Reciprocal cross-talk between Nod2 and TAK1 signaling pathways.

Mutations in the leucine-rich repeat (LRR) domain of Nod2 have been implicated in the pathogenesis of Crohn's disease, yet the function of Nod2 and regulation of the Nod2 pathway remain unclear. In this study, we determined that mitogen-activated protein kinase kinase transforming growth factor (TGF)-beta-activated kinase 1 (TAK1) interacts with Nod2 and is required for Nod2-mediated NF-kappaB activation. The dominant negative form of TAK1 abolished muramyl dipeptide-induced NF-kappaB activation in Nod2-expressing cells. Nod2, acting in a reciprocal manner, inhibited TAK1-induced NF-kappaB activation in RICK-deficient embryonic fibroblasts. Nod2 appears to interact with TAK1 through its LRR region to exert its inhibitory effect on TAK1-induced NF-kappaB activation. Further, wild-type LRR more effectively suppressed NF-kappaB activation induced by TAK1 than LRR with a 3020insC mutation. Considered together, these findings demonstrate a critical role for TAK1 in Nod2-mediated innate immune responses and reveal a novel function for Nod2 in the regulation of the TAK1 signaling pathway.

Mutations in the leucine-rich repeat (LRR) domain of Nod2 have been implicated in the pathogenesis of Crohn's disease, yet the function of Nod2 and regulation of the Nod2 pathway remain unclear. In this study, we determined that mitogen-activated protein kinase kinase transforming growth factor (TGF)-␤-activated kinase 1 (TAK1) interacts with Nod2 and is required for Nod2-mediated NF-B activation. The dominant negative form of TAK1 abolished muramyl dipeptide-induced NF-B activation in Nod2-expressing cells. Nod2, acting in a reciprocal manner, inhibited TAK1-induced NF-B activation in RICK-deficient embryonic fibroblasts. Nod2 appears to interact with TAK1 through its LRR region to exert its inhibitory effect on TAK1-induced NF-B activation. Further, wild-type LRR more effectively suppressed NF-B activation induced by TAK1 than LRR with a 3020insC mutation. Considered together, these findings demonstrate a critical role for TAK1 in Nod2-mediated innate immune responses and reveal a novel function for Nod2 in the regulation of the TAK1 signaling pathway.
Crohn's disease is a chronic inflammatory bowel disease with an estimated prevalence of 1 in 1000 in Western countries (1,2). Despite its unknown etiology, research indicates a strong association between Crohn's disease and mutation of the Nod2 (CARD15) gene (3)(4)(5)(6)(7)(8)(9). A member of the CED/APAF1 superfamily of apoptosis regulatory proteins (11), Nod2 contains two N-terminal CARD domains, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeat (LRR) 1 regions (10). Nod2 interacts with RICK via CARD-CARD interaction. A recent study shows that Nod2 activates NF-B and that RICK is essential for this process (11,12). Embryonic cells from RICK knockout mice (RICKϪ/Ϫ) are deficient in Nod2-mediated NF-B activation, which suggests that RICK involvement in signaling occurs downstream of Nod2 (11,12). Muramyl dipeptide (MDP) enhances Nod2-mediated NF-B activation (13,14), which suggests that Nod2 is a general sensor of MDP. Interestingly, the level of MDP-induced NF-B activation is less in cells expressing mutant Nod2 than in cells expressing wild-type Nod2 (13,14). These results suggest that Nod2 mutation confers susceptibility to Crohn's disease by dysregulation of NF-B (5). The NF-B family of transcription factors exerts pleiotropic effects on the regulated expression of many genes involved in inflammation (15)(16)(17). The importance of NF-B in Crohn's disease is manifested by dysregulation of NF-B and enhanced production of proinflammatory cytokines (17).
