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• W2039344765 abstract "Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis, NFκB activation, and cytokine regulation. In this study we identified a novel human protein, CARD-8, which contains a C-terminal CARD domain with high similarity to the CARD domain of caspase-1/ICE. We demonstrate that CARD-8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1β generation in the THP-1 monocytic cell line. CARD-8 binds also to ICEBERG and pseudo-ICE, two other recently identified proteins, which bind to the CARD domain of caspase-1 and negatively regulate its activity. Reverse transcriptase-PCR analysis revealed that CARD-8 is expressed mainly in monocytes, placenta, lymph nodes, and spleen. This pattern of expression is consistent with caspase-1 expression in the same cells and tissues. CARD-8 was also found to negatively regulate NF-κB activation by TNF-α stimulation and by ectopically expressed RICK, suggesting that this protein may control cell survival. Consistent with these results, stable expression of CARD-8 in U937 or THP-1 cells sensitizes the cells to differentiation-induced apoptosis. Overexpression of CARD-8 can also induce apoptosis in transfected cells. The results suggest that CARD-8 represents a new signaling molecule involved in the regulation of caspase-1 and NF-κB activation. Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis, NFκB activation, and cytokine regulation. In this study we identified a novel human protein, CARD-8, which contains a C-terminal CARD domain with high similarity to the CARD domain of caspase-1/ICE. We demonstrate that CARD-8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1β generation in the THP-1 monocytic cell line. CARD-8 binds also to ICEBERG and pseudo-ICE, two other recently identified proteins, which bind to the CARD domain of caspase-1 and negatively regulate its activity. Reverse transcriptase-PCR analysis revealed that CARD-8 is expressed mainly in monocytes, placenta, lymph nodes, and spleen. This pattern of expression is consistent with caspase-1 expression in the same cells and tissues. CARD-8 was also found to negatively regulate NF-κB activation by TNF-α stimulation and by ectopically expressed RICK, suggesting that this protein may control cell survival. Consistent with these results, stable expression of CARD-8 in U937 or THP-1 cells sensitizes the cells to differentiation-induced apoptosis. Overexpression of CARD-8 can also induce apoptosis in transfected cells. The results suggest that CARD-8 represents a new signaling molecule involved in the regulation of caspase-1 and NF-κB activation. Apoptosis or programmed cell death is an indispensable process for normal development and homeostasis (1.Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12927) Google Scholar, 2.Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6728) Google Scholar). Dysregulation of apoptosis has been correlated with degenerative diseases, autoimmune disorders, and cancer. Although the death signals that initiate the apoptotic program can originate from a number of sources, in most cases they lead to the activation of a family of cysteine proteases known as caspases (3.Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar, 4.Alnemri E.S. J. Cell. Biochem. 1997; 64: 33-42Crossref PubMed Scopus (290) Google Scholar, 5.Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar), which execute the apoptotic program. Despite the overall structural similarity and cleavage specificities shared by all caspases, not all caspases have a primary function in apoptosis. For instance, caspase-1 (also known as interleukin (IL) 1The abbreviations used are: ILinterleukinICEIL-1β-converting enzymeCARDcaspase-associated recruitment domainCARD-8FLfull-length CARD-8GSTglutathioneS-transferaseHAhemagglutininRT-PCRreverse transcriptase-PCRELISAenzyme-linked immunosorbent assayPMAphorbol 12-myristate 13-acetateLPSlipopolysaccharideTNFtumor necrosis factorNF-κBnuclear factor κBINFγinterferon γNTDN-terminal domainTRAILTNF-related apoptosis-inducing ligandzbenzyloxycarbonylFMKfluoromethylketoneAMC7-amino-4-methyl coumarinPIPESN-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)-1β-converting enzyme (ICE)) plays a key role in inflammatory response by cleaving pro-IL-1β and pro-IL-18 into active secreted cytokines (6.Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. et al.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2232) Google Scholar, 7.Cerretti D.P. Kozlosky C.J. Mosley B. Nelson N. Van Ness K. Greenstreet T.A. March C.J. Kronheim S.R. Druck T. Cannizzaro L.A. et al.Science. 1992; 256: 97-100Crossref PubMed Scopus (1007) Google Scholar, 8.Ghayur T. Banerjee S. Hugunin M. Butler D. Herzog L. Carter A. Quintal L. Sekut L. Talanian R. Paskind M. Wong W. Kamen R. Tracey D. Allen H. Nature. 1997; 386: 619-623Crossref PubMed Scopus (1049) Google Scholar, 9.Gu Y. Kuida K. Tsutsui H. Ku G. Hsiao K. Fleming M.A. Hayashi N. Higashino K. Okamura H. Nakanishi K. Kurimoto M. Tanimoto T. Flavell R.A. Sato V. Harding M.W. Livingston D.J. Su M.S. Science. 1997; 275: 206-209Crossref PubMed Scopus (1026) Google Scholar). At low concentrations IL-1β is a local inflammatory mediator of the activation of mononuclear cells and endothelial cells. However, at high concentrations IL-1β exerts potentially lethal systemic effects including fever, chills, and shock (10.Dinarello C.A. Chest. 1997; 112 (suppl.): 321-329Abstract Full Text Full Text PDF Scopus (414) Google Scholar, 11.O'Reilly M. Newcomb D.E. Remick D. Shock. 1999; 12: 411-420Crossref PubMed Scopus (69) Google Scholar). Caspase-1 has also been implicated in the Death receptor CD95/Fas apoptotic pathway because thymocytes derived from caspase-1-deficient animals are partially resistant to CD95-induced apoptosis (12.Kuida K. Lippke J.A. Ku G. Harding M.W. Livingston D.J. Su M.S. Flavell R.A. Science. 1995; 267: 2000-2003Crossref PubMed Scopus (1461) Google Scholar). interleukin IL-1β-converting enzyme caspase-associated recruitment domain full-length CARD-8 glutathioneS-transferase hemagglutinin reverse transcriptase-PCR enzyme-linked immunosorbent assay phorbol 12-myristate 13-acetate lipopolysaccharide tumor necrosis factor nuclear factor κB interferon γ N-terminal domain TNF-related apoptosis-inducing ligand benzyloxycarbonyl fluoromethylketone 7-amino-4-methyl coumarin N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) The caspase-associated recruitment domain (CARD) is a conserved homology domain, which mediates protein-protein interactions between key apoptotic signaling molecules. The CARD domain is present in the nematode CED-4 and mammalian Apaf-1 and is used to recruit CED-3 and caspase-9, respectively (13.Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 14.Chinnaiyan A.M. Chaudhary D. O'Rourke K. Koonin E.V. Dixit V.M. Nature. 1997; 388: 728-729Crossref PubMed Scopus (164) Google Scholar), into the apoptosome. After recruitment into the apoptosome these caspases undergo autocatalytic processing and become fully active. The CARD domain is also present in the prodomains of several other caspases including human caspase-1, -2, -4, and -5, and mouse caspase-1, -2, -11, and -12. The CARD of caspase-1 mediates its interaction with the CARDs of RICK and Ipaf/CARD-12 (15.Geddes B.J. Wang L. Huang W.J. Lavellee M. Manji G.A. Brown M. Jurman M. Cao J. Morgenstern J. Merriam S. Glucksmann M.A. DiStefano P.S. Bertin J. Biochem. Biophys. Res. Commun. 2001; 284: 77-82Crossref PubMed Scopus (97) Google Scholar, 16.Poyet J.L. Srinivasula S.M. Tnani M. Razmara M. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2001; 276: 28309-28313Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar), two adaptor molecules that have been implicated in the activation of caspase-1. The CARD of caspase-1 also mediates its interaction with the dominant-negative CARD-only proteins ICEBERG and pseudo-ICE, which block caspase-1 activation (17.Humke E.W. Shriver S.K. Starovasnik M.A. Fairbrother W.J. Dixit V.M. Cell. 2000; 103: 99-111Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 18.Druilhe A. Srinivasula S.M. Razmara M. Ahmad M. Alnemri E.S. Cell Death Differ. 