Correspondence to Daniel K. Podolsky: dpodolsky{at}partners.org
Abstract
Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)B in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-
B. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-
B activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-
B activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.
Introduction
Nucleotide oligomerization domain (NOD) 2/caspase activation and recruitment domain (CARD) 15 was the first gene linked to the risk of Crohn's disease (CD; Hugot et al., 2001; Ogura et al., 2001a). NOD2 expression has been found in intestinal epithelial cells (Ogura et al., 2001b; Gutierrez et al., 2002; Hisamatsu et al., 2003), including Paneth cells (Berrebi et al., 2003; Rosenstiel et al., 2003) and monocytes. NOD2 recognizes and reacts to the bacterial component muramyl dipeptide L-Ala, D-Glx (MDP-LD) via its COOH-terminal leucine-rich repeat domain (Girardin et al., 2003; Inohara et al., 2003). In contrast, the 3020insC mutant form of NOD2, which is associated with CD, does not respond to MDP stimulation (Bonen et al., 2003; Chamaillard et al., 2003; Girardin et al., 2003; Inohara et al., 2003). NOD2 activates the nuclear translocation of the transcription nuclear factor (NF)B, and RICK (RIP-like interacting caspase-like apoptosis regulatory protein kinase)/RIP2 is essential for this process (Chin et al., 2002; Kobayashi et al., 2002). After the activation of NOD proteins, RICK interacts through homophilic CARDCARD interaction and leads to the nuclear translocation of NF-
B (Bertin et al., 1999; Inohara et al., 1999; Ogura et al., 2001b; Kobayashi et al., 2002). RICK/RIP2 is involved in the NOD2-dependent activation of NF-
B, and NOD2 activation leads to the ubiquitinylation of the NF-
B essential modulator (NEMO), which is a key component of the NF-
B signaling complex (Abbott et al., 2004). Recent studies do not clearly conclude whether NOD2 is a regulator of TLR2-mediated responses that may be deregulated in the presence of the NOD2 3020insC mutant (Watanabe et al., 2004; Kobayashi et al., 2005; Maeda et al., 2005). Direct interaction between NOD2 and TGF-ßactivated kinase 1 and NOD2 and GRIM-19 have also been shown, and each can regulate NOD2-mediated NF-
B activation (Chen et al., 2004; Barnich et al., 2005).
The subcellular localization and trafficking of NOD2/CARD15 during MDP stimulation, which is the minimal bioactive component of the bacterial peptidoglycan, remain unclear. A recent study described regulatory regions and characterized critical residues of NOD2 that are involved in NF-B activation after MDP stimulation (Tanabe et al., 2004). Several mutations of NOD2 leading to the loss of MDP recognition involve amino acid residues that are predicted to be located in the
-helix/turn regions that form the convex face of the leucine-rich repeat. In this study, we demonstrate that NOD2 can be directed to the intracellular vesicle compartment and cell surface membranes in intestinal epithelial cells. In these cells, the membrane association of NOD2 is necessary for NF-
B activation after MDP stimulation. The ability of NOD2 to function as a bacterial recognition molecule depends on the COOH-terminal protein motif.
Results and discussion
Cellular localization of endogenous NOD2/CARD15 in intestinal epithelial cells
Expression of NOD2/CARD15 was assessed by Western blot analysis in several intestinal epithelial cell lines as well as in COS7 and HEK293 cells. Colo205, SW480, HT29, and LS174 exhibited a strong expression of endogenous NOD2. In contrast, T84 and Caco-2 expressed little or no NOD2 protein. No endogenous expression was observed in COS7 or HEK293 cells (Fig. 1 A). NOD2 that was expressed in HT29 did not contain any of the three common mutations that are associated with CD, and stimulation by MDP-LD induced a 6.2-fold increase in NF-B activity, indicating functional NOD2 in this cell line (Fig. 1 B).
