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. Nakajima (Osaka City University Medical School, 5Ј-cccatgctacatttgccgaa-3Ј; IL-8 forward, 5Ј-cagttttgccaaggagtgctaa-3Ј and Osaka, Japan), and S. Miyatake (Tokyo Metropolitan Institute of Medical reverse, 5Ј-aacttctccacaaccctctgc-3Ј; CXCL2 forward, 5Ј-aaccgcctgctgagc Science, Tokyo, Japan), respectively. Reporter plasmids carrying NF-␬B cccat-3Ј and reverse, 5Ј-ccttcaggaacagccaccaa-3Ј; ␤-actin forward, 5Ј-tc binding sites (pNF-␬B-Luc) were purchased from Stratagene, and those cctggagaagagctacga-3Ј and reverse, 5Ј-aaagccatgccaatctcatc-3Ј; FOS for- Ј Ј Ј Ј by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. carrying IFN-stimulated response elements (ISREs) (pISRE-TA-Luc) and ward, 5 -caccgacctgcctgcaagat-3 and reverse, 5 -cagctcgggcagtggcactt-3 ; ␥-activated sequences (GAS) (pGAS-TA-Luc) were purchased from BD JUNB forward, 5Ј-ccacctcccgtttacaccaa-3Ј and reverse, 5Ј-gccttgagcgtct Clontech. Expression plasmids for a short isoform of caspase-like apopto- tcacctt-3Ј; JUN forward, 5Ј-cgaactgcacagccagaaca-3Ј and reverse, 5Ј-tcct sis regulatory protein (CLARP-S), a constitutive active mutant of CARD12 gggactccatgtcgat-3Ј; RELB forward, 5Ј-agctacggcgtggacaagaa-3Ј and re- (CARD12⌬LRR, aa 1–457); dominant-negative mutants of FADD (aa 80– verse, 5Ј-atcgcggggctacgtggctt-3Ј; NFKBIA forward, 5Ј-ccgagactttcgaggaaat 208), caspase-8B (C377S), caspase-9 (C287S), and c-Jun (aa 123–334); a-3Ј and reverse, 5Ј-tcagcccctttgcactcataa-3Ј; and NFKBIZ forward, 5Ј-gcac and a proteasome-resistant I␬B␣ mutant (S32A, S36A) were described aggtgaacaccacaga-3Ј and reverse, 5Ј-gagctaatacggtggagctct-3Ј. previously (12, 17, 18). Results Small-interfering RNAs (siRNAs) ASC induces AP-1 activation, leading to IL-8 promoter

http://classic.jimmunol.org The FADD-, caspase-8-, and RICK-targeting siRNAs were described pre- activation viously (17). ASC-targeting siRNA and control siRNA were purchased We previously established HEK293 cell-derived stable cell line from Dharmacon (si-GENOM D-004378-03 and D-001210-05, respec- tively) or Invitrogen (Stealth Select RNAi HSS147064 and Stealth RNAi (MAIL8) that expresses ASC and C12N2, and found that MDP Negative Control Low GC Duplex, respectively). MAIL8 cells were trans- stimulation induces the C12N2-mediated oligomerization of fected with double-stranded siRNA using Lipofectamine 2000 or ASC and ASC-mediated NF-␬B activation, leading to IL-8 pro- RNAiMAX (Invitrogen), according to the manufacturer’s protocol. In duction in this cell line (12). In this response, ASC activation some experiments, cells were transfected with reporter plasmids using lin-

Downloaded from ␬ ear polyethyleneimine 12 h later. Forty-eight hours after the initial trans- and NF- B activation occur primarily in a same cell, so that the fection, the cells were used for assays, unless otherwise specified. NF-␬B activation does not depend on externalization or secre- tion of a certain factor that can act in a paracrine manner. In this Reporter assay meaning, the ASC-mediated NF-␬B activation is a cell-auton- Reporter assays were performed, as described previously (12). In brief, omous response. In the current study, we first sought to clarify cells were transfected with one of the firefly luciferase reporter plasmids further the molecular mechanisms of ASC-mediated IL8 gene and a control plasmid (pRL-TK) constitutively expressing Renilla lucif- expression. Although some sublines of MAIL8 are killed by erase, using linear polyethyleneimine. The firefly and Renilla luciferase apoptosis upon MDP stimulation (20), many MAIL8 sublines as activities were measured 24 h after the transfection using the dual-lucif- well as the parental HEK293 cell line are resistant to ASC- erase reporter assay system (Promega). The fold induction of luciferase activity ϭ experimental relative luciferase activity/relative luciferase ac- mediated apoptosis. To avoid the influence of cell death on tivity of unstimulated cells or vector control, where relative luciferase ac- ASC-mediated gene expression, we used an apoptosis-resistant tivity ϭ firefly luciferase activity/Renilla luciferase activity. subline (MAIL8-102) throughout this study. The IL8 promoter region contains three important cis-acting el- Western blotting ements for IL8 gene transcription, namely the NF-␬B, AP-1, and Western blotting was conducted, as described previously (17, 18). NF-IL-6 binding sites (16), and IL8 transcription requires either The Journal of Immunology 7657

FIGURE 1. ASC activates the IL8 promoter through AP-1 activation in MAIL8 cells, and the ASC-induced AP-1 activation is an NF-␬B-independent and cell-autonomous response. A, MAIL8 cells were transfected with a reporter plasmid carrying a luciferase gene driven by the wild-type IL8 promoter or this promoter mutated at the NF-␬B, AP-1, or NF-IL-6 binding site, and were stimulated with 10 ng/ml MDP or left unstimulated during the last 16 h of a 32-h culture. B, MAIL8 cells were cotransfected with the wild-type IL8 promoter reporter plasmid and an expression plasmid for a dominant-negative mutant of c-Jun (cJun-DN) or the DNA binding domain of Gal4 (Gal4-DB), and stimulated as in A. C, MAIL8 cells were transfected with an AP-1 reporter plasmid and stimulated as in A. D, HEK293 cells were transfected with an empty vector, or expression vectors for C12N2 and/or ASC as indicated together with an AP-1 reporter plasmid, and then stimulated as in A. E, MAIL8 cells were sequentially transfected with siRNA (control or ASC-targeting siRNA) and an AP-1 reporter plasmid with a 12-h interval. Cells were then stimulated as in A. Knockdown of ASC expression was confirmed by Western blot. GAPDH serves as an internal control to show equal loading. F, HEK293 cells were transfected with an empty vector, or expression vectors for by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. CARD12⌬LRR and/or ASC as indicated together with an AP-1 reporter plasmid. AP-1 activity was determined 24 h after the transfection. G, MAIL8 cells were transfected with an AP-1 reporter plasmid and stimulated as in A, except that the cells were pretreated with or without 10 ␮M MG132 for 1 h before MDP stimulation. H, MAIL8 cells were cotransfected with the AP-1 reporter plasmid together with either empty vector or an expression plasmid for I␬B␣-SR, and stimulated as in A. I, MAIL8 cells and HEK293 cells were cultured separately in two wells and transfected with an empty vector or an AP-1 reporter plasmid, as indicated. Twelve hours later, the cells in the two wells were mixed and stimulated with 10 ng/ml MDP or left unstimulated during the last 16 h of a further 24-h culture. A–I, IL8 promoter and AP-1 activation was evaluated by a luciferase reporter assay.

