title: Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli.
abstract: Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl(-)/HCO3(-) exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl(-)/HCO3(-) exchange activity was measured as DIDS-sensitive (125)I(-) uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl(-)/HCO3(-) exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; Department of Physiology, University of Illinois at Chicago, Chicago, Illinois;
authors.forename: Tarunmeet
authors.initials: T
authors.lastname: Gujral
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois;
authors.forename: Anoop
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois;
authors.forename: Shubha
authors.initials: S
authors.lastname: Priyamvada
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois;
authors.forename: Seema
authors.initials: S
authors.lastname: Saksena
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois;
authors.forename: Ravinder K
authors.initials: RK
authors.lastname: Gill
authors.affiliation: Gastroenterology and Nutrition, Department of Medicine, Loyola University, Maywood, Illinois.
authors.forename: Kim
authors.initials: K
authors.lastname: Hodges
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and.
authors.forename: Waddah A
authors.initials: WA
authors.lastname: Alrefai
authors.affiliation: Gastroenterology and Nutrition, Department of Medicine, Loyola University, Maywood, Illinois.
authors.forename: Gail A
authors.initials: GA
authors.lastname: Hecht
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and pkdudeja@uic.edu.
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Dudeja
mesh_terms: D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D015153:Blotting, Western; D018938:Caco-2 Cells; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D054308:Enteropathogenic Escherichia coli; D004927:Escherichia coli Infections / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D000076988:Sulfate Transporters
chemical_list: D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D002966:Clathrin; C081011:SLC26A3 protein, human; C489860:Slc26a3 protein, mouse; D000076988:Sulfate Transporters
keywords: DRA recycling; EPEC infection; clathrin; endocytosis; exocytosis; microtubules