Understanding of the regulation of Nod2-mediated NF-B activation remains limited. Whether cross-talk exists between the Nod2 pathway and other signaling pathways of inflammatory cytokines is still not known. In this study, we examined the regulatory effect of TAK1, TBK1, Ubc13, Rip, and MEKK1, proteins implicated in NF-B activation, on Nod2-mediated NF-B activation. Only the dominant negative form of TAK1 (TAK1DN) suppressed Nod2-induced NF-B activation. TAK1DN also inhibited MDP-induced NF-B activation in Nod2-expressing cells. In vitro and in vivo coimmunoprecipitation and confocal microscopic analysis confirmed a biochemical interaction between Nod2 and TAK1. Interestingly, Nod2 had a negative regulatory effect on TAK1-induced NF-B activation. The LRR region of Nod2 inhibited TAK1-induced NF-B activation. This is the first report of Nod2 and TAK1 interaction and evidence of reciprocal cross-talk between the Nod2 and inflammatory cytokine signaling pathways.

EXPERIMENTAL PROCEDURES
Cell Culture-HEK293T and COS cells were grown in Dulbecco's modified Eagle's medium, and LS174T cells were grown in minimum Eagle's medium. Both media were supplemented with 10% fetal bovine serum and penicillin, and cultures were maintained in a humidified atmosphere of 95% air and 5% CO 2 . Embyronic fibroblasts derived from RICKϪ/Ϫ and wild-type littermates were kindly provided by Dr. Richard Flavell (11).
Immunoprecipitation and Immunoblotting-HEK293T cells were cotransfected with pHA-TAK1 and pLPCX-FLAG-Nod2 using the transfection reagent FuGENE 6 (Roche Diagnostics). LS174T cells were treated with tumor necrosis factor-␣ (5 ng/ml) for 20 min to induce Nod2 expression. Cells were lysed on ice for 30 min in lysis buffer containing 20 mM Hepes (pH 7.2), 0.5% Triton X-100, 12.5 mM 2-glycerophosphate, 150 mM NaCl, 1.5 mM MgCl 2 , 2 mM dithiothreitol, 2 mM EGTA, 10 mM NaF, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and complete proteinase inhibitor (Roche Diagnostics). Lysates were immunoprecipitated with the indicated antibodies for 2 h at 4°C. The immune complexes were precipitated with protein G-agarose (Invitrogen) for 1 h at 4°C, washed with lysis buffer, resolved in 4 -20% gradient SDS-PAGE, and analyzed by immunoblotting. All immunoblots were developed by an enhanced chemiluminescence imaging assay developed by Amersham Biosciences. Anti-HA and anti-FLAG antibodies were obtained from Santa Cruz Biotechnology and Strategene.
In Vitro GST Pull-down Assay-GST, GST⅐CARD, and GST⅐LRR fusion proteins were each expressed in the Escherichia coli BL21 strain and purified. TAK1 proteins were synthesized by in vitro transcription and translation using rabbit reticulocyte lysates (Promega) and [ 35 S]methionine (Amersham Biosciences), and incubated in lysis buffer with the indicated GST fusion proteins for 4 h at 4°C. The beads were washed four times with lysis buffer, resolved in 4 -20% gradient SDS-PAGE, and visualized by autoradiography.
Immunofluorescence Microscopy-For immunofluorescence studies, COS cells were transfected with the indicated expression constructs. To detect FLAG-Nod2 and HA-TAK1, cells were stained 24 h after transfection with anti-FLAG rabbit polyclonal antibody and anti-HA monoclonal antibody, respectively, and incubated with Alexa 488 goat antirabbit antibody or Alexa 594 goat anti-mouse antibody (Molecular Probes). Images were captured by an Olympus FV300 confocal laser scanning microscope.
NF-B Activation Assays-Cells were cotransfected with RSV-KB-Luc (100 ng), a reporter construct encoding the luciferase reporter gene B-luc under the control of a minimal promoter with an NF-B-binding site; RSV-␤-galactosidase (50 ng), an expression construct containing ␤-galactosidase; and an indicated amount of each expression construct, by FuGENE 6, in triplicate. Cell lysates were prepared 24 -36 h after transfection, and the relative luciferase activity was determined according to the manufacturer's instructions (Promega). Results were normalized for transfection efficiency on the basis of ␤-galactosidase activity.