the combination of the NF-␬B and NF-IL-6 or of the NF-␬B and rately (Fig. 1D), indicating that C12N2 and ASC cooperatively http://classic.jimmunol.org AP-1 binding sites, depending on the type of cell or stimulation transduce the MDP signal into AP-1 activation. Furthermore, (21, 22). To determine which elements contribute to the ASC- knockdown of ASC expression using ASC-targeting siRNA abro- mediated IL8 promoter activation, we used a luciferase reporter gated MDP-induced AP-1 activation in MAIL8 cells (Fig. 1E), gene construct under the control of the minimal essential promoter confirming that the observed AP-1 activation was mediated region of the IL8 gene (–133 to ϩ44 bp), which contains all three by ASC. elements, and a series of constructs with a mutation in each of the MDP stimulation induced NF-␬B and AP-1 activation also in an

Downloaded from elements. Consistent with our above-described previous findings, apoptosis-sensitive subline of MAIL8 cells (supplemental Fig. ASC activation (induced by MDP stimulation) resulted in potent S1),5 indicating that these responses are not restricted to apoptosis- transcriptional activity of the wild-type IL8 promoter in MAIL8 resistant sublines. To exclude the possibility that ASC could me- cells, and a mutation in the NF-␬B binding site diminished the diate AP-1 activation only in the experimental system using the promoter activity. In addition, a mutation in the AP-1, but not the C12N2 chimera protein, a LRR-truncated form of CARD12 NF-IL-6 binding site caused a severe reduction in the ASC-in- (CARD12⌬LRR) that can constitutively activate ASC was used duced IL8 promoter activation (Fig. 1A). Furthermore, a c-Jun in place of C12N2 (Fig. 1F). As expected, coexpression of dominant-negative mutant that inhibits AP-1 activity inhibited the CARD12⌬LRR and ASC in HEK293 cells induced AP-1 activa- ASC-induced IL8 promoter activity, whereas the Gal4 DNA bind- tion, whereas either one alone did not. These results further ing domain used as a control did not significantly affect the activity support the notion that ASC has a potential to mediate AP-1 (Fig. 1B). Consistent with these results, MDP stimulation also in- activation. duced the expression of an AP-1-reporter construct carrying tan- dem TREs (Fig. 1C). MDP induced the AP-1 activation in HEK293 cells only when ASC and C12N2 were expressed simul- taneously, but not when these components were expressed sepa- 5 The online version of this article contains supplemental material. 7658 MECHANISM AND REPERTOIRE OF ASC-MEDIATED GENE EXPRESSION

FIGURE 2. ASC activates APRE-reporter gene expression by a non- cell-autonomous mechanism involving NF-␬B activation. A, MAIL8 cells were transfected with a reporter plasmid carrying APREs, ISREs, GAS, or NF-AT binding sites, and stimulated with 10 ng/ml MDP or left unstimu- lated during the last 16 h of a 32-h culture. The observed weak induction of the NF-AT reporter gene by MDP was not reproducible. B, MAIL8 cells were transfected with the APRE reporter plasmid and stimulated as in A, except that the cells were pretreated with or without 10 ␮M MG132 for 1 h FIGURE 3. Caspase-8 is essential for the ASC-induced AP-1 activa- before MDP stimulation. C, MAIL8 cells and HEK293 cells were cultured tion. A, MAIL8 cells were cotransfected with the AP-1 reporter plasmid separately in two wells and transfected with an empty vector or an APRE and an expression plasmid for a dominant-negative mutant of caspase-8 or reporter plasmid, as indicated. Twelve hours later, the cells in the two wells -9 (casp8-DN or casp9-DN), and stimulated with 10 ng/ml MDP or left were mixed and stimulated with 10 ng/ml MDP or left unstimulated during unstimulated during the last 16 h of a 32-h culture. B, MAIL8 cells were the last 16 h of a further 24-h culture. A–C, Reporter activity was deter- cotransfected with the AP-1 reporter plasmid and an expression plasmid for mined by luciferase assay. CLARP-S or a dominant-negative mutant of FADD (FADD-DN), and stimulated as in A. C, MAIL8 cells were cotransfected with the AP-1 re- porter plasmid and siRNA-targeting caspase-8, FADD, or RICK (10 nM), Pretreatment with the proteasome inhibitor MG132 and expres- and stimulated with 10 ng/ml MDP or left unstimulated during the last 16 h sion of a proteasome-resistant mutant of I␬B␣ (I␬B␣-SR) that in- of a 48-h culture. D, MAIL8 cells were transfected with the AP-1 reporter hibits NF-␬B activation failed to inhibit the MDP-induced AP-1 plasmid and stimulated as in A, except that the cells were pretreated with or without Z-VAD-fmk (pan-caspase inhibitor), Ac-YVAD-cmk

by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. activation in MAIL8 cells (Fig. 1, G and H). Conversely, the c-Jun ␬ (caspase-1 inhibitor), z-IETD-fmk (caspase-8 inhibitor), or Z-AAD-cmk dominant-negative mutant failed to inhibit NF- B activation under (granzyme B inhibitor) for 1 h before MDP stimulation (20 ␮M). A–D, the same conditions (data not shown). Thus, the MDP-induced AP-1 activation was evaluated by a luciferase reporter assay. NF-␬B and AP-1 activation were independent of each other. To clarify whether MDP induces AP-1 activation in a manner that is cell autonomous or dependent on a factor externalized on the cell surface or in the medium, MAIL8 or HEK293 cells was strongly inhibited by MG132 and I␬B␣-SR (Fig. 2B and data were transfected with an AP-1 reporter plasmid, and these cell not shown). Furthermore, potent APRE activity was induced in lines were then cocultured in the presence or absence of MDP. HEK293 cells cocultured with MAIL8 cells upon MDP stimula- http://classic.jimmunol.org MDP potently induced AP-1 activation in MAIL8 cells, but only tion (Fig. 2C). These results indicate that the ASC-mediated APRE weak AP-1 activation was observed in HEK293 cells under these activation was mainly mediated by an externalized factor, whose conditions (Fig. 1I), indicating that MDP induced AP-1 activation production depended on NF-␬B activation. largely in a cell-autonomous manner. These data together with our previous results (12) indicate that Caspase-8 is involved in the ASC-mediated AP-1 activation the oligomerization of ASC induced by C12N2 upon MDP stim- Because caspase-8 plays an important role in ASC-mediated

Downloaded from ulation causes the cell-autonomous activation of AP-1 and NF-␬B NF-␬B activation (12), we investigated whether it is also involved independent of each other, and AP-1 and NF-␬B then synergisti- in the ASC-mediated AP-1 activation. The expression of a cally induce the transcriptional activity of the IL8 promoter. caspase-8 dominant-negative mutant or of CLARP (also called FLIP, a natural inhibitor protein for caspase-8), but not of domi- ASC-mediated STAT3 activation depends on an externalized nant-negative mutants of caspase-9 or FADD, inhibited the MDP- factor induced AP-1 activation in MAIL8 cells (Fig. 3, A and B). Con- To investigate whether the activation of ASC induces other in- sistent with this, the knockdown of caspase-8, but not of FADD flammatory transcription factors, we introduced reporter genes car- expression by their corresponding siRNAs inhibited this response rying tandem APREs (STAT3 binding sites), ISREs (ISGF-3 bind- (Fig. 3C). In addition, the knockdown of RICK, which is essential ing sites), GAS (STAT1 binding site), or NF-AT binding sites in for NOD2-mediated NF-␬B activation (23, 24), did not affect this MAIL8 cells. MDP induced the expression of the APRE reporter response. Furthermore, the pan-caspase inhibitor z-VAD-fmk and gene, but not of the other reporter genes (Fig. 2A), indicating that caspase-8 inhibitor z-IETD-fmk inhibited this response, whereas the activation of ASC induces STAT3, but not STAT1, ISGF-3 (a the caspase-1 inhibitor Ac-YVAD-cmk and granzyme B inhibitor complex of STAT1, STAT2, and IFN regulatory factor 9), or z-AAD-cmk did not significantly affect it (Fig. 3D). These results NF-AT activity. Unlike the AP-1 activation, the APRE activation indicate that the catalytic activity of caspase-8 is required for the The Journal of Immunology 7659

