Mechanism of ASC-mediated apoptosis: Bid-dependent apoptosis in type II cells

Apoptosis-associated speck-like protein containing aCARD (ASC) is an adaptormolecule that mediatesapoptotic and inflammatory signals, and implicated intumor suppression. However, the mechanism of ASC-mediatedapoptosishasnotbeenwellelucidated.Here,weinvestigated the molecularmechanisms of ASC-mediatedapoptosis in several cell lines using a caspase recruitmentdomain 12-Nod2 chimeric protein that transduces thesignal from muramyl dipeptide into ASC-mediatedapoptosis. Experiments using dominant-negative mutants,small-interfering RNAs and peptide inhibitors for cas-pases indicated that caspase-8 was generally requiredfor ASC-mediated apoptosis, whereas a requirement forcaspase-9 depended on the cell type. In addition, caspase-like apoptosis-regulatory protein (CLARP)/Fas-like in-hibitor protein, a natural caspase-8 inhibitor, suppressedASC-mediated apoptosis, and Clarp / mouse embryo-nic fibroblasts were highly sensitive to ASC-mediatedapoptosis. Bax-deficient HCT116 cells were resistant toASC-mediated apoptosis as reported previously, althoughwe failed to observe colocalization of ASC and Bax incells.LikeFas-ligand-inducedapoptosis,theASC-mediatedapoptosis was inhibited by Bcl-2 and/orBcl-XL in type-IIbut not type-I cell lines. Bid was cleaved upon ASCactivation, and suppression of endogenous Bid expressionusing small-interfering RNAs in type-II cells reducedthe ASC-mediated apoptosis. These results indicatethat ASC, like death receptors, mediates two types ofapoptosisdependingonthecelltype,inamannerinvolvingcaspase-8.Oncogene(2007) 26, 1748–1756.doi:10.1038/sj.onc.1209965;published online 11 September 2006Keywords: apoptosis;ASC;CARD12;caspase;Bid;BaxIntroductionASC (also called TMS1) is a cytoplasmic adaptorprotein consisting of a pyrin domain (PYD) and acaspase recruitment domain (CARD). This adaptorprotein links CARD12 (also called Ipaf or CLAN), amember of the Apaf-1-like proteins, to signal-transduc-tion pathways leading to apoptosis and nuclear factor-kappa B (NF-kB) activation (Masumoto et al., 2003).ASC also links several PYD-containing members of theApaf-1-like proteins (called NALPs or PYPAFs) withcaspase-1, and induces the caspase-1-mediated matura-tion of inflammatory cytokines, such as interleukin(IL)-1b (Martinon et al., 2002; Wang et al., 2002).Accordingly, ASC-deficient macrophages fail to secreteIL-1b and resist cell death upon intracellular infectionwith Salmonella(Mariathasan etal., 2004).Another line of studies has shown that the expressionof ASC is downregulated in various malignant tumors(McConnell and Vertino, 2004). In addition, the genesfor ASC and CARD12 are targets of p53, and arerequired for the apoptosis induced by p53 andchemotherapeutic drugs in some human cells (Ohtsukaet al., 2004; Sadasivam et al., 2005). Thus, theCARD12–ASC axis seems to play an important role intumor suppression and chemosensitivity of cancer cells.Several research groups have studied the molecularmechanism of ASC-mediated apoptosis, but the resultshave been controversial. It was first reported that adominant-negative mutant for caspase-9 but notcaspase-8 inhibits the apoptosis induced by overexpres-sing ASC in human embryonic kidney (HEK)293 cells(McConnell and Vertino, 2000). However, a dominant-negative mutant of caspase-8 but not caspase-9 inhibitsthe apoptosis induced by the coexpression of ASC andCARD12 or by the forced oligomerization of ASC in293T cells (Masumoto etal., 2003). It was also reportedthat ASC interacts with Bax and recruits it to themitochondria, implying the caspase-9-dependent path-way (Ohtsuka etal., 2004).The lack of information regarding the upstreammolecules that activate CARD12 or other ASC-activat-ing Apaf-1-like proteins has prevented us from investi-gating the functions of ASC under physiologicalconditions. (While we were revising this paper, aphysiological stimulus for CARD12 was reported to