REGULATION OF HUMAN KERATINOCYTE GENE EXPRESSION BY TPA

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The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) is a potent tumor promoter and causes epidermal hyperplasia in dermatological systems. In human keratinocytes, TPA induces epidermal differentiation and suppresses ornithine decarboxylase (ODC) activity without causing any concomitant changes in ODC mRNA levels. In contrast, in mouse skin, TPA is a proliferative agent, and causes increases in ODC activity, mRNA levels, and gene transcription rates; this increase in ODC activity appears to be necessary for tumor promotion, suggesting an important role for ODC in carcinogenesis. TPA thus has opposite effects in human and mouse, and the goal of these studies is to elucidate the mechanisms whereby TPA suppresses ODC gene expression in human keratinocytes. As TPA decreases ODC synthetic rates, and also appears to decrease ODC protein stability, we propose that TPA regulates ODC gene expression by regulating ODC mRNA translatability and by altering ODC enzyme half-life. The mechanisms of these effects will be studied by (1) elucidating the effects of TPA on the expression of transfected human ODC cDNA in human and mouse keratinocytes, (2) assessing the role of the ODC mRNA 51-noncoding (5'-nc) region in mediating the effect of TPA on ODC mRNA translatability by transfection of 5'-nc region deletants, (3) elucidating if the effect of TPA on ODC mRNA translatability is mediated by protein binding to the ODC mRNA 5'-nc region using RNA-protein UV cross-linking assays, (4) determining which sequences in the ODC protein mediate its destabilization by TPA, by transfection of probein-coding region deletants and determining enzyme half-life, and (5) isolating the protein(s) interacting with the ODC sequences that mediates the effect of TPA on ODC enzyme half-life through co-immunoprecipitation with ODC enzyme or through conventional protein purification techniques. We will thus obtain an understanding of the mechanisms by which TPA causes changes in ODC gene expression in human keratinocytes, and how, in mouse skin. TPA can cause tumor-promotion, whereas in human keratinocytes, TPA can cause cellular differentiation. This information will give us greater insight into skin cell proliferation, thereby contributing to the development of rational treatments of human epidermal disease.