FIGURE 4. JNK and p38 activities are required for the ASC-induced AP-1 activation and IL-8 production. A, MAIL8 cells were stimulated with 10 ng/ml MDP for the indicated periods. The whole-cell lysates were then assayed by Western blotting using Abs against phosphorylated or entire JNK1/2, p38, or ERK. Whole-cell lysates after UV treatment (500 J/m2) were used as a positive control for phosphorylated JNK1/2 and p38. B and C, MAIL8 cells were transfected with the AP-1 reporter plasmid and pretreated with the indicated MAPK inhibitors (20 ␮M) for 1 h, and further stimulated with MDP (10 ng/ml) for 16 h. AP-1 activation was evaluated by a luciferase reporter assay. D and E, MAIL8 cells were pretreated with the indicated MAPK inhibitors (20 ␮M) for 1 h and further stimulated with MDP (10 ng/ml) for 16 h. The amount of IL-8 in the culture supernatant was determined by ELISA. F, MAIL8 cells were transfected with the NF-␬B reporter plasmid and then treated as in B. NF-␬B activity was evaluated by a luciferase reporter assay. G, MAIL8 cells were pretreated with the indicated caspase inhibitors (20 ␮M) for 1 h and further stimulated with MDP (10 ng/ml) for 16 h. The whole-cell lysates were then assayed by Western blotting using Abs against phosphorylated or entire JNK1/2. H, MAIL8 cells were pretreated with the indicated MAPK or caspase inhibitors (20 ␮M) for 1 h and further stimulated with MDP (10 ng/ml) for 16 h. Cell lysates were then analyzed for caspase-8 activity using fluorogenic caspase-8 substrate, IETD-7-amino-4-trifluoromethyl coumarin. RFU, relative fluorescent units. B–H, SP600125, JNK inhibitor; PD98059, MEK inhibitor; SB202190 and SB203580, p38 inhibitors; SB202474, control substance; z-VAD-fmk, pancaspase-inhibitor; z-IETD-fmk, caspase-8 inhibitor. by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd.

ASC-mediated AP-1 activation, whereas caspase-1, caspase-9, interrelated. In MAIL8 cells, pan-caspase-inhibitor z-VAD-fmk FADD, and RICK are not essential for this response. inhibited MDP-induced JNK phosphorylation (Fig. 4G), whereas none of MAPK inhibitors inhibited MDP-induced caspase-8 acti- Role of MAPKs in the ASC-mediated AP-1 activation and IL-8 vation detected using fluorogenic substrate of caspase-8 (Fig. 4H). production These results indicate that caspase-8 activation is upstream of Different MAPKs play important roles in AP-1 activation in dif- MAPK activation. ferent contexts. Therefore, we investigated which MAPKs are ac- http://classic.jimmunol.org tivated in MAIL8 cells upon MDP stimulation. Western blots us- Profiling of the gene expression induced by ASC activation ing a pair of phosphorylated form-specific and pan-specific Abs Finally, the profiling of ASC-induced genes by microarray analy- against each of the three major types of MAPKs (namely ERK1/2, ses was performed (original datasets of microarray analyses are JNK1/2, and p38) indicated that MDP induced phosphorylation of available at CIBEX (http://cibex.nig.ac.jp/index.jsp, accession no. JNK and p38, but not of ERK (Fig. 4A). To determine the contri- CBX41) upon publication). The expression of 75 and 23 genes was bution of these MAPKs to ASC-mediated AP-1 activation and found to be increased and decreased more than 2-fold, respec-

Downloaded from IL-8 production, we tested the effects of MAPK and MAPK kinase tively, in MAIL8 cells, but not in control KBG cells (HEK293- inhibitors. Consistent with the observed phosphorylation of JNK derived cell line stably expressing a NF-␬B-GFP reporter con- and p38, inhibitors for these MAPKs (SB202190 and SB203580 struct) upon MDP stimulation (supplemental Table S1).5 The top for p38, and SP600125 for JNK), but not a control substance 30 genes that were up-regulated in MDP-stimulated MAIL8 cells, (SB202474), inhibited the MDP-induced AP-1 activation and IL-8 but not in MDP-stimulated KGB cells, are listed in Table I. In production in MAIL8 cells (Fig. 4, B–E), whereas an inhibitor for contrast, 81 genes and 46 genes were, respectively, increased and MAP/ERK kinase (PD98059) failed to inhibit these responses decreased more than 2-fold, when the KBG cells were stimulated (Fig. 4, C and E). These results indicate that JNK and p38 are with TNF-␣ (supplemental Table S2).5 Cluster analysis indicated involved in the ASC-mediated AP-1 activation that is required for that some, but not all of the genes up-regulated by ASC activation optimal IL-8 production. None of these inhibitors affected the were also up-regulated by TNF-␣ stimulation (Fig. 5A; see also MDP-induced NF-␬B activation in MAIL8 cells (Fig. 4F), con- Table I and supplemental Table S1).5 The genes up-regulated by firming that ASC-mediated NF-␬B activation is independent of both ASC activation and TNF-␣ stimulation included components AP-1 activation. or inhibitors of NF-␬B, and inflammatory cytokines. In contrast, Because ASC-mediated AP-1 activation was dependent on both the genes that were specifically induced by ASC activation in- caspase-8 and MAPKs, we investigated how these dependencies cluded components of AP-1, and stress-related genes. The gene 7660 MECHANISM AND REPERTOIRE OF ASC-MEDIATED GENE EXPRESSION

