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A. P. Saravanan, C. Y. Gao, B. Tripathi, P. Zelenka; Cell-Cell Junctions in a Cdk5-Deficient Corneal Epithelial Cell Line. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2593.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the role of Cdk5 at corneal epithelial cell-cell junctions using a Cdk5-deficient corneal epithelial cell line.
A blasticidin-inducible vector for expression of a Cdk5-specific short hairpin RNA (ShCdk5) was generated by recombination and packaged into non-replicative lentiviral particles for transduction of human corneal limbal epithelial (HCLE) cells. Blasticidin resistant cells were isolated for analysis. Immunoblotting and immunofluorescence were used to examine expression of Cdk5, E-cadherin, p120 and beta-catenin. Cdk5 activity was inhibited with 15microM olomoucine. The trafficking of p120 at cell-cell junctions was analyzed using TIRF microscopy of pEGFP-p120 transfected cells. The immunofluorescence of E-cadherin at cell-cell borders and in the cytoplasm was quantified using Image-Pro Plus.
Immunoblotting showed that expression of Cdk5 in the ShCdk5 stable cell line was suppressed by 90%. E-cadherin levels were slightly reduced, with a concomitant increase in an E-cadherin degradation product of 29KDa. In contrast, p120 catenin levels were increased two-fold. Similar changes in E-cadherin and p120 expression were seen in cells treated with the Cdk5 inhibitor, olomoucine. Analysis of E-cadherin immunofluorescence intensity in ShCdk5 cells showed 40% reduction in the ratio of border to internal localization, as previously seen in olomoucine treated cells. TIRF analysis of pEGFP-p120 in live transfected cells revealed trafficking of in p120-containing vesicles to cell junctions. The tortuosity of the path of such vesicles was greater in control cells than in ShCdk5 cells.
Suppression of Cdk5 expression with ShRNA reduces border localization and increases internalization and degradation of E-cadherin, confirming previous results obtained with olomoucine and indicating a role for Cdk5 in stabilizing cell-cell junctions. Increased degradation of E-cadherin in ShCdk5 cells is accompanied by accumulation of p120 and altered trafficking of p120-containing vesicles. Effects of Cdk5 on p120 may be mediated via a Cdk5 consensus site (SPAR) at S655.
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