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

Abstract

Background & Aims Diarrhea associated with inflammatory bowel diseases (IBD) has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with IBD has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in intestines of mice, and studied the mechanisms of these effects. Methods We performed quantitative reverse transcription PCR, immunofluorescence, and immunoblot analyses of Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/ml for 6–24 hrs). We purified nuclear extracts and quantified NF-κB activation and DNA binding. We isolated intestinal crypts from C57/BL6 mice, cultured enteroids, incubated these with TNF (50 ng/ml, 24 hrs), and quantified mRNAs. DRA-mediated exchange of Cl− for HCO3− was measured by uptake of 125I. Expression of the NFKB inhibitor alpha (NFKBIA, also called IKBA) was knocked down in Caco-2 or HT-29 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin precipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6J mice were injected with TNF (5 μg/mouse for 3–6 hrs) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. Results Incubation of IECs with TNF reduced expression of DRA. Knockdown of IKBA in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA mRNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin precipitation assays, p65 bound directly to the promoter of DRA, at the regions of −935 of −629 and −375 to −84. Injection of mice with TNF or incubation of crypt-derived organoids with TNF reduced their expression of DRA mRNA and protein. Conclusions In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl− to HCO3− exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for IBD-associated diarrhea.

SLC26A3 (DRA) prevents TNF-alpha-induced barrier dysfunction and dextran sulfate sodium-induced acute colitis

“…We observed that mutation (−1137 to −1128 bp) of the DRA promoter region disrupted the TNF-α-induced reduction in DRA-LUC activity compared with the WT control, suggesting that the TNF-α-induced inhibition of DRA promoter activity is mediated by NF-кB. Our finding is consistent with a recent study by Kumar et al, who also showed that TNF-α decreases DRA expression by activating NF-кB [43]. There are indirect signaling pathways between TNF-α and DRA.…”

cAMP stimulates SLC26A3 activity in human colon by a CFTR-dependent mechanism that does not require CFTR activity

“…26-29 Its molecular size as revealed by Western blot varies and this is probably due to heterogenous glycosylation in different cell systems and animal species. 26-30 We report here that human DRA in HEK293/DRA cells, Caco-2 cells and differentiated proximal colonoids appears as two bands; the upper band is slightly above 102 kDa and the lower band is slightly above 76 kDa, and as previously demonstrated, both of these bands are glycosylated. 19 While the distribution of DRA throughout the human GI tract both horizontally and vertically has been described and there is no debate that it functions as a Cl - /HCO 3 - exchanger with 1:1 stoichiometry, there continues to be confusion relating to its acute regulation, particularly in digestive physiology and in the pathophysiology of cAMP-driven secretory diarrheal diseases.…”

Lipophilicity of the Cystic Fibrosis Drug, Ivacaftor (VX-770), and Its Destabilizing Effect on the Major CF-causing Mutation: F508del

“…Lipophilicity of VX-770 Affects F508del-CFTR Stability reduce SLC26A3 expression through direct binding to NF-kB (Kumar et al, 2017). Hence, it is possible that the destabilizing effect of VX-770 that we observed on SLC26A3 may modify the ORKAMBI response in the intestinal tissues.…”