Protein Kinase Assays-Autophosphorylates of TAK1 were assayed as described previously (27). Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25°C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl 2 , and 5 Ci of [␥-32 P]ATP (3000 Ci/mmol). Samples were fractionated by 4 -20% SDS-PAGE and visualized by autoradiography.
Nod2 siRNA Experiments-A double-stranded oligonucleotide was designed to contain a sequence derived from the 5Ј end of human Nod2 open reading frame (nucleotides 1187-1207): 5Ј-AAGACAUCUUCCA-GUUACUCC-3Ј in forward and reverse orientation (Qiagen). Nod2 siRNA and a control siRNA were transfected into cells using Oligofectamine (Invitrogen) according to the manufacturer's instructions.

TAK1 Regulates Nod2-induced NF-B Activation-
To delineate the pathways that regulate Nod2 function, we studied the effect of several proteins implicated in NF-B activation on Nod2induced NF-B activation. HEK293T cells were transfected with Nod2 plus the dominant negative forms of TAK1, TBK1, Ubc13, Rip, or MEKK1. Interestingly, only TAK1DN, exerting its effect in a dose-dependent manner, inhibited Nod2-induced NF-B activation ( Fig. 1, A and B). The other proteins and the control vector had no effect ( Fig. 1A; MEKK1 data not shown), thus implicating TAK1 in the regulation of Nod2 function.
TAK1 Regulates MDP-induced NF-B Activation in Nod2expressing Cells-Recent studies suggest that Nod2 serves as an intracellular receptor for the bacterial product MDP (11,12). Further, MDP enhances Nod2-mediated NF-B activation.
To determine whether TAK1 regulates MDP-induced NF-B activation, HEK293T cells were transfected with Nod2 and TAK1DN, and 8 h later, stimulated with MDP (100 ng/ml). TAK1DN effectively inhibited MDP-induced NF-B activation in Nod2-transfected HEK293T cells; the control vector had minimal effect (Fig. 2). These results show that MDP-induced NF-B activation in Nod2-expressing cells requires activation of TAK1.
TAK1 Interacts with Nod2-TAK1 regulation of Nod2-mediated NF-B activation suggested an interaction between Nod2 and TAK1. To investigate this possibility, FLAG-Nod2 and HA-TAK1 were coexpressed in HEK293T cells and immunoprecipitated with anti-HA antibody. Immune complexes were subsequently resolved by SDS-PAGE. Western blot analysis with anti-FLAG antibody showed that the TAK1-precipitated complexes contained Nod2 (Fig. 3A, left). Nod2 and TAK1 interaction was confirmed by the immunoprecipitation of FLAG-Nod2 and Western blot analysis for HA-TAK1 (Fig. 3A, right). To verify interaction between endogenous TAK1 and Nod2 proteins, coprecipitation studies were performed with lysate from cells of the colonic epithelial cell line LS174T and cells of the human monocytic cell line THP-1. Both cell lines are known to express Nod2 (28,29). TAK1 was readily detected in Nod2 immunoprecipitates ( Fig. 3B) but not in the immunoprecipitates prepared using a control serum. These results confirm that Nod2 is able to associate with TAK1 in vivo.
To localize the region of Nod2 that interacts with TAK1, HEK293T cells were cotransfected with FLAG-tagged Nod2 containing only the C-terminal LRR domain (Nod2-LRR) and HA-tagged TAK1 (HA-TAK1). Coprecipitation of LRR with TAK1 (Fig. 3A, left and right) indicated an interaction between the Nod2 LRR domain and TAK1. To confirm this interaction, full-length TAK1 was translated in vitro and incubated with GST⅐LRR, GST⅐CARD, or GST protein. TAK1 bound to GST⅐LRR but not GST⅐CARD or GST protein (Fig. 3C). These results suggest that Nod2 binds to TAK1 via its LRR domain.
To determine which region of TAK1 interacts with Nod2, GST⅐LRR fusion protein was incubated with different regions of TAK1 translated in vitro. Full-length TAK1 and two deletion mutants (aa 1-303 and aa 1-403) bound to GST⅐LRR but not GST (Fig. 3D). In contrast, the C-terminal of TAK1 (aa 286 -632) did not bind to GST⅐LRR. These results suggest that the N-terminal of TAK1 (aa 1-303) is sufficient for Nod2 binding.
