Inhibition of RICK/Nuclear Factor-κB and p38 Signaling Attenuates the Inflammatory Response in a Murine Model of Crohn Disease*

Nuclear factor-κB (NF-κB) is the main target of anti-inflammatory therapies in human chronic inflammatory bowel diseases (IBD), Crohn disease, and ulcerative colitis. This study investigates the molecular anti-inflammatory mechanisms of SB203580, an inhibitor of the mitogen-activated protein kinase p38. The murine trinitrobenzene sulfonic acid (TNBS)-induced colitis was used as an established model of human Crohn disease. Here we show that SB203580 improved the clinical condition, reduced intestinal inflammation, and suppressed mRNA levels of pro-inflammatory cytokines elevated upon induction of colitis. Besides p38 kinase activity, the “classical” IκB-dependent NF-κB pathway was strongly up-regulated during colitis induction, whereas the “alternative” was not. SB203580 treatment resulted in a drastic down-regulation of p38 and NF-κB activity. The molecular analysis of NF-κB activation revealed that Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK), a key component of a pathway leading to NF-κB induction, is also strongly inhibited by SB203580. In contrast, SB203580 had no effect on the colitis-induced activation of other potential NF-κB-activating kinases such as protein kinase Cθ (PKCθ), mixed lineage kinase 3, and the oncogene product Cot/TPL2. Thus, the inhibitory effect of SB203580 on NF-κB activation is to a large extent mediated by RICK inhibition. RICK is the effector kinase of the intracellular receptor of bacterial peptidoglycan NOD. Because bacterial products are suggested to be the key pathogenic agents triggering IBD, inhibition of the NOD/RICK pathway may serve as a novel target of future therapies in human IBD.

Crohn disease is characterized by chronic intestinal inflammation frequently relapsing with clinical manifestations including diarrhea, blood in the stool, abdominal pain, and weight loss. The etiology of Crohn disease is so far unknown (1) but epidemiological and linkage studies suggest a genetic predisposition of the patient and the involvement of environmen-tal factors (1,2). Furthermore, several observations strongly implicate a pathogenic role of the intestinal flora during the initiation process of the immunological dysregulation (3)(4)(5). At present, treatment of Crohn disease is limited to 5-aminosalicylates, corticosteroids, and immunosuppressants such as azathioprine or 6-mercaptopurine (1). Because of the lack of specific and curative treatments with limited toxicity or side effects, there is still a great demand for developing effective therapeutic approaches.
NF-B 1 is one of the most important transcription factors for the induction of genes mediating innate and adaptive immunity (6), and is also the key transcription factor for pro-inflammatory responses in inflammatory bowel disease (IBD) (7). The NF-B system consists of homo-or heterodimers that are retained in the cytoplasm upon association with an inhibitory IB protein (8). In the "classical" pathway of NF-B activation, the IB kinase complex (IKK) is activated and leads to phosphorylation and polyubiquitination of IB thereby marking this NF-B inhibitor for degradation in the 26 S proteasome. Protein kinase C (PKC) and the MAP3K Cot/TPL2 and mixed lineage kinase 3 (MLK3) are important activators of the IKK complex and therefore of NF-B (9 -11). The recently described "alternative" activation pathway of NF-B involves the processing of p100 to p52 (12)(13)(14).
One pathway critical for IBD and activation of NF-B is dependent on NOD proteins (15,16). NOD proteins are intracellular receptors for bacterial peptidoglycan and play a pivotal role in the innate immune system (17). Therefore, it is reasonable to suggest that NOD-mediated signal transduction pathways may serve as an initiation point for a variety of inflammatory responses in bacterial triggered "autoimmune" diseases. In fact, genome-wide screens have identified NOD2/caspase-activation recruitment domains (CARD15) as the first susceptibility gene for Crohn disease (15,16). The pathogenic role of mutations within the NOD2 gene in patients with Crohn disease is supported by functional studies showing an inappropriate response to bacterial components with respect to NF-B activation within the innate immune system (16). Those NOD2 mutations (i.e. the 3020InsC mutant) lead to a truncated NOD2 protein because of a frameshift. This results in the inability to recognize bacterial lipopolysaccharide (LPS), an abolished NF-B activation (16), and finally in a persistent intestinal inflammation because of the strongly reduced TNF-␣-induced NOD2 gene transcription (2,18). Furthermore, a direct link has been shown between NOD signaling and NF-B activation (19). Recently, DLG5 as a scaffolding protein involved in the maintenance of epithelial integrity has been mapped as another candidate susceptibility gene for Crohn disease. This function of DLG5 may be another indication for the role of intestinal flora in IBD, because a shredded epithelial bowel layer is a potential prerequisite for bacterial triggered disease induction (20).
