| Keratinocytes are the major cell type in the human epidermis. Basal layer cells maintain proliferation potential. As the daughter cells leave the basal layer and move outward towards the surface of the skin, they lose the ability to proliferate and initiate the differentiation program. This process requires a balance between keratinocyte proliferation, differentiation, and apoptosis. The goal of this thesis is to probe the molecular mechanisms of the regulation of human epidermal keratinocyte survival and differentiation using in vitro cell culture approach.; Previous studies have shown that nPKCs, including PKCdelta, activate keratinocyte differentiation and increase human involucrin gene expression. This increase is mediated via a p38 MAPK signaling cascade. In this thesis, I study the role of individual tyrosine residue on PKCdelta-dependent involucrin gene expression using mutagenesis and promoter activity assay. My studies show that individual mutation of PKCdelta tyrosine residue 52, 64, 155,187 or 565 does not reduce the ability of PKCdelta to increase hINV promoter activity. However, simultaneous mutation of these five tyrosine residues markedly reduces activity. This reduction of activity is associated with decreased phosphorylation of PKCdelta at tyrosine residue 311. Moreover, restoration of any one of the tyrosine residues in the five tyrosine mutant restores tyrosine 311 phosphorylation and biological activity. These studies suggest that tyrosine 311 phosphorylation increases the ability of PKCdelta to drive human involucrin gene expression; multiple tyrosine residues influence the phosphorylation status of PKCdelta at tyrosine 311.; DeltaNp63alpha is an important regulator of apoptosis in epidermal keratinocytes. My studies show that DeltaNp63alpha overexpression in primary human epidermal keratinocytes causes decreased cell proliferation and increased apoptosis by modulating the expression of cell cycle regulatory proteins and Bcl-2 family of proteins. Furthermore, p53 levels are increased in the DeltaNp63alpha-expressing cells and p53 overexpression reproduces features of DeltaNp63alpha phenotype, which suggests that DeltaNp63alpha-induced apoptosis may be partially mediated via increased level of p53.; Abbreviations: PKC, protein kinase C; MAPK, mitogen-activated protein kinase; hINV, human involucrin. |
