NOD2-nitric oxide-responsive microRNA-146a activates Sonic hedgehog signaling to orchestrate inflammatory responses in murine model of inflammatory bowel disease - PubMed (original) (raw)
NOD2-nitric oxide-responsive microRNA-146a activates Sonic hedgehog signaling to orchestrate inflammatory responses in murine model of inflammatory bowel disease
Devram Sampat Ghorpade et al. J Biol Chem. 2013.
Abstract
Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disorder of the intestine. The interactions between enteric bacteria and genetic susceptibilities are major contributors of IBD etiology. Although genetic variants with loss or gain of NOD2 functions have been linked to IBD susceptibility, the mechanisms coordinating NOD2 downstream signaling, especially in macrophages, during IBD pathogenesis are not precisely identified. Here, studies utilizing the murine dextran sodium sulfate model of colitis revealed the crucial roles for inducible nitric-oxide synthase (iNOS) in regulating pathophysiology of IBDs. Importantly, stimulation of NOD2 failed to activate Sonic hedgehog (SHH) signaling in iNOS null macrophages, implicating NO mediated cross-talk between NOD2 and SHH signaling. NOD2 signaling up-regulated the expression of a NO-responsive microRNA, miR-146a, that targeted NUMB gene and alleviated the suppression of SHH signaling. In vivo and ex vivo studies confirmed the important roles for miR-146a in amplifying inflammatory responses. Collectively, we have identified new roles for miR-146a that established novel cross-talk between NOD2-SHH signaling during gut inflammation. Potential implications of these observations in therapeutics could increase the possibility of defining and developing better regimes to treat IBD pathophysiology.
Keywords: Inflammation; Inflammatory Bowel Disease; Innate Immunity; Macrophages; MicroRNA; NOD-like Receptors (NLR); Nitric Oxide; Signal Transduction; Signaling.
Figures
FIGURE 1.
NOD2 pathway activates SHH signaling. A and B, mouse peritoneal macrophages were stimulated with MDP at the indicated concentrations. A and B show transcript and protein level changes in the SHH activation markers and nuclear translocation of GLI1. Med, medium; PCNA, proliferating cell nuclear antigen. C and D, time kinetics analysis of SHH signaling activation at both transcript (C) and protein levels (D) upon stimulation of NOD2 pathway is shown. E and F, macrophages were treated with PP2, a RIP2 kinase inhibitor, prior to MDP treatment and status of SHH signaling induction, and activation was assayed at transcript (E) or protein levels (F). G, RAW 264.7 cells were transfected with siRNAs specific to Nod2, Rip2, and Tak1, and activation of SHH signaling was assayed by immunoblotting. NT, nontargeted siRNA. *, p < 0.05 versus control; **, p < 0.05 versus MDP treatment (one-way ANOVA).
FIGURE 2.
NO mediates NOD2-driven activation of SHH signaling. A, macrophages were treated with various concentrations of MDP as indicated. mRNA levels of Inos were assayed by quantitative real time RT-PCR, and change in nitrate production was measured using Griess reagent. B, macrophages were treated with MDP, and kinetics of iNOS expression and NO production were assayed by immunoblotting and Griess reagent, respectively (mean ± S.E., n = 3). Med, medium. C and D, iNOS expression and NO production were analyzed by inhibition of NOD2 signaling by PP2 (C) or _Nod2_-, _Rip2_-, and _Tak1_-specific siRNAs (D). *, p < 0.05 versus control; **, p < 0.05 versus MDP treatment or MDP-treated nontargeted (NT) siRNA (one-way ANOVA). E and F, iNOS null and WT macrophages were treated with MDP or SIN1, and the activation status of SHH signaling was monitored at the transcript (E) and protein levels (F). WT, wild type; _iNOS_−/−, iNOS knock-out. G, BAY 11-7082, an IκB inhibitor, was used to monitor activation of SHH signaling upon activation of NOD2 pathway by MDP or with exogenous supply of NO by SIN1. *, p < 0.05 versus control; **, p < 0.05 versus WT MDP treatment (one-way ANOVA).
FIGURE 3.
NOD2 pathway modulates NUMB expression to activate SHH signaling. A and B, expression of NUMB in macrophages with MDP treatment at the indicated (A) time points and (B) concentrations was assessed using immunoblotting. Med, medium. C and D, macrophages were treated with various concentrations of MDP (C) or at different time points (D) as indicated. The change in mRNA levels of Numb was assayed by quantitative real time RT-PCR. E and F, _Nod2_-, _Rip2_-, and _Tak1_-specific siRNA-transfected RAW 264.7 macrophages (E) or PP2-pretreated macrophages (F) were analyzed for NUMB expression. NT, nontargeted siRNA. G, WT or iNOS null macrophages were treated with MDP or SIN1, and expression of NUMB upon MDP treatment was determined. WT, wild type; _iNOS_−/−, iNOS knock-out. H and I, pcDNA3 NUMB-transfected macrophages were assessed for activation of SHH signaling at transcript levels (H) as well as at protein levels and by nuclear translocation of GLI1 (I). *, p < 0.05 versus control; **, p < 0.05 versus pcDNA3 MDP treatment (one-way ANOVA).
