ANALYSIS OF THE UPSTREAM SIGNALLING PATHWAY CONTROLLING DELTANP63ALPHA PROTEIN STABILITY AND FUNCTION

1. Abstract The p63 transcription factor, homolog to the p53 tumor suppressor, plays a crucial role in epidermal and limb development. Dominant mutations in the p63 gene give rise to several human congenital syndromes characterized by skin, craniofacial and limb defects. One of the syndromes caused by p63 mutations is the Split-Hand/Foot Malformation-IV (SHFM-IV) syndrome, characterized by the loss of central rays of hands and feet. These developmental defects are due to failure of Apical Ectodermal Ridge (AER) development. The correct limb outgrowth and patterning is guarantee by the expression of key molecules including Fybroblast Growth Factor 8 (FGF8), p63 and the DLX5 and DLX6 transcription factors. In this context, the study of the molecular mechanisms regulating p63 stability and function is fundamental for understanding the molecular bases of the SHFM-IV pathogenesis: indeed p63 as been proposed to be one of the crucial regulators of limb and epidermal development. Little is known on the post-translational modifications and the upstream signalling pathway controlling ΔNp63α functions, one of the most expressed p63 isoform in epithelial tissues and in the AER cells. The projects performed during my PhD thesis achieved to the identification of FGF8 as a crucial regulator of ΔNp63α stability and activity in human osteosarcoma and keratinocyte cell lines. FGF8 determined also ΔNp63α protein stabilization in mice embryonic limb buds put in culture at Embryonic day 10.5 (E10.5). In particular, treatments with FGF8 of human osteosarcoma cell lines (U2OS) and human keratinocytes (HaCat), activate the tyrosine kinase c-Abl, leading to ΔNp63α phosphorylation and consequent acetylation by the p300 acetyl-transferase, promoting ΔNp63α stabilization and transcriptional activation. Moreover, I have found that p300 interacts with ΔNp63α determing its acetylation on lysine K193E, in vitro. Interestingly, this regulatory cascade is not active on the natural ΔNp63αK193E mutant associated to the SHFM-IV syndrome. Indeed, the ΔNp63αK193E mutant displays promoter specific altered DNA binding activity that results in altered expression of ΔNp63α target genes involved in limb development (like Perp, Ikkα and DLX5 gene) (Manuscript in preparation). One of the mechanism by which FGF8 promotes ΔNp63α stability and activation, is inhibiting its interaction with Pin1, a prolyl isomerase known to positively regulate p53 and p73 in response to DNA damage stress. In particular, PIN1 has an opposite effect on ΔNp63α respect to p53 and p73: it promotes ΔNp63α degradation through the proteasome pathway. Moreover, ΔNp63α mutant proteins, associated with SHFM-IV or EEC syndromes, characterized by limb defects, are not degraded by PIN1 overexpression. These data were confirmed also by in vivo experiments on PIN1 Knock-Out (KO) mice, where lack of PIN1 expression caused the accumulation of p63 in the embryonic limbs and ectoderm compared to wild-type littermates. Moreover, I found that FGF8 is a downstream target of the transcription factor Dlx5. Indeed, in the limb buds of both p63 and DLX5;DLX6 KO mice, the AER is poorly stratified and FGF8 expression is severely reduced. All these data suggest that DLX5, ΔNp63α, FGF8 and PIN1 participate in a regulatory loop essential for AER stratification, normal patterning and morphogenesis of the limb buds (1). The work performed during my PhD contributes to a better understanding of the regulatory mechanisms controlling ΔNp63α function and stability. We have identified FGF8 as a crucial upstream signal required for ΔNp63α activation and stabilization during limb development: mutations or altered expression of regulators in this pathway leads to abnormal limb development and onset of pathogenesis.