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Originally published In Press as doi:10.1074/jbc.M107811200 on January 30, 2002

J. Biol. Chem., Vol. 277, Issue 16, 13952-13958, April 19, 2002
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CARD-8 Protein, a New CARD Family Member That Regulates Caspase-1 Activation and Apoptosis*

Marjaneh RazmaraDagger , Srinivasa M. SrinivasulaDagger §, Lin Wang, Jean-Luc PoyetDagger , Brad J. Geddes, Peter S. DiStefano, John Bertin||, and Emad S. AlnemriDagger **

From the Dagger  Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and  Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139

Received for publication, August 14, 2001, and in revised form, January 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis, NFkappa B 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-1beta 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-kappa B activation by TNF-alpha 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-kappa B activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Apoptosis or programmed cell death is an indispensable process for normal development and homeostasis (1, 2). 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 (3-5), 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)1-1beta -converting enzyme (ICE)) plays a key role in inflammatory response by cleaving pro-IL-1beta and pro-IL-18 into active secreted cytokines (6-9). At low concentrations IL-1beta is a local inflammatory mediator of the activation of mononuclear cells and endothelial cells. However, at high concentrations IL-1beta exerts potentially lethal systemic effects including fever, chills, and shock (10, 11). 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).

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, 14), 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, 16), 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, 18). 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, 20).

In addition to its role in apoptosis, the CARD domain mediates interactions of several upstream components of the NF-kappa B 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 (21-23). Similarly, the CARD-containing protein Bcl-10, which is implicated in the activation of NF-kappa B 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, 24-27).

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-kappa B activation and sensitize cells to apoptosis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cell Culture-- 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 beta -mercaptoethanol.

Plasmid Construction and cDNA Cloning-- 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-- In vitro binding assays with CARD-8 and CARD proteins were performed as described previously (28). Briefly, CARD-8FL and CARD-8-CARD were expressed in DH5-alpha 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.

Transfection, Immunoprecipitation, and Immunoblot Analysis-- 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.

Recombinant Adenovirus Construction and Infection Protocol-- Full-length CARD-8 was subcloned into the adenovirus transfer vector pLE11phi . 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.

Apoptosis Assays-- 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, 30).

Infection of THP-1 Cells and Assay of IL-1beta -- 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). 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-1beta . To assay for IL-1beta , we incubated the cells (1 × 106 cells/ml) for 4 h with INFgamma and then for 14 h with 1 µg/ml of LPS. Media of the cells were then used to quantify IL-1beta by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Assay of NF-kappa B Activation-- NF-kappa B activation was performed using a luciferase reporter gene. 293 cells were transfected with 5× kappa B-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). In certain experiments, cells were treated with hTNF-alpha for 5 h prior to harvesting. To normalize for transfection efficiency, all lysates were assayed for beta -galactosidase activity. Data represent the average of at least three different individual experiments.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Identification of CARD-8-- To identify and characterize new proteins involved in regulation of apoptosis, inflammation, and NF-kappa B 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.


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  		Fig. 1.   Amino acid sequence and domain structure of CARD-8. A, domain structure of CARD-8 with the predicted amino acid sequence. The CARD domain (residues 341-431) near the carboxyl terminus of the protein is underlined. B, the amino acid sequence of the CARD domain of CARD-8 was aligned with several other CARDs. Identical residues are indicated in black shading.

Tissue and Cell Line Distribution-- 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, 16). These results suggest that these genes may be under similar transcriptional regulation.


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  		Fig. 2.   Expression of CARD-8 mRNA in adult human tissues and cell lines. Total RNA samples from different human tissues (A) and cell lines (B) were amplified by RT-PCR with primers specific for CARD-8FL and beta -actin and then analyzed on agarose gel and stained with ethidium bromide. Plasmid containing CARD-8 was used as a control template for the PCR reaction.

Identification of CARD-8 Interacting Proteins-- 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 NFkappa B 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).


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  		Fig. 3.   In vitro interaction of CARD-8 and CARD-8-CARD with other CARD proteins. A, GST, CARD-8FL-GST, or CARD-8-CARD-GST proteins bound to glutathione-Sepharose beads were incubated with in vitro-translated 35S-labeled caspase-1, pseudo-ICE, ICEBERG, BCL-10, or CRADD. The interactions were analyzed by SDS-PAGE and autoradiography. B, GST or caspase-1-CARD-GST were incubated with in vitro-translated 35S-labeled CARD-8FL and then analyzed as above. C, GST, CARD-8-CARD-GST, pseudo-ICE-GST, or Apaf-1-CARD-GST proteins were incubated with in vitro-translated 35S-labeled caspase-1 or caspase-9 as indicated. The interactions were analyzed as in A. The binding data are representative of at least three different experiments.

