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Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Barnich N, Aguirre JE, Reinecker HC, Xavier R, Podolsky DK - J. Cell Biol. (2005)

Bottom Line: To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants.Membrane targeting and subsequent NF-kappaB 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-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

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)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. 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-kappaB 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-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

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Ligand-induced NF-κB activation and IL-8 release are dependent on NOD2 membrane association. (A) Expression of NOD2 and mutants without tag that were transfected into HEK293 cells was determined by Western blot analysis using NOD2 antiserum HM2563. (B) HEK293 cells were transfected with 1 ng NOD2 expression vector or mutants together with 1 μg MDP-LD. NF-κB activity was determined by using an NF-κB luciferase reporter assay and was normalized with renilla after 18 h of transfection. Fold increase of NF-κB activation was determined by comparing untransfected and nonstimulated HEK293 with MDP. (C) IL-8 released in the supernatant of HEK293 cells that were transfected with mutants (M) of NOD2 and stimulated 18 h with 1 μg MDP-LD was measured by ELISA. Error bars represent SEM of at least four separate experiments. NT, nontransfected. *, P < 0.05.
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fig4: Ligand-induced NF-κB activation and IL-8 release are dependent on NOD2 membrane association. (A) Expression of NOD2 and mutants without tag that were transfected into HEK293 cells was determined by Western blot analysis using NOD2 antiserum HM2563. (B) HEK293 cells were transfected with 1 ng NOD2 expression vector or mutants together with 1 μg MDP-LD. NF-κB activity was determined by using an NF-κB luciferase reporter assay and was normalized with renilla after 18 h of transfection. Fold increase of NF-κB activation was determined by comparing untransfected and nonstimulated HEK293 with MDP. (C) IL-8 released in the supernatant of HEK293 cells that were transfected with mutants (M) of NOD2 and stimulated 18 h with 1 μg MDP-LD was measured by ELISA. Error bars represent SEM of at least four separate experiments. NT, nontransfected. *, P < 0.05.

Mentions: NOD2 is known to activate the nuclear transcription factor NF-κB after MDP-LD stimulation, and the NOD2 3020insC mutant's ability to activate NF-κB is impaired (Girardin et al., 2003). The inability of the NOD2 mutant to respond to MDP-LD and activate NF-κB in CD is paradoxical, considering that NF-κB 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-κB activation, whereas mutants 1–7 and 11 induced less than a 3-fold increase in NF-κB. However, mutants 8–10 and 12, which are still membrane associated, activated NF-κB. No NF-κB 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-κB 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-κB activation (Fig. 4 C). These results demonstrate that the membrane targeting of NOD2 in intestinal epithelial cells is required for NF-κB activation upon the recognition of MDP. NOD2 binds to RIP2, a cytoplasmic protein, via a CARD–CARD 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.


Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Barnich N, Aguirre JE, Reinecker HC, Xavier R, Podolsky DK - J. Cell Biol. (2005)

Ligand-induced NF-κB activation and IL-8 release are dependent on NOD2 membrane association. (A) Expression of NOD2 and mutants without tag that were transfected into HEK293 cells was determined by Western blot analysis using NOD2 antiserum HM2563. (B) HEK293 cells were transfected with 1 ng NOD2 expression vector or mutants together with 1 μg MDP-LD. NF-κB activity was determined by using an NF-κB luciferase reporter assay and was normalized with renilla after 18 h of transfection. Fold increase of NF-κB activation was determined by comparing untransfected and nonstimulated HEK293 with MDP. (C) IL-8 released in the supernatant of HEK293 cells that were transfected with mutants (M) of NOD2 and stimulated 18 h with 1 μg MDP-LD was measured by ELISA. Error bars represent SEM of at least four separate experiments. NT, nontransfected. *, P < 0.05.
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Related In: Results  -  Collection

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fig4: Ligand-induced NF-κB activation and IL-8 release are dependent on NOD2 membrane association. (A) Expression of NOD2 and mutants without tag that were transfected into HEK293 cells was determined by Western blot analysis using NOD2 antiserum HM2563. (B) HEK293 cells were transfected with 1 ng NOD2 expression vector or mutants together with 1 μg MDP-LD. NF-κB activity was determined by using an NF-κB luciferase reporter assay and was normalized with renilla after 18 h of transfection. Fold increase of NF-κB activation was determined by comparing untransfected and nonstimulated HEK293 with MDP. (C) IL-8 released in the supernatant of HEK293 cells that were transfected with mutants (M) of NOD2 and stimulated 18 h with 1 μg MDP-LD was measured by ELISA. Error bars represent SEM of at least four separate experiments. NT, nontransfected. *, P < 0.05.
Mentions: NOD2 is known to activate the nuclear transcription factor NF-κB after MDP-LD stimulation, and the NOD2 3020insC mutant's ability to activate NF-κB is impaired (Girardin et al., 2003). The inability of the NOD2 mutant to respond to MDP-LD and activate NF-κB in CD is paradoxical, considering that NF-κB 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-κB activation, whereas mutants 1–7 and 11 induced less than a 3-fold increase in NF-κB. However, mutants 8–10 and 12, which are still membrane associated, activated NF-κB. No NF-κB 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-κB 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-κB activation (Fig. 4 C). These results demonstrate that the membrane targeting of NOD2 in intestinal epithelial cells is required for NF-κB activation upon the recognition of MDP. NOD2 binds to RIP2, a cytoplasmic protein, via a CARD–CARD 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.

Bottom Line: To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants.Membrane targeting and subsequent NF-kappaB 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-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

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)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. 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-kappaB 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-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

Show MeSH
Related in: MedlinePlus