The mitogen-activated protein kinase (MAPK) p38 is another crucial mediator of inflammation (21)(22)(23)(24). p38 directly controls the activity of several transcription factors relevant to the production of pro-inflammatory cytokines, which are up-regulated in IBD. Furthermore, p38 can modulate a number of different steps in the inflammatory cascade including the transcriptional regulation of the genes encoding the key cytokines of IBD TNF-␣, IL-1, and interferon ␥ in monocytes and lymphocytes, the degranulation of neutrophils, and the expression of receptors essential for neutrophil activation and chemotaxis like the CXC receptors (25)(26)(27). The expression of this receptor is strongly inhibited by p38 MAPK inhibitors (28). Additionally, upstream kinases of p38 like the MAPK kinase MKK3 as well as downstream substrates such as the activator of transcription factor 2 (ATF-2) or MAPK-activated protein kinase 2 are by itself potent regulators of genes for Th1-derived cytokines like TNF-␣, which are all up-regulated in Crohn disease (26, 27, 29 -31). In a murine knock-out model, elimination of MAPK-activated protein kinase 2 led to a dramatic reduction of TNF-␣ in response to lipopolysaccharide (32).
Because the p38 gene as well as other candidate genes has been localized to the IBD susceptibility region 3, much attention has been focused on p38 as a key kinase maintaining intestinal inflammation in IBD (21,33,34). p38 displays the strongest activity compared with other MAPK within the inflamed intestinal mucosa of IBD patients (21,35,36). A recent pilot study investigating the efficiency of the combined p38 and JNK inhibitor CNI-1493 in patients with Crohn disease demonstrated a significant clinical improvement in moderate to severe active disease (35).
In this study, we investigated the anti-inflammatory effects of the p38 MAPK inhibitor SB203580 on signal transduction pathways involving NF-B and p38 in a murine model of Crohn disease. We show that SB203580 has highly beneficial antiinflammatory effects resulting in a significant suppression of the transcription of pro-inflammatory cytokines as well as the induction of p38 and the classical but not the alternative NF-B activation pathway. The inhibition of NF-B by SB203580 is probably mediated by the blocked NOD/RICK pathway. Thus, dual inhibition of p38 and NOD/RICK may lead to new treatment options with very limited side effects in chronic inflammatory conditions like human IBD.

EXPERIMENTAL PROCEDURES
Animals and Experimental Design-Female BALB/c mice (6 -8 weeks old) ϳ19 -22 g in body weight were obtained from Harlan-Winkelmann (Borchen, Germany) and given ad libitum access to water and standard rodent food before and during the study. Mice were weighted and randomized into groups of 7. Clinical assessments and histological scoring of colitis were performed in a blinded fashion. Experiments were performed after Ն3 days following arrival of the animals. To induce colitis, 0.1 ml of a 2.5% (w/v) TNBS solution in 50% ethanol (Sigma) was slowly administered into the colon of lightly anesthetized mice via a thin catheter (PE-50, BD Biosciences, Heidelberg, Germany) attached to a 0.5-ml syringe (37). The catheter tip was inserted 5-6 cm proximal to the anal verge, and the mouse was held in a vertical position for 1 min after instillation. Using this procedure, Ͼ95% of the mice retained the TNBS-ethanol enema. However, if an animal quickly (i.e. Ͻ10 min) excluded this solution, it was omitted from the remainder of the study. Treatment with 5 mol of SB203508/kg of body weight in 200 l of 0.9% NaCl solution was given intraperitoneally twice daily beginning 12 h after TNBS administration and continuing until death. For control experiments, bowel tissue was analyzed from: 1) completely untreated animals, 2) animals receiving vehicle injections (3% Me 2 SO) without rectal challenge, or 3) mice receiving SB203580 (Calbiochem, Schwalbach, Germany) injections in the same manner but treated with 0.9% saline intracolonically instead of TNBS.
Macroscopic Assessment and Histological Analysis of Colitis-Daily weight, physical condition, stool consistency, and water/food consumption were determined. Animals were sacrificed by CO 2 inhalation at days 1, 2, 3, 5, 7, and 10 (n ϭ 7 in each group per day). After sacrifice, the colon was quickly removed, opened longitudinally, and gently cleared of stool. Macroscopic assessment of the disease activity was scored according to a previously established scoring system (38) as follows: 0: no ulcer, no inflammation; 1: no ulcer, local hyperemia; 2: ulceration without hyperemia; 3: ulceration and inflammation at one site only; 4: two or more sites of ulceration and inflammation; and 5: ulceration extending more than 2 cm. Subsequently, samples of colonic tissue were either fixed in 4% buffered formalin, embedded in paraffin, and 4-m thick serial step sections were stained with hematoxylineosin (HE) or snap-frozen in liquid nitrogen in OCT embedding medium and stored at Ϫ80°C until usage. Microscopic scoring was performed according to Elson et al. (39).