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).


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  		Fig. 4.   Interaction of CARD-8 with other CARD proteins in transfected 293 cells. A and B, 293T cells were cotransfected with expression constructs for T7-caspase-1-C287A, FLAG-CARD-8FL, and T7-ICEBERG or T7-pseudo-ICE. 36 h after transfection cells were lysed, and the lysates were immunoprecipitated with FLAG antibody. The immunoprecipitates were immunoblotted with anti-T7 or FLAG antibodies as indicated. The binding data in A and B are representative of at least three different experiments.

Effect of CARD-8 on Processing of Caspase-1-- The concept that oligomerization of caspases promotes autoprocessing has been investigated in different studies (33, 34). These studies also suggest that self-association occurs through the prodomain of these caspases (35). 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.


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  		Fig. 5.   Effect of CARD-8 on the processing of caspase-1 and IL-beta secretion. A, 293T cells were transiently transfected with empty vector or HA-CARD-8FL, FLAG-caspase-1 wild type, and FLAG-RICK in different combinations as indicated. 36 h after transfection total lysates were fractionated by SDS-PAGE and detected by Western blot analysis using anti-FLAG-M2/M5. The p35 and p18 processed fragments of caspase-1 are indicated. B, 293 cells were cotransfected with pro-IL-1beta together with empty vector, CARD-8FL, ICEBERG, caspase-1, caspase-1 plus CARD-8FL, or caspase-1 plus ICEBERG expression constructs as indicated. After 36 h, total lysates from the transfected cells were fractionated by SDS-PAGE and detected by Western blot analysis using anti-FLAG-M2/M5 (upper panel). The p35 processed fragments of caspase-1 are indicated. The culture media were also assayed for IL-1beta by ELISA (lower panel). C, THP-1 cells stably infected with control retroviral vector (control) or retroviral vector encoding HA-CARD-8FL (CARD-8) were treated with 1 µg/ml LPS for 1 h. S100 extracts were prepared from total cell lysates, and the total amount of the proteins in the samples was normalized. The S100 extracts were incubated with 100 µM fluorogenic caspase-1 tetrapeptide substrate Ac-WEHD-AMC. Production of AMC was monitored in the indicated times at room temperature by spectrofluorimetry. D, THP-1 cells stably infected with control retroviral vector (control) or retroviral vector encoding HA-CARD-8FL (CARD-8) were treated with INFgamma for 4 h and then LPS for 3 or 15 h as indicated. Cells were lysed, and the total cellular lysates were assayed by Western blotting for endogenous IL-1beta processing using an anti-IL-1beta polyclonal antibody. NT, non-treated control cells. E, THP1 cells were infected with an empty retroviral vector (control) or a retroviral vector encoding an HA-tagged CARD-8FL (CARD-8). After selection in G418 (1 mg/ml), stable THP1 cells were assayed for CARD-8 expression by Western blot analysis with an HA antibody (inset). THP1 cells were then treated with IFNgamma and LPS for 18 h. IL-1beta secretion in the culture media was quantified by ELISA. F, THP-1 cells stably infected with control retroviral vector (control) or retroviral vector encoding HA-CARD-8FL (CARD-8) were lysed and incubated with HA.II monoclonal antibody (Babco). The complexes were bound to protein G-Sepharose, and then eluted by boiling in SDS sample buffer. The eluted proteins were resolved by SDS-PAGE and detected by Western analysis with caspase-1 or HA antibodies. The data in A-F are representative of at least three different experiments.

Effect of CARD-8 on Interleukin-1beta Generation by Caspase-1-- IL-1beta 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-1beta generation by caspase-1. To test this hypothesis we measured the effect of ectopically expressed CARD-8 on IL-1beta secretion from 293T cells transiently transfected with wild type caspase-1 and IL-1beta 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-1beta 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-1beta 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).

CARD-8 Is a Negative Regulator of NF-kappa B Activation-- Several CARD-containing proteins are critical regulators of proinflammatory cytokine-induced NF-kappa B activation (18, 21, 26, 36-38). Because our data suggest that CARD-8 is a negative regulator of IL-1beta 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-kappa B activation by the proinflammatory cytokine TNF-alpha . As shown in Fig. 6A, ectopic expression of the full-length CARD-8 or its isolated NTD significantly decreased NF-kappa B activation by TNF-alpha . Interestingly, expression of the isolated CARD domain of CARD-8 had an opposite effect and resulted in enhancement of TNF-alpha -induced NF-kappa B activation. These results indicate that the NTD is responsible for the observed NF-kappa B inhibition by CARD-8. CARD-8 was also able to suppress NF-kappa B activation by ectopically expressed RICK, a known activator of NF-kappa B (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-kappa B activation (39). However, we were unable to reproduce the finding that CARDINAL (CARD-8) interacts with IKKgamma /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-1beta generation and NF-kappa B activation levels.