Table I. Top 30 genes induced by ASC activation descriptions and/or gene ontology terms provided by the manufac- turer suggested that ϳ23, 21, and 16% of the 75 genes up-regu- MAIL8-MDPa KBG-MDPb KBG-TNFc lated in MAIL8 after MDP stimulation were related to transcrip- tion, inflammation, and apoptosis/cell death, respectively (Fig. Gene Name Indd (LR, LRE)e Ind (LR, LRE) Ind (LR, LRE) 5B). The induction of mRNAs for inflammatory cytokines (TNF, f f NFKBIA 6.56 (0.82, 0.08) 1.04 (0.02, 0.05) 5.48 (0.74, 0.07) IL-6, IL-8, and CXCL2) and components or regulators of AP-1 TNF 6.38f (0.81, 0.08) 1.00 (0.00, 0.08) 2.19f (0.34, 0.06) ATF3 5.33f (0.73, 0.07) 1.29 (0.11, 0.05) 1.19 (0.08, 0.05) and NF-␬B (Fos, c-Jun, JunB, RelB, I␬B␣, and I␬B␨) was con- f FOS 4.92 (0.69, 0.07) 0.89 (Ϫ0.05, 0.09) 0.83 (Ϫ0.08, 0.08) firmed by RT-PCR analyses (Fig. 5, C and D). MDP-induced ex- IL6 4.92f (0.69, 0.12) 1.24 (0.09, 0.06) 2.95f (0.47, 0.11) RELB 4.71f (0.67, 0.07) 1.05 (0.02, 0.05) 6.81f (0.83, 0.08) pression of these mRNAs was markedly reduced when ASC ex- f f IER3 4.50 (0.65, 0.07) 1.20 (0.08, 0.05) 2.13 (0.33, 0.06) E DUSP1 4.47f (0.65, 0.07) 1.03 (0.01, 0.05) 1.38 (0.14, 0.05) pression was suppressed using ASC-targeting siRNA (Fig. 5 ), RPL22L1 4.35f (0.64, 0.07) 1.09 (0.04, 0.05) 1.13 (0.05, 0.05) indicating that the MDP-induced gene expression in this system is RRP1B 4.24f (0.63, 0.08) 1.15 (0..06, 0.06) 1.42 (0.15, 0.07) TLE3 4.10f (0.61, 0.07) 1.37 (0.14, 0.06) 1.21 (0.08, 0.06) ASC dependent. JUNB 3.85f (0.59, 0.07) 1.20 (0.08, 0.05) 1.64f (0.22, 0.05) DEPDC7 3.60f (0.56, 0.10) 1.41 (0.15, 0.07) 1.38 (0.14, 0.07) NR4A1 3.58f (0.55, 0.07) 1.31 (0.12, 0.05) 1.04 (0.02, 0.05) Discussion CALCA 3.51f (0.55, 0.06) 1.40 (0.15, 0.05) 3.59f (0.56, 0.06) JUN 3.39f (0.53, 0.06) 1.12 (0.05, 0.05) 0.82 (Ϫ0.09, 0.05) One of the obstacles to investigate the molecular mechanisms of PKHD1L1 3.33f (0.52, 0.10) 1.32 (0.12, 0.07) 1.56 (0.19, 0.08) the ASC-induced gene expression under physiological conditions USP43 3.31f (0.52, 0.09) 1.06 (0.02, 0.06) 1.95f (0.29, 0.08) C8orf4 3.29f (0.52, 0.09) 1.14 (0.06, 0.05) 3.62f (0.56, 0.10) is that there is no specific stimulus for ASC. Actually, many stim- LMX1A 3.27f (0.51, 0.09) 1.45 (0.16, 0.06) 2.04f (0.31, 0.07) uli for ASC also activate TLRs that have a potential to induce a TRIM31 3.15f (0.50, 0.08) 1.24 (0.09, 0.06) 6.31f (0.80, 0.22) PIP5K1B 3.11f (0.49, 0.12) 1.13 (0.05, 0.07) 1.13 (0.05, 0.07) variety of genes. In addition, although macrophages have been IL8 3.08f (0.49, 0.06) 1.11 (0.05, 0.05) 1.40 (0.15, 0.05) used to investigate physiological functions of ASC, activation of THBS2 3.05f (0.48, 0.11) 1.18 (0.07, 0.05) 2.55f (0.41, 0.09) ␤ FNBP4 3.03f (0.48, 0.06) 0.95 (Ϫ0.02, 0.05) 1.35 (0.13, 0.05) ASC induces caspase-1-mediated IL-1 production, and thus, an MYST1 2.99f (0.48, 0.07) 1.19 (0.07, 0.06) 1.16 (0.06, 0.06) observed ASC-mediated response may be secondary to IL-1␤ pro- MAMDC4 2.95f (0.47, 0.12) 0.81 (Ϫ0.09, 0.06) 1.23 (0.09, 0.06) CXCL2 2.93f (0.47, 0.07) 1.09 (0.04, 0.06) 1.62f (0.21, 0.06) duction in this type of cells. To overcome these problems, we C12orf30 2.92f (0.47, 0.07) 1.32 (0.12, 0.06) 1.45 (0.16, 0.06) previously established MAIL8 cells. Importantly, the parental f Ϫ CDYL 2.88 (0.46, 0.06) 0.94 ( 0.03, 0.05) 1.19 (0.08, 0.06) HEK293 cells do not express NOD2, and do not respond to MDP. a,b,c MAIL8a or KBG cellsb,c were stimulated with 10 ng/ml MDPa,b or 10 ng/ml Furthermore, all the MDP-induced responses observed to date (i.e., TNF-␣c for 16 h. ␬ d Ind, Induction (fold) ϭ normalized red fluorescent intensity (corresponding to apoptosis, NF- B activation, and AP-1 activation) in MAIL8 cells the mRNA expression level of stimulated cells)/normalized green fluorescent inten- were ASC dependent (12, 20) (Fig. 1D). In addition, MAIL8 cells sity (corresponding to the mRNA expression level of unstimulated cells). as well as HEK293 cells express no or little TLRs or caspase-1. e ϭ LR, Log ratio log10 (normalized red fluorescent intensity/normalized green fluorescent intensity); LRE, the most conservative log ratio error using a SE propa- Thus, our experimental system is more suitable for investigating gation algorithm (Agilent) and a universal error model (Rosetta Biosoftware). the molecular mechanism of ASC-mediated responses than are f Significantly induced ( p Ͻ 0.001). by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 5. Profiling of the gene expression induced by ASC activation. A, Single-linkage hierarchical clustering with the Euclidean distance metric of the microarray data shown in Table I and supplemental Table S15 was performed for those genes that were induced more than 2-fold by MDP stimulation in MAIL8 cells, but not in KBG cells, using MultiExperiment Viewer v4.0 (19). B, Sets of genes that are related to transcription, inflammation, and/or cell death among 75 genes up-regulated upon ASC activation. C and D, The total RNA was purified from MAIL8 cells cultured with or without 10 ng/ml MDP for the indicated periods in C or 12 h in D. Induction of the mRNAs for inflammatory cytokines, AP-1, and NF-␬B-related genes was confirmed by RT-PCR analyses. E, MAIL8 cells were transfected with control and ASC-targeting siRNA twice at a 36-h interval. Cells were then stimulated with or without 10 ng/ml MDP during the last 12 h of another 36-h culture. The expression levels of indicated genes were then examined by RT-PCR. ACTB, ␤-actin; NFKBIA, I␬B␣; NFKBIZ, I␬B␨. The Journal of Immunology 7661