Blood Sampling-Blood (ϳ0.4 ml) was drawn intracardially and mixed with 50 l of 0.5 M EDTA. Blood samples were subjected to differential blood cell count analysis.
Extraction of Total RNA-A gut specimen obtained 5 cm proximal to the anus was used for extraction of total RNA using a recently described method (40). For each animal, 1 g of total RNA was transcribed into cDNA. Quantitative real-time reverse transcriptase-PCR was performed in an iCycler (Bio-Rad). The 30-l reaction mixture consisted of 15 l of HotStarTaq TM Master Mix, 1.2 l of the reverse transcriptase reaction, 0.3 l of SYBR Green I (1:10000; Molecular Probes, Eugene, OR), and 0.5 M of the specific primers (TIB Molbiol, Berlin, Germany). Initial denaturation and activation of Taq polymerase at 95°C for 15 min was followed by 40 cycles consisting of (i) denaturation at 94°C for 30 s, (ii) annealing at 60°C for 45 s for ␤-actin, TNF-␣, interferon ␥, IL-2, IL-12p35, IL-18, and at 55°C for IL-10, and (iii) elongation at 72°C for 30 s. The fluorescence intensity of the double strand-specific SYBR Green I, reflecting the amount of actually formed PCR product, was read real-time at the end of each elongation step. Specific initial template mRNA amounts were calculated by determining the time point of a standard curve (artificial units). ␤-Actin mRNA amounts were used to normalize the cDNA contents of the different samples. In addition, an aliquot of the PCR mixture was separated on a 1.8% agarose gel and stained with ethidium bromide.
For cellular subfractionation of proteins, tissue was carefully homogenized so that many viable single cells were obtained and re-suspended in 1 ml of ice-cold TBS buffer, centrifuged (10 min, 800 ϫ g at 4°C), and washed in buffer A (10 mM HEPES, pH 7.9, 10% glycerol, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 10 mM NaF, protease inhibitor mix-ture). After centrifugation, the pellet was resuspended in 0.2 ml of buffer A and 2 l of 12.5% (v/v) Nonidet P-40 was added, briefly incubated on ice, and centrifuged (10 min, 800 ϫ g at 4°C). The supernatant was used for cytoplasmic analyses. The pellet was washed again in buffer A. After centrifugation, pellets were resuspended in 40 l of buffer C (20 mM HEPES, pH 7.9, 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 10 mM NaF, protease inhibitor mixture) and incubated on a shaker for 1 h at 4°C. Subsequently, samples were centrifuged (10 min, 13,000 ϫ g at 4°C), the supernatant (nuclear protein extract) was transferred to liquid nitrogen, and stored at Ϫ80°C until usage. Protein concentration was measured as previously described prior to storage.
Immunofluorescence Microscopy-To identify the cellular source of activated p38, immunohistochemical studies were performed using gut specimens obtained 3-4 cm proximal to the anus. Cryosections were fixed in acetone for immunofluorescence microscopy. After washing in phosphate-buffered saline, nonspecific binding was blocked with 1% bovine serum albumin for 1 h prior to the addition of 10 g/ml phospho-p38 antibody, and incubated for 1 h at room temperature in a humidified chamber. After repeated washings in phosphate-buffered saline, the sections were incubated with a fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody for 1 h. The sections were washed again in phosphate-buffered saline and mounted in Vectashield media (Vector Labs, Burlingame, CA).
In Vitro Kinase Assays-RICK and p38 activity were assayed in an in vitro kinase activity using 0.2 mg of protein lysate and incubated with anti-RICK or anti-p38 (1 g/sample) by permanent shaking for 3 h at 4°C. Immune complexes were recovered with 50 l of a 50% protein A-Sepharose bead suspension (Amersham Biosciences) and incubated for 90 min at 4°C in a permanent shaker, respectively. Subsequently, immunoprecipitates were washed six times with lysis buffer and incubated with 1.5 g of myelin basic protein (MBP) for the RICK assay or 1.5 g of ATF-2 for the p38 assay in 20 l of kinase buffer (20 mM HEPES, pH 7.6, 2 mM EGTA, 20 mM MgCl 2 , 1 mM Na 3 VO 4 , 1 mM dithiothreitol, 0,1% Triton X-100, 0.25 mM ATP) for 30 min at 30°C. Samples were suspended in loading buffer and subjected to SDS-PAGE. ATF-2 and phospho-ATF-2 antibody were obtained from Cell Signaling (Frankfurt, Germany). Dephosphorylated MBP and phospho-MBP antibody were purchased from Upstate/Biomol. To prove a direct suppression of RICK activity by SB203580, the cell extract was incubated with SB203580 30 min prior to the in vitro kinase assay.
