REGULATION OF KERATINOCYTE SURVIVAL

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Homeostasis of multicellular organisms depends in part on the ability of cells to undergo controlled cell death or apoptosis. Conversely, disruption of pathways that induce apoptosis is often associated with tumor development. At present, the molecular control of epithelial cell survival is poorly understood except that activation of a tyrosine kinase (focal adhesion kinase) through substrate adhesion is known to be required. Our recent studies indicate that the epidermal growth factor-receptor (EGF-R) tyrosine kinase similarly supports survival of human normal keratinocytes but not of non-epithelial normal human cells, including melanocytes and fibroblasts. The proposed experiments are designed to define molecular pathways linking EGF-R activation to regulation of epithelial cell survival. In preliminary studies we observed that (i) EGF-R-dependent apoptosis is accelerated by detachment of keratinocytes from extracellular matrix; (ii) activation of the tyrosine kinase moiety of the EGF-R and activation of the Ras/Raf/MEK signaling cascade is required to protect keratinocytes from apoptosis; and (iii) blocking EGF-R is associated with down-regulation of the anti- apoptotic Bcl-xL molecule. Based on these results we hypothesize that signaling pathways triggered by tyrosine phosphorylation of the EGF-R are critical to keratinocyte survival and that EGF-R activation protects normal keratinocytes from apoptosis via regulation of members of the Bcl-2 protein family. To test these hypotheses we will: 1) investigate the effect of EGF-R blockade on mRNA and protein expression of members of the Bcl-2 protein family (Bcl-2, Bad, Bak, Bax) and death genes (ICE; CPP32); 2) determine by forced overexpression the functional contribution of Bcl-xL and other relevant Bcl-2 family members to keratinocyte apoptosis and, 3) examine EGF-R-dependent signal transduction pathways responsible for survival and up-regulation of Bcl- xL in keratinocytes. EGF-R dependent expression of relevant molecules will be assessed using established immunoprecipitation, Western and Northern blotting, RNAse protection, and RT-PCR methods. Forced gene expression is accomplished using advanced gene expression systems which enable regulatable gene expression in the immortalized keratinocyte cell line HaCaT and in normal keratinocytes. The long-term goal of these studies is to elucidate molecular pathways relevant to aberrant cell survival in epithelial malignancies.