Alcohol Consumption Increases Claudin‐18 Turnover in Alveolar Epithelial Cells

Abstract

Alveolar epithelial cell (AEC) tight junctions are crucial for maintaining barrier function between fluid filled tissues and the airspace in the lung. Failure of alveolar tight junctions leads to acute respiratory distress syndrome (ARDS), a frequently lethal condition characterized by airspace flooding in the lung. Chronic alcohol abuse is a great risk factor for ARDS. Alcohol consumption impairs alveolar barrier function by modulation of tight junction proteins, called claudins. Claudins are a family of tetraspan transmembrane proteins that confer specific barrier properties to epithelial cell monolayers by regulating tight junction permeability. We used a rodent model of chronic alcohol ingestion to investigate the effects of alcohol on claudins and barrier function. Primary AECs from alcohol and control fed rats were cultured on Transwell permeable supports and allowed to differentiate into model type I cells. Transepithelial resistance measurements (TER) and dye flux assays were used to determine barrier function in cultured AECs. Tight junction morphology was assessed by immunofluorescence. In response to alcohol, alveolar tight junctions showed altered claudin composition leading to an increase in paracellular leak and formation of projections perpendicular to tight junctions (“spikes”) when immunostained for claudin‐18. Quantification of cells containing tight junction spikes revealed that alcohol increases the portion of cells containing tight junction spikes, which correlated with decreased barrier function. Live cell imaging of AECs expressing YFP‐tagged claudin‐18 confirmed that vesicles containing tagged claudin‐18 fused with and pinched off of tight junction spikes. Therefore, we hypothesized that these tight junction spikes are sites of enhanced protein turnover which might then result in decreased barrier function. As endocytosis is an important regulator of claudin turnover, we examined the effect of treating cells with the inhibitor Dynasore that targets dynamin, a protein which has a role in the scission of endocytic vesicles. Treatment of AECs with Dynasore resulted in a significant decrease in the number of cells containing claudin‐18 spikes and also caused a concomitant decrease in paracellular flux of a 10 kDa marker, Texas Red dextran. Our data support a role for tight junction spikes in regulating paracellular permeability and suggest that dynamin or dynamin‐related proteins may be useful therapeutic targets to stabilize alveolar barrier function and impair the development of ARDS.Support or Funding InformationThis work was supported by Emory Alcohol and Lung Biology Center/National Institutes of Health (NIH) grant P50‐AA013757 (MK), R01‐HL116958 (MK), T32‐AA‐013528 (SAM), the German Academic Exchange Service (DAAD) (BS), R25‐GM099644 (SDW), and Emory+Children's Center of Excellence for Cystic Fibrosis Research (MK).