Aryl Hydrocarbon Receptor Activation Modulates Intestinal Epithelial Barrier Function by Maintaining Tight Junction Integrity

Abstract

Activation of Aryl hydrocarbon receptor (AhR) is involved in the control of intestinal mucosal homeostasis. Intestinal barrier dysfunction contributes to the development of many intestinal diseases, such as inflammatory bowel disease (IBD). In this study, we investigated the mechanisms of AhR activation in the maintenance of intestinal barrier function. Adult C57BL/6 mice were treated with dextran sulphate sodium (DSS) for 7 days, with or without 6-Formylindolo(3,2-b)carbazole (FICZ), a ligand of AhR. We found that AhR activation by FICZ attenuated the decreased TJ protein expression in the colonic mucosa of the DSS-induced mice. Further, the increase of both MLC phosphorylation and MLCK expression in the mice with DSS-induced colitis was also significantly inhibited by FICZ induced AhR activation. For in vitro experiments, Caco-2 cells were treated with tumour necrosis factor alpha (TNF-α)/interferon gamma (IFN-γ) for 48 h, with or without FICZ. AhR activation prevented TNF-α/IFN-γ-induced decrease in TER and morphological disruption of the TJs in Caco-2 monolayers. It also inhibited TNF-α/IFN-γ-induced increase in MLCK expression and MLC phosphorylation by suppression of NF-κB p65 signaling pathway. Thus, AhR-activating factors might have potential as therapeutic agents for the treatment of patients with IBD.

Keywords: Aryl hydrocarbon receptor; inflammatory bowel disease; intestinal barrier function; myosin light chain; tight junction.

Figure 1

FICZ attenuates the loss of intestinal barrier function during DSS-induced colitis. C57BL/6J WT mice were treated with 3% DSS for 7 days, and FICZ was administered to the mice for 5 days beginning at 2 days after the start of DSS administration. (A) The CYP1A1 mRNA level in colon homogenates. The data are presented as the mean ± SD (n = 5). **P<0.01 versus DSS group. (B) Colons from the control, DSS-treated and DSS+FICZ-treated groups were stained with haematoxylin-eosin. Magnification: ×100. (C) TER was measured to assess the permeability of the colonic epithelium on day 7 of the experiment. The data are presented as the mean ± SD (n = 5). *P<0.05 versus control group, ***P<0.001 versus DSS+FICZ group.

Figure 2

FICZ prevents DSS-induced disruption of TJ. (A) Expressions of TJ proteins (ZO-1, Occludin and Claudin-1) and CYP1A1 in the colonic mucosa were evaluated by western blotting. (B) Immunohistochemical analysis of TJ proteins in the colonic mucosa. Magnification: ×400.

Figure 3

The effects of FICZ and TNF-α/IFN-γ on the expression and localization of tight junction proteins in vitro. Caco-2 cell monolayers were treated with 20 ng/ml TNF-α and 10 ng/ml IFN-γ for 48 h, respectively, with or without FICZ. (A) TER was detected. The results are expressed as the mean ± SD (n = 3). **P<0.01. (B) Expression of TJ proteins was assessed by western blotting. The results are representative of three independent experiments. The subcellular localization of ZO-1 (C, green), Occludin (D, green) and Claudin-1 (E, green) was determined in Caco-2 cells treated as described above by immunofluorescence staining. Scale bar, 20 μM.

Figure 4

FICZ inhibits the increases in MLC phosphorylation and MLCK expression. (A) Caco-2 cell monolayers were treated with 20 ng/ml TNF-α and 10 ng/ml IFN-γ for 48 h, respectively, with or without FICZ. MLCK and pMLC expression was assessed by western blotting. (B) Expression of MLCK and pMLC in the colonic mucosa was evaluated by western blotting. The results are representative of three independent experiments. (C) Immunohistochemical analysis of MLCK and pMLC in the colonic mucosa. Magnification: ×400.

Figure 5

FICZ suppress TNF-α/IFN-γ induced activation of NF-κB p65. Caco-2 monolayers were exposed to 20 ng/ml TNF-α and 10 ng/ml IFN-γ for 30 min with or without FICZ. (A) The phosphorylation level of NF-κB p65 was assessed by western blotting. (B) Caco-2 monolayers were stained for NF-κB p65 by immunofluorescence. Scale bar, 20 μM.