NOD2-Nitric Oxide-responsive MicroRNA-146a Activates Sonic Hedgehog Signaling to Orchestrate Inflammatory Responses in Murine Model of Inflammatory Bowel Disease*

Background: Genetic variants of NOD2 are linked to inflammatory bowel disease (IBD) etiology. Results: DSS model of colitis in wild-type and inducible nitric-oxide synthase (iNOS) null mice revealed that NOD2-iNOS/NO-responsive microRNA-146a targets NUMB gene facilitating Sonic hedgehog (SHH) signaling. Conclusion: miR-146a-mediated NOD2-SHH signaling regulates gut inflammation. Significance: Identification of novel regulators of IBD provides new insights into pathophysiology and development of new therapy concepts. 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.

The nucleotide-binding oligomerization domain (NOD) 5 leucine-rich repeat containing receptor (NLR) protein family orchestrates intracellular surveillance to mediate innate immune responses and inflammation (1,2). Among the several NOD leucine-rich repeat-containing receptor proteins, NOD2 acts as an important innate sensor that detects cytosolic pathogen-associated molecular patterns as well as damage-associated molecular patterns. Muramyl dipeptide (MDP), a component of bacterial cell wall, is recognized by NOD2 to effectuate immune responses including the production of important cytokines such as IL-1␤, IL-6, TNF-␣, and IL-10 via NF-B (3). Interestingly, NOD2 plays a crucial role during several human disorders. Polymorphisms in NOD2 gene are shown to be associated with an assortment of inflammatory conditions such as IBDs (4,5). Thus, the functional attributes of NOD variants gain immense importance as critical regulators of inflammation.
IBDs are a group of chronic and relapsing inflammatory conditions of the intestinal lining that are characterized by abdominal pain, diarrhea, and severe rectal bleeding. IBDs are broadly categorized into two subtypes among which Crohn disease (CD) represents the most severe form of the pathogenesis (6,7). Notably, NOD2 is one of the first genes to be linked to IBD susceptibility, and more than 60 NOD2 genetic variants are reported to be associated with CD (8). NOD2 variants confer defective sensing of the agonist, MDP, and NF-B activation by either not executing the role to restrain inflammation (loss of function) or directly activating expression of several proinflammatory genes (gain of function) (9). Consistent with these observations, monocytes obtained from CD patients (3020insC NOD2 frameshift mutant) show decreased ability to produce inflammatory cytokines such as IL-1␤, IL-6, TNF-␣, and IL-10, suggesting that 3020insC CD-associated mutation results in loss of function. However, other studies have reported that gain of function variant-associated CD macrophages or epithelial cells show elevated NF-B activation and production of IL-1␤, IL-6, and TNF-␣ (10). Further, patients with CD respond paradoxically to anti-TNF-␣ treatments, presenting an interesting conundrum about the involvement of NOD2 variants in the pathogenesis of CD (9). Despite the wealth of information available regarding the identities of NOD2 variants, the precise role and dynamics of NOD2 signaling and its contribution in initiating and controlling inflammation remain unclear.
Intriguingly, the pathology of IBDs is a result of the complex association between genetic and environmental factors that lead to exaggerated inflammatory immune responses and the destruction of intestinal mucosa (7). The knowledge of cellular programming in initiation, development, and manifestation of IBDs is insufficiently understood. However, several evidences implicate a vital role for deregulated host responses to gut microbiota during IBDs. This evidence includes excessive superoxides and NO produced by immune cells such as macrophages that culminate in defective intestinal mucosal barrier functions (11). Although NO is an important secondary messenger that modulates normal cellular functions and gut homeostasis, the sustained production of NO results in detrimental effects (12). Further, inducible nitric-oxide synthase (iNOS)induced NO has been implicated to play a vital role in arbitrating the inflammatory response during colitis (13). Nevertheless, the studies on the molecular mechanism involved in NO-mediated immune homeostasis during IBD are scanty. In this regard, recent investigations have underscored the ability of NO to regulate key signaling networks including Sonic hedgehog (SHH) pathways (14). Remarkably, SHH signaling is associated with processes such as gut development and maintenance of gut homeostasis (15,16). Additionally, inflammatory environments often trigger increased expression of SHH ligands (17,18). In this perspective, the current study attempts to delineate the role for SHH signaling during the onset or establishment and the perpetuation of IBDs.
Here, we identified a novel cross-talk between NOD2 and SHH signaling pathways in macrophages. Notably, our data delineate a prominent role of miR-146a/SHH signaling in amplifying inflammatory responses and provide a mechanistic understanding of gain-of-function variants of NOD2 in the pathogenesis of human IBDs.

EXPERIMENTAL PROCEDURES
Cells and Mice-Primary macrophages were isolated from peritoneal exudates of C57BL/6 wild-type (WT) and iNOS Ϫ/Ϫ mice. Brewer thioglycollate (8%) was used to enrich macrophages in the peritoneal cavity of mice. The cells were cultured in DMEM (Life Technologies) containing 10% FBS (Sigma-Aldrich) for 6 -8 h, and adherent cells were used as peritoneal macrophages. The purity of these macrophages was confirmed by F4/80 staining using FACS and was found to be Ͼ95%. All transfection studies were carried out with murine RAW 264.7 macrophage-like cells. All studies involving mice were carried out after the approval from the Indian Institute of Science, Institutional Ethics Committee for Animal Experimentation as well as from the Institutional Biosafety Committee.
Quantification of miRNA Expression-For detection of miR-146a by quantitative real time RT-PCR, total RNA was isolated from treated or untreated macrophages. Quantitative real time RT-PCR for miR-146a was done using TaqMan miRNA assays (Applied Biosystems-Invitrogen) as per the manufacturer's instructions. U6 snRNA was used for normalization.

MicroRNA-146a Regulates SHH Signaling and Inflammation
difluoride membranes (Millipore, Billerica, MA) by the semidry transfer (Bio-Rad) method. The blots were blocked with 5% nonfat dry milk powder in TBST (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20) for 60 min to remove nonspecific binding. The blots were incubated overnight at 4°C with primary antibody followed by incubation with goat anti-rabbit-HRP or anti-mouse-HRP secondary antibody in 5% BSA for 2 h. The immunoblots were developed with enhanced chemiluminescence detection system (PerkinElmer Life Sciences) as per the manufacturer's instructions. All immunoblots are representatives of at least three independent experiments.
Nuclear and Cytosolic Subcellular Fractionation-Macrophages were harvested and gently resuspended in Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After incubation on ice for 15 min, cell membranes were disrupted with 10% Nonidet P-40. The cytosolic extract was separated by centrifugation at 13,000 rpm for 15 min at 4°C. The pellet was lysed with Buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF), and the nuclear protein extract was collected. The nuclear and cytosolic fractions were resolved on denaturing polyacrylamide gel, and further processing was done as mentioned as described under "Immunoblotting." In Vivo Studies In Mice Using Murine DSS Model of Colitis-The murine colitis model of intestinal inflammation was established using low molecular weight dextran sodium sulfate (DSS) as described below. WT and iNOS Ϫ/Ϫ mice were divided into two groups containing six mice each. The test group was administered drinking water supplemented with low molecular weight DSS solution (2.5%), whereas the control group of mice was fed with autoclaved water for 9 days. Mice were carefully monitored every day for clinical symptoms such as weight loss, bloody stools, and diarrhea. After 7, 8, or 9 days of DSS treatment, the clinical symptoms of IBD were scored in WT and their iNOS Ϫ/Ϫ littermates. The clinical scores were given as 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 Ͻ 0.05 versus control; **, p Ͻ 0.05 versus MDP treatment (one-way ANOVA). NOVEMBER 15, 2013 • VOLUME 288 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33039 follows: 0 ϭ no symptoms; 1 ϭ diarrhea; 2 ϭ rectal bleeding; and 4 ϭ death. At the end of DSS treatment, mice were euthanized, and colons and small intestines were dissected. The total length of colon in both groups of WT and iNOS Ϫ/Ϫ mice was measured, and colon was divided into three parts as ascending colon, transverse colon, and descending colon. Each of these samples was processed for total RNA isolation.