2001; 8: 649-657Crossref PubMed Scopus (147) Google Scholar). The CARD-containing caspases have been shown to play important roles in diseases through gene knockout studies in mice. For example, caspase-1 knockout mice exhibit marked resistance to endotoxin-induced sepsis. Caspase-2 and caspase-11 knockout mice show less tissue loss in stroke models (19.Bergeron L. Perez G.I. Macdonald G. Shi L. Sun Y. Jurisicova A. Varmuza S. Latham K.E. Flaws J.A. Salter J.C. Hara H. Moskowitz M.A. Li E. Greenberg A. Tilly J.L. Yuan J. Genes Dev. 1998; 12: 1304-1314Crossref PubMed Scopus (605) Google Scholar, 20.Wang S. Miura M. Jung Y.K. Zhu H. Li E. Yuan J. Cell. 1998; 92: 501-509Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar). In addition to its role in apoptosis, the CARD domain mediates interactions of several upstream components of the NF-κB signaling pathway that play a role in the activation of genes involved in immunity, inflammation, and apoptosis. The CARD-containing adaptor protein RICK interacts with the CARD proteins CARD-4/Nod1 and Nod2 to form a large complex that activates the IKK complex (21.Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 22.Ogura Y. Inohara N. Benito A. Chen F.F. Yamaoka S. Nunez G. J. Biol. Chem. 2001; 276: 4812-4818Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar, 23.Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Similarly, the CARD-containing protein Bcl-10, which is implicated in the activation of NF-κB in response to stimulation of the antigen receptors in T and B cells, forms protein complexes with the upstream CARD proteins CARD-9, CARD-10, CARD-11, and CARD-14 (21.Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 24.Poyet J.L. Srinivasula S.M. Alnemri E.S. J. Biol. Chem. 2001; 276: 3183-3187Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 25.Bertin J. Wang L. Guo Y. Jacobson M.D. Poyet J.L. Srinivasula S.M. Merriam S. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 276: 11877-11882Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 26.Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.L. Merriam S. Du M.Q. Dyer M.J. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2000; 275: 41082-41086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 27.Wang L. Guo Y. Ke X. Huang W.J. Poyet J.L. Manji G.A. Merriam S. Glucksmann M.A. DiStefano P.S. Alnemri E.S. Bertin J. J. Biol. Chem. 2001; 276: 21405-21409Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In this study we identified and characterized a new member of the CARD-containing family of proteins designated CARD-8, which binds to caspase-1 and negatively regulates its activity. CARD-8 can also negatively regulate NF-κB activation and sensitize cells to apoptosis. Cells were cultured either in Dulbecco's modified Eagle's medium (for 293T; Phoenix) or RPMI 1640 (for MCF-7; THP-1) supplemented with 10% fetal bovine serum, 200 μg/ml penicillin, and 100 μg/ml streptomycin sulfatein. RPMI 1640 for THP-1 also contained 10 mm PIPES, 1 mm sodium pyruvate, and 55 μm β-mercaptoethanol. The full-length open reading frame of CARD-8 (CARD-8FL) was cloned by PCR using CARD-8 adaptor primers in modified HA-pCI, FLAG-pCMV, pRSC-LacZ, pEGFP, or pMSCV neo vectors. CARD-8 NTD (residues 1–340) and CARD-8-CARD (residues 341–431) were constructed by inserting the indicated domains in the same vectors. In vitro binding assays with CARD-8 and CARD proteins were performed as described previously (28.Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar). Briefly, CARD-8FL and CARD-8-CARD were expressed in DH5-α bacteria as GST fusion proteins, and equal amounts of proteins were immobilized on glutathione-Sepharose (Amersham Biosciences).35S-labeled CARD-8FL, caspase-1, pseudo-ICE, ICEBERG, caspase-9, Bcl-10, and CRADD were prepared using the TNT-coupled transcription/translation kit (Promega) and incubated with the protein-bound Sepharose beads in 100 μl of binding buffer (50 mm Tris-HCl, pH 7.6, 120 mm NaCl, 0.5% Brij, and protease inhibitors) for 3 h. The beads were washed three times with the same buffer, boiled in SDS sample buffer, and visualized by SDS-PAGE and autoradiography. 293T cells (5 × 106) in 100-mm dishes were transiently transfected with the expression plasmid(s) using the LipofectAMINETM reagent (Invitrogen). Cells were lysed in a lysis buffer (50 mm Tris, pH 7.6, 150 mm NaCl, 0.1% Nonidet P-40) and incubated with anti-FLAG-M2/M5 monoclonal antibody (Sigma) or HA.11 monoclonal antibody (Babco). The immune complexes were precipitated with protein G-Sepharose, washed extensively, and boiled in SDS sample buffer. The proteins were resolved by SDS-PAGE and detected by Western blot analysis with a horseradish peroxidase-conjugated T7 antibody (Novagen), anti-HA-peroxidase (Roche Molecular Biochemicals), or anti-caspase-1 (Santa Cruz). The total lysates were also resolved by SDS-PAGE and detected by Western analysis using anti-FLAG-M2/M5, anti-HA-peroxidase, or T7 antibody. Full-length CARD-8 was subcloned into the adenovirus transfer vector pLE11φ. This placed the gene of interest under the transcriptional control of a tetracycline-regulated promoter. An internal ribosome entry site downstream to the gene of interest allowed a modified green fluorescent protein, KGFP (Kelly Theriault, Millennium Pharmaceuticals, Inc.), to be expressed off the same transcript. Adenovirus was generated by homologous recombination in 911 cells followed by plaque purification. VERO cells were infected with recombinant adenovirus expressing either CARD-8 or KGFP at a multiplicity of infection of 20. Cells were fixed 36 or 56 h after infection. The nuclei were then stained with Hoescht 33342, and the percentage of apoptotic versus healthy infected cells was scored. The apoptotic assays in MCF-7-Fas cells were performed as described previously (29.Srinivasula S.M. Ahmad M. Ottilie S. Bullrich F. Banks S. Wang Y. Fernandes-Alnemri T. Croce C.M. Litwack G. Tomaselli K.J. Armstrong R.C. Alnemri E.S. J. Biol. Chem. 1997; 272: 18542-18545Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 30.Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). The amphitropic packaging cell line Phoenix (G. P. Nolan's laboratory, Stanford University Medical Center, Stanford, CA) was transfected with pMSCV neo vectors using the calcium phosphate/chloroquine method (31.Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Crossref PubMed Scopus (672) Google Scholar). Forty-eight hours after transfection, the media of the cells containing retroviral particles were then collected and incubated with THP-1 cells (1 × 106 cells/well) in three cycles of infection in the presence of polybrene (Sigma). After changing the media, THP-1 cells were then selected using 1 mg/ml neomycin (Invitrogen). After 3 weeks of selection, viable cells were used for detection of protein and IL-1β. To assay for IL-1β, we incubated the cells (1 × 106 cells/ml) for 4 h with INFγ and then for 14 h with 1 μg/ml of LPS. Media of the cells were then used to quantify IL-1β by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). NF-κB activation was performed using a luciferase reporter gene. 293 cells were transfected with 5× κB-luciferase reporter, pRSC-LacZ plasmids, and various expression plasmids using the LipofectAMINETM method according to the manufacturer's instructions. 