Danny S Roh1, James L Funderburgh. 1. Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Abstract
PURPOSE: To determine how corneal endothelial (CE) cells respond to acute genotoxic stress through changes in connexin-43 (Cx43) and gap junction intercellular communication (GJIC). METHODS: 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. RESULTS: 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. DISCUSSION: 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.
PURPOSE: To determine how corneal endothelial (CE) cells respond to acute genotoxic stress through changes in connexin-43 (Cx43) and gap junction intercellular communication (GJIC). METHODS: 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. RESULTS: 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. DISCUSSION: 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.
Authors: Audrey A Chan; Andrew J Hertsenberg; Martha L Funderburgh; Mary M Mann; Yiqin Du; Katherine A Davoli; Jocelyn Danielle Mich-Basso; Lei Yang; James L Funderburgh Journal: PLoS One Date: 2013-02-21 Impact factor: 3.240
Authors: Uwe Knippschild; Marc Krüger; Julia Richter; Pengfei Xu; Balbina García-Reyes; Christian Peifer; Jakob Halekotte; Vasiliy Bakulev; Joachim Bischof Journal: Front Oncol Date: 2014-05-19 Impact factor: 6.244