Signal transduction in Epithelial Cells of the Lens and

National Eye Institute

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Abstract

This project investigates signal transduction pathways that regulate proliferation, migration, and differentiation of lens and corneal epithelial cells with the goal of expanding the basic understanding of these pathways and identifying enzymes that may be therapeutically targeted in pathological conditions. The primary concentration in this fiscal year has been to investigate the role of the proline-directed kinase, Cdk5, in regulating cell-cell and cell-matrix adhesion. One avenue of investigation has been to identify lens proteins that interact with Cdk5 or its activating proteins, p35 and p39, using the yeast two-hybrid technique. These studies have identified two proteins, muskelin and myosin essential light chain, as specific interactors of p39. Both of these proteins are involved in cytoskeletal regulation, and may participate in Cdk5-dependent regulation of cell-matrix or cell-cell adhesion. Studies that were in progress last fiscal year to determine the relationship between the Cdk5 activating protein, p39, and the essential myosin light chain were completed and submitted for publication this year. The results demonstrated that a site in the p39 N-terminus is responsible for this binding and revealed a previously unrecognized role for Cdk5 in regulating phosphorylation of myosin regulatory light chain. [unreadable] Previous studies of corneal epithelial wound closure indicated that inhibitors of Cdk5 enhance the rate of epithelial cell migration, suggesting that such inhibitors might be therapeutically useful in treating certain corneal epithelial defects. This possibility is being pursued in an ongoing collaboration with Dr. Mary Ann Stepp (GWU). Results of in vivo testing in mice have shown that dropwise administration of an inhibitor of Cdk5 activity significantly enhances wound closure. Further analysis of the consequences of this treatment is in progress.[unreadable] Analysis has proceeded on prreviously generated lines of transgenic mice that overexpress Cdk5 or a kinase inactive form of Cdk5 only in lens fibers under the direction of the betaB1 crystallin promoter. Immunostaining of lenses from these lines has shown that animals expressing the kinase inactive form has shown a strong accumulation of the gap junction protein, connexin 43, suggesting a role for Cdk5 in turnover of gap junctions. [unreadable] Studies of the role of Cdk5 in regulating E-cadherin junction formation in cornea are nearing completion and will soon be submitted for publication. These studies have that suppressing Cdk5 expression in cultured corneal epithelial cells with siRNA leads to a reduction in the fraction of E-cadherin associated with cell-cell boundaries in an intact epithelial sheet. Moreover, pharmacological inhibition of Cdk5 activity reduces the ability of dissociated corneal epithelial cells to attach to a surface coated with E-cadherin. Thus, Cdk5 seems to promote formation of E- cadherin junctions. To examine the mechanism of this effect, we used surface biotinylation to determine whether Cdk5 has an effect on either endocytosis or exocytosis of E-cadherin. The results indicate that Cdk5 activity is required for efficient exocytosis of E-cadherin, thus demonstrating a role for Cdk5 in E-cadherin trafficking.