24 h after transfection, cells were harvested and subjected to luciferase assay as described by Lin et al. (32.Lin X. Mu Y. Cunningham Jr., E.T. Marcu K.B. Geleziunas R. Greene W.C. Mol. Cell. Biol. 1998; 18: 5899-5907Crossref PubMed Google Scholar). In certain experiments, cells were treated with hTNF-α for 5 h prior to harvesting. To normalize for transfection efficiency, all lysates were assayed for β-galactosidase activity. Data represent the average of at least three different individual experiments. To identify and characterize new proteins involved in regulation of apoptosis, inflammation, and NF-κB activation, we searched the Millennium Pharmaceutical data base of expressed sequence tags (EST) for clones encoding CARD motifs. We identified an EST sequence encoding a novel CARD-containing protein with the calculated molecular mass of 48 kDa (Fig. 1A). A Blast search of the protein data base indicated that this protein contains at least two putative functional domains. The C-terminal region (residues 341–431) shares significant similarity to CARD motifs found in many apoptotic proteins, including those found in caspase-1/ICE (34% identity, 47% similarity) (Fig. 1B). The N-terminal domain has a high similarity to NAC/DEFCAP-L/CARD-7 (39% identity, 52% similarity) and contains several candidate phosphorylation sites including protein kinase C ((S/T)X(R/K)) sites at amino acids 72, 286, 313, and 416, casein kinase II ((S/T)X(D/E)) sites at 289, 376, 398, 414, and 416, and Map kinase/CDK ((S/T)P) sites at 187 and 289, which may serve to regulate CARD activity. This CARD-containing protein was designated CARD-8. To determine the tissue distribution of CARD-8 we performed RT-PCR analysis, using primers complementary to 5′ and 3′ of the CARD-8 open reading frame. RT-PCR analysis of multiple tissue and cell line mRNA revealed that CARD-8 is expressed mainly in placenta, spleen, lymph node, and bone marrow tissues and in the monocytic THP-1 cell line (Fig. 2, A and B). Similar distribution was observed for caspase-1, pseudo-ICE, and Ipaf (10.Dinarello C.A. Chest. 1997; 112 (suppl.): 321-329Abstract Full Text Full Text PDF Scopus (414) Google Scholar, 16.Poyet J.L. Srinivasula S.M. Tnani M. Razmara M. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2001; 276: 28309-28313Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). These results suggest that these genes may be under similar transcriptional regulation. Because CARD/CARD interactions are highly selective and most CARD-containing proteins segregate with discrete binding partners and modulate intracellular signaling pathways, we determined whether CARD-8 interacts with any of the known CARD proteins involved in apoptosis, inflammation, or NFκB activation. Based on the sequence homology between the prodomain of caspase-1 and the CARD domain of CARD-8, we tested whether CARD-8 could bind to caspase-1 and other CARD-containing proteins by in vitro GST pull-down assays. To this end,35S-labeled caspase-1, pseudo-ICE, ICEBERG, Bcl-10, and CRADD were incubated with GST fusion proteins of full-length CARD-8 (GST-CARD-8FL) and the isolated CARD domain of CARD-8 (GST-CARD-8-CARD). Among these proteins caspase-1 and pseudo-ICE interacted with both GST-CARD-8FL and the GST-CARD-8-CARD, whereas ICEBERG interacted only with the GST-CARD-8FL (Fig. 3A). Because pseudo-ICE shares high homology (∼93% identity) with the CARD domain of caspase-1, these observations suggest that CARD-8 interacts with caspase-1 through CARD-CARD interaction. This was confirmed by performing a reverse interaction between 35S-labeled CARD-8FL and an isolated caspase-1 CARD-GST fusion protein. Consistent with the above results, the isolated caspase-1 CARD-GST fusion protein but not the GST control was able to interact with CARD-8FL. (Fig. 3B). To further confirm the specificity of the CARD domain of CARD-8 toward caspase-1, we compared the binding of 35S-labeled caspase-1 and caspase-9 to the isolated CARD domain of CARD-8 (GST-CARD-8-CARD). CARD-8-CARD was only able to interact with caspase-1 but not with caspase-9 (Fig. 3C), indicating that CARD-8 specifically associates with caspase-1. Both caspase-1 and caspase-9 were able to interact with the GST-pseudo-ICE and GST-Apaf-1-CARD positive controls, respectively (Fig. 3C). To determine whether CARD-8 interacts with ICE, pseudo-ICE, and ICEBERG in transfected cells, T7-tagged caspase-1-C287A, pseudo-ICE, or ICEBERG were