TAK1 Colocalizes with Nod2-To ascertain the intracellular distribution of Nod2 and to determine whether Nod2 colocalizes with TAK1, we performed dual color immunofluorescence staining using COS-7 cells that were transiently cotransfected with constructs encoding FLAG-Nod2 and HA-TAK1. These cells were double-stained with anti-HA (Fig. 4A) and anti-FLAG antibodies (Fig. 4B). Confocal microscopic analysis revealed that both Nod2 and TAK1 localize mainly in the cell cytoplasm. An overlay of the staining images showed colocalization of both proteins (Fig. 4C).
Nod2 Expression Does Not Activate TAK1-Because dominant negative TAK1 suppressed Nod2-mediated NF-B activation, we tested whether Nod2 induces activation of NF-B by activating TAK1. TAB1 and IL-1 are known to activate TAK1 and to induce autophosphorylation of TAK1 (27). We therefore performed an in vitro phosphorylation assay to examine TAK1 activation, as described previously (27). HEK293T cells were transfected with TAK1 plus Nod2 or control vector. The positive control was HEK293T cells transfected with TAK1 and TAB1. TAK1 protein was immunoprecipitated with anti-TAK1 antibody and subjected to phosphorylation. Nod2 expression did not activate TAK1 (Fig. 5). These results indicate that Nod2 does not activate NF-B via the TAK1 pathway.
Coexpression of Nod2 and TAK1 Does Not Have a Synergistic Effect on NF-B Activation-The requirement of TAK1 activation for Nod2-induced NF-B activation led us to hypothesize that coexpression of Nod2 and TAK1 may exert a synergistic effect on NF-B activation. To test this hypothesis, we transfected HEK293T cells with TAK1, TAB1, and Nod2. HEK293T cells transfected with TAK1, TAB1, and control vector were used as the control. NF-B activation in HEK293T cells trans-fected with TAK1, TAB1, and Nod2 was comparable with that in the control cells. (Fig. 6). Further, the level of NF-B activation in cells transfected with TAK1, TAB1, and Nod2 tended to be lower than the sum of NF-B activation in cells transfected with only TAK1 and TAB1 and in cells transfected with only Nod2 (Fig. 6). Thus, Nod2 and TAK1 do not appear to exert a synergistic or additive effect on NF-B activation. Rather, a negative regulatory interaction between TAK1 and Nod2 is implied.
Nod2 Inhibits TAK1-induced NF-B Activation-Research has shown that RICK is a downstream molecule in the Nod2mediated NF-B activation pathway (11,12). Because Nod2 expression does not induce NF-B activation in RICKϪ/Ϫ fibroblasts (11,12), we used these cells to examine the effect of Nod2 on TAK1-induced NF-B activation. We first showed that the level of TAK1-induced NF-B activation was similar in wild-type and RICKϪ/Ϫ fibroblasts (Fig. 7, A and B), which suggests that TAK1-induced NF-B activation is not RICK-dependent. To study the effect of Nod2 expression on TAK1-induced NF-B activation, we transfected RICKϪ/Ϫ fibroblasts with TAK1, TAB1, and Nod2. RICKϪ/Ϫ fibroblasts transfected with TAK1, TAB1, and control vector were the control. The expression plasmids RSV-KB-Luc and RSV-␤-galactosidase were included in each transfection. As shown in Fig. 7C, we confirmed that Nod2 expression did not activate NF-B in RICKϪ/Ϫ fibroblasts. Further, Nod2 effectively suppressed TAK1-induced NF-B activation in RICKϪ/Ϫ fibroblasts; the control vector exerted no inhibitory effect. As seen in HEK293T cells, NF-B activation in RICK wild-type fibroblasts transfected with TAK1, TAB1, and Nod2 was comparable with that in RICK wild-type fibroblasts transfected with TAK1, TAB1, and control vector (data not shown). These results show that in the absence of RICK, Nod2 has a negative regulatory effect on TAK1-mediated activation of NF-B. In the presence of RICK, Nod2 may also suppress TAK1-mediated NF-B activation. However, the decreased NF-B activity is less obvious because NF-B activation induced by Nod2 compensates for the suppression of NF-B activity induced by TAK1. To confirm the negative role of Nod2 in TAK1-dependent NF-B activation, we reduced endogenous Nod2 