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D. S. Roh, J. L. Funderburgh; Connexin 43 and Gap Junction Intercellular Communication in Bovine Corneal Endothelial Cells After Mitomycin-C Treatment. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1804.
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Gap junction intercellular communication (GJIC) connects the cytoplasm of adjacent cells and allows the intercellular transfer of small molecules. Connexin proteins and GJIC can be rapidly altered in response to various cell stressors to prevent or enhance cell-cell communication. We recently reported (IOVS 2008 49:4837-4843) that mitomycin-C (MMC) induces DNA damage in corneal endothelial (CE) cells. The current study explores the effects of MMC treatment on CE cell-cell communication via gap junctions mediated by connexin 43 (Cx43).
Post-confluent bovine CE cells were treated with MMC at various concentrations and times. Relative levels of Cx43 mRNA were determined by qRT-PCR, total levels of protein were detected by Western blotting, and localization was determined by immunostaining. The generation of intracellular reactive oxygen species (ROS) was determined by the fluorescent probe CM-H2DCFDA and measured by spectrofluorometry. GJIC was assessed by loading cells with Calcein AM and performing fluorescence recovery after photobleaching (FRAP).
Total Cx43 protein in post-confluent CE cells increased within a few hours of MMC treatment while significant changes in mRNA levels were not detected during that time frame. Immunostaining demonstrated that MMC produced a decrease in Cx43 at the cell surface and an increase in the perinuclear region. GJIC measured by FRAP was not altered by MMC nor did MMC treatment generate significant levels of intracellular ROS. Over a longer 3 day period, CE cells treated with MMC also increased total Cx43 protein. Changes in Cx43 localization at the cell surface were also detected after MMC treatment during this longer time frame.
CE cells alter protein levels and subcellular localization of Cx43 in response to MMC stress, but appear to maintain functional GJIC. Cx43 increases do not appear to involve increased transcription but may involve post-translational mechanisms. Increased Cx43 protein and maintenance of GJIC may be protective responses to MMC-induced stress.
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