Repression of Connexin26 Hemichannel activity protects the Barrier Function of Respiratory Airway Epithelial Cells against LPS-induced Alteration

In respiratory airway epithelial cells lipopolysaccharide (LPS) treatment induced an enhancement of connexin 26 (Cx26) hemichannel activity shown by dye uptake experiments combined with siRNA knock-down of Cx26. This effect was already observed at infection relevant concentrations (≤ 10 ng/mL LPS) and involved tumor necrosis factor (TNF)-α- and Ca ²⁺ -dependent signaling. High concentrations (1 µg/mL LPS) reduced the transepithelial electrical resistance (TEER) of Calu-3 cells by 35 % within an application time of 3 h followed by a recovery. Parallel to barrier alteration, a reduced tight junction organization rate (TiJOR) of claudin-4 (CLDN4) by 75 % was observed within an application time of 3 h. After TEER recovery, CLDN4 TiJOR stayed reduced. Low concentrations (10 ng/mL LPS) required three times repeated application for barrier and CLDN4 TiJOR reduction by 30 %. The small molecule CVB4-57, newly published as a potential inhibitor of Cx26 hemichannels, mitigated the effects of LPS on the epithelial barrier function. Molecular docking studies revealed a potential interaction between CVB4-57 and Cx26 thereby reducing its hemichannel activity. We conclude that LPS-related enhancement of Cx26 hemichannel activity acts like a “molecular scar” that weakens the lung epithelium, which could be attenuated by agent targeting the Cx26 hemichannels.