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TA Nguyen, L Wagner, D Boyle, T Shinohara, D Zoukhi, D Takemoto; LEDGF Activation Of PKC And Gap Junction Disassembly In Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2342.
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Purpose: The decrease in PKCγ levels during galactosemia and in human diabetic lens would result in an increase in gap junction activity. Lens epithelium-derived growth factor (LEDGF) is known to enhance survival of lens epithelial cells (LECs) against stress. The goal of these studies is to determine how PKCγ activity is controlled through LEDGF in normal LECs. How this control of PKCγ regulates the phosphorylation of Connexin 43, the inhibition of gap junction activity, and the prevention of assembly of gap junctions in lens epithelial cells. Methods: A rabbit LEC line was grown in the absence or presence of LEDGF. Endogenous PKCγ total activity was determined by a PKC enzyme assay and cell location determined by cellular fractionation and Western blots, and by immunolabeling and confocal microscopy using specific PKCγ and Connexin 43 antisera. Specific PKCγ activity was determined by including pseudosubstrate inhibitors for PKCγ and PKCα (control). Physical and functional interactions between PKCγ and Connexin 43 were determined by co-immunoprecipitation and phosphorylation assays. Results: PKCγ protein was translocated from the cytosolic fractions to the membrane fractions upon addition of LEDGF at 10 ng/ml. In whole cell extract of N/N 1003A cells, co-immunoprecipitation assay showed a direct protein-protein interaction between PKCγ and Connexin 43. In the presence of LEDGF the activation of PKCγ enhanced the phosphorylation of Connexin 43 by 4-fold compared to the absence of LEDGF; however, pseudosubstrate inhibitors for PKCγ decreased the phosphorylation of Connexin 43 by 50% compared to the control. The addition of LEDGF for 30 minutes resulted in a 65% decrease in gap junction Connexin 43 at the cell surface. Conclusion: These results suggest that the activation of PKCγ by LEDGF plays a major role in gap junction assembly/disassembly, which may enhance survival of LECs against osmolarity-stress induced by high sugar concentration.
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