Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon

Abstract

Asghar MN, Priyamvada S, Nyström JH, Anbazhagan AN, Dudeja PK, Toivola DM. Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon. Am J Physiol Gastrointest Liver Physiol 310: G1147-G1154, 2016. First published April 28, 2016 doi:10.1152 doi:10. /ajpgi.00354.2015 are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8 Ϫ/Ϫ ) 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 Ϫ/Ϫ cecum and proximal and distal colon compared with K8 ϩ/ϩ , whereas the levels in ileum were normal. In K8 ϩ/Ϫ 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 Ϫ/Ϫ distal colon, while K8 ϩ/Ϫ displayed a similar but more patchy apical DRA distribution compared with K8 ϩ/ϩ . DRA was similarly decreased when K8 was knocked down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammationindependent manner. Taken together, the loss of DRA in the K8

Activation of Nuclear Factor−κB by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3

“…We had validated DRA antibodies utilizing DRA knockout 4 , keratin-8 knockout 16 and C. rodentium -infected mice 17 , where DRA expression is markedly reduced or is absent, as well as in Caco2 cells where DRA expression was knocked down by shRNA.…”

Apical cytoskeletons and junctional complexes as a combined system in epithelial cell sheets

“…Studies of certain mutations, as well as knockdown and knockout experiments, have revealed that IFs containing heteropolymerization of type I (K18–K20 and K23) and type II (K7 and K8) keratins have roles in epithelial apical processes, such as transport and barrier functions . Such IFs are often also associated with desmosomes, which are instrumental in integration of apical components for cellular functions .…”

“…99 Studies of certain mutations, as well as knockdown and knockout experiments, have revealed that IFs containing heteropolymerization of type I (K18-K20 and K23) and type II (K7 and K8) keratins have roles in epithelial apical processes, such as transport and barrier functions. 106,107 Such IFs are often also associated with desmosomes, which are instrumental in integration of apical components for cellular functions. 108 Since the TJ protein claudin-1 has been reported to be associated with keratin76, 106 the direct or indirect association of IFs with the TJapical complex is an obligatory subject for future study.…”

Epithelial Intermediate Filaments: Guardians against Microbial Infection?

“…A likely reason is that keratins fulfil a general scaffolding function for membrane proteins [ 122 ]. Very recently Asghar and co-workers [ 123 ] observed a complete loss of the apical localizing chloride transporter (DRA) explaining the strong diarrhea phenotype occurring in patients with inflammatory bowel disease. Furthermore, a direct connection between higher levels of microbiota-produced short chain fatty acids in stool and decreased levels of the monocarboxylate transporter 1 (MCT1) was shown in K8 −/− colon [ 124 ].…”