p63 is a p53 family member consisting of six isoforms, broadly characterized by the presence (TA) or absence (ΔN) or an amino-terminal transactivation domain. The generation of p63 null mice revealed the importance of p63 function in the development of ectodermally-derived structures and stratified epithelial tissue, including the epidermis. However, less is known about the requirement for p63 in tissue regeneration and the mechanisms by which p63 functions to promote cell proliferation. To investigate p63 function in a post-developmental context, we used siRNAs directed against p63 to downregulate p63 expression in regenerating human epidermis. Loss of p63 resulted in severe tissue hypoplasia and inhibited both stratification and differentiation in a cell-autonomous manner. Isoform-specific knockdown revealed that ΔNp63 isoforms are the main mediators of p63 effects; however, TAp63 isoforms may contribute to late differentiation. p63-deficient cells exhibited hypo-proliferation that was accompanied by an increase in p53 activity. Simultaneous p63 and p53 knockdown rescued the hypoplasia of p63-deficient tissue, suggesting that p63 and p53 antagonize each other to control keratinocyte proliferation. Combined p63 and p53 loss failed to restore differentiation, however, indicating that p63's effects on epidermal proliferation and differentiation are controlled by distinct mechanisms. To discover other potential downstream mediators of p63 function, we integrated expression profiling of p63-deficient cells with chromatin immunoprecipitation data characterizing direct p63 targets on a genome-wide scale. This approach identified c-Myc as a novel mediator of p63 function, and we confirmed that p63 binds within the c-Myc promoter to directly regulate its expression. Downregulation of c-Myc recapitulated the proliferation defect and a subset of gene expression changes observed with p63 knockdown, whereas overexpression of c-Myc in the context of p63 loss was able to partially rescue these effects. We determined that p53 and c-Myc function in distinct pathways downstream of p63 in regulating cell proliferation. Our results extend the importance of p63 in maintaining the proliferative and differentiation potential of developmentally mature human keratinocytes and identify two downstream mediators of p63 function.
|School Location:||United States -- California|
|Source:||DAI-B 70/03, Dissertation Abstracts International|
|Keywords:||Cell differentiation, Epidermal proliferation, Epidermis, Keratinocytes, p63|
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