more physiological experimental systems in which macrophages 20). Thus, this ASC-caspase-8 axis branches in at least three di- are stimulated with microbial components or microbes, which can rections. This signal transduction system is strikingly similar to the activate other pattern-recognition receptors, such as TLRs. Fas ligand-induced one, which is mediated by the Fas-FADD- We previously demonstrated that activation of ASC induces caspase-8 axis, in that the same three pathways are activated down- NF-␬B activation, which is essential for the MDP-induced IL-8 stream of caspase-8 (17). Nonetheless, FADD is not involved in production in MAIL8 cells. In this study, we showed for the first the ASC-mediated responses. Considering the structural similarity time that the activation of ASC also induces a cell-autonomous of FADD and ASC, it is likely that ASC plays an equivalent role AP-1 activation. This response is critical for the MDP-induced to FADD as an adaptor protein that acts as a bridge between an IL-8 production in MAIL8 cells. Because NF-␬B and AP-1 act upstream molecule (i.e., CARD12) and caspase-8. This notion is synergistically to induce expression of a variety of genes involved also supported by our previous finding that endogenous caspase-8 in immune responses, it is likely that the function of ASC to me- colocalizes with ASC upon MDP stimulation in MAIL8 cells (12). diate coordinated activation of these transcription factors in re- ASC activation induced a significantly higher level of AP-1 acti- sponse to pathogen recognition by pattern recognition receptors vation than did Fas ligand stimulation in MAIL8 cells (data not such as CARD12 and cryopyrin plays an important role in the shown), although caspase-8 plays an essential role in both cases. innate immune system. This notion is consistent with a recent re- This is probably because ASC activation induced the phosphory- port demonstrating that ASC plays an important role in NF-␬B lation of both p38 and JNK, which were involved in AP-1 activa- activation and cytokine production in human monocytic/macroph- tion (Fig. 5), whereas the Fas ligand-induced AP-1 activation agic cell lines infected by P. gingivalis (13). In contrast, it has been solely depends on JNK, and not on p38 (18). Thus, ASC and demonstrated that macrophages isolated from ASC-deficient mice FADD mediate similar signaling pathways, both using caspase-8, produce normal levels of TNF-␣ and/or IL-6 upon LPS stimulation but they are not exactly the same. This difference between their or microbial infection, suggesting that ASC is dispensable for cy- signals may be explained by the different components of the sig- tokine gene expression in mouse macrophages (7, 14, 15). How- naling complexes recruited by ASC and FADD. ever, the role of ASC in cytokine production from other cell types In conclusion, our results demonstrate a novel function for ASC in mice, especially those cells that do not express TLRs, still re- in the transcriptional regulation of inflammatory and cell death- mains to be determined. related genes in human cells, and thus provide new insights into The activation of ASC also induced the expression of an APRE- the role of ASC in the innate immune system. reporter gene, indicating STAT3 activation. A cell-autonomous assay (Fig. 2C) revealed that the latter response is not cell auton- Acknowledgments omous, but mediated by a factor that can act in a paracrine manner. We thank H. Kushiyama for secretarial and technical assistance. ␬ In addition, this response was suppressed by inhibiting NF- B Disclosures (Fig. 2B and data not shown) or AP-1 (data not shown), suggesting The authors have no financial conflict of interest. that the factor is produced in a NF-␬B- and AP-1-dependent man- ner. Although IL-6 is a well-known inducer for STAT3 activation, References and IL-6 mRNA was actually induced upon MDP stimulation in 1. Masumoto, J., S. Taniguchi, K. Ayukawa, H. Sarvotham, T. Kishino, N. Niikawa,

by guest on October 1, 2021. Copyright 2009 Pageant Media Ltd. MAIL8 cells (Table I and Fig. 5C), the MDP-induced APRE-re- E. Hidaka, T. Katsuyama, T. Higuchi, and J. Sagara. 1999. ASC, a novel 22-kDa porter gene expression in MAIL8 cells was not inhibited by neu- protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J. Biol. Chem. 274: 33835–33838. tralization with an anti-IL-6 mAb, and rIL-6 failed to induce the 2. Conway, K. E., B. B. McConnell, C. E. Bowring, C. D. Donald, S. T. Warren, and APRE-reporter gene expression in MAIL8 cells (data not shown). P. M. Vertino. 2000. TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast can- Thus, some other factor(s) must be responsible for the STAT3 cers. Cancer Res. 60: 6236–6242. activation. 3. Manji, G. A., L. Wang, B. J. Geddes, M. Brown, S. Merriam, A. Al Garawi, Consistent with the potent activation of three major inflamma- S. Mak, J. M. Lora, M. Briskin, M. Jurman, et al. 2002. PYPAF1, a PYRIN- ␬ containing Apaf1-like protein that assembles with ASC and regulates activation tion-associated transcription factors, NF- B, AP-1, and STAT3, of NF-␬B. J. Biol. Chem. 277: 11570–11575. microarray analyses indicated that the expression of a number of 4. Wang, L., G. A. Manji, J. M. Grenier, A. Al-Garawi, S. Merriam, J. M. Lora,

http://classic.jimmunol.org genes encoding inflammatory cytokines as well as components or B. J. Geddes, M. Briskin, P. S. DiStefano, and J. Bertin. 2002. PYPAF7, a novel PYRIN-containing Apaf1-like protein that regulates activation of NF-␬B and regulators of NF-␬B and AP-1 was up-regulated upon ASC acti- caspase-1-dependent cytokine processing. J. Biol. Chem. 277: 29874–29880. vation. These results further supported the idea that ASC plays an 5. Grenier, J. M., L. Wang, G. A. Manji, W. J. Huang, A. Al-Garawi, R. Kelly, A. Carlson, S. Merriam, J. M. Lora, M. Briskin, et al. 2002. Functional screening important role in inflammatory responses through not only of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-␬B caspase-1 activation, but also the transcriptional regulation of a and caspase-1. FEBS Lett. 530: 73–78. variety of inflammatory factors in human cells. Interestingly, a 6. Martinon, F., K. Burns, and J. Tschopp. 2002. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of Downloaded from subset of ASC-induced genes, including FOS, JUNB, and JUN, all proIL-␤. Mol. Cell 10: 417–426. of which encode components of AP-1, was not significantly in- 7. Mariathasan, S., K. Newton, D. M. Monack, D. Vucic, D. M. French, W. P. Lee, duced upon TNF-␣ stimulation in MAIL8 cells. One likely reason M. Roose-Girma, S. Erickson, and V. M. Dixit. 2004. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218. for the failure of TNF-␣ to induce some ASC-inducible genes is 8. Kanneganti, T. D., N. Ozoren, M. Body-Malapel, A. Amer, J. H. Park, L. Franchi, that, unlike ASC activation, TNF-␣ stimulation induces only mar- J. Whitfield, W. Barchet, M. Colonna, P. Vandenabeele, et al. 2006. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. ginal AP-1 activation in HEK293-derived cell lines, including Nature 440: 233–236. MAIL8 cells (data not shown). The microarray analyses also in- 9. Kanneganti, T. D., M. Body-Malapel, A. Amer, J. H. Park, J. Whitfield, dicated that ASC induces a number of cell death-related genes, L. Franchi, Z. F. Taraporewala, D. Miller, J. T. Patton, N. Inohara, and G. Nunez. 2006. Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to including TNF, NRA41 (also called NUR77; Table I), and BBC3 viral infection and double-stranded RNA. J. Biol. Chem. 281: 36560–36568. (also called PUMA; Fig. 5B). Thus, ASC may be involved in ap- 10. Mariathasan, S., D. S. Weiss, K. Newton, J. McBride, K. O’Rourke, optosis not only through the activation of caspase-8, but also by M. Roose-Girma, W. P. Lee, Y. Weinrauch, D. M. Monack, and V. M. Dixit. 2006. Cryopyrin activates the inflammasome in response to toxins and ATP. inducing the expression of proapoptotic genes. Nature 440: 228–232. In this study, we found that the activation of ASC induces AP-1 11. Masumoto, J., T. A. Dowds, P. Schaner, F. F. Chen, Y. Ogura, M. Li, L. Zhu, T. Katsuyama, J. Sagara, S. Taniguchi, et al. 2003. ASC is an activating adaptor in a caspase-8-dependent manner. Caspase-8 also plays important for NF-␬B and caspase-8-dependent apoptosis. Biochem. Biophys. Res. Commun. roles in ASC-mediated apoptosis and NF-␬B activation (11, 12, 303: 69–73. 7662 MECHANISM AND REPERTOIRE OF ASC-MEDIATED GENE EXPRESSION

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Supplementary Table S1. List of genes induced or suppressed by ASC-activation in MAIL8 but not KBG cells. infllammatory/ apoptosis Gene Name Description MAIL8-MDP1) KBG-MDP2) KBG-TNF3) transcription immune cell death response

