CARD6 Is Interferon Inducible but Not Involved in Nucleotide-Binding Oligomerization Domain Protein Signaling Leading to NF-κB Activation

Abstract

We have previously reported the cloning and characterization of CARD6, a caspase recruitment domain (CARD)-containing protein that is structurally related to the interferon (IFN)-inducible GTPases. CARD6 associates with microtubules and with receptor-interacting protein 2 (RIP2). RIP2 mediates NF-B activation induced by the intracellular nucleotide-binding oligomerization domain (NOD) receptors that sense bacterial peptidoglycan. Here we report that the expression of CARD6 and RIP2 in bone marrow-derived macrophages is rapidly induced by beta IFN and gamma IFN. This IFN-induced upregulation of CARD6 is suppressed by lipopolysaccharide (LPS), in contrast to LPS's enhancement of IFN-induced RIP2 upregulation. We generated CARD6-deficient (CARD6 ؊/؊ ) mice and carried out extensive analyses of signaling pathways mediating innate and adaptive immune responses, including the NOD pathways, but did not detect any abnormalities. Moreover, CARD6؊/؊ mice were just as susceptible as wild-type mice to infection by Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Candida albicans, lymphocytic choriomeningitis virus, or mouse adenovirus type 1. Thus, although structural and in vitro analyses strongly suggest an important role for CARD6 in immune defense, the physiological function of CARD6 remains obscure.The caspase recruitment domain (CARD) is a homotypic protein-protein interaction module that links components of signal transduction pathways implicated in the regulation of apoptosis or adaptive or innate immunity (2, 37). Although much progress has been made in assigning precise roles to most CARD-containing proteins, the functions of the 1,037-amino-acid (aa) human and 1,175-aa mouse CARD6 proteins are still unknown. CARD6 has a unique structure in that it contains the CARD at the N terminus, a glutamic acid-rich region following the CARD, and a proline-rich region at the C terminus (7). CARD6 also harbors a ϳ350-aa region with similarity to upregulated gene 4 (URG4), a protein that is induced in response to hepatitis Bx antigen overexpression and exerts a positive effect on proliferation (36). Both CARD6 and URG4 share structural features with members of the multifaceted, interferon (IFN)-inducible GTPase (IFNiGTPase) superfamily, which contains some of the proteins most abundantly induced during cell-autonomous immune responses (6).We are only now beginning to understand the functions of the IFNiGTPases (reviewed in reference 24). The Mx proteins were among the first IFNiGTPases identified and were shown to comprise cell-autonomous factors conferring resistance to viral infection (13). Three other groups of IFNiGTPases are the p47 GTPases, the guanylate-binding-protein GTPases, and the very large inducible GTPases (VLIGs). Members of the p47 GTPase family interact with phagocytic and secretory pathways to oppose infection by enforcing disposal and presentation of bacterial and protozoan antigens (22). The functions of the guanylate-binding proteins and the VLIGs are still not clear. The prototypical VLIG...

Caspase Recruitment Domain 6 Protects Against Cardiac Hypertrophy in Response to Pressure Overload

“…6 Notably, CARD6 is expressed ubiquitously in various tissues including the heart, 5 but it is unclear whether its expression levels are altered under pathological stimulus. In the present study, we found that in response to hypertrophic stress, CARD6 expression was increased progressively in adaptive hypertrophy (the first 2 and 4 weeks after hypertrophy stimuli) and then markedly decreased in maladaptive cardiac remodeling (the past 4 weeks).…”

“…A detailed Methods section is available in the online-only Data Supplement, which includes detailed methods on the following: Reagents, Human Heart Samples, Animal Models and Procedures, 6 Echocardiography Evaluation, 13,14 Histological Analysis, Quantitative Real-Time Polymerase Chain Reaction (PCR) and Western Blotting, Cultured Neonatal Rat Cardiomyocytes (NRCMs) and Recombinant Adenoviral Vectors, [14][15][16] and Statistical Analysis.…”

“…8 In addition, CARD6 regulates the innate and adaptive immune responses, and it plays critical roles in infectious diseases. 6 Interestingly, an increasing number of studies has supported the involvement of immune and tumor modulatory factors in modulating cardiac hypertrophy, such as signal regulatory protein-α, interferon regulatory factor 7, Ras-association domain family 1 isoform A, and A20. [9][10][11][12] These functional impacts of modulatory factors on cardiac hypertrophy and pathological remodeling suggest that CARD6 may exert prominent regulatory effects on cardiac pathology.…”

“…5 CARD6 expression can be induced by interferon in a variety of cell types, including T lymphocytes, B lymphocytes, thymocytes, and splenocytes. 6 Once expressed, CARD6 either transmits or amplifies the intracellular signals to downstream effector molecules, which activate or suppress intracellular signal pathways to regulate biological processes. 5,7 Previous studies have implicated CARD6 as a tumor activator gene in gastric, colorectal, and esophageal cancers.…”

Acidic nuclear phosphoprotein 32kDa (ANP32)B-deficient mouse reveals a hierarchy of ANP32 importance in mammalian development

“…The growth of primary MEFs in culture was analyzed as previously described (35). Purified B cells were treated with 1 μg/mL LPS for 24 h. Purified T cells were stimulated by seeding on plates precoated with anti-CD3 and anti-CD28 antibodies as previously described (45). Statistical differences in cell growth were assessed using Student's t test.…”