MicroRNA-146a Regulates SHH Signaling and Inflammation
Transfection Studies-RAW 264.7 macrophage cells were transfected with 100 nM siRNA or miRNA mimic using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Transfection efficiency was found to be more than 50% in all the experiments as determined by counting the number of siGLO lamin A/C-positive cells in a microscopic field using a fluorescent microscope. 48 h after transfection, respective experiments were performed as indicated. Nod2, Rip2, Tak1, Shh, Gli1, and control siRNAs were obtained from Dharmacon (Waltham, MA) as siGENOME TM SMARTpool reagents, which contain a pool of four different double-stranded RNA oligonucleotides. miR-146a mimic, miR-146a inhibitor (Anti-miR TM ), and control mimics were purchased from Ambion-Invitrogen. RAW 264.7 macrophages were transiently transfected with the following constructs using low molecular weight polyethylenimine (PEI) (Sigma-Aldrich): pcDNA3 vector, pcDNA3 SHH vector, pcDNA3 NUMB vector, pGVP2-NUMB 3Ј-UTR luciferase reporter construct, pGVP2-NUMB 3Ј-UTR reverse luciferase reporter construct, WT miR-146a promoter luciferase construct, and mutant miR-146a promoter luciferase for binding sites of transcription factors NF-B, c-ETS, PU.1, c-Myc, HSF2, and Oct-1 constructs. 48 h after transfection, the cells were treated as described and processed as required.
Luciferase Assays-RAW 264.7 macrophages were transfected with NUMB 3Ј-UTR construct, NUMB 3Ј-UTR reverse construct, along with ␤-galactosidase vector and indicated miRNA mimics or treatments. After 48 h of transfection, cells were lysed in reporter lysis buffer (Promega, Madison, WI) and assayed for luciferase activity using luciferase assay reagent. The results were normalized for transfection efficiencies by assay of ␤-galactosidase activity. 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 Ϯ S.E., n ϭ 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 Ͻ 0.05 versus control; **, p Ͻ 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 Ϫ/Ϫ , iNOS knock-out. G, BAY 11-7082, an IB 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 Ͻ 0.05 versus control; **, p Ͻ 0.05 versus WT MDP treatment (one-way ANOVA).
Statistical Analysis-Levels of significance for comparison between samples were determined by the Student's t test distribution and one-way ANOVA. The data in the graphs are expressed as the mean Ϯ S.E., and p values Ͻ 0.05 were defined as significant. GraphPad Prism 3.0 software (GraphPad software, San Diego, CA) was used for all the statistical analyses.

iNOS/NO Mediates NOD2-SHH Signaling Cross-talk-NOD2
signaling is a central cytosolic surveillance pathway as well as a crucial regulator of inflammation (2). However, NOD2-mediated molecular regulators of inflammatory responses have not been clearly identified and studied. In this regard, recent studies have indicated a strong role for SHH signaling during inflammation (19). We thus analyzed the potential role of SHH signaling as a NOD2-responsive pathway. The SHH signaling activation is marked by induced expression of SHH, glioma-associated oncogene family zinc finger (GLI)1, GLI2, smoothened (SMO), Patched 1 (PTCH1), and nuclear translocation of transcriptional activator GLI1 (20). Treatment of macrophages by MDP, a NOD2 agonist, showed robust induction as well as activation of SHH signaling with increase in NOD2 signaling activation (Fig. 1, A-D). To ascertain the involvement of NOD2-arbitrated RIP2-TAK1 signaling in controlling SHH pathway, the expression levels of SHH signaling molecules were assayed in the presence of the RIP2-specific inhibitor, PP2. The inactivation of RIP2 kinase activity by PP2 showed significant reduction in MDP-triggered SHH signaling (Fig. 1, E and F). The utilization of Nod2-, Rip2-, and Tak1-specific siRNAs validated the involvement of NOD2 signaling in regulating the activation of SHH signaling (Fig. 1G). Together, these observations strongly suggest that NOD2 signaling holds the capacity to control activation of SHH signaling.
