A Novel Role of SLC26A3 in Maintenance of Intestinal Barrier Function

Abstract

Inflammatory Bowel Disease (IBD) is a global health burden currently affecting around 3 million people in the United States with increasing incidence worldwide. The pathogenesis of IBD is still unclear due to the multifactorial nature of the disease. Compromised intestinal barrier is one of the most critical early events linked to the onset of intestinal inflammation. In this regard, SLC26A3 or DRA (Down Regulated in Adenoma) is an essential transporter for chloride absorption in the mammalian intestine and has been identified as an IBD susceptibility gene in certain cohorts. Further, DRA levels are markedly decreased in colonic mucosa of IBD patients and in mouse models of gut inflammation. Also, DRA KO mice are more susceptible to DSS colitis. However, the mechanisms underlying increased susceptibility to inflammation by loss of DRA are not fully understood.AimsTo examine the impact of loss of DRA (in DRA KO mice) on epithelial barrier function and expression of key tight junction and adherens junction proteins and elucidate underlying mechanisms.MethodsWild type and DRA KO mice (8–10 weeks old, M/F) were used. Protein expression was measured by immunoblotting and immunofluorescence. FITC‐dextran flux was examined utilizing Ussing chamber.ResultsDRA KO mice exhibited an increase in colonic paracellular permeability (~ 5 fold, p<0.05) indicating impaired barrier function. This was associated with a decrease in levels of TJ protein ZO‐1 and occludin (~ 65%, p<0.05) and major AJ protein, E‐cadherin (~ 60%, p<0.05). Also, the expression of pore forming, Claudin 2 was significantly upregulated in DRA KO mouse colon (~ 3 fold, p<0.05). No significant changes were observed in claudin 1 or 3. Since mouse intestine is a complex physiological system, we next examined if DRA silencing in a model epithelial Caco‐2 cell line, affects barrier function and tight junction protein expression. shRNA Knock‐down of DRA in Caco‐2 cells resulted in a marked decrease in TEER (~ 40%, p<0.05) and occludin expression (~ 60%, p<0.05). Similarly, enteroids derived from DRA KO mice exhibited decreased expression of occludin and E‐cadherin proteins (~ 50%, p<0.05). There was no alteration in mRNA levels of TJ/AJ proteins in DRA KO mice indicating role of post‐transcriptional mechanisms. To understand the mechanisms underlying these alterations, we next examined the expression of RNA‐binding proteins that are known to regulate the translation of occludin and E‐cadherin. Our results showed a marked increase in expression of CUGBP1 (a negative regulator of TJ/AJ proteins) and a marked decrease in expression of intact HuR (a positive regulator).ConclusionOur studies suggest a novel role of a membrane chloride transporter DRA in maintenance of intestinal barrier function and thus a down regulation of this protein may play a key role in pathogenesis of IBD.Support or Funding InformationSupported by NIDDK and Department of Veterans AffairsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.