Regulation of gap junction intercellular communication in primary canine lens epithelial cells: role of protein kinase C.
OBJECTIVE
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 x 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 x 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 Cx43 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 x 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.
Long
Amy C
AC
Ohio State University, Interdisciplinary Nutrition Program, The Ohio State University, Columbus, Ohio, USA.
Colitz
Carmen M H
CM
Bomser
Joshua A
JA
D000818:Animals; D002450:Cell Communication / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000378:metabolism*; D015151:Immunoblotting; D007527:Isoenzymes / Q000502:physiology; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity
D018031:Connexin 43; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate