# Repression of Connexin26 hemichannel activity protects the barrier function of respiratory airway epithelial cells against LPS-induced alteration

## TL;DR

This study shows that LPS increases Cx26 hemichannel activity in airway cells, weakening the epithelial barrier, and that this effect can be reduced by a Cx26 inhibitor.

## Contribution

The study identifies Cx26 hemichannels as a target for mitigating LPS-induced epithelial barrier dysfunction in respiratory cells.

## Key findings

- LPS increases Cx26 hemichannel activity in a TNF-α- and Ca2+-dependent manner.
- The small molecule CVB4-57 reduces LPS-induced barrier dysfunction by inhibiting Cx26 hemichannels.
- LPS reduces tight junction organization of claudin-4, with effects persisting even after barrier recovery.

## Abstract

In respiratory airway epithelial cells, lipopolysaccharide (LPS) treatment induced an enhancement of connexin 26 (Cx26) hemichannel activity shown by dye uptake experiments after siRNA-mediated knock-down of Cx26. This effect was already observed at infection relevant concentrations (≤ 10 ng/mL LPS) and involved tumor necrosis factor alpha (TNF-α)- and Ca2+-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 reduction 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 agents targeting Cx26 hemichannels.

The online version contains supplementary material available at 10.1186/s12964-025-02228-6.