A1 Refereed original research article in a scientific journal
Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon
Authors: Asghar MN, Priyamvada S, Nystrom JH, Anbazhagan AN, Dudeja PK, Toivola DM
Publisher: AMER PHYSIOLOGICAL SOC
Publication year: 2016
Journal: American Journal of Physiology : Gastrointestinal and Liver Physiology
Journal name in source: AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
Journal acronym: AM J PHYSIOL-GASTR L
Volume: 310
Issue: 11
First page : G1147
Last page: G1154
Number of pages: 8
ISSN: 0193-1857
eISSN: 1522-1547
DOI: https://doi.org/10.1152/ajpgi.00354.2015
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.
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.
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