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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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Expression and cellular localization of endogenous NOD2. (A) Western blot analysis using rabbit NOD2 antiserum HM2559 and anti–β-actin. 10 μg of total protein from different intestinal epithelial cells (IEC), COS7, and HEK293 cell lines were loaded onto 4–12% Tris-glycine gel. (B) NF-κB activation in HT29 and Caco-2 cells after stimulation with 1 μg/ml MDP-LD or MDP-LL using NF-κB luciferase reporter assay and normalization with renilla after 18 h of transfection. Error bars represent SEM of at least four separate experiments. *, P < 0.05. (C) Confocal microscopy examination of endogenous NOD2 in HT29 and Caco-2 cells using rabbit NOD2 antiserum HM2559 or preimmune serum and anti–rabbit Texas red as a secondary antibody. Cytosolic and surface membrane localization (arrows) of NOD2 in HT29 cells is shown. Bar, 20 μm.
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fig1: Expression and cellular localization of endogenous NOD2. (A) Western blot analysis using rabbit NOD2 antiserum HM2559 and anti–β-actin. 10 μg of total protein from different intestinal epithelial cells (IEC), COS7, and HEK293 cell lines were loaded onto 4–12% Tris-glycine gel. (B) NF-κB activation in HT29 and Caco-2 cells after stimulation with 1 μg/ml MDP-LD or MDP-LL using NF-κB luciferase reporter assay and normalization with renilla after 18 h of transfection. Error bars represent SEM of at least four separate experiments. *, P < 0.05. (C) Confocal microscopy examination of endogenous NOD2 in HT29 and Caco-2 cells using rabbit NOD2 antiserum HM2559 or preimmune serum and anti–rabbit Texas red as a secondary antibody. Cytosolic and surface membrane localization (arrows) of NOD2 in HT29 cells is shown. Bar, 20 μm.

Mentions: 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-κB activity, indicating functional NOD2 in this cell line (Fig. 1 B).


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)

Expression and cellular localization of endogenous NOD2. (A) Western blot analysis using rabbit NOD2 antiserum HM2559 and anti–β-actin. 10 μg of total protein from different intestinal epithelial cells (IEC), COS7, and HEK293 cell lines were loaded onto 4–12% Tris-glycine gel. (B) NF-κB activation in HT29 and Caco-2 cells after stimulation with 1 μg/ml MDP-LD or MDP-LL using NF-κB luciferase reporter assay and normalization with renilla after 18 h of transfection. Error bars represent SEM of at least four separate experiments. *, P < 0.05. (C) Confocal microscopy examination of endogenous NOD2 in HT29 and Caco-2 cells using rabbit NOD2 antiserum HM2559 or preimmune serum and anti–rabbit Texas red as a secondary antibody. Cytosolic and surface membrane localization (arrows) of NOD2 in HT29 cells is shown. Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171381&req=5

fig1: Expression and cellular localization of endogenous NOD2. (A) Western blot analysis using rabbit NOD2 antiserum HM2559 and anti–β-actin. 10 μg of total protein from different intestinal epithelial cells (IEC), COS7, and HEK293 cell lines were loaded onto 4–12% Tris-glycine gel. (B) NF-κB activation in HT29 and Caco-2 cells after stimulation with 1 μg/ml MDP-LD or MDP-LL using NF-κB luciferase reporter assay and normalization with renilla after 18 h of transfection. Error bars represent SEM of at least four separate experiments. *, P < 0.05. (C) Confocal microscopy examination of endogenous NOD2 in HT29 and Caco-2 cells using rabbit NOD2 antiserum HM2559 or preimmune serum and anti–rabbit Texas red as a secondary antibody. Cytosolic and surface membrane localization (arrows) of NOD2 in HT29 cells is shown. Bar, 20 μm.
Mentions: 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-κB activity, indicating functional NOD2 in this cell line (Fig. 1 B).

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