The Journal of Immunology Mechanism and Repertoire of ASC-Mediated Gene Expression1 Mizuho Hasegawa,2* Ryu Imamura,* Kou Motani,* Takumi Nishiuchi,† Norihiko Matsumoto,* Takeshi Kinoshita,* and Takashi Suda3* Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor molecule that mediates inflammatory and apoptotic signals. Although the role of ASC in caspase-1-mediated IL-1␤ and IL-18 maturation is well known, ASC also induces NF-␬B activation and cytokine gene expression in human cells. In this study, we investigated the molecular mechanism and repertoire of ASC-induced gene expression in human cells. We found that the specific activation of ASC induced AP-1 activity, which was required for optimal IL8 promoter activity. ASC activation also induced STAT3-, but not STAT1-, IFN-stimulated gene factor 3- or NF-AT-dependent reporter gene expression. The ASC-mediated AP-1 activation was NF-␬B-independent and primarily cell-autonomous response, whereas the STAT3 activation required NF-␬B activation and was mediated by a factor that can act in a paracrine manner. ASC-mediated AP-1 activation was inhibited by chemical or protein inhibitors for caspase-8, caspase-8-targeting small-interfering RNA, and p38 and JNK inhibitors, but not by a caspase-1 inhibitor, caspase-9 or Fas-associated death domain protein (FADD) dominant-negative mutants, FADD- or RICK-targeting small-interfering RNAs, or a MEK inhibitor, indicating that the ASC-induced AP-1 activation is mediated by caspase-8, p38, and JNK, but does not require caspase-1, caspase-9, FADD, RICK, or ERK. DNA microarray analyses identified 75 genes that were induced by ASC activation. A large proportion of them was related to transcription (23%), inflammation (21%), or cell death (16%), indicating that ASC is a potent inducer of inflammatory and cell death-related genes. This is the first report of ASC-mediated AP-1 activation and the repertoire of genes induced downstream of ASC activation. The Journal of Immunology, 2009, 182: 7655–7662. he caspase recruitment domain (CARD)4-containing teractions. In this context, ASC resembles Fas-associated death protein, apoptosis-associated speck-like protein con- domain protein (FADD), an adaptor protein that recruits T taining a CARD (ASC; also called TMS-1), was orig- caspase-8 to death receptors. In fact, ASC has been found to inally identified as a protein that forms large aggregates (called recruit caspase-1 to several members of the nucleotide-binding specks) in HL-60 human leukemia cells treated with chemo- domain and leucine-rich repeat (LRR)-containing proteins therapeutic agents (1), and as the product of a gene that is (NLRs), including cryopyrin (also called NLRP3), and to in- silenced in cancer cells by DNA methylation (2). Thus, ASC ␤ by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. duce caspase-1-mediated IL-1 maturation (3–6). Because has been implicated in apoptosis and tumor suppression. ASC these NLRs function as cytoplasmic sensors for invading bac- consists of an N-terminal pyrin domain and a C-terminal teria and viruses in cells, and because macrophages from ASC- CARD. Pyrin domain and CARD belong to the death domain- deficient mice are defective in the production of IL-1␤ upon fold domains, which engage in homophilic protein-protein in- microbial infection, ASC is considered to be an important mo- lecular component of the innate immune system (7–10). In terms of the proinflammatory activity of ASC, it has been *Division of Immunology and Molecular Biology, Cancer Research Institute, and †Division of Functional Genomics, Advanced Science Research Center, Kanazawa demonstrated that ASC by itself or in combination with NLRs, University, Kanazawa, Ishikawa, Japan including cryopyrin and CARD12 (also called NLRC4, IPAF, http://classic.jimmunol.org Received for publication February 8, 2008. Accepted for publication April 5, 2009. CLR2.1, and CLAN), induces NF-␬B activation when ex- The costs of publication of this article were defrayed in part by the payment of page pressed in HEK293 cells by genetic reconstitution (3, 11, 12). charges. This article must therefore be hereby marked advertisement in accordance Consistent with this, the knockdown of ASC expression by with 18 U.S.C. Section 1734 solely to indicate this fact. RNA interference in human monocytic/macrophagic cell lines 1 This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (Cancer) from the Ministry of Education, Culture, Sports, Science, and Tech- results in reduced NF-␬B activation as well as diminished IL-8 nology, the Japanese Government. and TNF-␣ production upon Porphyromonas gingivalis infec- Downloaded from 2 Current address: Department of Pathology, University of Michigan Medical School, tion (13), indicating that ASC plays an essential role in the Ann Arbor, MI 48109. transcriptional activation of inflammatory cytokine genes in hu- 3 Address correspondence and reprint requests to Dr. Takashi Suda, Division of Im- munology and Molecular Biology, Cancer Research Institute, Kanazawa University, man