Constitutive androstane receptor regulates the intestinal mucosal response to injury

Abstract

Background and purpose: The pathogenesis of the inflammatory bowel diseases (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), involves aberrant interactions between a genetically susceptible individual, their microbiota and environmental factors. Alterations in xenobiotic receptor expression and function are associated with increased risk for IBD. Here, we have assessed the role of the constitutive androstane receptor (CAR), a xenobiotic receptor closely related to the pregnane X receptor, in the regulation of intestinal mucosal homeostasis.

Experimental approach: CAR expression was assessed in intestinal mucosal biopsies obtained from CD and UC patients, and in C57/Bl6 mice exposed to dextran sulphate sodium (DSS; 3.5% w/v in drinking water) to evoke intestinal inflammation and tissue damage. CAR-deficient mice were exposed to DSS and mucosal healing assessed. Modulation of wound healing by CAR was assessed in vitro. The therapeutic potential of CAR activation was evaluated, using 3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP), a selective rodent CAR agonist.

Key results: CAR expression was reduced in CD and UC samples, compared with expression in healthy controls. This was reproduced in our DSS studies, where CAR expression was reduced in colitic mice. CAR-deficient mice exhibited reduced healing following DSS exposure. In vitro, CAR activation accelerated intestinal epithelial wound healing by enhancing cell migration. Lastly, treating mice with TCPOBOP, following induction of colitis, enhanced mucosal healing.

Conclusion and implications: Our results support the notion that xenobiotic sensing is altered during intestinal inflammation, and suggest that CAR activation may prove effective in enhancing mucosal healing in patients with IBD.

Figure 1

The expression of the CAR is reduced during intestinal inflammation. (A) The expression of NR1I3, the gene that encodes the CAR, is significantly reduced in intestinal mucosal biopsies obtained from UC and CD patients (n = 8 health control; n = 8 UC; n = 10 CD) with active mild to moderate inflammation. (B) The expression of ABCB1, a CAR target gene that encodes the MDR1 protein, is significantly reduced in samples from UC and CD patients. (C, D) The expression of Nr1i3 and Abcb1a is significantly reduced in colonic tissues isolated from DSS‐treated mice (male mice; 10 weeks of age; 2.5% w/v in drinking water for 7 days) compared to non‐DSS treated control mice. (n = 5 control; n = 5 DSS‐treated mice). (E) The CAR is expressed in intestinal crypts isolated from the colon and ileum of male C57/Bl6 mice (10 weeks of age). (F) The expression of the CAR is reduced in the colonic tissue of DSS‐treated mice as assessed by western blot. (G) The pooled densitometry of CAR expression in control versus DSS‐treated mice expressed as a percentage of β‐actin expression (n = 5 control; n = 5 DSS‐treated mice). * P < 0.05, significantly different from control; human data are expressed as mean ±SD; mouse data are expressed as mean ±SEM.

Figure 2

CAR−/− mice exhibit delayed recovery from DSS‐induced colitis. (A) The body weight of WT C57/Bl6 and CAR−/− mice treated with DSS (male; 10 weeks of age; 2.5% w/v; in the drinking water) for 5 days and then allowed to recovery for 7 days with normal drinking water. * denotes P < 0.05, WT + DSS and CAR−/− + DSS significantly different from Naïve WT and Naïve CAR−/− groups. (B) Colonic tissue MPO levels assessed at experimental day 12, after 7 days of recovery from DSS‐induced colitis. * P < 0.05, WT + DSS and CAR−/− significantly different from Naïve WT and Naïve CAR−/−. (C, D) Representative images of colonic tissues (distal colon nearest the centre of the roll) isolated from mice at experimental day 12. Blind histological scoring of the histological samples assessing the magnitude of colonic tissue inflammation (E) and architectural changes (F). # P < 0.05, significantly different from Naïve WT and Naïve CAR−/−; * P < 0.05, significantly different from DSS WT; (n = 8 Naïve WT; n = 8 Naïve CAR−/−; n = 8 DSS‐treated WT mice; n = 8 DSS‐treated CAR−/− mice).

Figure 3

The CAR is expressed in the Caco‐2 cell line and its activation enhances intestinal epithelial wound closure. (A) Representative western blot depicting the expression of the CAR in Caco‐2 cells at various time‐points post‐confluence compared to the positive control (human liver tissue lysate – ab29889; Abcam). (B) Representative images depicting the extent of wound closure at 12 and 24 h post‐treatment with various concentration of CITCO, a selective CAR agonist, or the DMSO vehicle (Control). (C) The pooled data from the experiments depicted in panel B expressed as a percentage of the original wound. * P < 0.05, 50 nM CITCO significantly different from all other groups; ^ P < 0.05, 25 nM significantly different from Control; n = 8 separate experiments. (D) 50 nM CITCO evokes the expression of ABCB1 in Caco‐2 cells. * P < 0.05, significantly different from all groups; n = 5. (E) The CAR selective inverse agonist 17α‐ethynyl‐3,17β‐estradiol (EE2; 10 μM) reduced the basal/non‐stimulated wound closure between 19–24 h. (F) EE2 pretreatment significantly attenuates CITCO‐induced responses. In panel E, * denotes P < 0.05 compared to control; In panel F, * P < 0.05, significantly different from 50 nM CITCO group; n = 6 separate experiments.