Induced genes

Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, involved in transcription regulation by NFKBIA inhibiting nuclear localization and DNA binding of transcription 6.56 1.04 5.48 14) 1 activator NFkB in unstimulated cells; mutated in Hodgkin lymphoma Homo sapiens tumor necrosis factor (TNF superfamily, member TNF 6.38 1.00 2.19 1 1 2) (TNF), mRNA Activating transcription factor 3, binds cAMP and AP-1 responsive elements, able to activate or repress transcription, ATF3 5.33 1.29 1.19 1 1 and may respond to radiation and oxidative stress, possesses intrinsic oncogenic and metastatic potential V-fos homolog, transcription factor that forms a heterodimer with JUN and stimulates transcription of genes containing FOS 4.92 0.89 0.83 1 1 activator protein-1 (AP-1) regulatory elements, may be involved in development of tumors of the skin, bone, lung, and breast Interleukin 6 (interferon-beta 2), cytokine that promotes the proliferation of T and B cells and final maturation of B-cells; IL6 4.92 1.24 2.95 1 implicated in multiple sclerosis and the persistence of multiple myeloma and B-chronic lympohcytic leukemia Reticuloendotheliosis viral (v-rel) oncogene related B, a member of the NF kappa B/Rel family of transcriptional regulators that RELB heterodimerizes with an NF-kappa B subunit and regulates 4.71 1.05 6.81 1 1 NF-kappa B transcriptional activity, involved in the immune response Immediate early response 3, a radiation-inducible protein that may regulate cell growth, apoptosis, and T-cell homeostasis, IER3 4.50 1.20 2.13 1 1 may also play roles in inflammation and keratinocyte growth and differentiation Dual specificity phosphatase 1, stress factor-induced non-membrane spanning phosphatase, inactivates MAP kinases, DUSP1 acts in chemotaxis and perhaps heat shock and oxidative stress 4.47 1.03 1.38 responses, overexpressed in breast, colon, and prostate epithelial tumors Protein with high similarity to ribosomal protein L22 (human RPL22), which is a component of the large 60S ribosomal RPL22L1 4.35 1.09 1.13 subunit that binds heparin, member of the ribosomal L22e family Protein of unknown function, has very strong similarity to RRP1B 4.24 1.15 1.42 uncharacterized human KIAA0179 Transducin-like enhancer of split 3 (homolog of Drosophila E(sp1)), expressed in the same pattern as NOTCH and may act TLE3 4.10 1.37 1.21 1 as part of the signaling pathway, may function in cell determination and epithelial differentiation Jun B proto-oncogene, member of the Jun family of DNA binding proteins, binds to AP-1 sites in genomic DNA to JUNB regulate transcription, plays a role in cellular proliferation, 3.85 1.20 1.64 1 1 differentiation, and expression of viral genomes, including HIV-1 DEPDC7 Homo sapiens novel 58.3 KDA protein (LOC91614), mRNA 3.60 1.41 1.38 Nuclear receptor subfamily 4 group A member 1, transcription factor that induces apoptosis through mitochondrial targeting, NR4A1 3.58 1.31 1.04 1 1 involved in response to stress via regulation of the hypothalamus-pituitary-adrenal axis Retired, replaced by A_23_P301846, was Precursor of calcitonin and calcitonin gene-related peptide, neuropeptide hormones CALCA 3.51 1.40 3.59 1 which regulate blood pressure, apoptosis, cell proliferation, and are probably involved in nociception, may have roles in … V-jun homolog, proto-oncoprotein and transcription factor that functions as a homodimer or in association with FOS in the JUN AP-1 complex, involved in apoptosis; may have a role in 3.39 1.12 0.82 1 1 1 retinitis pigmentosum, renal cell carcinoma, and IgA nephropathy Protein containing three IPT or TIG domains, which contain PKHD1L1 3.33 1.32 1.56 immunoglobulin-like folds Retired, was Member of the ubiquitin carboxyl-terminal hydrolase family 2, contains a ubiquitin carboxyl-terminal I_1100757 hydrolases family 2 domain, has a region of low similarity to a 3.31 1.06 1.95 region of human USP4, which removes ubiquitin from ubiquitin-conjugated … Chromosome 8 open reading frame 4, thyroid cancer 1, a C8orf4 ubiquitously expressed protein; overexpression is associated 3.29 1.14 3.62 with papillary thyroid carcinoma Homo sapiens LIM homeobox transcription factor 1, alpha LMX1A 3.27 1.45 2.04 1 (LMX1A), transcript variant 6A, mRNA Member of the B-box zinc finger family, contains a C3HC4 type (RING) zinc finger, which may mediate protein-protein TRIM31 interactions, has a region of low similarity to a region of 3.15 1.24 6.31 tripartite motif protein 10 (hematopoietic RING finger 1, mouse Trim10) Phosphoinositide-4-phosphate 5-kinase I beta, a member of a PIP5K1B family of kinases that are responsible for the synthesis of 3.11 1.13 1.13 PtdIns-4,5-P2; may be associated with Friedreichs ataxia Interleukin 8, a cytokine that plays a role in chemoattraction and IL8 activation of neutrophils, involved in immune and inflammatory 3.08 1.11 1.40 1 responses Retired, was Thrombospondin 2, an adhesive molecule that binds heparin and plays roles in cell adhesion and inhibition of THBS2 3.05 1.18 2.55 1 angiogenesis, may have roles in development and response to viruses; lack of expression is important in the enhancement of ... Retired, replaced by A_23_P395595, was Protein with strong similarity to formin binding protein 4 (mouse Fnbp4), which is FNBP4 3.03 0.95 1.35 an SH3/SH2 adaptor protein, contains two WW domains, which bind proline-rich peptides MYST histone acetyltransferase 1, an acetyltransferase with activity toward histones H2A, H3, and H4, MYST1 2.99 1.19 1.16 chromodomain-containing member of the MYST family of histone acetyltransferases Protein containing 7 MAM domains, and 7 low-density lipoprotein (LDL) receptor class A domains, has a region of low I_963399 2.95 0.81 1.23 similarity to a region of low density lipoprotein receptor-related protein (human LRP1) Homo sapiens chemokine (C-X-C motif) ligand 2 (CXCL2), CXCL2 2.93 1.09 1.62 1 mRNA Homo sapiens hypothetical protein FLJ13089 (FLJ13089), C12orf30 2.92 1.32 1.45 mRNA Chromodomain protein Y chromosome-like, a histone CDYL acetyltransferase that is involved in histone H4 hyperacetylation 2.88 0.94 1.19 and the histone-to-protamine transition during spermatogenesis Butyrophilin 2, member of the butyrophilin family, which is a BTN2A2 subset of the immunoglobulin superfamily, with 2.88 1.34 1.38 1 immunoglobulin, transmembrane, and B30.2 domains BBC3 Homo sapiens BCL2 binding component 3 (BBC3), mRNA 2.88 1.40 1.10 1 Homo sapiens hypothetical protein LOC285636 (LOC285636), LOC285636 2.87 1.35 1.02 mRNA Protein with high similarity to odorant receptor (mouse Ors25), which transduces responses for molecules with related chemical OR5H2 2.83 1.20 1.44 structures but differing odors, member of the rhodopsin family of G protein-coupled receptors (GPCR) Leukocyte platelet-activating factor receptor, a putative HUMNPIIY seven-transmembrane domain receptor that binds the 2.82 1.08 1.81 1 20 phospholipid, platelet-activating factor Protein containing five Kelch motif domains and a a BTB (BR-C, ttk and babor) or POZ (Pox virus and zinc finger) C16orf44 domain, which are found in some DNA and actin binding 2.79 1.27 1.31 proteins and may mediate protein-protein interactions, has low similarity to human ... Homo sapiens NADH dehydrogenase 2 (MTND2), nuclear gene MTND2 2.79 0.97 1.49 encoding mitochondrial protein, mRNA LOC153222 Homo sapiens adult retina protein (LOC153222), mRNA 2.78 1.31 1.34 Protein containing ten C2H2 type zinc finger domains, which bind nucleic acids, has a region of weak similarity to a region of PRDM10 2.77 0.93 1.96 PR domain containing 4 (rat Prdm4), which may play a role in nerve growth factor-induced cell differentiation Barttin, a chloride channel beta subunit that may play roles in salt reabsorption in the kidney and potassium recycling in the BSND 2.75 0.96 0.87 ear; mutations in the corresponding gene cause antenatal Bartter syndrome with sensorineural deafness and kidney failure Retired, replaced by A_23_P406376, was Member of the RNB-like family, which may have exonuclease activity, has low DIS3L2 similarity to S. cerevisiae Dis3p, which is a ran-binding protein 2.69 1.23 1.67 involved in mitotic control that is a component of the exosome 3-5 ... Retired, replaced by A_23_P354704, was Sialyltransferase 8A, an alpha-2,8-sialyltransferase which functions as ganglioside D3 ST8SIA1 2.65 1.26 2.34 1 synthase, involved in cell proliferation, apoptosis, and probably neuron differentiation; altered expression is associated ... Zinc finger protein 143, a zinc-finger transcriptional activator of ZNF143 small nuclear (snRNA) and snRNA-type genes transcribed by 2.64 0.91 1.50 1 RNA polymerases II and III C5orf26 Protein of unknown function 2.61 1.10 1.03 Heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA-binding protein 1 37kD), an RNA and DNA-binding HNRPD 2.59 1.15 0.51 1 protein which binds to AU-rich elements in the 3 untranslated regions of mRNAs and mediates their degradation Protein containing four cystatin domains, has low similarity to alpha 2-HS-glycoprotein (human AHSG), which inhibits insulin FETUB receptor tyrosine kinase and may act in bone metabolism, 2.59 1.19 1.71 1 1 monocyte recruitment, cell death, cell adhesion, and the immune response Vasodilator-stimulated phosphoprotein, an actin binding-bundling protein involved in cytoskeletal regulation, VASP 2.59 0.97 0.91 binds cytoskeletal proteins, involved in integrin signaling, cell adhesion, may be involved in inhibition of platelet activation

