Pregnane X receptor agonists enhance intestinal epithelial wound healing and repair of the intestinal barrier following the induction of experimental colitis
Graphical abstract
Introduction
The inflammatory bowel diseases (IBD; Crohn’s disease – CD; ulcerative colitis – UC) are complex diseases of the gastrointestinal tract thought to be caused by aberrant inflammatory responses to luminal antigens/microbes in genetically susceptible individuals (Xavier and Podolsky, 2007). Patients with IBD exhibit alterations in innate immune function, enhanced T cell-dependent inflammation and defects in intestinal epithelial barrier function, each thought to contribute to IBD pathogenesis in an interdependent fashion (Xavier and Podolsky, 2007). As such, therapies designed to inhibit the inflammatory responses in IBD are effective, however the clinical efficacy of these agents is associated with enhanced mucosal wound healing and ulcer restitution (Iacucci and Ghosh, 2011, Turner, 2009).
Maintenance of the intestinal epithelial barrier is key to optimal gastrointestinal health. Increased permeability is associated with IBD (Blair et al., 2006, Heller et al., 2005, Zeissig et al., 2007), thus studying the mechanisms that regulate intestinal epithelial growth/survival and epithelial barrier function may provide insight into the pathogenesis of IBD and provide new therapeutic targets for its treatment. The PXR, a ligand-activated nuclear receptor, is a key regulator of xenobiotic metabolism that is expressed in various regions of the gastrointestinal tract and has been implicated in the regulation of metabolic pathways in intestinal epithelial cells (IECs) (Cheng et al., 2012). The PXR can be activated by a number of diverse ligands including steroids, bile acids, antibiotics and other exogenous compounds (Staudinger et al., 2013). As such, the PXR is often coined a “xenobiotic sensor” and master regulator of detoxification pathways throughout the gastrointestinal tract, through its ability to upregulate the expression of key proteins involved in metabolism including specific cytochrome P450 enzymes and various transporters (Jana and Paliwal, 2007, Staudinger et al., 2013).
Several genome-wide association studies have reported that specific single nucleotide polymorphisms (SNPs) in coding and non-coding regions of the PXR gene are associated with increased risk for CD and UC (Dring et al., 2006, Glas et al., 2011, Martinez et al., 2007). Furthermore, aberrant PXR expression has been observed in the colonic mucosa of patients with IBD (Langmann et al., 2004). Interestingly, PXR-deficient mice exhibit spontaneous small intestinal inflammation (Zhou et al., 2006) and selective PXR agonists can dampen the inflammatory response in experimental models of colitis (Dou et al., 2013, Shah et al., 2007). Furthermore, rifaximin, an antibiotic used to treat IBD and a gut specific human PXR agonist (Ma et al., 2007), attenuates experimental colitis in humanized PXR mice (Cheng et al., 2010). In each case, the anti-inflammatory effects observed in experimental colitis were attributed to PXR-dependent inhibition of NFkappaB driven cytokine/chemokine production (Cheng et al., 2010, Dou et al., 2013, Shah et al., 2007).
In addition to regulating gene transcription and directly inhibiting NFkappaB signaling, activation of the PXR has been linked to intracellular signaling pathways associated with cytoskeletal rearrangement, cell motility and cell proliferation. Kodama and Negishi (2011) reported that the activation of the PXR triggered cytoskeletal rearrangement enhancing cell migration in a p38 MAP kinase-dependent fashion (Kodama and Negishi, 2011). Furthermore, rifampicin, a prototypical PXR agonist, enhanced the migration of colonic epithelial cell lines through the activation of FGF19-dependent pathways (Wang et al., 2011). Taken together these data suggest that the activation of the PXR may promote cell proliferation and migration, two of the key processes involved in epithelial wound healing.
Given that alterations in the intestinal epithelial barrier play a key role in the induction of inflammation in experimental colitis, and that PXR activation can drive the cellular responses associated with the wound healing process, we posit that activation of the PXR may provide its anti-colitic effects, in part, through its ability to enhance the intestinal epithelial barrier by accelerating the wound healing process.
In the current manuscript we report that PXR agonists enhance intestinal epithelial wound healing by triggering cell migration in a p38 MAP kinase-dependent fashion, an effect that occurs in the absence of increased cell proliferation. Furthermore, oral administration of a mouse specific PXR agonist attenuates the intestinal barrier defect observed in experimental colitis, an effect that occurs independent of the inflammatory status.
Section snippets
Reagents
Human PXR agonists: rifaximin, rifampicin and SR12813 (all from Sigma Aldrich) were dissolved in sterile DMSO to stock concentrations of 10 mM. Volumes of stock solutions were added to culture media to reach the appropriate experimental concentration (10 μM). Matching volumes of sterile DMSO were added to culture media for experimental vehicle controls. p38 MAP kinase inhibitor: SB202190 (Sigma Aldrich) was dissolved in sterile DMSO to stock concentrations of 10 mM. Volumes of stock solutions were
Results
In the current study we sought to assess whether activation of the PXR could enhance intestinal epithelial wound healing. PXR agonists rifaximin, rifampicin and SR12813 each enhanced wound closure in Caco-2 monolayers (Fig. 1A; summarized in Fig. 1C), an effect most evident after 24 h of treatment. Interestingly, pretreating the cells with a p38 MAP kinase inhibitor (SB202190; 10 μM) significantly attenuated the wound closure induced by rifaximin, rifampicin and SR12813 (Fig. 1B; summarized in
Discussion
In the current study we provide evidence that PXR agonists can enhance wound healing in IEC monolayers through the activation of p38 MAP kinase-dependent cell migration. Wound healing triggered by PXR agonists occurred in the absence of increased cell proliferation. In our in vivo studies, treating mice with the rodent specific PXR agonist PCN reversed the intestinal barrier dysfunction observed in experimental colitis and enhanced barrier recovery independent of the inflammatory status of the
Acknowledgements
J.T. is funded by a summer studentship from the Crohn’s and Colitis Foundation of Canada/Canadian Association of Gastroenterology. S.A.H. is supported by the Dr. Lloyd Sutherland Professorship in IBD/GI Research. S.A.H. has also receives support from the CCFC Chair in IBD Research held by Dr. Keith Sharkey. The Snyder Institute for Chronic Diseases has provided institutional infrastructure support for these studies.
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