macrophages, although studies involving ASC-deficient 13-1 Takaramachi, Kanazawa, Ishikawa, 920-0934 Japan. E-mail address: sudat@ mice have indicated that ASC is not essential for the expression kenroku.kanazawa-u.ac.jp of cytokine genes and NF-␬B activation induced by microbial 4 Abbreviations used in this paper: CARD, caspase recruitment domain; APRE, acute- infection in murine macrophages (7, 14, 15). phase response element; ASC, apoptosis-associated speck-like protein containing a CARD; cmk, chloromethylketone; CLARP-S, a short isoform of caspase-like apoptosis To investigate the molecular mechanism of ASC-mediated regulatory protein; FADD, Fas-associated death domain protein; fmk, fluoromethylk- NF-␬B activation, we previously developed a chimeric protein etone; GAS, ␥-activated sequence; ISRE, IFN-stimulated response element; LRR, leucine-rich repeat; MDP, muramyl dipeptide; NLR, nucleotide-binding domain and (C12N2) consisting of the CARD from CARD12 and the nucle- LRR-containing protein; NOD, nucleotide-binding oligomerization domain; siRNA, otide-binding oligomerization domain (NOD) and LRRs from small-interfering RNA; TRE, 12-O-tetradecanoylphorbol-13-acetate-responsive element. NOD2 (also called NLRC2), a sensor for muramyl dipeptide Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 (MDP) (12). The C12N2 and ASC-expressing HEK293-derived www.jimmunol.org/cgi/doi/10.4049/jimmunol.0800448 7656 MECHANISM AND REPERTOIRE OF ASC-MEDIATED GENE EXPRESSION cell line (MAIL8) produces IL-8 upon MDP stimulation in a man- Measurement of IL-8 ␬ ner dependent on NF- B activation. Using this cell line, we dem- The amount of IL-8 in the culture supernatant was determined using the onstrated that caspase-8 is required for ASC-mediated NF-␬B ac- BD OptEIA set (BD Pharmingen). tivation and IL-8 production (12). In this study, to clarify the mechanism of ASC-mediated tran- Measurement of caspase-8 activity scriptional regulation of cytokine genes, we explored the ASC- Caspase-8 activity in cell lysates was determined using caspase-8 fluoro- mediated activation of the IL8 promoter and transcription fac- metric assay kit (MBL), according to the manufacturer’s protocol. tors. In addition, we performed microarray analyses to obtain an Microarray analysis overview of the genes that are up- or down-regulated upon ASC activation. Microarray analyses were performed according to the Agilent 60-mer Oligo Microarray Processing Protocol (Agilent). Total RNA was purified from MAIL8 or KBG cells cultured with or without 10 ng/ml MDP or Materials and Methods TNF-␣ for 16 h. The quality of RNA samples was assessed with the RNA Reagents 6000 Nano LabChip kit using Bioanalyzer 2100 (Agilent). Cy3 (for un- stimulated samples)- and Cy5 (for stimulated samples)-labeled cRNAs MDP was purchased from Sigma-Aldrich, and rTNF-␣ from Genzyme. were prepared using the low RNA input linear amplification kit (Agilent). Z-VAD-fluoromethylketone (fmk), z-AAD-chloromethylketone (cmk), Various mixtures of Cy3- and Cy5-labeled cRNAs were purified using the and the MAPK inhibitor set (PD98059, SB202190, SB203580, and RNeasy RNA purification kit (Qiagen), and then applied onto the Human SB202474) were from Calbiochem, and the JNK inhibitor SP600125 was 1A (v2) 22 k Oligo Microarray (Agilent) loaded with 20,173 DNA 60- from Alexis Biochemicals. mers, including those from 18,716 human genes. Hybridized and washed arrays were scanned using a microarray scanner G2565BA (Agilent). Mi- Cell lines croarray images were analyzed with Feature Extraction Software v7.5 (Agilent). The significance of the log ratio was assessed by computing the The KBG cell line is a HEK293-derived stable transfectant of a NF-␬B-GFP most conservative log ratio error and significance value ( p value), using a reporter construct. The MAIL8 cell line is a KBG-derived stable transfectant SE propagation algorithm (Agilent) and a universal error model (Rosetta expressing ASC and the FLAG-tagged C12N2 chimeric protein (12). In this Biosoftware). Single-linkage hierarchical clustering with the Euclidean dis- study, we used an apoptosis-resistant subline (MAIL8-102). tance metric was performed using MultiExperiment Viewer v4.0 (19). Plasmids RT-PCR Luciferase reporter plasmids carrying the IL8 promoter (Ϫ133-Luc) and DNA-free RNA was purified from cells using the RNeasy mini kit and its mutants were described previously (16, 17). Reporter plasmids RNase-free DNase set (Qiagen), and cDNA was prepared using an oligo- carrying 12-O-tetradecanoylphorbol-13-acetate-responsive elements (dT) primer and Superscript II reverse transcriptase (Invitrogen), accor- (TREs) (2ϫTRE-Luc), acute-phase response elements (APREs) ding to the manufacturer’s protocol. The following primers were used for (5ϫAPREsp-Luc), and NF-AT binding sites (pNFAT72-Luc) were pro- PCR: TNF forward, 5Ј-aaggacaccatgagcactga-3Ј and reverse, 5Ј-cgtttgg vided by K. Yoshioka (Kanazawa University Cancer Research Institute, gaaggttggatgtt-3Ј; IL-6 forward, 5Ј-ctccaggagcccagctatga-3Ј and reverse, Kanazawa, Japan), K.