CUGBP1 Homo sapiens CUG triplet repeat, RNA binding protein 1 2.56 1.00 1.08 (CUGBP1), mRNA FLJ21908, a soluble proapoptotic protein, induces apoptosis of T-cells, B-cells, and neuronal cell lines via activation of FLJ21908 caspases, induced in HIV-infected monocytes, upregulated in 2.55 1.01 1.25 1 brain and lymph nodes of patients with HIV-1-associated dementia Neural precursor cell expressed developmentally down-regulated 4-like, a putative ubiquitin-protein ligase that NEDD4L interacts with serum glucocorticoid regulated kinase (SGK) and 2.55 1.21 1.19 sodium channel EnaC (SCNN1A), regulates epithelial sodium absorption Homo sapiens hypothetical protein FLJ20294 (FLJ20294), FLJ20294 2.47 0.84 1.45 mRNA Homo sapiens hypothetical protein FLJ25084 (FLJ25084), SASP 2.44 1.06 1.37 mRNA Retinitis pigmentosa 1 (autosomal dominant), a photoreceptor protein with a potential protein kinase domain, expression is RP1 regulated by retinal oxygen levels; mutation in the 2.44 1.09 0.87 corresponding gene causes one form of autosomal dominant retinitis pigmentosa FEM1 beta, member of a family of proteins that have structural FEM1B similarity to C. elegans FEM-1 protein that functions in sex 2.44 1.21 1.27 determination, has ankyrin repeats Protein of unknown function, has strong similarity to LOC162427 2.40 0.83 2.59 uncharacterized mouse 1300010M03Rik Protein of unknown function, has high similarity to TAIP-2 2.40 1.06 1.55 uncharacterized human C12orf22 RIT1 Homo sapiens Ras-like without CAAX 1 (RIT1), mRNA 2.39 1.12 1.09 Protein of unknown function, has a region of high similarity to a region of chondroitin sulfate proteoglycan 4 LOC84664 melanoma-associated (human CSPG4), which is an adhesive 2.39 1.06 1.84 extracellular matrix protein that promotes melanoma cell growth and invasion CREB binding protein, a coactivator for several transcription factors that binds to the cAMP-regulated enhancer (CRE) and CREBBP 2.37 1.03 1.09 1 has histone acetyltransferase activity; mutations in the corresponding gene cause Rubinstein-Taybi syndrome SMIF gene product, a transcriptional co-activator that interacts with the Smad4 protein (human MADH4) and contributes to DCP1A transcriptional activation by Smad4, may be part of the 2.36 0.98 1.11 1 transforming growth factor-beta (human TGFB1) signaling pathway SRY box 4, transcriptional activator, member of the SYR related HMG box family, may be involved in apoptosis, aberrant SOX4 transcriptional regulation in colon carcinoma, and in the 2.34 1.10 1.00 1 1 commitment to the differentiated state in normal and malignant mammary glands Homo sapiens POU domain, class 2, transcription factor 1 POU2F1 2.33 0.82 1.01 1 (POU2F1), mRNA Molecule possessing ankyrin repeats induced by NFKBIZ lipopolysaccharide, ankyrin repeat-containing protein that may 2.27 0.82 0.93 1 1 play a role in signal transduction Growth arrest and DNA-damage-inducible alpha, a DNA damage-inducible protein that is a downstream target of p53 GADD45A (TP53) and is involved in DNA repair, cell cycle arrest, and 2.24 1.03 0.88 inhibition of cell growth; interacts with PCNA, CDC2 and p21 (CDKN1A) Retired, replaced by A_23_P320250, was Protein containing a myosin head domain (motor domain), has moderate similarity to MYOHD1 2.20 0.76 1.10 a region of myosin I (C. elegans HUM-2), which is a class V unconventional myosin heavy chain General transcription factor IIIC polypeptide 4, a subunit of DNA-binding subcomplex TFIIIC2, displays histone GTF3C4 2.20 1.03 1.24 1 H3-specific acetyltransferase activity, may recruit TFIIIB and RNA polymerase III to the pre-initiation complex

Protein with moderate similarity to microrchidia (mouse Morc), MORC2 which is a male germ cell-specific protein involved in 2.17 1.01 1.04 1 spermatogenesis and germ cell apoptosis and is associated with infertility due to spermatogenic arrest in early meiotic prophase I

Tyrosylprotein sulfotransferase 2, a type II transmembrane protein that catalyzes transfer of a sulfuryl group from TPST2 3-phosphoadenosine 5-phosphosulfate to tyrosine residues in 2.12 1.01 1.33 proteins, a modification process that promotes protein-protein interactions Serine palmitoyltransferase long chain base subunit 2, member of the aminolevulinate synthase superfamily, catalyzes the first SPTLC2 step in ceramide formation, involved in epidermal cell response 2.11 0.73 0.94 1 to UV exposure and in leukemia cell ectopside-induced apoptosis CHK1 checkpoint homolog (S. pombe), protein kinase that is required for the mitotic G2 checkpoint in response to DNA CHEK1 2.11 0.96 1.21 damage caused by radiation, inhibits mitotic entry after DNA damage via a mechanism that involves the phosphatase CDC25 Homo sapiens nucleolar and coiled-body phosphoprotein 1 NOLC1 2.08 0.87 1.15 (NOLC1), mRNA Inner centromere protein antigens (135kD and 155kD), a passenger protein that targets Survivin (BIRC5) and AIRK2, INCENP associates with HP1 protein, plays a role in cytokinesis, 2.07 1.00 1.03 chromosome segregation, and cleavage furrow formation at metaphase and anaphase Mitogen-inducible gene 6, putative tumor suppressor expressed MIG-6 in a cell cycle dependent manner, activates c jun N terminal 2.07 0.91 1.00 kinase (human MAPK10), may mediate ERBB2 signaling Protein containing an RNA recognition motif (RRM, RBD, or SR140 RNP) and a Surp module (SWAP domain), which may bind 2.04 0.98 1.00 RNA, has low similarity to uncharacterized C. elegans C07H6.4 Sjogren syndrome scleroderma autoantigen 1, functions as an SSSCA1 autoantigen in scleroderma and Sjogren syndrome, may have a 2.02 0.69 0.93 role in mitosis; overexpressed in scleroderma Cumulative number: 17 16 12 % of total MDP-induced genes: 23% 21% 16%

