Abstract
Purpose: :
To explore the role of Cdk5 at corneal epithelial cell-cell junctions using a Cdk5-deficient corneal epithelial cell line.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: cell adhesions/cell junctions • cornea: epithelium