Annual Report 2001 This project investigates signal transduction pathways that regulate survival, growth, and differentiation in the lens and corneal epithelium. Recent studies have focused on the signaling pathway linking EGF receptor activation with c-fos transcription in the lens and on the functions of Cdk5, a member of the cyclin dependent kinase family, in both lens and corneal epithelium. Our previous studies of lens epithelial cells have shown that 12(S)HETE, a lipoxygenase metabolite of arachidonic acid, is required for EGF-dependent activation of PKCalpha and PKCbeta.. Further studies of this pathway have now demonstrated that exogenous 12(S)HETE alone leads to activation of these two PKC isoforms and of PKCgamma, an isoform that is not activated in the presence of EGF. We are now investigating the possibility that EGF-dependent signals may prevent 12(S)HETE-dependent activation of PKCgamma. Since we have also shown that 12(S)HETE-dependent activation of PKCalpha and PKCbeta is necessary but not sufficient for c-fos transcription in lens epithelial cells, we are investigating sequences in the c-fos gene that mediate the response to PKC. Our studies of Cdk5 suggest that this enzyme may regulate cell adhesion, migration, and survival in a variety of non-neuronal cells, including lens and corneal epithelial cells. To investigate the possible mechanism of these effects in the lens, cDNA library has been constructed from E18 embryonic rat lenses for yeast two-hybrid screening using Cdk5, p35, and p39 as 'baits'. Interacting clones are being characterized. To extend our previous studies of Cdk5 and p35 in corneal epithelial cells, we have stably transfected a mouse corneal epithelial cell line, A(6)1, with Cdk5 or Cdk5T33 and have used these lines for in vitro studies of cell adhesion and migration. Results indicate that Cdk5 promotes cell adhesion to a fibronectin or collagen matrix and inhibits cell migration, as measured by an in vitro scratch assay. Similarly, we have found that overexpression of Cdk5 in the corneal epithelium of transgenic mice retards corneal wound healing. However, this inhibitory effect on cell migration seems to be relatively specific for corneal epithelial cells. In several other cell types that we have examined, overexpression of Cdk5 increases, rather than decreases, the rate of cell migration. Possible explanations for this cell-type specific difference are being explored. Finally, we have found that Cdk5 overexpression induces apoptosis in COS1 cells and in certain other cell types. This effect is also produced by the Cdk5 activator, p39, but not by the alternative activator, p35, or by the kinase deficient form of Cdk5, Cdk5T33. Cdk5-transfected COS1 cells were found to release a substance to the medium that causes apoptosis even in non-transfected COS1 cells. The nature of this substance is being examined.
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