Ca2+‐ activated Cl− secretion in murine distal colon is inhibited in acute dextran sulfate sodium (DSS)‐induced inflammation via down‐regulation of TMEM16A

Abstract

BackgroundMouse colonic epithelium exhibits Ca2+‐activated Cl− conductance (CaCC), which is mediated in large part by a Cl− channel known as TMEM16A (a.k.a. ANO1). Dextran sulfate sodium (DSS)‐ treated mice have been previously shown in the literature to have a dramatically reduced CaCC when treated with carbamoylcholine (CCH; a muscarinic receptor agonist). However, this finding predates the identification of TMEM16A as the primary mediator of CaCC, and was thus attributed to dysfunctional Na‐K ATPase and/or basolateral K+ channel activity. To date, the effects of DSS treatment on TMEM16A itself have not been determined. Furthermore, a cytokine involved in DSS pathology, transforming growth factor β (TGF‐β), has been shown to suppress TMEM16A expression in epithelial cell lines (T84 and HAEC). It is therefore plausible that DSS treatment causes a decrease in TMEM16A protein expression in mouse colon, and this phenomenon is potentially mediated by the effects of TGF‐β.Hypotheses1.) DSS‐induced inflammation causes a down‐regulation of TMEM16A expression, contributing significantly to reduced CaCC. 2.) Increased TGF‐β signaling in DSS‐induced inflammation results in suppression of TMEM16A.MethodsBALB/c mice were given water with or without 5% DSS for 7 days, followed by 1 day of regular water. Disease Activity Index (DAI) scores, colonic length measurements and inflammatory cytokine profiles were recorded. Distal colonic epithelium was harvested either for short‐circuit current (ISC) recordings in an Ussing chamber or for qRT‐PCR and Western blot analyses. T84 cells were grown into monolayers and treated with one of a series of DSS‐associated cytokines for 48 hours. ISC recordings from T84 monolayers were taken and cells were subsequently used for qRT‐PCR and Western blot analyses.ResultsWe found that DSS‐treated mice exhibit a substantially reduced secretory response to CCH compared to control (−1.4 vs 13.4 μA/cm2), which was corroborated by a decrease in TMEM16A protein (70%) and mRNA (81%) expression. T84 cells treated with TGF‐β, but not other DSS‐associated cytokines, also showed a reduction in CCH‐induced Cl− secretion (109.71 vs 170.1 μA/cm2), as well as TMEM16A protein expression (32%) compared to control.ConclusionsDSS‐induced inflammation suppresses the expression of TMEM16A in mice at the transcript and protein level. We speculate that this effect is potentially mediated by TGF‐β signaling in the colonic epithelium.Support or Funding InformationNIH 10019896.1.1006856RThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.