Felicia F. Chen

A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease

Crohns disease is a chronic inflammatory disorder of the gastrointestinal tract, which is thought to result from the effect of environmental factors in a genetically predisposed host. A gene location in the pericentromeric region of chromosome 16, IBD1, that contributes to susceptibility to Crohns disease has been established through multiple linkage studies, but the specific gene(s) has not been identified. NOD2, a gene that encodes a protein with homology to plant disease resistance gene products is located in the peak region of linkage on chromosome 16 (ref. 7). Here we show, by using the transmission disequilibium test and case-control analysis, that a frameshift mutation caused by a cytosine insertion, 3020insC, which is expected to encode a truncated NOD2 protein, is associated with Crohns disease. Wild-type NOD2 activates nuclear factor NF-κB, making it responsive to bacterial lipopolysaccharides; however, this induction was deficient in mutant NOD2. These results implicate NOD2 in susceptibility to Crohns disease, and suggest a link between an innate immune response to bacterial components and development of disease.

Nod2, a Nod1/Apaf-1 Family Member That Is Restricted to Monocytes and Activates NF-κB

Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding domain, which regulate apoptosis and/or NF-κB activation. Nod2, a third member of the family, was identified. Nod2 is composed of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1 were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor κB (NF-κB) activation, which required IKKγ and was inhibited by dominant negative mutants of IκBα, IKKα, IKKβ, and IKKγ. Nod2 interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-κB activity induced by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to activate a NF-κB signaling pathway.

Expression of NOD2 in Paneth cells: a possible link to Crohn’s ileitis

Background and aims: Genetic variation in NOD2 has been associated with susceptibility to Crohn’s disease (CD) and specifically with ileal involvement. The reason for the unique association of NOD2 mutations with ileal disease is unclear. To identify a possible link, we tested expression of NOD2 in intestinal tissue of CD patients and controls. Patients and methods: Fifty five specimens of ileum or colon from 21 CD patients, seven ulcerative colitis (UC) patients, and five controls with pathology other than CD or UC were stained for NOD2 using an immunoperoxidase method. Results: Using a monoclonal antibody against NOD2 developed in our laboratory, we detected uniform expression of NOD2 in terminal ileum Paneth cells from controls and patients as well as in metaplastic Paneth cells in the colon. Mechanical purification showed enriched expression of NOD2 mRNA in ileal crypts. In Paneth cells, NOD2 was located in the cytosol in close proximity to the granules that contain antimicrobial peptides. We detected minimal NOD2 in the villous epithelium of the ileum or in the colonic epithelium from both CD patients and controls. Conclusions: These results suggest a role for NOD2 in the regulation of Paneth cell mediated responses against intestinal bacteria and a plausible mechanism to explain the selective association of NOD2 mutations with ileal disease. The impaired capacity of CD associated mutations to sense luminal bacteria may result in increased susceptibility to certain gut microbes.

ASC is an activating adaptor for NF-κB and caspase-8-dependent apoptosis

ASC is a pro-apoptotic protein containing a pyrin domain (PD) and a caspase-recruitment domain (CARD). A previous study suggests that ASC interacts with Ipaf, a member of the Apaf-1/Nod1 protein family. However, the functional relevance of the interaction has not been determined. Here, we report that co-expression of ASC with Ipaf or oligomerization of ASC induces both apoptosis and NF-kappa B activation. Apoptosis induced through ASC was inhibited by a mutant form of Caspase-8 but not by that of Caspase-1. The PD of ASC physically interacted with Caspase-8 as well as with pyrin, the familial Mediterranean fever gene product. Caspase-8 deficiency rescued mouse fibroblasts from apoptosis induced by ASC oligomerization. Pyrin disrupted the interaction between ASC and Caspase-8, and inhibited both apoptosis and NF-kappa B activation induced by ASC. These findings suggest that ASC is a mediator of NF-kappa B activation and Caspase-8-dependent apoptosis in an Ipaf signaling pathway.

A Role for Erbin in the Regulation of Nod2-dependent NF-κB Signaling

Nod2 is an intracellular sensor of a specific bacterial cell wall component, muramyl dipeptide, and activation of Nod2 stimulates an inflammatory response. Specific mutations of Nod2 have been associated with two inflammatory diseases, Crohn disease and Blau syndrome, and are thought to contribute to disease susceptibility through altering Nod2 signaling. Association of disease with inappropriate activation of Nod2 highlights the importance of proper regulation of Nod2 activity. However, little is known about specific regulation of the Nod2 pathway. We performed a biochemical screen to discover potential regulators of Nod2 and identified Erbin, a protein involved in cell polarity, receptor localization, and regulation of the mitogen-activated protein kinase pathway, as a novel Nod2-interacting protein. In our studies, we demonstrate specific interaction of Erbin and Nod2 both in vitro and in vivo and characterize the regions required for interaction in both proteins. We found that Nod2-dependent activation of NF-κB and cytokine secretion is inhibited by Erbin overexpression, whereas Erbin-/- mouse embryo fibroblasts show an increased sensitivity to muramyl dipeptide. These studies identify Erbin as a regulator of Nod2 signaling and demonstrate a novel role for Erbin in inflammatory responses.

