Specifically Sized Hyaluronan (35 kDa) Prevents Ethanol-Induced Disruption of Epithelial Tight Junctions Through a layilin-Dependent Mechanism in Caco-2 Cells.

Abstract

BACKGROUND Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35kDa (HA35) protects mice from short term ethanol-induced liver injury. This protection was associated with maintenance of the co-localization of zonula occludins-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. METHODS Female C57BL/6J mice were fed an ethanol containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of ethanol feeding. Intestinal morphology and tight junction integrity was assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with ethanol. Localization of tight junction proteins, FITC-dextran permeability and transepithelial electrical resistance (TEER) were evaluated. RESULTS While short-term ethanol did not result in any apparent changes in the gross morphology of the intestine, co-localization of ZO-1 and occludin at tight junctions was decreased in proximal and distal colon. HA35 prevented these effects of ethanol. In differentiated Caco-2 cells, ethanol decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, ethanol also decreased TEER. Pre-treatment with HA35 prevented these changes. When the hyaluronan receptor Layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from ethanol-induced loss of tight junctions. CONCLUSION Taken together, these data indicate that HA35 interacts with Layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term ethanol exposure. This article is protected by copyright. All rights reserved.