addition to the reduction in p63 transcript and protein levels during differentiation, we also observed a decrease in p53 transcript and protein. The reduction in p53 and p63 expression correlated with an increase in expression of the cell cycle regulatory proteins p21 and 14-3-3σ. Using Gal4 fusion proteins, we determined that the p63 protein represses transcription and that the ΔNp63α splice variant has the highest activity. In addition, in vitro and in vivo DNA-binding assays showed that ΔNp63α binds to both p53 response elements in the p21 and 14-3-3σ promoters with p63 occupancy at p21 site 2 and 14-3-3σ sites 1 and 2 decreasing as cells differentiated.
Consistent with previously published reports (41
), we observed a decrease in p63 transcript and protein during differentiation. Analysis of the limited number of keratinocytes in the p63−/−
mouse showed expression of epithelial terminal differentiation markers (58
), suggesting that epithelial defects were due to the lack of cell survival and/or proliferation and not to impaired terminal differentiation. In support of this, a recent study in zebrafish using antisense oligonucleotides demonstrated that the ΔNp63 splice variant(s) were required for epithelial proliferation (30
). These model systems suggest a role for p63 in maintaining the survival or proliferation of basal keratinocytes and, in conjunction with our HEK data, indicate that the loss of ΔNp63α facilitates the growth arrest associated with differentiation.
We determined that p63 migrated as multiple phospho-forms by SDS-polyacrylamide gel electrophoresis (PAGE), suggesting that phosphorylation is a mechanism by which the p63 protein is regulated. This hypothesis is supported by the findings that phosphorylation is a key posttranslational modification for regulation of p53 (49
). However, ΔNp63α lacks a transactivation domain where many of the regulatory phosphorylation sites are found in p53. Future studies are required to identify the phosphoresidues in ΔNp63α, upstream kinases, and phosphorylation-dependent associated proteins.
It has been suggested that ΔNp63α-mediated repression can occur through direct protein-protein interaction, and several groups have examined the association of p63 proteins encoded by the various splice variants with other p53 family members. Davison et al. and Irwin et al. determined that p63 and p73 can form homodimers or have weak heterotypic interactions through their oligomerization domain but do not interact with the p53 oligomerization domain (10
). Kojima et al. found similar results by using a yeast two-hybrid system (28
). Further, several studies have shown that p63 and p53 can interact through the core/DNA-binding domain (21
). One consequence of p53 association with ΔNp63α may be caspase-dependent degradation of select ΔNp63 proteins (p40 and ΔNp63α) (44
). The significance of these findings remains to be determined in the context of proliferating and differentiating epithelial cells.
Through the use of Gal4 fusion proteins, we determined that the C-terminal domain of ΔNp63α is involved in transcriptional repression. Further, single amino acid substitutions within the SAM domain of ΔNp63α resulted in reduced transcriptional repression. Similar results were obtained for the ΔNp63α proteins containing SAM domain mutations by using a p53-reporter assay and cotransfections of the mutant proteins with p53 or TA-p63γ (35
). Chi et al. (8
) have shown that the SAM domain of p73 contains a folded, globular α-helical structure and suggest that this domain interacts with additional, as-yet-uncharacterized signaling proteins. However, the SAM domains of p73 and p63 are monomeric and do not interact with one another, leaving the possibility that the p63 SAM domain may play a role in recruiting transcriptional corepressors to select target genes, and these protein-protein interactions are disrupted in individuals with Hay-Wells syndrome (35
). Consistent with this hypothesis is our finding that ΔNp63α proteins containing the Hay-Wells mutations bind DNA with the same relative affinity as the wild-type protein. Studies have also demonstrated that a frameshift mutation found in ectrodactyl, ectodermal dysplasia, and cleft lip patients, causing loss of the SAM domain and carboxy-terminal sequence, results in the total loss of transcriptional repressive ability (5
). Taken together, the data suggest that the carboxy-terminal region of ΔNp63α containing the SAM domain plays an integral role in ΔNp63α-mediated transcriptional repression.