Regulation of cryopyrin/Pypaf1 signaling by pyrin, the familial Mediterranean fever gene product.

Cryopyrin, a member of the Nod protein family mutated in familial cold urticaria and Muckle-Wells syndrome, has been recently implicated in inflammation. However, the mechanism of activation and regulation of the cryopyrin signaling pathway remains poorly understood. We report here that co-expression of cryopyrin with its binding partner, ASC, induced both apoptosis and NF-kappaB activation. This signaling was mimicked by oligomerization of ASC, suggesting that cryopyrin activates downstream targets as reported for other Nod family members. Notably, pyrin, the product of the familial Mediterranean fever gene, inhibited cryopyrin-mediated apoptosis and NF-kappaB activation by disrupting the cryopyrin-ASC interaction. These results provide evidence for a cryopyrin signaling pathway activated through the induced proximity of ASC, which is negatively regulated by pyrin.

Caspy, a Zebrafish Caspase, Activated by ASC Oligomerization Is Required for Pharyngeal Arch Development*

The pyrin domain was identified recently in multiple proteins that are associated with apoptosis and/or inflammation, but the physiological and molecular function of these proteins remain poorly understood. We have identified Caspy and Caspy2, two zebrafish caspases containing N-terminal pyrin domains. Expression of Caspy and Caspy2 induced apoptosis in mammalian cells that were inhibited by general caspase inhibitors. Biochemical analysis revealed that both Caspy and Caspy2 are active caspases, but they exhibit different substrate specificity. Caspy, but not Caspy2, interacted with the zebrafish orthologue of ASC (zAsc), a pyrin- and caspase recruitment domain-containing protein identified previously in mammals. The pyrin domains of both Caspy and zAsc were required for their interaction. Furthermore, zAsc and Caspy co-localized to the “speck” when co-transfected into mammalian cells. Enforced oligomerization of zAsc, but not simple interaction with zAsc, induced specific proteolytic activation of Caspy and enhanced Caspy-dependent apoptosis. Injection of zebrafish embryos with a morpholino antisense oligonucleotide corresponding tocaspy resulted in an “open mouth” phenotype associated with defective formation of the cartilaginous pharyngeal skeleton. These studies suggest that zAsc mediates the activation of Caspy, a caspase that plays an important role in the morphogenesis of the jaw and gill-bearing arches.

Genetic variation and activity of mouse Nod2, a susceptibility gene for Crohn’s disease

Genetic variation in human Nod2 has been associated with susceptibility to Crohns disease. The mouse Nod2 locus is located at chromosome 8 and composed of 12 exons, 11 of which encode the Nod2 protein. Sequence analysis of Nod2 from 45 different strains of Mus musculus and Mus spretus revealed extensive polymorphism involving all exons of Nod2. Of the 140 polymorphic sites identified, 68 were located in the coding region, of which 28 created amino acid substitutions in Nod2. Expression of mouse Nod2 activated NF-kappaB and conferred responsiveness to bacterial components, an activity that was deficient in mutants corresponding to those associated with susceptibility to Crohns disease. These studies demonstrate a conserved role for Nod2 in the response to bacterial components and suggest that selective evolutionary pressure exerted by pathogens may have contributed to the high level of variability of Nod2 sequences in both humans and mice.

Cutting Edge: Crohn's Disease-Associated Nod2 Mutation Limits Production of Proinflammatory Cytokines To Protect the Host from Enterococcus faecalis-Induced Lethality

Nucleotide-binding oligomerization domain 2 (Nod2) mutations including L1007fsinsC are associated with the development of Crohn’s disease (CD). These CD-associated Nod2 mutations are common in healthy white populations, suggesting that they may confer some protective function, but experimental evidence is lacking. Using a mouse strain that expresses Nod22939iCstop, the equivalent of the L1007fsinsC mutation, we found that macrophages homozygous for Nod22939iCstop are impaired in the recognition of muramyl dipeptide and Enterococcus faecalis, a commensal bacterium that is a common cause of sepsis-associated lethality in humans. Notably, Nod2 deficiency and homozygocity for Nod22939iCstop were associated with reduced production of TNF-α and IL-6 and lethality after systemic infection with E. faecalis despite normal bacteria loads. Consistently, inhibition of TNF-α signaling protected wild-type mice from E. faecalis-induced lethality. These results suggest that the same Nod2 mutation can increase the susceptibility to CD, but also protect the host from systemic infection by a common enteric bacterium.

Protein Kinase C-associated Kinase (PKK) Mediates Bcl10-independent NF-κB Activation Induced by Phorbol Ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKCβ. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-κB (NF-κB) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-κB activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-κB activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-κB. A catalytic inactive PKK mutant inhibited NF-κB activation induced by phorbol ester and Ca2+-ionophore, but it did not block that mediated by tumor necrosis factor α, interleukin-1β, or Nod1. Inhibition of NF-κB activation by dominant negative PKK was reverted by co-expression of PKCβI, suggesting a functional association between PKK and PKCβI. PKK-mediated NF-κB activation required IKKα and IKKβ but not IKKγ, the regulatory subunit of the IKK complex. Moreover, NF-κB activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-κB signaling pathway that is independent of Bcl10 and IKKγ.