expression through RNA-mediated interference. Fig. 7D shows a marked decrease in exogenous Nod2 induced by expression of Nod2-specific siRNA, evidence that Nod2-specific siRNA can efficiently reduce Nod2 expression. NF-B activity was reduced in LS174T cells transfected with Nod2-specific siRNA as compared with cells transfected with control siRNA, which suggests that Nod2 siRNA effectively suppresses NF-B activation induced by endogenous Nod2 (Fig. 7E). Interestingly, TAK1 induced higher NF-B activation in LS174T cells pretreated with Nod2-specific siRNA than in LS174T cells pretreated with control siRNA (Fig. 7F). These results show that Nod2 siRNA expression potentiates TAK1-induced NF-B activation and further support the observation that Nod2 negatively regulates TAK1induced NF-B activation.
LRR Domain of Nod2 Suppresses TAK1-induced NF-B Activation-Previous studies have shown that Nod2 activates NF-B activation through the CARD domain; the LRR domain of Nod2 does not activate NF-B (10). Because the LRR region of Nod2 interacts with TAK1 (Fig. 3), we examined its effect on TAK1-induced activation of NF-B. HEK293T cells were transfected with LRR, TAK1, and TAB1. As shown in Fig. 8A, LRR effectively suppresses TAK1-induced NF-B activation; the inhibitory effect of the control vector is insignificant. Nod2 with deletion of LRR exerts no inhibitory effect (data not shown).  3. Nod2 interacts with TAK1. A, Nod2 coimmunoprecipitates with TAK1. HEK293T cells were cotransfected with plasmid pCMV-HA-TAK1 (4 g) plus 4 g of DNA pLPCX-FLAG-Nod2, control vector pLPCX-FLAG, or pLPCX-FLAG-LRR. Immunoprecipitations (IP) were performed with anti-HA antibodies and subjected to Western blot (IB) analysis using anti-FLAG antibodies to detect FLAG-Nod2 or FLAG-LRR (left). Immunoprecipitations were also performed using anti-FLAG antibodies and subjected to Western blot analysis using anti-HA antibodies to detect TAK1 (right). All experiments were repeated three times with equivalent results. B, coprecipitation of endogenous TAK1 and Nod2. LS174T and THP-1 cell lysates were prepared as described under "Experimental Procedures." Immunoprecipitations were performed with anti-Nod2 antibodies or control Ig and subjected to Western blot analysis using anti-TAK1 antibodies (top panel). The membranes were reprobed with anti-Nod2 (middle panel). Aliquots of cell lysates were immunoblotted with anti-TAK1 (bottom panel). C, the LRR region of Nod2 interacts with TAK1. Full-length TAK1 translated in vitro and labeled with 35 S was incubated with GST⅐LRR fusion protein, GST⅐CARD, or GST protein. Input TAK1 indicates 1/10 of 35 S-labeled protein used in each GST pull-down assay. All experiments were repeated two times with equivalent results. D, the N-terminal of TAK1 interacts with the LRR region of Nod2. Full-length (FL) and truncated forms of TAK1 (aa 1-303, 1-403, and 286 -632) were generated by in vitro transcription and translation and incubated with GST⅐LRR or GST protein. Input TAK1 indicates 1/10 of 35 S-labeled protein used in each GST pull-down assay. All experiments were repeated three times with equivalent results. and TAB1 plus wild-type LRR or mutant LRR. Mutant LRR was less effective than wild type at suppressing TAK1-induced NF-B activation even at comparable levels of protein expression (Fig. 8B). These results suggest that mutation in the LRR region of Nod2 may affect the ability of Nod2 to regulate the TAK1 signaling pathway. DISCUSSION Research has demonstrated that mutations in the Nod2 gene are associated with Crohn's disease. However, regulation of Nod2 function remains unclear. In this study, we showed that activation of TAK1 is required for Nod2-associated NF-B activation. TAK1 is known to be activated by multiple proinflammatory cytokines (18 -24). Proinflammatory cytokines IL-1, tumor necrosis factor-␣, and IL-18 have been shown to activate NF-B via activation of TAK1. These results suggest that crosstalk exists between inflammatory cytokines and the Nod2 pathway through TAK1.
