Rapid Changes in Connexin-43 in Response to Genotoxic Stress Stabilize Cell–Cell Communication in Corneal Endothelium

Abstract

To determine how corneal endothelial (CE) cells respond to acute genotoxic stress through changes in connexin-43 (Cx43) and gap junction intercellular communication (GJIC). Cultured bovine CE cells were exposed to mitomycin C or other DNA-damaging agents. Changes in the levels, stability, binding partners, and trafficking of Cx43 were assessed by Western blot analysis and immunostaining. Live-cell imaging of a Cx43-green fluorescent protein (GFP) fusion protein was used to evaluate internalization of cell surface Cx43. Dye transfer and fluorescent recovery after photobleaching (FRAP) assessed GJIC. After genotoxic stress, Cx43 accumulated in large gap junction plaques, had reduced zonula occludens-1 binding, and displayed increased stability. Live-cell imaging of Cx43-GFP plaques in stressed CE cells revealed reduced gap junction internalization and degradation compared to control cells. Mitomycin C enhanced transport of Cx43 from the endoplasmic reticulum to the cell surface and formation of gap junction plaques. Mitomycin C treatment also protected GJIC from disruption after cytokine treatment. These results show a novel CE cell response to genotoxic stress mediated by marked and rapid changes in Cx43 and GJIC. This stabilization of cell-cell communication may be an important early adaptation to acute stressors encountered by CE.