Statistical Analysis-The data are expressed as mean Ϯ S.E. Significance of differences between treatment groups and controls were assessed by analysis of variance and a Bonferroni-Dunn procedure as a post-hoc test. Statistical significance of the difference between drugtreated and vehicle-treated groups was established at a probability of p Ͻ 0.05.

SB203580 Represses TNBS-induced Colitis in Mice-Previ-
ous studies showed that rectal administration of TNBS induces colitis resembling human IBD (37,42,43) and this well established murine model is commonly used to screen pharmacological agents (37, 44 -46). Additionally, recently initiated clinical trials using MAPK inhibitors like SB203580 were very successful in the treatment of patients with Crohn disease refractory to standard medication (35). Based on these studies, we explored the possibility that administration of SB203580 can reduce TNBS-induced intestinal inflammation of the murine colon. We found that BALB/c mice subjected to intrarectal TNBS administration reproducibly developed colitis with diarrhea accompanied by severe weight loss, multiple mucosal erosions, and ulcerative colitis with a maximum at 2-3 days following TNBS administration (Fig. 1A). In the treatment groups, SB203580 was administered to mice 12 h after TNBS/ ethanol enema when the first signs of colitis were notable. The peak of clinical disease occurred always between days 2 and 3 but the recovery from colitis was markedly enhanced by SB203580 treatment as reflected by the clinical data and reduced macroscopic score (Fig. 1, A and B). Control mice treated with rectal NaCl solution (n ϭ 10) or intraperitoneal vehicle (3% Me 2 SO) alone failed to develop wasting disease and appeared healthy. Analysis of weight loss and daily food and water consumption revealed a reduced intake of food from day 1 to 3 in non-treated versus the SB203580-treated mice. Thus, these mice also had a lower weight reduction (Fig. 1A). The daily assessment of weight and the evaluation of feeding behavior indicated no effect of SB203580 on food or water intake compared with controls (Fig. 1A). The colons of TNBS-treated mice removed at days 1, 2, 3, 5, 7, and 10 (n ϭ 7/day) revealed FIG. 1. Effect of SB203508 on TNBS-induced colitis. A, schematic presentation of daily water and food intake as well as weight loss of the mice over the experimental period. Values are mean Ϯ S.D. of 7 animals/group/day. Results for TNBS-administered mice with SB203508 treatment were compared with mice without SB203580 treatment (*, p Ͻ 0.05). B, colitis was induced in BALB/c mice by rectal administration of 0.1 ml of TNBS (2.5% (w/v)) in 50% ethanol followed by a 10-day observation period. Values are mean Ϯ S.D. of 7 animals/group/day. Results for TNBS-administered mice with SB203508 treatment were compared with mice without SB203580 treatment (*, p Ͻ 0.05). C, differential blood cell count was measured with intracardially drawn blood immediately obtained after sacrifice. Values are mean Ϯ S.D. of 7 animals/group/day. Results for TNBS-administered mice with SB203508 treatment were compared with mice without SB203580 treatment (*, p Ͻ 0.05). hyperemia and inflammation, whereas in mice additionally treated with SB203580 (n ϭ 7/day) the macroscopic signs of inflammation were reduced. The colons of control mice showed no such macroscopic signs of inflammation (Fig. 1B).
Differential white blood cell count showed a dramatic increase of monocytes and granulocytes in the peripheral blood after induction of colitis starting at day 1 with a maximum at day 3. The number of monocytes was significantly reduced between days 2 and 3 in the SB203580-treated animals, whereas peripheral granulocytes were not significantly altered upon treatment (Fig. 1C).
SB203580 Administration Reduces the Histological Score of TNBS-induced Colitis-Histologic analysis in formalin-fixed HE-stained sections during the course of TNBS-induced colitis revealed a strong infiltration of the mucosa and submucosa with lymphocytes, macrophages, and neutrophilic granulocytes at days 1 and 2. A transmural diffuse inflammation was detected starting at day 1 associated with deep ulcerations, edema, thickened colon wall, loss of goblet cells, and the presence of some granulomas ( Fig. 2A). Colonic inflammation slowly resolved after day 3 but could be detected until the end of the study at day 10. SB203580 treatment clearly reduced the impairment of the glandular architecture and the infiltration of macrophages, lymphocytes, and neutrophils between days 2 and 10 ( Fig. 2A). From day 2 onward, the histological signs of colitis as reflected by a lower histological score were significantly reduced in SB203580-treated mice (Fig. 2B). SB203580 alone did not result in any changes of the intestinal mucosa (data not shown).