TAK1 may play a role in host defense against infection. Null mutations in the Drosophila dTAK1 gene reveal a conserved function for TAK1 in the control of rel/NF-B-dependent innate immune responses (31). dTAK1 mutant flies do not produce antibacterial peptides and are highly susceptible to Gramnegative bacterial infection (31). The bacterial product lipopolysaccharide activates multiple protein kinases via TAK1 (32). The bacterium, nontypeable Haemophilus influenzae, uses the TAK1-NIK-IB-related kinase-␤/␥-IB␣ pathway to mediate NF-B-dependent transcription of MUC2 mucin, which is a primary innate defensive response for mammalian airways and intestines (33). In the current study, we showed that the dominant negative form of TAK1 abolished MDP-induced NF-B activation in Nod2-expressing cells, which suggests that TAK1 plays an important role in regulating the host response to MDP. Further, these results elucidate a novel mechanism by which TAK1 regulates innate immune responses.
The underlying mechanism involving Nod2 mutation and the pathogenesis of Crohn's disease is still unclear. Mutant Nod2 is found to elicit a weaker response in NF-B activation (3). Further, MDP less effectively induces NF-B activation in cells transfected with mutant Nod2 than in cells transfected with wild-type Nod2 (13,14). Why a lower level of NF-B activation leads to chronic inflammation in Crohn's disease remains unclear. In this study, we have shown for the first time that Nod2 has a negative regulatory effect on TAK1-induced NF-B activation. In RICKϪ/Ϫ cells, Nod2 markedly suppresses TAK1mediated NF-B activation, suggesting that Nod2 may act as negative regulator of TAK1-mediated NF-B activation.
Previous studies have shown that Nod2 expression, which mainly occurs in monocytes (10), can be readily detected without stimulation (10). In comparison, the expression of RICK in monocytes occurs only after treatment with lipopolysaccharide (11). These results suggest that Nod2 and RICK expression occur at different temporal points. Therefore, Nod2 may have a dual function: 1) in the absence of RICK, Nod2 may suppress TAK1-induced activation of NF-B, and 2) in the presence of RICK, Nod2 may serve as an intracellular receptor for MDP to activate NF-B. Inflammatory mediators such as lipopolysaccharide may regulate Nod2 function by modulating the expression of RICK.
Emerging research has shown that TAK1-mediated NF-B activation plays an important role in IL-1-and tumor necrosis factor-␣-induced secretion of proinflammatory cytokines (34). Our finding that Nod2 suppresses TAK1-mediated NF-B activation suggests that Nod2 may exert a negative regulatory effect on TAK1-mediated inflammation. The discovery that the LRR region of Nod2 suppresses TAK1-induced NF-B activation suggests that Nod2 may inhibit TAK1-mediated NF-B activation via its LRR domain. Further, the LRR mutant was a less effective inhibitor of TAK1-mediated NF-B activation. Considered together, these results raise the possibility that mutations in the LRR domain may reduce the capability of Nod2 to suppress TAK1-mediated NF-B activation, which, in turn, may lead to a dysregulated TAK1-associated inflammation.
