title: Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon.
abstract: Keratins (K) are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8(-/-)) mice develop a Th2-type colonic inflammation, epithelial hyperproliferation, and mild diarrhea caused by a keratin level-dependent decrease in short-circuit current and net sodium and chloride absorption in the distal colon. The lack of K8 leads to mistargeting or altered levels of membrane proteins in colonocytes; however, the main transporter responsible for the keratin-related ion transport defect is unknown. We here analyzed protein and mRNA levels of candidate ion transporters CFTR, PAT-1, NHE-3, and DRA in ileum, cecum, and proximal and distal colon. Although no differences were observed for CFTR, PAT-1, or NHE-3, DRA mRNA levels were decreased by three- to fourfold and DRA protein was almost entirely lost in K8(-/-) cecum and proximal and distal colon compared with K8(+/+), whereas the levels in ileum were normal. In K8(+/-) mice, DRA mRNA levels were unaltered, while decreased DRA protein levels were detected in the proximal colon. Immunofluorescence staining confirmed the loss of DRA in K8(-/-) distal colon, while K8(+/-) displayed a similar but more patchy apical DRA distribution compared with K8(+/+) DRA was similarly decreased when K8 was knocked down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammation-independent manner. Taken together, the loss of DRA in the K8(-/-) mouse colon and cecum explains the dramatic chloride transport defect and diarrheal phenotype after K8 inactivation and identifies K8 as a novel regulator of DRA.
authors.affiliation: Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University and Turku Center for Disease Modeling, University of Turku, Turku, Finland; and.
authors.forename: M Nadeem
authors.initials: MN
authors.lastname: Asghar
authors.affiliation: University of Illinois at Chicago, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois.
authors.forename: Shubha
authors.initials: S
authors.lastname: Priyamvada
authors.affiliation: Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University and Turku Center for Disease Modeling, University of Turku, Turku, Finland; and.
authors.forename: Joel H
authors.initials: JH
authors.lastname: Nyström
authors.affiliation: University of Illinois at Chicago, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois.
authors.forename: Arivarasu Natarajan
authors.initials: AN
authors.lastname: Anbazhagan
authors.affiliation: University of Illinois at Chicago, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois.
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Dudeja
authors.affiliation: Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University and Turku Center for Disease Modeling, University of Turku, Turku, Finland; and dtoivola@abo.fi.
authors.forename: Diana M
authors.initials: DM
authors.lastname: Toivola
mesh_terms: D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017920:Antiporters / Q000235:genetics* / Q000378:metabolism; D018938:Caco-2 Cells; D003106:Colon / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053533:Keratin-8 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism; D000076988:Sulfate Transporters; D027981:Symporters / Q000235:genetics / Q000378:metabolism
chemical_list: D026905:Amino Acid Transport Systems; D017920:Antiporters; D053533:Keratin-8; C486595:Krt8 protein, mouse; D012333:RNA, Messenger; C000613385:SLC9A3 protein, human; C489860:Slc26a3 protein, mouse; C483904:Slc36a1 protein, mouse; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000076988:Sulfate Transporters; D027981:Symporters; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: DRA; chloride; colon; epithelium; ion transport; keratin; sodium