LEDGF activation of PKC γ and gap junction disassembly in lens epithelial cells

Abstract

Lens epithelium-derived growth factor (LEDGF) has been shown to enhance survival of lens epithelial cells (LECs) against stress. The objectives of these studies are to determine how LEDGF controls PKC γ activity in normal LECs: how this control of PKC γ regulates the phosphorylation of Connexin 43, the inhibition of gap junction activity, and the prevention of assembly of gap junctions in LECs. A rabbit LEC line, N/N1003A, was grown in the absence or presence of LEDGF. PKC γ protein was translocated from the cytosolic fractions to the membrane fractions upon addition of LEDGF at 10 ng ml −1. In whole cell extracts of N/N1003A cells, co-immunoprecipitation assays showed a protein–protein interaction between PKC γ and Connexin 43. In the presence of LEDGF the activation of PKC γ enhanced the phosphorylation of Connexin 43 by four-fold compared to the absence of LEDGF. The addition of LEDGF for 30 min resulted in a 65% decrease in gap junction Connexin 43 at the cell surface and a 70% decrease in gap junction activity. These results suggest that the activation of PKC γ 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.