Figure 4

Activation of the CAR enhances cell movement and p38 MAP kinase activation in Caco‐2 intestinal epithelial cells. (A) Representative images of cell movement tracks in Caco‐2 monolayers over the course of 24 h of treatment with various concentration of CITCO, a selective CAR agonist, or the DMSO vehicle (Control). (B) The average length of cell movement tracks quantified from the images depicted in panel A. Units of Length were assessed in 6–8 cells per separate experiment and the average plotted in this graph. * P < 0.05, significantly different from Control and 25 nM CITCO; n = 8 separate experiments. (C) Caco‐2 cells exhibit enhanced migration across a porous membrane (8 μm pore diameter) when treated with CITCO (50 nM), as assessed with xCELLigence Real Time Impedance Analysis. Vehicle control or CITCO were place in both the apical and basolateral chambers to avoid the generation of a concentration gradient. * P < 0.05, significantly different from Control; n = 6 separate experiments. (D) Representative Western blot depicting the induction of p38 MAP kinase phosphorylation following stimulation of the CAR by CITCO (50 nM). This blot is representative of four separate experiments. (E) The pooled densitometric analysis of CITCO‐induced p38 MAP kinase phosphorylation in Caco‐2 cells. * P < 0.05, significantly different from Control (DMSO vehicle); n = 6 separate experiments.

Figure 5

Activation of the CAR in Caco‐2 cells does not induce proliferation, nor does it activate the ERK1/2 MAP kinase signalling cascade. Proliferation of Caco‐2 cells in response to varying concentrations of CITCO, a selective CAR agonist, as assessed by crystal violet staining. In (A), the Control group was treated with serum‐free OptiMEM (A) while all other groups were assayed in 5% FBS‐containing OptiMEM with the depicted CITCO concentration for 24 h. * P < 0.05, significantly different from Control (serum‐free media); n = 7 separate experiments. In (B), the Control group was treated with serum‐free OptiMEM while the experimental groups were assayed in serum‐free OptiMEM with the depicted CITCO concentration for 24 h. * P < 0.05, significantly different from Control (serum‐free media); n = 5 separate experiments. (B) Representative western blot depicting the levels of ERK1/2 MAP kinase phosphorylation following stimulation of the CAR by CITCO (50 nM). This blot is representative of 6 separate experiments. (C) The pooled densitometric analysis of CITCO‐induced ERK1/2 MAP kinase phosphorylation in Caco‐2 cells. n = 6 separate experiments.

Figure 6

Inhibition of p38 MAP kinase signalling attenuates CITCO‐induced enhancement of wound closure and cell movement in Caco‐2 intestinal epithelial monolayers. (A) Representative images depicting the extent of wound closure at 12 and 24 h post‐treatment with CITCO (50 nM) in the presence and absence of SB202190 (10 μM), a selective inhibitor of p38 MAP kinase. (B) The pooled data from the experiments depicted in panel A expressed as a percentage of the original wound. * P < 0.05, CITCO + SB202190 significantly different from CITCO alone; n = 7 separate experiments. (C) Representative images of cell movement tracks in Caco‐2 monolayers over the course of 24 h of treatment with CITCO (50 nM) in the presence and absence of SB202190 or the DMSO vehicle (Control). (D) The average length of cell movement tracks quantified from the images depicted in panel A. Units of Length were assessed in 6–8 cells per separate experiment and the average plotted in this graph. * P < 0.05, significantly different from all groups; # P < 0.05, significantly different from CITCO (50 nM); n = 7 separate experiments.

Figure 7

Activation of the CAR does not modify the acute response to DSS‐induced colitis. Mice were treated with the selective CAR agonist TCPOBOP (male; 10 weeks of age; 3 mg·kg⁻¹/oral gavage in corn oil) or vehicle and exposed to DSS (2.5% w/v in drinking water) for 7 days. (A) The body weight of C57/Bl6 mice treated with DSS for 7 days; n = 7 per group. (B) Colonic tissue MPO levels assessed at after 7 days of DSS exposure; n = 7 per group.

Figure 8

Activation of the CAR enhanced recovery from DSS‐induced colitis. (A) The body weight of C57/Bl6 mice treated with DSS (male; 10 weeks of age; 2.5% w/v; in the drinking water) for 5 days and then allowed to recovery for 7 days with normal drinking water while treated with a selective mouse CAR agonist (TCPOBOP; 3 mg·kg⁻¹; oral gavage; daily) or vehicle. # P < 0.05, DSS + Vehicle and DSS + TCPOBOP significantly different from Naïve and TCPOBOP groups. (B) Colonic tissue MPO levels assessed at experimental day 12, after 7 days of TCPOBOP treatment during recovery from DSS‐induced colitis. # P < 0.05, significantly different from Naïve and TCPOBOP; * P < 0.05, significantly different from DSS + Vehicle; n = 5 per group. (C, D) Representative images of colonic tissues (distal colon nearest the centre of the roll) isolated from mice at experimental day 12. Blind histological scoring of the histological samples assessing the magnitude of colonic tissue inflammation (E) and architectural changes (F). # P < 0.05, significantly different from Naïve and TCPOBOP; * P < 0.05, significantly different from DSS + Vehicle; n = 5 per group.