Regulation of Gap Junction Intercellular Communication in Primary Canine Lens Epithelial Cells: Role of Protein Kinase C

Abstract

Purpose: Gap junction intercellular communication (GJIC) is important in maintaining lens epithelial cell homeostasis and reductions in GJIC may be associated with the development of cataract. Protein kinase C (PKC) activation can disrupt gap junction communication via phosphorylation of connexin 43 (C×43) proteins that compose gap junction channels. This study examined the role of PKC activation in modulating GJIC in a primary canine lens epithelial cell (LEC) line. Methods: TPA (12-O-tetradecanoyl-phorbol-acetate), a potent PKC activator and inhibitor of GJIC, was utilized in the present study. Primary cultures of canine LEC were treated with TPA (0–1000 ng/ml) for 0.5 hr and GJIC was assessed by scrape loading/dye transfer (SL/DT), and immunoblotting to detect phosphorylation of C×43 protein. Inhibition of general and calcium-dependent PKC activity was achieved by pretreatment of cells with GF109203X and Gö6976, respectively. Results: Treatment with TPA (1–1000 ng/ml) significantly decreased GJIC in canine LEC as assessed by SL/DT. Pretreatment with 10 and 100 ng/ml TPA decreased GJIC by 80% as compared to controls and increased C×43 phosphorylation as assessed by immunoblotting. Pretreatment of cells with GF109203X and Gö6976, partially restored TPA-inhibited GJIC by 40% and 60%, respectively, and reduced C×43 phosphorylation. Expression of calcium dependent PKC isoforms was detected in canine whole lens and LEC. Conclusions: Treatment with TPA significantly reduces GJIC in canine LEC. These effects are mediated, in part, by activation of calcium-dependent PKC isoforms. Primary canine LEC are a useful model in the study of the molecular mechanisms involved in GJIC and cataractogenesis.