Recent studies have demonstrated that NOD2 signaling activation in macrophages increases the expression of TNF-␣, IL-6, COX-2, and iNOS (21,22). Further, NO initiates and mediates cross-talks with other signaling pathways including NOTCH and WNT signaling (21,23). Thus, we hypothesized that NOD2-induced iNOS expression and sustained production of NO could be vital to arbitrate cross-talks with SHH signaling. From this perspective, kinetics analysis of iNOS expression in MDPtreated macrophages showed an induced expression of iNOS as  NOVEMBER 15, 2013 • VOLUME 288 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33041 well as NO production (Fig. 2, A and B). The loss of NOD2 signaling functions by perturbations mediated by pharmacological inhibitor of RIP2 or specific siRNAs to Nod2, Rip2, and Tak1 confirmed NOD2 signaling-dependent iNOS expression and NO production (Fig. 2, C and D and data not shown). This affirms NO as a mediator of NOD2 signaling. We further evaluated the role of NOD2-responsive NO to activate SHH signaling. Interestingly, iNOS null macrophages showed compromised ability to trigger NOD2-induced activation of SHH signaling as compared with WT macrophages (Fig. 2, E and F). The inadequacy of iNOS null macrophages to activate SHH signaling was not a result of generalized defect as the addition of an NO donor, SIN1, showed augmented activation of SHH signaling in iNOS null macrophages, which is comparable with that of WT macrophages (Fig. 2F and data not shown).

MicroRNA-146a Regulates SHH Signaling and Inflammation
NOD2 receptors have been known to utilize NF-B to regulate expression of downstream genes (24). To dissect the role of NF-B during NOD2-iNOS/NO-mediated activation of SHH signaling, macrophages were treated with MDP or SIN1 in the presence or absence of a NF-B inhibitor, BAY 11-7082 (which prevents IB-␣ phosphorylation). As represented in Fig. 2G, inhibition of NF-B activity showed a significant decrease in SHH signaling activation. As a proof of concept, we monitored NF-B translocation from cytosol to nucleus with MDP or SIN1 treatment of macrophages (data not shown). These data conclusively suggest NO as a potential link for NOD2-SHH signaling cross-talk.
NOD2 Attenuates NUMB Gene Activity-The binding of SHH ligand to PTCH1 receptors derepresses PTCH1-mediated suppression of SMO that culminates into destabilization of inhibitory complex and nuclear translocation of GLI1 to the promoters of SHH-responsive genes (20). However, checks and balances in terms of activation of SHH signaling are carefully regulated by the NUMB-mediated negative regulatory loop. 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 Ͻ 0.05 versus control; **, p Ͻ 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 Ϫ/Ϫ , 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 Ͻ 0.05 versus WT control; **, p Ͻ 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 Ϯ S.E., n ϭ 3). *, p Ͻ 0.05 versus WT control; **, p Ͻ 0.05 versus WT MDP (one-way ANOVA).

MicroRNA-146a Regulates SHH Signaling and Inflammation
Recent studies have implicated NUMB in GLI1 degradation through ITCH (Itchy E3 ubiquitin protein ligase)-dependent ubiquitination, sequestering active forms of GLI1 transcription factors and suppression of GLI transcriptional activity (25,26). In this perspective, we analyzed the NOD2-mediated regulation of NUMB gene expression. The dosage dependence and time kinetics analysis demonstrated the progressive decrease in the NUMB protein levels with increase in NOD2-MDP engagement (Fig. 3, A and B). However, there was no significant change observed at transcript levels of NUMB (Fig. 3, C and D). Further, NOD2 signaling interference by Nod2-, Rip2-, or Tak1-specific siRNAs or PP2 led to the significant alleviation of MDP-induced decrease in NUMB levels (Fig. 3, E and F). In agreement with the previous results, the exogenous supply of NO by SIN1 showed a decrease in NUMB protein levels, whereas iNOS null macrophages failed to exhibit such effects (Fig. 3G). Gain-of-function studies were undertaken to further validate the role of NUMB in regulating NOD2-driven SHH signaling. The activation of SHH signaling in response to MDP was severely compromised in the NUMB protein overexpressing macrophages as compared with that of control cells (Fig. 3, H and I) as analyzed by expression of SHH, GLI1, GLI2, SMO, PATCH1, and nuclear translocation of transcriptional activator GLI1. Altogether, these results summarize and confirm that NOD2-iNOS/NO-triggered SHH signaling activation is mediated through down-regulation of NUMB protein.