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  		Fig. 6.   CARD-8 inhibits TNF-alpha - and RICK-induced NF-kappa B activation. A, 293 cells were transfected with 5× kappa B-luciferase reporter together with either empty vector or expression constructs for FLAG-CARD-8FL, FLAG-CARD-8NTD, and FLAG-CARD-8-CARD. 24 h after transfection, cells were either left untreated or incubated with TNF-alpha for 5 h. Cells were then collected and lysed, and the luciferase activity in the cell lysates was determined. pRSC-LacZ was included in all transfection reactions to normalize the transfection efficiency. Total cell lysates also were analyzed by immunoblotting with anti-FLAG horseradish peroxidase (HRP) antibody (upper panel). B, 293 cells were transfected with a C-terminal FLAG-tagged RICK expression construct alone or with FLAG-CARD-8FL in the presence of 5× kappa B-luciferase reporter and pRSC-LacZ constructs. The luciferase assays were performed 24 h post-transfection and normalized to beta -galactosidase activity for evaluating the transfection efficiency. Total cell lysates were also analyzed by immunoblotting with an anti-FLAG HRP antibody (upper panel). The data in A and B are representative of at least three different experiments.

CARD-8 Is an Apoptotic Protein-- The NF-kappa B signaling pathway is essential for cell survival (40). Phorbol 12-myristate 13-acetate (PMA)-induced differentiation of the promonocytic cell line U937 is associated with persistent NF-kappa B activation (41). Inhibition of NF-kappa B activation by a dominant negative Ikappa B-alpha mutant or the NF-kappa B inhibitor pyrrolidine dithiocarbamate during PMA-induced differentiation leads to cell death (41, 42). These findings suggest that NF-kappa B activation is essential for survival of U937 cells induced to differentiate with PMA. Consistent with the ability of CARD-8 to inhibit NF-kappa B 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-kappa B activation.


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  		Fig. 7.   CARD-8 is an apoptotic protein. A, U937 and THP-1 cells stably infected with control retroviral vector (control) or retroviral vector encoding HA-CARD-8FL (CARD-8) were treated with 50 and 200 nM phorbol 12-myristate 13-acetate, respectively. After 50 (U937) or 24 h (THP-1) the cellular viability was determined by trypan blue dye exclusion assay and represented as the percentage of dead cells relative to the total cells counted. The percentages of dead cells in the untreated cultures (5-8%) were subtracted from the percentages obtained with PMA-treated cultures. CARD-8 expression was determined by Western blot analysis using anti-HA horseradish peroxidase antibody (insets). B, MCF-7 cells were transfected with HA-tagged CARD-8 and control vector. 24 h after transfection cells were stained with calcein AM and EthD-1 using LIVE/DEAD Viability/Cytotoxicity kit (Molecular Probes, Eugene, OR) and then visualized under a fluorescent microscope. Note the red-stained cells (EthD-1 panels), which indicate dead cells. C, MCF-7 cells were transiently transfected with the green fluorescent protein construct along with either empty vector or FADD, CRADD, Bcl-10, and CARD-8FL expression vectors. After 36 h of transfection, dead cells were visualized and counted by fluorescent microscopy. D, 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 then were stained with Hoescht 33342, and the percentage of apoptotic versus healthy transfected cells was then scored.

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.


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  		Fig. 8.   Characterization of CARD-8-induced apoptosis. A, MCF-7 were transiently transfected with pRSC-LacZ-CARD-8FL construct and treated with zVAD-FMK or cotransfected with pRSC-LacZ-CARD-8FL and Bcl-xL, p35, CrmA, or caspase-9-C287A expression constructs. After 36 h cells were stained in situ for beta -galactosidase activity using the beta -Gal staining kit from Invitrogen and scored as described under "Experimental Procedures." B and C, 293T cells were transfected with empty vector alone or CARD-8FL. After 36 h, S100 extracts were prepared from total cell lysates, and the total amount of proteins in the samples was normalized. The S100 extracts were incubated with Ac-DEVD-AFC in the presence (B) or absence (C) of cytochrome c and dATP for the indicated periods of time. Generation of the fluorogenic AFC was measured (relative fluorescence units (RFU) continuously over time by spectrofluorimetry). The data in A-C are representative of at least three different experiments.