Since ΔNp63α has been identified as the primary splice variant expressed at the protein level in epithelial cells, several questions remain to be addressed. In particular, what target genes does ΔNp63α regulate and which of these genes are coordinately regulated by p53? Our in vitro DNA-binding assays and ChIP analyses support the hypothesis that p53 and p63 can coordinately bind target genes such as p21 and 14-3-3σ. These results are in agreement with those of Flores et al. showing that increased association of both p53 and p63 with p21, mdm2, PERP, and NOXA promoters in mouse embryo fibroblasts expressing E1A after DNA damage (19
If ΔNp63α functions as a transcriptional repressor in vivo, as our Gal4 fusion experiments support, then protein levels, promoter binding affinity, and coassociated proteins are likely factors involved in this coordinate regulation of downstream target genes. Similar to our results, Weinberg et al. reported that p53 transcript and protein decreased during differentiation whereas p21 promoter activity increased (55
). Does this increase reflect an elevation of p53 activity, an elevation of the activity of other transcriptional activators, or the loss of ΔNp63α repressor activity at the promoter? In support of a role for other transcriptional activators, several studies show that Sp1 and Sp3 can transcriptionally activate the p21 promoter (29
) and transcriptional regulators such as these may activate the p21 promoter when p53 levels are decreased during keratinocyte differentiation. In support of the theory that loss of ΔNp63α repressor activity at the p21 promoter allows p53 or other transactivators to act unopposed, Liefer et al. showed a decrease in p63 in mouse keratinocytes in vitro and mouse epidermis in vivo after UV-B exposure (32
), a treatment which leads to elevated p53 transcriptional activity (33
). Further, ectopic expression of ΔNp63α in the mouse epidermis resulted in decreased UV-B-induced apoptosis (32
), a phenotype thought to be primarily dependent on p53 activity (61
). Our findings that p63 and p53 can bind the same promoter elements in vivo support the role of ΔNp63α acting coordinately with p53 to regulate select target genes during keratinocyte proliferation and differentiation. Our observations also favor the possibility that p53 and ΔNp63α compete for consensus DNA-binding sites with p53 having a relatively higher binding affinity than ΔNp63α for select promoters, such as p21.
Previous studies and findings reported here support the following model. When rapidly proliferating basal epithelial cells are exposed to cell stress, increased p53 protein combined with the higher binding affinity of p53 for select promoter sites displaces ΔNp63α. Further, as is the case after exposure of keratinocytes to UV radiation, the p63 protein levels decrease. These events lead to subsequent transactivation of genes whose products are involved in growth arrest and apoptosis. In the absence of cell stress, constitutively expressed ΔNp63α protein levels exceed those of p53, and thus select target gene promoters are repressed, allowing for continued proliferation of keratinocytes in the basal layer where ΔNp63α is localized in stratified epithelium. During differentiation, both p53 and ΔNp63α levels decrease; however, it is the loss of ΔNp63α-mediated repression of select target genes that plays a role in differentiation. This model is consistent with the the proposed oncogenic role of p63 overexpression in squamous cell carcinomas of the head and neck (9
) and the observations that ectopic expression of the p40AIS
splice variant in Rat 1a cells results in increased growth of these cells in soft agar and athymic, nude mice (25
). However, as suggested above, it is likely that ΔNp63α also regulates gene expression independently of p53, since mutation of p53 and amplification of p63 both occur during genesis of squamous cell carcinomas (25
) (J. Sniezek and J. Pietenpol, unpublished results). Clearly, additional experimentation is required to further link p63 biochemistry to biology and to determine the interplay of p63 and p53 signaling pathways. New technologies, including in vivo DNA-binding assays and mass spectrometry, will aid in the identification of key posttranslational modifications, associated proteins, and novel target genes that are regulated by p63.