NOD2-induced miR-146a Potentiates SHH Signaling Activation by Targeting NUMB Gene-The down-regulation of NUMB upon NOD2 signaling at the protein levels but not at the transcript levels implicated the possible intervention of post-transcriptional regulations mediated by microRNAs. In this regard, NUMB has been identified as a potential target of miR-146a (27). Besides, miR-146a is one of the first identified inflammatory miRNAs whose expression was found to be elevated during various inflammatory conditions (28,29). Further, extensive bioinformatic algorithms validated that miR-146a targets an 8-mer site located at residues spanning 2899 -2906 in mouse NUMB 3Ј-UTR. In this perspective, we analyzed the contribution of miR-146a, if any, in modulating NOD2/NO-mediated SHH signaling. As illustrated in Fig. 4A, miR-146a levels increased gradually in macrophages with increasing doses of MDP. NOD2 signaling interference with PP2 showed significant reduction in MDP- FIGURE 5. miR-146a modulates SHH signaling by targeting NUMB. A-C, pGVP2-NUMB 3Ј-UTR luciferase reporter construct or pGVP2-NUMB 3Ј-UTR reverse luciferase reporter construct (3Ј-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 3Ј-UTR luciferase activity (mean Ϯ S.E., n ϭ 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). NOVEMBER 15, 2013 • VOLUME 288 • NUMBER 46 induced expression of miR-146a (Fig. 4B). Further, iNOS null macrophages showed compromised ability to induce miR-146a expression. Concordantly, SIN1-induced expression of miR-146a was comparable in both iNOS null and WT macrophages (Fig. 4C). The miR-146a promoter luciferase reporter assays validated the crucial role of NOD2-iNOS signaling in the expression of miR-146a. (Fig. 4, D and E). Promoter luciferase analysis with WT and mutant miR-146a promoters revealed NF-B along with HSF2, PU.1, Oct1, c-ETS, and c-Myc as vital transcription factors that orchestrate NOD2-mediated expression of miR-146a (Fig. 4F).

MicroRNA-146a Regulates SHH Signaling and Inflammation
To ascertain the miR-146a-NUMB interactions, we utilized WT NUMB 3Ј-UTR and NUMB 3Ј-UTR reverse (luciferase construct that harbors NUMB 3Ј-UTR in reverse direction). As represented in Fig. 5, A and B, MDP or SIN1 treatment considerably reduced WT NUMB 3Ј-UTR luciferase activity, but not NUMB 3Ј-UTR reverse luciferase activity. Importantly, enforced miR-146a expression through miR-146a mimics markedly reduced NUMB 3Ј-UTR luciferase activity as well as NUMB protein levels, validating NUMB as a direct target of miR-146a (Fig. 5C). The positive regulation of SHH signaling by miR-146a was confirmed by enforced expression of miR-146a in macrophages, which triggered activation of SHH signaling (Fig. 5, D and E).
iNOS/NO-mediated SHH Signaling Is Necessary for IBD Immunopathology in Vivo-To establish the in vivo significance of NOD2-iNOS/NO-miR-146a-SHH signaling-induced inflammatory responses, we utilized the well established DSSinduced colitis murine model of mucosal inflammation. The DSS model mimics human IBD with respect to its etiology, pathogenesis, and therapeutic response (30,31).
iNOS and its product NO are key regulators of acute and chronic phases of inflammatory responses during IBDs (13). The use of iNOS/NO inhibitors has been shown to be effective in reducing inflammation during IBD (32). In agreement with these previous studies (13,32), we observed that DSS-administered WT mice showed significant decrease in body weight and survival as well as colon length with DSS treatment as compared with results in iNOS null mice (data not shown). Because IBDs are characterized by elevated proinflammatory cytokines that play key roles in orchestrating remittance and exacerbation of inflammatory responses, we analyzed the cytokine milieu or the inflammatory signatures of DSS-mediated inflamed colons. As shown in Fig. 7, A-D, WT mice exhibited elevated levels of Il-12, Tnf-␣, Il-6, Ccl5, and Cxcl9. On the contrary, iNOS null mice failed to effectuate inflammatory responses on DSS treatment, suggesting the essential requirement of iNOS/NO to mediate inflammatory responses in IBDs.