Expression of Pro-inflammatory Cytokines in TNBS-induced Colitis Is Reduced by SB203580 -Intestinal inflammation in
Crohn disease is characterized by a Th1-mediated immune response. Therefore, we evaluated the cytokine profile in TNBS-induced colitis and the effects of SB203580. The mRNA expression of TNF-␣, interferon ␥, IL-2, IL-12p35, IL-18, and IL-10 in the bowel tissue was analyzed using real-time quantitative PCR. These experiments showed that mRNA concentrations of pro-inflammatory cytokines strongly increased between days 1 and 2 and reached a maximum around day 3. SB203580 drastically down-regulated the mRNA levels of these cytokines (Fig. 3). The time course of these cytokines at the transcriptional level during increasing and resolving inflammation in bowel tissue of untreated and SB203580-treated animals is shown in Fig. 3. Collectively, these findings confirm an anti-inflammatory effect of SB203580 in murine TNBSinduced colitis. Control animals treated with SB203580 alone displayed the same mRNA levels as control animals receiving vehicle treatment only.
SB203580 Inhibits Activation of p38 -We visualized the activity of p38 MAPK in inflamed bowel tissue by immunofluorescence microscopy using phospho-specific antibodies. p38 was activated in lymphocytes and macrophages, the main cell types mediating early pro-inflammatory responses in human Crohn disease (Fig. 4A). The severity of inflammation detected by immunohistological analysis correlated with the macroscopic and histologic score in SB203580-treated and nontreated animals. No lesions or significant p38 activity were observed in control mice.
Because SB203580 targets only the ATP-binding of p38, but does not interfere with p38 phosphorylation (47), we assessed the activity of p38 by immunoprecipitation using a p38-specific were seen starting at day 1 and were manifested in extensive ulcerations, transmural edema, and inflammation accompanied with a diffuse infiltration of lymphocytes and granulocytes. A resolution of inflammatory infiltrates was observed starting at days 3-5. Treatment with SB203508 reduced mucosal injury, edema, and infiltration of inflammatory cells in the colonic bowel. The histology of the distal large intestine in the control group without and with daily intraperitoneal injections of SB203508 was regular (HE staining, magnification ϫ20). B, histological scoring was performed semi-quantitatively in HEstained sections. Values are mean Ϯ S.D. of 7 animals/group/day. Results for TNBS-administered mice with SB203508 treatment were compared with mice without SB 203580 treatment (*, p Ͻ 0.05).

FIG. 3. SB 203580 inhibits pro-inflammatory cytokine mRNA synthesis.
Descendent colon was removed from untreated mice (control) as well as TNBS-and TNBS ϩ SB203580-treated mice. The tissue was homogenized in TRIzol as described under "Experimental Procedures." Amounts of colonic cytokine mRNAs were quantified using real-time reverse transcriptase-PCR. Values are mean Ϯ S.E. (n ϭ 7 mice/group/day; *, p Ͻ 0.05 TNBS-treated mice with versus without SB203580 treatment). antibody and bowel tissue protein extracts. Subsequently, we performed an in vitro kinase assay using ATF-2 as substrate followed by immunoblotting with phospho-specific ATF-2 antibodies. A strong increase of p38 activity was detected after day 1 with a maximum between days 2 and 5. This activation was strongly down-regulated by SB203580 during the course of colonic inflammation (Fig. 4B).
SB203580 Inhibits the Classical but Not the Alternative NF-B Activation Pathway-Because SB203580 inhibited the transcription of the pro-inflammatory cytokines in the bowel wall of mice with TNBS-induced colitis, we wanted to examine whether this effect is mediated by NF-B, a key transcription factor in inflammatory processes. Cytoplasmic and nuclear extracts were prepared from bowel tissue. First, we analyzed the phosphorylation of IKK, IB␣ degradation, and the nuclear translocation of p65 by immunoblot analysis. These parameters served as a readout for NF-B activation. We found a peak of IKK␣/␤ phosphorylation and of IB␣ degradation at days 2 and 3 after the induction of colitis by TNBS administration (Fig. 5A). In addition, nuclear translocation of p65 showed in general the same kinetics as IB␣ degradation (Fig. 5, A and  B). However, the peak of p65 translocation is delayed compared with IB␣ degradation. SB203580 almost completely inhibited IKK activation, IB␣ degradation, and p65 translocation (Fig.  5, A and B). This might be an indication that SB203580 is not only specific for p38 but also affects NF-B activation during intestinal inflammation via so far unknown targets.
