April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Cell-Cell Junctions in a Cdk5-Deficient Corneal Epithelial Cell Line
Author Affiliations & Notes
  • A. P. Saravanan
    LMDB, NEI/NIH, Bethesda, Maryland
  • C. Y. Gao
    LMDB, NEI/NIH, Bethesda, Maryland
  • B. Tripathi
    LMDB, NEI/NIH, Bethesda, Maryland
  • P. Zelenka
    LMDB, NEI/NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  A.P. Saravanan, None; C.Y. Gao, None; B. Tripathi, None; P. Zelenka, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 2593. doi:
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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)

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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 

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