FIGURE 4.
NOD2/NO axis regulates expression of miR-146a. A, miR-146a expression was determined in macrophages stimulated with MDP using quantitative real time RT-PCR. B, macrophages pretreated with RIP2 kinase inhibitor, PP2, were assayed for miR-146a expression after MDP treatment. *, p < 0.05 versus control; **, p < 0.05 versus MDP treatment (one-way ANOVA). Med, medium. C, iNOS null or WT macrophages were treated with MDP or SIN1, and expression levels of miR-146a were analyzed as described. WT, wild type; _iNOS_−/−, iNOS knock-out. D and E, miR-146a promoter-transfected cells were treated with the indicated concentrations of MDP (D) or with 1400W, iNOS activity inhibitor, prior to MDP stimulation (E), and miR-146a promoter luciferase activity was measured. Alternatively, cells were stimulated with SIN1 in panel E, and miR-146a promoter activity was assayed. *, p < 0.05 versus WT control; **, p < 0.05 versus WT MDP (one-way ANOVA). RLU, relative luciferase unit. F, WT miR-146a promoter luciferase construct or mutant miR-146a promoter luciferase constructs for binding sites of indicated transcription factors were used to assess MDP-induced miR-146a promoter reporter activity by luciferase assay (mean ± S.E., n = 3). *, p < 0.05 versus WT control; **, p < 0.05 versus WT MDP (one-way ANOVA).
FIGURE 5.
miR-146a modulates SHH signaling by targeting NUMB. A–C, pGVP2-NUMB 3′-UTR luciferase reporter construct or pGVP2-NUMB 3′-UTR reverse luciferase reporter construct (3′-UTR is in reverse orientation)-transfected macrophages were treated with MDP (A) or SIN1 (B) or co-transfected with miR-146a mimics (C) to analyze NUMB 3′-UTR luciferase activity (mean ± S.E., n = 3) (one-way ANOVA) or NUMB protein levels. Med, medium; RLU, relative luciferase unit. D and E, miR-146a mimic-transfected macrophages were assessed for activation of SHH signaling at protein (D) and transcript levels (E).
FIGURE 6.
miR-146a-mediated SHH signaling regulates inflammatory responses. A, pcDNA3 SHH-transfected macrophages were assessed for expression of proinflammatory genes, Il-12, _Tnf-_α, Il-6, Ccl5, and Cxcl9. B, macrophages pretreated with pharmacological inhibitors of SHH signaling, cyclopamine (inhibits SMO), and betulinic acid (inhibits GLI), were analyzed for the indicated genes after MDP stimulation. **, p < 0.05 versus control; *, p < 0.05 versus MDP (one-way ANOVA). Med, medium. C, expression of MDP-induced proinflammatory genes was analyzed in macrophages transfected with miR-146a inhibitor and Control inhibitor. *, p < 0.05 versus MDP + Control inhibitor (MDP+Control inh) (one-way ANOVA). D and E, miR-146a mimic-transfected macrophages were either treated with SHH signaling-specific pharmacological inhibitor such as cyclopamine or betulinic acid (D) or co-transfected with _Shh_- or _Gli1_-specific siRNA (E). Quantitative real time RT-PCR for analysis of inflammatory cytokines Il-12, _Tnf-_α, Il-6, Ccl5, and Cxcl9 was performed. *, p < 0.05 versus miR-146a mimic (one-way ANOVA). NT, nontargeted siRNA.
FIGURE 7.
Inflammatory responses during DSS-induced IBD immunopathology are controlled by iNOS/NO-miR-146a-SHH signaling. A–H, the expression levels of proinflammatory genes, Il-12, _Tnf-_α, Il-6, Ccl5, and Cxcl9 (A–D) and SHH signaling markers expression (E–H) were measured along ascending colon (AC), transverse colon (TC), descending colon (DC), and small intestine (SI) in WT and iNOS−/− animals (n = 6 each) using quantitative real time RT-PCR. *, p < 0.05 versus DSS-treated WT mice (Student's t test). I–L, quantitative real time RT-PCR analysis of miR-146a after DSS treatment of WT or iNOS null animals (n = 6) in ascending colon, transverse colon, descending colon, and small intestine is shown. *, p < 0.05 versus WT control; **, p < 0.05 versus WT DSS (one-way ANOVA). WT, wild type; iNOS−/−, iNOS knock-out.
FIGURE 8.
Molecular mechanism of DSS-induced colitis in murine model. NOD2-triggered iNOS/NO regulates miR-146a expression through transcription factors, NF-κB, PU.1, HSF2, and Oct-1. miR-146a thus produced targets Numb mRNA, leading to reduced expression of NUMB and activation of SHH signaling. Overall, NOD2/NO pathway establishes cross-talk with SHH pathway via miR-146a-mediated degradation of NUMB to control inflammatory responses.
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