To exclude the possibility that the inhibitory effect of SB203580 is because of a negative effect on other NF-B activating pathways mediated by PKC, Cot/TPL2, or MLK3, which are known to phosphorylate and thereby induce the IKKs (9 -11), we performed activation studies by immunoblot analysis using phospho-specific antibodies. All three kinases were activated by phosphorylation upon induction of the in-flammatory process (Fig. 5C). Furthermore, this activation could not be suppressed by administration of SB203580 (Fig.  5C). Thus, based on our data, these signaling kinases are not involved in the SB203580-mediated down-regulation of NF-B activity and the release of pro-inflammatory cytokines in TNBS-induced colitis in mice.
Additionally, we analyzed the alternative pathway of NF-B activation involving p100 processing to active p52. Immunoblot analysis was performed using antibodies recognizing both, the p100 precursor as well as the p52 product. However, in contrast to the classical activation pathway of NF-B, this pathway was not up-regulated during TNBS-induced colitis and hence, was not affected by SB203580 (data not shown).

FIG. 4. SB203580 inhibits p38 activation during TNBS-induced colitis.
A, immunofluorescence microscopy of the descendent colon was performed using fluorescein isothiocyanate-coupled phospho-specific antibodies against p38 (see "Experimental Procedures") in non-TNBS exposed animals (left, 0 days) and in mice administered with TNBS enema (right, 3 days). B, p38 was immunoprecipitated from protein extracts of the descendent colon of untreated (control) or TNBS-treated BALB/c mice that were either pretreated with SB203580 or left untreated (n ϭ 7 mice/group/day). The immunocomplexes were subjected to an in vitro kinase assay using ATF-2 as substrate followed by immunoblotting with an anti-phospho-ATF-2 antibody. To control equal loading, immunoblotting with p38-specific antibodies was performed. All results are representative of three independent experiments.

FIG. 5. SB203580 directly inhibits RICK/NF-B in TNBS-induced colitis.
A, protein extracts from the bowel of TNBS-treated BALB/c mice were analyzed by immunoblotting using phospho-specific IKK and IB␣-specific antibodies. Mice (n ϭ 7 mice/group/day) were either left untreated or administered with SB203580 for the indicated times. To control equal loading, blots were re-probed with extracellular signal-regulated kinase (ERK) 1/2 antibody. B, cytoplasmic (c) and nuclear (n) protein extracts were prepared from bowel tissue and immunoblotted with a p65-specific antibody. C, protein extracts from the bowel of TNBS-treated Balb/c mice were analyzed by immunoblotting using phospho-specific PKC, Cot/TPL2, and MLK3 antibodies showing that SB203580 does not affect their activity. Mice (n ϭ 7 mice/group/ day) were either left untreated or exposed to TNBS for the indicated times. To control equal loading, blots were re-probed with ERK 1/2 antibody. D, RICK was immunoprecipitated with a RICK-specific antibody from whole cellular extracts of bowel tissue from TNBS-administered Balb/c mice, which were either left untreated or treated with SB203580 (n ϭ 7 mice/group/day). The immunocomplexes were subjected to an in vitro kinase assay using MBP as substrate for RICKdependent phosphorylation followed by immunoblotting with an anti-phospho-MBP antibody. To control equal loading, the blots were re-probed with RICK antibody. All results are representative of three independent experiments. E, RICK was immunoprecipitated with a RICK-specific antibody from whole cellular extracts of bowel tissue from untreated Balb/c mice (lane 1) or from TNBSadministered Balb/c mice at day 3 (lane 2 and 3). The cell extract was incubated with SB203580 30 min prior to the kinase assay (lane 3).
RICK Is a NF-B Activating Kinase Inhibited by SB203580 during TNBS-induced Colitis-To analyze putative targets of SB203580, we studied molecular components regulated by NOD proteins that may exert a role in NF-B activation. NOD as an intracellular receptor for bacterial peptidoglycan probably plays a crucial role in the pathogenesis of IBD by triggering intestinal inflammation (3-5, 17, 48). Initial studies have shown that NF-B becomes activated following a transient expression of NOD1 and NOD2 in mammalian cells. This activation required the integrity of the CARD domains (41). RICK physically interacts with NOD1 through CARD-CARD interactions (47,49) and has been shown to be the main effector kinase of NOD1. It also interacts with and regulates the IKK complex finally leading to NF-B activation (47). Therefore, RICK is an interesting candidate to regulate NF-B via the NOD signaling pathway.