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The COOH-terminal domain of NOD2 facilitates membrane targeting
Given the fact that the NOD2 3020insC mutant, which is associated with CD, did not respond to MDP-LD stimulation, the membrane association that was observed for NOD2 led us to examine the subcellular localization of the NOD2 3020insC mutant. Confocal microscopic examination of Caco-2 and COS7 cells that were transfected with Flag or GFP-tagged NOD2 was performed to achieve finer delineation of the spatial distribution of NOD2. Confocal analysis showed that the expressed protein Flag-NOD2 is located in the cytosol and also appears enriched close to the plasma membrane, recapitulating the distribution of endogenous NOD2 (Fig. 2 B). In contrast to wild-type NOD2, Flag-NOD2 3020insC mutant was only present in the cytosol and vesicular compartment. The loss of membrane targeting of the NOD2 3020insC mutant was confirmed by Western blot analysis after separation of cytosolic and membrane fractions. The relative purity of cytosolic and membrane fractions was confirmed by Western blot analysis using antibody against E-cadherin as a membrane marker and antibody antilactate dehydrogenase as a cytosolic marker (Fig. 2 C). The ratio of the Flag-tagged NOD2 3020insC protein between membrane and cytosolic fractions was significantly decreased compared with that of the NOD2 wild-type protein, taken as one (Fig. 2 C). GFP-tagged and Flag-tagged NOD2 colocalized in Caco-2 cells (Fig. 2 D), indicating that the nature of the NH2-terminal tag did not affect cellular localization. However, GFP-tagged NOD2 and Flag-tagged NOD2 3020insC mutant did not colocalize near the plasma membrane, confirming the specific membrane association of the NOD2 wild type. This membrane association was confirmed by using antiE-cadherin antibody, a membrane marker. As shown in Fig. 2 E, the GFP-NOD2 wild type colocalized with E-cadherin in Caco-2 cells.
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NF-B activation by MDP-LD is dependent on the membrane targeting of NOD2
NOD2 is known to activate the nuclear transcription factor NF-B after MDP-LD stimulation, and the NOD2 3020insC mutant's ability to activate NF-
B is impaired (Girardin et al., 2003). The inability of the NOD2 mutant to respond to MDP-LD and activate NF-
B in CD is paradoxical, considering that NF-
B is responsible for the induction of a large number of inflammatory mediators (O'Neill, 2004). To investigate whether the membrane targeting of NOD2 is required for MDP response, we transfected HEK293 cells with NOD2 wild type and different COOH-terminal mutants without tag. NOD2 expression was confirmed by Western blot analysis using antiserum HM2563 against NOD2 (Fig. 4 A). As shown in Fig. 4 B, NOD2 wild type induced a 47-fold increase in NF-
B activation, whereas mutants 17 and 11 induced less than a 3-fold increase in NF-
B. However, mutants 810 and 12, which are still membrane associated, activated NF-
B. No NF-
B activation was observed after stimulation with an MDP L-Ala, L-Glx (MDP-LL)inactive form (unpublished data). The release of IL-8 by HEK293 cells transfected with NOD2 and selected mutants after MDP-LD stimulation was determined by ELISA to confirm the NF-
B activation. The amount of IL-8 found in the supernatant of HEK293 cells that were transfected with NOD2 and stimulated with MDP-LD (10.55 pg/ml) was significantly increased compared with untransfected cells (0.47 pg/ml). The amount of IL-8 released in the supernatant of HEK293 cells that were transfected with the selected NOD2 mutants correlated with NF-
B activation (Fig. 4 C). These results demonstrate that the membrane targeting of NOD2 in intestinal epithelial cells is required for NF-
B activation upon the recognition of MDP. NOD2 binds to RIP2, a cytoplasmic protein, via a CARDCARD interaction (Kobayashi et al., 2002), suggesting that signaling of NOD2 at the membrane implies either NOD2 redistribution to the cytoplasm after MDP-LD stimulation or recruitment of RIP2 to the membrane.
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Materials and methods
Cell culture and transfection
Caco-2, T84, HT29, HCT116, Colo205, SW480, LS174, COS7, and HEK293 cells were obtained from the American Type Culture Collection and were cultured as described previously (Hisamatsu et al., 2003). For NF-B assay or protein expression, cells were transfected using LipofectAMINE 2000 (Invitrogen), and for immunostaining experiments, cells were transfected using TransIT Transfection Reagent Kit (Mirus Bio Corp.) according to the manufacturer's protocols.
Construction of expression plasmids
Flag-tagged (pCMVtag2c-NOD2) and GFP-tagged (pEGFPC1-NOD2) NOD2 mammalian expression vectors were described previously (Hisamatsu et al., 2003). NOD2 mammalian expression vector without tag was provided by M. Chamaillard (Institut National de la Santé et de la Recherche Medicale, Lille, France). All NOD2 mutants were generated by PCR using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's protocols and were sequenced. Expression was confirmed by Western blot analysis using mouse anti-Flag M2 mAb (Sigma-Aldrich) or rabbit antiserum HM2559 or HM2563 against NOD2 (Hisamatsu et al., 2003).
