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CY Gao, S Negash, PS Zelenka; Cdk5 Regulates Corneal Epithelial Cell Adhesion and Migration in vitro . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4211.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Overexpression of cyclin dependendent kinase 5 (CDK5) in the corneal epithelium of transgenic mice has been shown to retard wound healing. To study the possible mechanism of this effect, we have investigated the functions of CDK5 in a corneal epithelial cell line. Methods: A conditionally transformed cell line, A6(1), derived from corneal epithelial cells of the ImmortoMouse, was stably transfected with CDK5 or a kinase-inactive form, CDK5-T33. Alternatively, cells were infected with adenovirus vectors carrying CDK5 or CDK5-T33 constructs. Immunoprecipitation, cellular fractionation, and immunoblotting were used to examine expression and distribution of CDK5 and its endogenous regulatory subunit, p35. The effect of CDK5 on cell adhesion was quantified by determining the centrifugal force needed to dislodge cells from a fibronectin-coated plate. Migration was assessed by image analysis following in vitro scrape wounding. Results: CDK5 and p35 co-immunoprecipitated from whole cell extracts, demonstrating the presence of potentially active complexes. Stable transfection with CDK5 significantly increased the proportion of vinculin in the cytoskeletal fraction, while increasing both the number of cells adhering to fibronectin and the strength of adhesion. In contrast, stable transfection with CDK5-T33 did not significantly affect adhesion, suggesting that the kinase activity of the transfected CDK5 is required. Since higher levels of CDK5-T33 may exert a dominant negative effect on endogenous CDK5 activity by sequestering the available p35, we also used adenovirus vector infection to achieve higher levels of CDK5-T33 expression. Under these conditions, adhesion was significantly decreased, implying that endogenous CDK5 activity regulates adhesion. Following in vitro scrape-wounding of stably transfected cells, re-occupation of the wound area was significantly decreased by CDK5 and increased by CDK5-T33. Conclusion: CDK5 increases corneal epithelial cell adhesion by promoting formation of vinculin-containing adhesion complexes and/or their attachment to the cytoskeleton. The strengthening of cell adhesion by CDK5 is likely to be an important factor in retarding cell migration.
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