transfected together with FLAG-tagged CARD-8 into 293T cells. Total cell lysates were immunoprecipitated with the FLAG antibody, and the immunoprecipitated products were analyzed by Western blotting with the T7 antibody. As shown in Fig. 4A, caspase-1, pseudo-ICE, and ICEBERG were all able to associate with CARD-8. Of note, pseudo-ICE and ICEBERG were able to compete with caspase-1 for binding to CARD-8 because there was less binding of caspase-1 to CARD-8 in the presence of these two proteins (Fig. 4, A and B). The concept that oligomerization of caspases promotes autoprocessing has been investigated in different studies (33.Gu Y. Wu J. Faucheu C. Lalanne J.L. Diu A. Livingston D.J. Su M.S. EMBO J. 1995; 14: 1923-1931Crossref PubMed Scopus (132) Google Scholar, 34.Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar). These studies also suggest that self-association occurs through the prodomain of these caspases (35.Van Criekinge W. Beyaert R. Van de Craen M. Vandenabeele P. Schotte P. De Valck D. Fiers W. J. Biol. Chem. 1996; 271: 27245-27248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Activation of many large prodomain initiator caspases is mediated by association with their respective upstream adaptor molecules. Inhibiting and/or displacing these upstream activators will result in decreased activation of the interacting caspases. Overexpression of caspase-1 leads to its oligomerization and autoprocessing. RICK is an adaptor molecule that has been shown to enhance the activation of caspase-1 by promoting its oligomerization. To determine the effect of CARD-8 on the processing of caspase-1, 293T cells were transiently transfected with FLAG-tagged wild type caspase-1 and FLAG-tagged RICK together with or without CARD-8FL. As shown in Fig. 5A, cotransfection of CARD-8 with caspase-1 significantly decreased the 35-kDa processed form of caspase-1 that results from the autoprocessing of caspase-1 itself. Cotransfection of CARD-8 with caspase-1 and RICK also diminished the RICK-induced processing of caspase-1 into its 35- and 18-kDa fragments. These results suggest that CARD-8 interferes with caspase-1 activation possibly by preventing its oligomerization or its association with its adaptor molecule RICK. IL-1β secretion is one of the consequences of caspase-1 activation. Because CARD-8 binds to caspase-1 and interferes with its activation, we reasoned that it should also decrease IL-1β generation by caspase-1. To test this hypothesis we measured the effect of ectopically expressed CARD-8 on IL-1β secretion from 293T cells transiently transfected with wild type caspase-1 and IL-1β precursor. As shown in Fig. 5B, CARD-8 significantly diminished processing/activation of caspase-1 into its 35-kDa fragment in the transfected 293 cells. As a consequence, IL-1β generation was also inhibited by CARD-8 expression in these cells (Fig. 5B). Similar results were obtained with the caspase-1 activation inhibitor ICEBERG (Fig. 5B). Stable expression of CARD-8 in the THP-1 monocytic cell line significantly decreased caspase-1 activity in response to LPS stimulation in extracts of these cells (Fig. 5C). IL-1β processing and generation were also diminished by CARD-8 in these cells (Fig. 5, D and E). These effects of CARD-8 may be attributed to its ability to bind to caspase-1 and interfere with its activation and/or activity. Consistent with this, the stably transfected CARD-8 was able to immunoprecipitate the endogenous caspase-1 from the THP-1 cells (Fig. 5F). Several CARD-containing proteins are critical regulators of proinflammatory cytokine-induced NF-κB activation (18.Druilhe A. Srinivasula S.M. Razmara M. Ahmad M. Alnemri E.S. Cell Death Differ. 2001; 8: 649-657Crossref PubMed Scopus (147) Google Scholar, 21.Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 26.Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.L. Merriam S. Du M.Q. Dyer M.J. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2000; 275: 41082-41086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 36.Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 37.Thome M. Hofmann K. Burns K. Martinon F. Bodmer J.L. Mattmann C. Tschopp J. Curr. Biol. 1998; 8: 885-888Abstract Full Text Full Text PDF PubMed Google Scholar, 38.Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar). Because our data suggest that CARD-8 is a negative regulator of IL-1β generation, which is a major pathway in the proinflammatory cytokine response, we decided to test the possibility that CARD-8 may also negatively regulate NF-κB activation by the proinflammatory cytokine TNF-α. As shown in Fig. 6A, ectopic expression of the full-length CARD-8 or its isolated NTD significantly decreased NF-κB activation by TNF-α. Interestingly, expression of the isolated CARD domain of CARD-8 had an opposite effect and resulted in enhancement of TNF-α-induced NF-κB activation. These results indicate that the NTD is responsible for the observed NF-κB inhibition by CARD-8. CARD-8 was also able to suppress NF-κB activation by ectopically expressed RICK, a known activator of NF-κB (Fig. 6B). These results are consistent with the recent findings of Bouchier-Hayes et al. who demonstrated that CARDINAL (CARD-8) can inhibit multiple pathways of NF-κB activation (39.Bouchier-Hayes L. Conroy H. Egan H. Adrain C. Creagh E.M. MacFarlane M. Martin S.J. J. Biol. Chem. 2001; 276: 44069-44077Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, we were unable to reproduce the finding that CARDINAL (CARD-8) interacts with IKKγ/NEMO (data not shown). Combined, the above results suggest that CARD-8 might be an important negative regulator of the proinflammatory cytokine response by acting at both the IL-1β generation and NF-κB activation levels. The NF-κB signaling pathway is essential for cell survival (40.Lee R. Collins T. Circ. Res. 2001; 88: 262-264Crossref PubMed Scopus (64) Google Scholar). Phorbol 12-myristate 13-acetate (PMA)-induced differentiation of the promonocytic cell line U937 is associated with persistent NF-κB activation (41.Hida A. Kawakami A. Nakashima T. Yamasaki S. Sakai H. Urayama S. Ida H. Nakamura H. Migita K. Kawabe Y. Eguchi K. Immunology. 2000; 99: 553-560Crossref PubMed Scopus (43) Google Scholar). Inhibition of NF-κB activation by a dominant negative IκB-α mutant or the NF-κB inhibitor pyrrolidine dithiocarbamate during PMA-induced differentiation leads to cell death (41.Hida A. Kawakami A. Nakashima T. Yamasaki S. Sakai H. Urayama S. Ida H. Nakamura H. Migita K. Kawabe Y. Eguchi K. Immunology. 2000; 99: 553-560Crossref PubMed Scopus (43) Google Scholar, 42.Pennington K.N. Taylor J.A. Bren G.D. Paya C.V. Mol. Cell. Biol. 2001; 21: 1930-1941Crossref PubMed Scopus (70) Google Scholar). These findings suggest that NF-κB activation is essential for survival of U937 cells induced to differentiate with PMA. Consistent with the ability of CARD-8 to inhibit NF-κB activation, we found that U937 and THP-1 cells stably transfected with a CARD-8 expression construct (but not an empty vector) undergo apoptosis in response to PMA-induced differentiation (Fig. 7A). These results indicate that CARD-8 may play an apoptotic role during cellular differentiation by inhibiting NF-κB activation. To determine whether transient overexpression of CARD-8 can induce apoptosis in MCF-7 cells, we transfected MCF-7 cells with HA-tagged CARD-8 or an empty vector and then stained the transfected cells with EthD-1, which stains selectively the nuclei of damaged or dead cells but not healthy cells. We also stained these cells with calcein AM, which stains preferentially live cells more intensely than dead cells, because live cells contain more estrase than dead cells to convert the nonfluorescent calcein AM to the intensely fluorescent calcein. As shown in Fig. 7B, the CARD-8 transfected cells showed significantly more EthD-1 and less calcein staining than the empty vector transfected cells, indicating that CARD-8 can indeed induce cell death in MCF-7 cells. The apoptotic