Confocal microscopy
HT29, Caco-2, or COS7 cells were seeded on sterile permanox coverslips and were transfected after 24 h with various NOD2 constructs. After 48 h, cells were washed twice with ice-cold PBS, fixed 10 min with cold methanol at 20°C, washed three times with ice-cold PBS, and blocked 30 min with 5% donkey serum in PBS. Cells were stained using rabbit NOD2 antiserum HM2559 and Texas redconjugated antirabbit IgG (Vector Laboratories) secondary antibodies for endogenous NOD2 or using mouse anti-Flag M2 mAb (Sigma-Aldrich) and FITC-conjugated antimouse IgG secondary antibodies. The coverslips were mounted in Vectashield (Vector Laboratories) and examined at RT with a confocal microscope (model Radiance 2000; Bio-Rad Laboratories) using multitracking (line switching) for two-color imaging (40x). Image acquisition was performed with LaserSharpScanning software (Bio-Rad Laboratories). For vesicular staining, anticalnexin rabbit pAb (StressGen Biotechnologies) was used in COS7 cells followed by Texas redconjugated antirabbit IgG secondary antibodies.
NF-B activation assay and detection of cytokine IL-8 in the supernatant of transfected cells
HEK293 cells were transfected overnight with 1 ng NOD2, 10 ng pIV luciferase reporter plasmid, and 1 ng of renilla plasmid together with 1 µg MDP-LD or MDP-LL (Sigma-Aldrich). After 18 h, luciferase activity was measured by using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions and was normalized to renilla activity.
The amount of human cytokine IL-8 released in the supernatant of HEK293 cells that were transfected with 1 ng NOD2 or different NOD2 mutants was determined by ELISA (BD Biosciences). The optical density was determined at a wavelength of 450 nm, and cytokine concentrations in picograms per milliliter were assessed according to the manufacturer's instructions.
Protein extraction and immunoblotting experiments
Cells were seeded on 6-well plates and were transfected 24 h later with 1 µg DNA. Medium was removed, and 300 µl of lysis buffer (1% Triton X-100, 0.1 M NaCl, 10 mM Hepes, pH 5.6, 2 mM EDTA, 4 mM Na3VO4, and 40 mM NaF) supplemented with protease inhibitor cocktail (Complete Mini; Roche Diagnostics) was added. Lysates were harvested and passed through a 21-gauge needle 10 times. The cytosolic fraction was obtained by centrifugation at 10,000 g for 30 min at 4°C. The pellet was resuspended in 200 µl of lysis buffer containing 1% SDS and was sonicated twice for 20 s. After 5 min of centrifugation at 10,000 g, the resulting supernatant was collected. The protein concentration was determined by using the DC Protein Assay Kit (Bio-Rad Laboratories).
10 µg of proteins from all intestinal epithelial cells were separated on 412% Tris-glycine gels (Invitrogen), blotted onto polyvinylidene difluoride membranes, and stained for NOD2 using rabbit NOD2 antiserum HM2559. Proteins from Caco-2 cells that were transfected with Flag-tagged NOD2 and all mutants were separated on 412% Tris-glycine gel (Invitrogen), blotted onto polyvinylidene difluoride membranes, and stained using anti-Flag M2 mAb (Sigma-Aldrich), antiE-cadherin mAb (BD Biosciences), antilactate dehydrogenase (Abcam), or antiß-actin (C-11; Santa Cruz Biotechnology, Inc.).
Statistical analysis
For the analysis of significant differences in NF-B activation or IL-8 release, a t test was used. All experiments were repeated at least four times. P
0.05 was considered to be statistically significant.
Acknowledgments
We are grateful to Dr. Mathias Chamaillard for NOD2 construct and to Dr. Ian Rosenberg, Jiri Kalabis, Jesus Yamamoto-Furusho, Emiko Mizoguchi, and Kathryn Devaney for helpful discussions and critical reading of the manuscript.
This work was supported by grants DK54429, DK33506 (to H.-C. Reinecker); and DK069344, DK043351, and DK07191 from the National Institutes of Health (to D.K. Podolsky).
Submitted: 25 February 2005
Accepted: 1 June 2005
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