Antonini, Dario (2011) Role of p63 in skin biology: its function in cell proliferation and differentiation. [Tesi di dottorato] (Unpublished)

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p63, a p53 family member, plays an essential role in epidermal development controlling several biological functions, some of which remain poorly understood. Using a global gene expression analysis, I contributed to the identification of novel downstream targets and signaling pathways regulated by p63. We firstly identified a set of genes not specifically expressed in epidermis (non-epidermal genes) that are indirectly suppressed by p63. In parallel, p63 sustains Bone Morphogenetic Protein (BMP) signaling by directly suppressing transcription of the inhibitory Smad7, and by inducing Bmp7. In the absence of p63, BMP signaling is compromised and leads to ectopic expression of the non-epidermal genes in vitro and in vivo. Reactivation of BMP signaling by exogenous stimuli suppresses ectopic expression of non-epidermal genes in the absence of p63. These data indicate that p63 prevents ectopic expression of non-epidermal genes by a mechanism involving activation of BMP signaling. In our genome-wide analysis we also observed that many cell cycle genes were positively regulated by p63. I found that loss of p63 in keratinocytes causes cell cycle arrest both in vitro and in vivo. Thus, I investigated how cell cycle genes are affected by p63. I identified a new mechanism through which p63 favors cell cycle progression repressing two members of a microRNA family: miR-34a and miR-34c. In the absence of p63, I observed increased levels of miR-34a and miR-34c in primary mouse keratinocytes and in mouse embryonic skin. p63 directly binds to p53-consensus sites in both miR-34a and miR-34c gene promoters and inhibits their activity. Keratinocytes are arrested in the G1-phase of the cell cycle in the absence of p63 and have reduced levels of two cell cycle regulators, cyclin D1 and cyclin-dependent kinase 4 (Cdk4), that are known targets of miR-34. Importantly, concomitant downregulation of miR-34a and miR-34c in the absence of p63 restores cell cycle progression and the expression of cyclin D1 and Cdk4. These data demonstrate that p63 sustains cell cycle progression in keratinocytes not only by previously defined mechanisms, such as repression of the CDK inhibitor p21 Cip1/Waf1, but also by directly repressing components of the miR-34 family. In conclusion, during my PhD program I uncovered two novel mechanisms through which p63 regulates cell cycle progression and tissue identity in epidermis.

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