RICK activity was assessed during the experimental colitis following immunoprecipitation of RICK from protein extracts derived from bowel tissue. RICK phosphorylates MBP in an in vitro kinase assay (Fig. 5D). Using this assay, we showed that RICK is dramatically activated during development of experimentally induced colitis. In contrast to the other NF-B activating kinases analyzed (Fig. 5C), SB203580 treatment of the mice could almost completely inhibit the activation of RICK (Fig. 5D). Because this inhibitory effect was also achieved upon SB203580 addition directly to the cellular extracts of TNBStreated mice, we postulate a direct suppressive effect of the SB203580 inhibitor on RICK activity (Fig. 5E). This indicates that RICK might be a crucial kinase involved in NF-B activation in the course of IBD. DISCUSSION Because the exact molecular mechanisms of the reduced intestinal inflammation by p38 inhibitors are unsolved so far, we sought to analyze the impact of SB203580 on: 1) the transcription of key cytokines for the development of IBD, 2) NF-B activation pathways, and 3) the NOD/RICK signal transduction pathway. The TNBS model of colitis in mice shares important similarities with human Crohn disease such as transmural inflammation, lymphocyte infiltration, Th1-dominated cytokine profile, and stricture formation (37,42,43). Thus, this model is very suitable to study anti-inflammatory agents during the course of developing and resolving inflammation.
We were able to demonstrate a clinical improvement of TNBS-induced colitis in mice treated with SB203580 as reflected in the clinical data, in the macroscopic and the histological disease score. This is in line with other studies using p38 inhibitors and different models of inflammation (e.g. animal models for arthritis, pulmonary inflammation, and other immune challenging procedures). In all cases, these inhibitors attenuated disease activity, mortality, or reduced pro-inflammatory cytokine concentrations (50,51). However, a recent study did not show uniformly beneficial effects of SB203580 (52). This might be, at least in part, because of a different experimental design, like double TNBS enemas, and because of the inability of SB203580 to inhibit TNF-␣ synthesis at the rather low concentration used in this specific study. The findings of this study are not only in contrast to our but also to other in vitro studies using human monocytes stimulated with lipopolysaccharide, where SB203580 inhibited the production of IL-1 and TNF-␣ (53,54). Furthermore, it is known that different BALB/c mice strains have variations in their susceptibility to chemically induced colitis (55).
TNF-␣ plays a central role in mucosal inflammation and is likely the key regulator of the inflammatory cascade in IBD (56). It is regulated by p38 (24) and AU-rich elements in the 3Ј-untranslated region of the TNF-␣ gene are necessary for p38-mediated regulation. Mice develop chronic inflammatory diseases when these AU-rich regions are deleted in the genome resulting in an increased TNF-␣ expression (57). p38 inhibitors in wild type mice can reduce the TNF-␣ synthesis, whereas AU-rich element-deficient mice did not respond to the drug (58). Our results clearly demonstrate that SB203580 down-regulates the expression of TNF-␣ as well as the expression of other pro-inflammatory cytokines (26,30) confirming a general anti-inflammatory effect of SB203580 in the inflamed bowel tissue.
We next wanted to know whether SB203580 had a suppressive effect on p38 as well as other pro-inflammatory pathways such as NF-B, a potent transcription factor regulating cellular immune responses and inflammation (6). We observed that both p38 and NF-B are dramatically activated during the development of TNBS-induced colitis and this induction was strongly inhibited by SB203580 treatment. A suppressive effect of SB203580 was only observed for the classical NF-B activation pathway based on the IB degradation by the 26 S proteasome (6). The more recently discovered so-called alternative NF-B activation pathway characterized by NIK/IKK␣dependent processing of the p100 precursor to active p52 (12,13) was not activated and, therefore, not affected by SB203580. This pathway was investigated, because p100 processing preferentially generates a different subset of NF-B dimers, mainly p52/RelB. The two activation pathways of NF-B might differentially influence inflammatory responses in IBD. Studies in transgenic mice suggest that the alternative activation pathway of NF-B increases chemokine concentrations, and the susceptibility to inflammation in general (59,60).
A fundamental finding of the current study is the suppression of the classical NF-B activation by SB203580. It is very improbable that the inhibitory effect of SB203580 on this NF-B activation pathway is indirect, e.g. mediated by the decrease of cytokines because of p38 inhibition. Cytokine synthesis is not regulated by p38 alone. NF-B is always involved in the regulation of pro-inflammatory cytokine genes. The more likely explanation would be that SB203580 has a broader range of target protein kinases. The only known impact of p38 on NF-B activity is by phosphorylation of p65/RelA, which increases the transcriptional activity of p65 containing NF-B dimers (61). However, we also found that SB203580 blocks the nuclear translocation of p65, which is not known to be influenced by p38. Our data strongly suggest that SB203580 directly or indirectly suppresses kinases involved in NF-B activation.
One strong candidate for such a novel target of SB203580mediated inhibition during IBD development is the serine/ threonine protein kinase RICK. We focused our studies on this kinase for three reasons. 1) As shown by a very recent proteome-wide study, RICK is a high affinity target of SB203580 (62). 2) RICK is known to interact with and activate IKK␣ (47).