In summary, we have shown that TAK1 regulates Nod2mediated NF-B activation. TAK1 modulates MDP-induced NF-B activation in Nod2-expressing cells. These results re-veal a novel mechanism by which TAK1 regulates an innate immune response. These results also indicate that cross-talk exists between the Nod2 and proinflammatory cytokine pathways. Proinflammatory cytokines may regulate Nod2-mediated NF-B by activating TAK1. Also, this is the first evidence that Nod2 exerts an inhibitory effect on TAK1-induced NF-B activation. The LRR domain of Nod2 appears to be involved in Nod2 suppression. The ability of Nod2 to interact physically and functionally with TAK1 represents a novel interaction FIG. 7. Nod2 suppresses TAK1-induced NF-B activation. A, TAK1 induces NF-B activation in RICKϪ/Ϫ fibroblasts. RICKϪ/Ϫ fibroblasts were seeded in 12-well plates (10 5 /well). Reporter constructs with TAK1 (25 ng) and TAB1 (2 ng) and without (none) were cotransfected into cells by FuGENE 6. B, TAK1 activates NF-B equally well in both RICKϪ/Ϫ and wild-type fibroblasts. RICK wild type (wild, shaded bar) and RickϪ/Ϫ (null, solid bar) fibroblasts were transfected with the indicated amounts of TAK1 and TAB1, along with the reporter constructs. Luciferase activity was determined 24 h after transfection and normalized on the basis of ␤-galactosidase activity. All experiments were performed in triplicate and repeated two times with equivalent results. C, Nod2 inhibits TAK1-induced NF-B activation in RICKϪ/Ϫ fibroblasts. RICKϪ/Ϫ fibroblasts were transfected with Nod2 alone (LPCX-Nod2, 50 ng) or with TAK1 (25 ng) and TAB1 (2 ng) plus the indicated amounts of Nod2 (LPCX-Nod2, 0, 25, 50 ng) or control vector (LPCX, 25, 50 ng). Luciferase activity was determined 24 h after transfection and normalized on the basis of ␤-galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent results. *, p Ͻ 0.01 (Nod2 versus control vector). D, Nod2 siRNA efficiently blocks expression of transfected Nod2. HEK293T cells were transiently transfected with Nod2-specific siRNA or control siRNA, and 24 h later, they were transfected with FLAG-Nod2. Cell lysates were prepared 24 h after the Nod2 transfection and subjected to SDS-PAGE and immunoblotting by anti-FLAG antibody. The arrows indicate the Nod2 and nonspecific (NS) bands. E, expression of Nod2 siRNA suppresses endogenous NF-B activity. LS174T cells were transfected with either Nod2-specific siRNA or control siRNA plus the reporter constructs. Luciferase activity was determined 24 h after transfection as in C. All experiments were performed in triplicate and repeated three times with equivalent results. *, p Ͻ 0.05. F, expression of Nod2 siRNA potentiates TAK1-induced NF-B activation. LS174T cells were transfected with Nod2-specific siRNA or control siRNA. These cells were then transfected with TAK1 (25 ng), TAB1 (2 ng), and the reporter constructs 24 h after the siRNA transfection. Luciferase activity was analyzed as in C. The results represent the mean of two independent experiments. Increase in NF-B activation in the control siRNA group was arbitrarily set at 1. between a Nod protein and a mitogen-activated protein kinase kinase. Further understanding of Nod2 function and regulation may shed light on the pathogenesis of Crohn's disease. FIG. 8. Inhibitory effect of the LRR region on TAK1-induced NF-B activation. A, the LRR region of Nod2 suppresses TAK1-induced NF-B activation. HEK293T cells were transfected with TAK1 (25 ng) and TAB1 (2 ng) plus LRR in the amounts indicated. HEK293T cells transfected with identical amounts of TAK1 and TAB1 and control vector were used as the control. Luciferase activity was determined 24 h after transfection and normalized on the basis of ␤-galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent results. B, mutant LRR is less effective than wild-type LRR at suppressing TAK1-mediated NF-B activation. HEK293T cells were transfected with TAK1 (25 ng) and TAB1 (2 ng) plus wild-type or mutant LRR in the amounts indicated (left). Luciferase activity was determined 24 h after transfection and normalized on the basis of ␤-galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent results. *, p Ͻ 0.05 (LRR versus mutant LRR). A cell lysate fraction used for the luciferase assay was subjected to Western blot analysis to determine the expression of TAK1, TAB1, and LRR, both wild type and mutant (right).