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). 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). 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-kappa B 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-1beta secretion. RT-PCR studies revealed that CARD-8 has the same pattern of expression as caspase-1. CARD-8 can also negatively regulate NF-kappa B 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-1beta production), NF-kappa B activation, and apoptosis.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA85421 and AG14357 (to E. S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF322184.

§ Special Fellow of the Leukemia and Lymphoma Society.

** To whom correspondence may be addressed: Thomas Jefferson University, Kimmel Cancer Inst., Bluemle Life Sciences Bldg., Rm. 904, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4632; Fax: 215-923-1098; E-mail: E_Alnemri@lac.jci.tju.edu.

|| To whom correspondence may be addressed: Millennium Pharmaceuticals, Inc., 640 Memorial Dr., Cambridge, MA 02139. Tel.: 617-679-7215; Fax: 617-679-7071; E-mail: bertin@mpi.com.

Published, JBC Papers in Press, January 30, 2002, DOI 10.1074/jbc.M107811200

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; ICE, IL-1beta -converting enzyme; CARD, caspase-associated recruitment domain; CARD-8FL, full-length CARD-8; GST, glutathione S-transferase; HA, hemagglutinin; RT-PCR, reverse transcriptase-PCR; ELISA, enzyme-linked immunosorbent assay; PMA, phorbol 12-myristate 13-acetate; LPS, lipopolysaccharide; TNF, tumor necrosis factor; NF-kappa B, nuclear factor kappa B; INFgamma , interferon gamma ; NTD, N-terminal domain; TRAIL, TNF-related apoptosis-inducing ligand; z, benzyloxycarbonyl; FMK, fluoromethylketone; AMC, 7-amino-4-methyl coumarin; PIPES, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239-257[Medline] [Order article via Infotrieve]
2. Wyllie, A. H., Kerr, J. F., and Currie, A. R. (1980) Int. Rev. Cytol. 68, 251-306[Medline] [Order article via Infotrieve]
3. Hengartner, M. O. (2000) Nature 407, 770-776[CrossRef][Medline] [Order article via Infotrieve]
4. Alnemri, E. S. (1997) J. Cell. Biochem. 64, 33-42[CrossRef][Medline] [Order article via Infotrieve]
5. Green, D. R. (1998) Cell 94, 695-698[CrossRef][Medline] [Order article via Infotrieve]
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.. (1992) Nature 356, 768-774[CrossRef][Medline] [Order article via Infotrieve]
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.. (1992) Science 256, 97-100[Abstract/Free Full Text]
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., and Allen, H. (1997) Nature 386, 619-623[CrossRef][Medline] [Order article via Infotrieve]
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., and Su, M. S. (1997) Science 275, 206-209[Abstract/Free Full Text]
10. Dinarello, C. A. (1997) Chest 112 (suppl.), 321-329
11. O'Reilly, M., Newcomb, D. E., and Remick, D. (1999) Shock 12, 411-420[CrossRef][Medline] [Order article via Infotrieve]
12. Kuida, K., Lippke, J. A., Ku, G., Harding, M. W., Livingston, D. J., Su, M. S., and Flavell, R. A. (1995) Science 267, 2000-2003[Abstract/Free Full Text]
13. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[CrossRef][Medline] [Order article via Infotrieve]
14. Chinnaiyan, A. M., Chaudhary, D., O'Rourke, K., Koonin, E. V., and Dixit, V. M. (1997) Nature 388, 728-729[CrossRef][Medline] [Order article via Infotrieve]
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., and Bertin, J. (2001) Biochem. Biophys. Res. Commun. 284, 77-82[CrossRef][Medline] [Order article via Infotrieve]
16. Poyet, J. L., Srinivasula, S. M., Tnani, M., Razmara, M., Fernandes-Alnemri, T., and Alnemri, E. S. (2001) J. Biol. Chem. 276, 28309-28313[Abstract/Free Full Text]
17. Humke, E. W., Shriver, S. K., Starovasnik, M. A., Fairbrother, W. J., and Dixit, V. M. (2000) Cell 103, 99-111[CrossRef][Medline] [Order article via Infotrieve]
