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.
- ion transport
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