activity of CARD-8 in MCF-7 cells was not as potent as that of the death domain-containing adaptor molecule FADD, but it was comparable with the activities of the CARD-containing adaptor molecules CRADD/RAIDD and Bcl-10 (Fig. 7C). Consistent with these results, CARD-8 can also potentiate Fas, TNF, and TRAIL-induced apoptosis in MCF-7 cells (data not shown). To determine the apoptotic activity of CARD-8 in another cell line we infected VERO cells with a recombinant adenovirus expressing either CARD-8 or KGFP. The percentage of apoptotic and control cells was scored using Hoescht 33342. As shown in Fig. 7D, CARD-8-overexpressing cells exhibited significantly more apoptotic nuclei after 36 or 56 h compared with control cells (15%versus 5 and 45% versus 12%, respectively), thus confirming the above observations in MCF-7 cells. To gain insight into the mechanisms by which CARD-8 induces apoptosis, we tested the effects of five inhibitors of apoptosis on MCF-7 cells transfected with CARD-8 (Fig. 8A). The direct caspase inhibitors zVAD-FMK, baculovirus p35, and CrmA were able to inhibit CARD-8-induced apoptosis suggesting that CARD-8 induces apoptosis via activation of caspases. Bcl-xL and caspase-9-C287A can also inhibit CARD-8-induced apoptosis suggesting that CARD-8 activates caspases by activating the Apaf-1-caspase-9 apoptotic complex. Our data are in complete contrast to recent findings, which indicated that TUCAN (CARD-8) is an antiapoptotic protein that inhibits caspase-9 activation by binding to the CARD region of procaspases-9 (43.Pathan N. Marusawa H. Krajewska M. Matsuzawa S. Kim H. Okada K. Torii S. Kitada S. Krajewski S. Welsh K. Pio F. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 32220-32229Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). As shown in Fig. 3C we did not see significant binding of CARD-8 to procaspases-9. In addition, S100 extracts prepared from 293 cells transfected with a CARD-8 expression construct had significantly more caspase cleaving activity than the control empty vector S100 extracts in the presence or absence of cytochrome c and dATP (Fig. 8, B and C). These observations indicate that overexpression of CARD-8 does not inhibit activation of caspases in S100 extracts by cytochrome c and dATP as suggested recently (43.Pathan N. Marusawa H. Krajewska M. Matsuzawa S. Kim H. Okada K. Torii S. Kitada S. Krajewski S. Welsh K. Pio F. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 32220-32229Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). On the contrary, CARD-8 overexpression can indeed activate caspases, which may explain its ability to induce apoptosis in transfected cells. These effects may be all related to its ability to inhibit the NF-κB survival pathway in the transfected cells. In conclusion we have identified and characterized the function of a new member of the human CARD-containing family of proteins. The CARD domain of CARD-8 has a high degree of homology to the CARD domain of caspase-1 and can bind to caspase-1 and its related proteins pseudo-ICE and ICEBERG. CARD-8 attenuates ICE activity and thereby decreasing IL-1β secretion. RT-PCR studies revealed that CARD-8 has the same pattern of expression as caspase-1. CARD-8 can also negatively regulate NF-κB activation by diverse stimuli, suggesting that this protein may control cell survival. Consistent with these results stable expression of CARD-8 sensitizes cells to differentiation-induced apoptosis. Furthermore, overexpression of CARD-8 can induce apoptosis in transfected cells. Although the precise function of CARD-8 is not clear, the results suggest that it may function as an adaptor molecule regulating caspase-1 activation (IL-1β production), NF-κB activation, and apoptosis. AF322184" @default.
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• W2039344765 title "CARD-8 Protein, a New CARD Family Member That Regulates Caspase-1 Activation and Apoptosis" @default.
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