18. Druilhe, A., Srinivasula, S. M., Razmara, M., Ahmad, M., and Alnemri, E. S. (2001) Cell Death Differ. 8, 649-657[CrossRef][Medline] [Order article via Infotrieve]
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., and Yuan, J. (1998) Genes Dev. 12, 1304-1314[Abstract/Free Full Text]
20. Wang, S., Miura, M., Jung, Y. K., Zhu, H., Li, E., and Yuan, J. (1998) Cell 92, 501-509[CrossRef][Medline] [Order article via Infotrieve]
21. Inohara, N., Koseki, T., Lin, J., del Peso, L., Lucas, P. C., Chen, F. F., Ogura, Y., and Nunez, G. (2000) J. Biol. Chem. 275, 27823-27831[Abstract/Free Full Text]
22. Ogura, Y., Inohara, N., Benito, A., Chen, F. F., Yamaoka, S., and Nunez, G. (2001) J. Biol. Chem. 276, 4812-4818[Abstract/Free Full Text]
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., and DiStefano, P. S. (1999) J. Biol. Chem. 274, 12955-12958[Abstract/Free Full Text]
24. Poyet, J. L., Srinivasula, S. M., and Alnemri, E. S. (2001) J. Biol. Chem. 276, 3183-3187[Abstract/Free Full Text]
25. Bertin, J., Wang, L., Guo, Y., Jacobson, M. D., Poyet, J. L., Srinivasula, S. M., Merriam, S., DiStefano, P. S., and Alnemri, E. S. (2001) J. Biol. Chem. 276, 11877-11882[Abstract/Free Full Text]
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., and Alnemri, E. S. (2000) J. Biol. Chem. 275, 41082-41086[Abstract/Free Full Text]
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., and Bertin, J. (2001) J. Biol. Chem. 276, 21405-21409[Abstract/Free Full Text]
28. Ahmad, M., Srinivasula, S. M., Wang, L., Talanian, R. V., Litwack, G., Fernandes-Alnemri, T., and Alnemri, E. S. (1997) Cancer Res. 57, 615-619[Abstract/Free Full Text]
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., and Alnemri, E. S. (1997) J. Biol. Chem. 272, 18542-18545[Abstract/Free Full Text]
30. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489[CrossRef][Medline] [Order article via Infotrieve]
31. Kinsella, T. M., and Nolan, G. P. (1996) Hum. Gene Ther. 7, 1405-1413[Medline] [Order article via Infotrieve]
32. Lin, X., Mu, Y., Cunningham, E. T., Jr., Marcu, K. B., Geleziunas, R., and Greene, W. C. (1998) Mol. Cell. Biol. 18, 5899-5907[Abstract/Free Full Text]
33. Gu, Y., Wu, J., Faucheu, C., Lalanne, J. L., Diu, A., Livingston, D. J., and Su, M. S. (1995) EMBO J. 14, 1923-1931[Medline] [Order article via Infotrieve]
34. Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., and Alnemri, E. S. (1998) Mol. Cell. 1, 949-957[CrossRef][Medline] [Order article via Infotrieve]
35. Van Criekinge, W., Beyaert, R., Van de Craen, M., Vandenabeele, P., Schotte, P., De, Valck, D., and Fiers, W. (1996) J. Biol. Chem. 271, 27245-27248[Abstract/Free Full Text]
36. Srinivasula, S. M., Ahmad, M., Lin, J. H., Poyet, J. L., Fernandes-Alnemri, T., Tsichlis, P. N., and Alnemri, E. S. (1999) J. Biol. Chem. 274, 17946-17954[Abstract/Free Full Text]
37. Thome, M., Hofmann, K., Burns, K., Martinon, F., Bodmer, J. L., Mattmann, C., and Tschopp, J. (1998) Curr. Biol. 8, 885-888[CrossRef][Medline] [Order article via Infotrieve]
38. Kelliher, M. A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B. Z., and Leder, P. (1998) Immunity 8, 297-303[CrossRef][Medline] [Order article via Infotrieve]
39. Bouchier-Hayes, L., Conroy, H., Egan, H., Adrain, C., Creagh, E. M., MacFarlane, M., and Martin, S. J. (2001) J. Biol. Chem. 276, 44069-44077[Abstract/Free Full Text]
40. Lee, R., and Collins, T. (2001) Circ. Res. 88, 262-264[Free Full Text]
41. Hida, A., Kawakami, A., Nakashima, T., Yamasaki, S., Sakai, H., Urayama, S., Ida, H., Nakamura, H., Migita, K., Kawabe, Y., and Eguchi, K. (2000) Immunology 99, 553-560[CrossRef][Medline] [Order article via Infotrieve]
42. Pennington, K. N., Taylor, J. A., Bren, G. D., and Paya, C. V. (2001) Mol. Cell. Biol. 21, 1930-1941[Abstract/Free Full Text]
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., and Reed, J. C. (2001) J. Biol. Chem. 276, 32220-32229[Abstract/Free Full Text]

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