Su1944 Probiotic Bifidobacterium Species Stimulate Human SLC26A3 Gene Function and Expression in In Vitro and In Vivo Models

Kumar, Anoop; Hecht, Cameron; Priyamvada, Shubha; Anbazhagan, Arivarasu Natarajan; Alakkam, Anas; Borthakur, Alip; Gill, Ravinder K.; Alrefai, Waddah A.; Dudeja, Pradeep K.

doi:10.1016/s0016-5085(12)62083-6

Cited by 2 publications

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“…Ready-made SDS-PAGE gels were procured from Bio-Rad (Hercules, CA). DRA antibodies were raised against the C-terminal amino acid sequence INTNGGLRNRVYEPVETKF of DRA (Accession number: BC025671) as previously described 15 .…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Methodsmentioning

confidence: 99%

“…DRA-mediated Cl − /HCO 3 − exchange activity was measured as DIDS-sensitive 125 I − uptake in the base-loaded cells as described previously by us 7, 15 .…”

Section: Methodsmentioning

confidence: 99%