Recent studies have implicated a crucial role of SHH signaling in intestinal inflammation (19). As represented in Fig. 2, E-G, SHH signaling activation is dependent on iNOS activity. Thus, we explored the possible role for iNOS/NO in activation of SHH signaling in the current in vivo model for IBDs. In accordance with the above observations (Fig. 7, A-D), DSSadministered WT mice showed significant increase in SHH signaling activation. However, iNOS null mice did not exhibit  's t test). I-L, quantitative real time RT-PCR analysis of miR-146a after DSS treatment of WT or iNOS null animals (n ϭ 6) in ascending colon, transverse colon, descending colon, and small intestine is shown. *, p Ͻ 0.05 versus WT control; **, p Ͻ 0.05 versus WT DSS (one-way ANOVA). WT, wild type; iNOS Ϫ/Ϫ , iNOS knock-out. NOVEMBER 15, 2013 • VOLUME 288 • NUMBER 46 SHH activation even with DSS administration (Fig. 7, E-H). Corroborating these results, DSS-administered iNOS null mice failed to induce miR-146a expression with DSS administration along the ascending colon, transverse colon, descending colon, and small intestine as compared with results in WT mice (Fig. 7, I-L). Together, our observations suggest that iNOS/NO-miR-146a-mediated SHH signaling activation presents a new mechanism for modulations of inflammatory diseases such as IBD.

DISCUSSION
NOD2 is a cytoplasmic surveillance sensor that governs homeostasis through regulation of immune responses, inflammation, and apoptosis (33)(34)(35). Several studies implicate NOD2 polymorphism in CD, the major form of IBDs. Strikingly, these variants of NOD2 have been documented not only in CD patients but also in healthy individuals, underscoring the presence of predisposition for development of CD (36). Although surplus NOD2 signaling during certain kinds of CD is evidenced, the role of the NOD2 downstream signaling network under such conditions is imprecisely understood. The deeper insight into the NOD2-activated molecular signaling is crucial for understanding NOD2-driven inflammation and designing target-specific pharmacological agents to treat inflammation. In this perspective, our current investigation identifies NO-triggered miR-146a as a novel downstream messenger of NOD2 signaling in modulating SHH signaling and inflammatory responses.
NO often acts as a key signaling molecule that defines critical rate-limiting steps for pathogenesis of chronic or acute inflammation across varying cellular contexts (37). Significantly, excessive production of NO in response to a variety of patho-logic or homeostatic stimuli leads to modulation of several cellular processes (38 -40). Although NO is implicated in IBD responses, its role in inflammatory modulation remains inadequately understood. On the other hand, SHH signaling is a fundamental pathway that directs gut development and maintains gut homeostasis through epithelial signaling (16). Studies have also implicated elevated SHH signaling during inflammatory diseases (19,41). In accordance with these observations, we explored the possible link between NO and SHH signaling. NOD2-responsive NO was found to induce SHH signaling in macrophages. Importantly, the in vivo DSS model revealed a possible NOD2 gain-of-function scenario of IBD as excessive NO-dependent SHH responses were observed in mice.
Unrestrained SHH signaling during inflammatory disorders recurrently entails the high risk of neoplastic transformations (42). In this context, the system of checks and balances that diligently titers the SHH signaling by several negative regulators such as NUMB assumes central importance. Corroborating our previous results, we found diminished levels of NUMB protein due to post-transcriptional regulation by miR-146a, which in itself is a consequence of NOD2/NO signaling. Supporting this, characterized inflammatory miRNAs such as miR-146a are implicated in various inflammatory diseases such as IBD, systemic lupus erythematosus, Sjögren syndrome, and rheumatoid arthritis (43). Furthermore, miR-146a expression correlates with iNOS expression as documented in one study (29). Interestingly, NO is known to mediate regulatory effects by modulating the activity of several miRNAs, suggesting a critical contribution of reactive nitrogen intermediates in modulating post-transcriptional modifications (44,45). In summary, our work demonstrates that NOD2-driven inflammation is critically regulated by NO-responsive miR-146a that facilitates activation of SHH signaling by targeting NUMB expression. These molecular events contribute to the expression of inflammatory genes such as IL-12, TNF-␣, IL-6, CCL-5, and CXCL-9 (Fig. 8). The in vivo DSS-induced colitis murine model presented unique roles for NO-dependent miR-146a and SHH signaling during IBD especially similar to that exemplified by the NOD2 gain-of-function variant. We believe that various novel molecular regulators identified in the current study would thus serve as potential therapeutic targets.