3) RICK is a prominent effector kinase of NOD proteins (17), which, as mentioned above, are intracellular receptors of bacterial peptidoglycan and play a crucial role in IBD pathogenesis (15)(16)(17). RICK, like p38 and NF-B was strongly activated in the course of experimentally induced colitis and this activation was also drastically reduced by SB203580 treatment of the mice. Our findings give support to the notion that a pathway triggered by bacteria, involving NOD proteins as well as RICK, and finally leading to NF-B activation, plays a pivotal role in IBD pathogenesis. In vitro studies demonstrated a failure of NF-B activation because of NOD2 mutations (16). However, to our knowledge evidence is still missing that patients carrying one of these NOD2 mutations have a decreased NF-B activation in their bowel mucosa. Additionally, it is not unlikely that other NF-B activating signal transduction pathways in addi-tion to the one analyzed in this study might also activate the underlying pathogenic immune response against the so far unidentified pathogen(s) in Crohn disease. This view is supported by epidemiological data showing that the majority of patients have no NOD2 mutations and virtually all patients with IBD exhibit strongly increased NF-B activation patterns (63). Thus, because RICK is so far the only accepted effector kinase regulated by NOD, our findings indicate that a pathway triggered by bacteria involving NOD proteins as well as RICK can be responsible for the inappropriate NF-B activation in IBD pathogenesis (Fig. 6).
Are other potential NF-B-activating kinases also targeted by SB203580 or is RICK unique in this respect? To answer this question we analyzed whether the activity of PKC, Cot/TPL2, and MLK3 are suppressed by SB203580 treatment of mice. All three kinases were induced during the development of inflammation, but none was inhibited by SB203580 in the inflamed bowel tissue. This finding is in line with a proteome-wide study showing that RICK is the only NF-B-activating kinase representing a high affinity target for SB203580 (62). Furthermore, this inhibitory effect of SB203580 on RICK is direct. This was demonstrated by in vitro kinase assays where the inhibitor was directly added to the cellular extracts. In summary, by excluding a possible role of other signaling kinases and by confirming a direct effect of SB203580 on the activation of RICK in the inflamed bowel tissue, our results strengthen the view that RICK plays an essential role in IBD development.
Corticosteroids and 5-aminosalicylic acid derivatives are the most widely used agents for the treatment of acute and chronic IBD (1). However, a significant number of patients are unresponsive or show an insufficient response to these drugs and may suffer from severe side effects (64). Therefore, patients with steroid-dependent or steroid-refractory courses of IBD as well as with therapy-refractory fistulas require an alternative treatment. Other therapeutic options like azathioprine or 6-mercaptopurine are limited by their delayed response or by the suppression of the bone marrow. A recent pilot study by Hommes et al. (35) has shown that MAPK inhibitors are among the most promising substances in the treatment of moderate to severe IBD without severe long-term side effects.
Taken together, SB203580 reduced the macroscopic signs of intestinal inflammation corresponding to improved clinical performance. Investigating the underlying molecular mechanisms, SB203580 was shown to significantly down-regulate pro-inflammatory cytokines at the transcriptional level probably because of a significant inhibition of p38 and the classical but not of the alternative pathway of NF-B activation. Our data suggest that the inhibitory effect of SB203580 on NF-B could be mediated by RICK inhibition. Therefore, the "p38 inhibitor" SB203580 is acting on different signal transduction pathways. Considering the significant beneficial effects of SB203580, RICK might be a promising new molecular target to inhibit inflammation associated with IBD in patients refractory to standard immunosuppressive therapy. Because NF-B is the target for all conventionally used drugs for anti-inflammatory therapy of Crohn disease (1), RICK inhibitors like SB203580 might serve as a new class of anti-inflammatory agents either as primary or secondary therapy with a well acceptable profile of side effects. However, the exact characterization of signal transduction leading to NF-B activation in Crohn disease and the importance of NOD-associated CARD proteins like RICK need to be further analyzed. Additionally, further investigations are needed to evaluate the importance of the dual inhibition of p38 and RICK as a general therapeutic mechanism. Acknowledgments-We thank Christine Wolf for excellent technical assistance and Dr. Jonathan Lindquist for critical reading of the manuscript. This triggers p38 MAPK activity via the upstream kinases MKK3/6 and NF-B activation via the classical IB-dependent pathway mediated by the IKK complex. RICK is a kinase upstream of IKK, which is able to activate the IKK complex. RICK activity is also induced upon colitis induction. SB203580 inhibits p38 MAPK as well as RICK activity. Suppression of RICK leads to down-regulation of NF-B. The solid arrows indicate direct activation of downstream targets and the dotted arrows indicate indirect or not yet defined activation processes.