Suppressed genes

Short-chain alcohol dehydrogenase family member, a putative alcohol dehydrogenase that is upregulated during monocyte to DHRS2 0.49 1.11 0.77 dendritic cell differentiation and synthesized in hepatoblastoma following treatment with butyrate Glypican 3, member of the glypican family of heparan sulfate proteoglycans, may play a role in embryogenesis, may have an GPC3 0.48 1.13 0.47 anticancer effect in ovary and mesothelial tissue; mutations in the gene are associated with Simpson-Golabi-Behmel syndrome Frizzled homolog 10, a predicted member of the frizzled family of plasma membrane receptors, positively regulates WNT FZD10 signaling and embryonic axis duplication, and increased 0.46 1.05 0.91 expression may lead to carcinogenesis, including colorectal and breast cancer Oculocutaneous albinism II, an integral membrane protein that may be involved in melanosome pH regulation; gene deletion or OCA2 alteration causes oculocutaneous albinism II and is associated 0.45 0.99 0.70 with hypopigmentation in Prader-Willi and Angelman syndromes Malonyl-CoA decarboxylase, converts malonyl-CoA to acetyl-CoA and CO2, cytoplasmic form regulates malonyl-CoA MLYCD abundance, peroxisomal form may degrade intraperoxisomal 0.44 1.04 0.87 malonyl-CoA; mutations may lead to cardiomyopathy, seizures, and developmental delay Androgen induced protein, upregulated by androgen, may AIG-1 0.44 1.24 0.86 function in androgen-regulated hair follicle growth Heme oxygenase (decycling) 1, inducible isoform that cleaves the heme ring at the alpha methene bridge to form biliverdin and HMOX1 carbon monoxide, involved in oxidative stress response; 0.44 1.04 0.82 corresponding gene mutation is associated with heme oxygenase deficiency Protein of unknown function, has moderate similarity to C3orf31 0.44 1.01 0.92 uncharacterized C. elegans Y71F9B.2 Transcription factor BMAL2, basic helix-loop-helix/PAS domain transcription factor, heterodimerizes with other factors, ARNTL2 regulates circadian oscillation of plasminogen activator inhibitor 0.43 1.00 0.76 1 1 (PAI1), may be involved in morning onset of myocardial infarction Homo sapiens a disintegrin-like and metalloprotease (reprolysin ADAMTS13 type) with thrombospondin type 1 motif, 13 (ADAMTS13), 0.43 0.90 0.73 transcript variant 1, mRNA Phosphorylase kinase alpha 2 (liver), the alpha subunit of liver phosphorylase kinase, a regulatory enzyme involved in glycogen PHKA2 0.43 1.21 0.88 breakdown, associated with X-linked liver glycogenosis subtypes 1 and 2 (X-linked phosphorylase b kinase deficiency) Member of the class I cysteinyl (C) tRNA synthetase family, has low similarity to human CARS, which is a cysteinyl-tRNA CARS2 synthetase that aminoacylates cognate tRNA with cysteine for 0.42 0.92 0.97 protein biosynthesis and interacts with elongation factor 1 gamma (EEF1G) SH2-containing inositol phosphatase, hydrolyzes Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3, interacts with SRC (SRC) or SHC (SHC1) INPP5D 0.40 1.23 0.93 1 proteins in signal transduction pathways; corresponding gene may be associated with Lowes oculocerebrorenal syndrome Origin recognition complex subunit 1-like, a chromatin-binding protein required for initiation of DNA replication, may mediate ORC1L 0.39 1.00 2.74 chromatin acetylation by its association with HBO1 acetyltransferase, does not associate with the core ORC complex Megalencephalic leukoencephalopathy with subcortical cysts 1, implicated in megalencephalic leukoencephalopathy with MLC1 subcortical cysts, a developmental and neurological disorder, 0.38 0.78 0.52 and periodic catatonia, a familial subtype of catatonic schizophrenia Alpha-galactosidase A (alpha-D-galactoside galactohydrolase), hydrolyzes glycosphingolipids to release alpha-D-galactosyl GLA 0.37 0.82 1.09 residues; mutation of the corresponding gene causes Fabry disease BCDIN3D Protein of unknown function [292-aa form] 0.36 0.81 0.80 Protein containing a sulfate transporter and antisigma factor SLC26A10 0.35 0.86 0.84 antagonist (STAS) domain, which may bind NTPs Protein containing a SET domain, which are involved in SETD5 0.26 0.81 0.90 protein-protein interactions MTCO2 Homo sapiens cytochrome c oxidase II (MTCO2), mRNA 0.25 1.27 1.07 1 Homo sapiens RNA binding motif protein 12 (RBM12), RBM12 0.24 0.70 0.98 transcript variant 1, mRNA Rgr Homo sapiens Ral-GDS related protein Rgr (Rgr), mRNA 0.24 1.08 0.90 Calsenilin-like protein, member of the calsenilin-KChIP protein family, has four putative EF-hand motifs, interacts with KCNIP4 presenilins 1 (PSEN1) and 2 (PSEN2), binds calcium, binds and 0.21 1.04 0.99 1 modulates properties of the Kv4.2 voltage-gated K+ channel (rat Kcnd2) Cumulative number: 4 0 0 1-3) Fold-induction estimated by microarray analyses of gene expression compared with corresponding unstimulated cells. MAIL8 (1) or KBG cells (2,3) were stimulated with 10 ng/ml MDP (1,2), or 10 ng/ml TNF-α (3) for 16 hr. 4) 1 indicates the corresponding term or related terms were found in the gene description and/or GO-terms. Supplementary Table S2. List of genes induced or suppressed by TNF-stimulation in KBG cells. GeneName Description KBG/TNF1) KBG/MDP2) Induced genes Reticuloendotheliosis viral (v-rel) oncogene related B, a member of the NF kappa B/Rel family of transcriptional regulators that RELB heterodimerizes with an NF-kappa B subunit and regulates 6.80 1.05 NF-kappa B transcriptional activity, involved in the immune response Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, involved in transcription regulation by NFKBIA inhibiting nuclear localization and DNA binding of transcription 5.49 1.04 activator NFkB in unstimulated cells; mutated in Hodgkin lymphoma Transcription factor binding to IGHM enhancer 3, binds to the immunoglobulin heavy-chain enhancer; chromosomal TFE3 translocations lead to the formation of chimeric TFE3 fusion 4.48 1.08 proteins involved in kidney carcinoma and alveolar soft part sarcoma Retired, replaced by A_23_P301846, was Precursor of calcitonin and calcitonin gene-related peptide, neuropeptide hormones I_929724 3.59 1.40 which regulate blood pressure, apoptosis, cell proliferation, and are probably involved in nociception, may have roles in ...

Cytokine A2, CC chemokine that attracts monocytes, memory T-cells, natural killer cells and endothelial cells, plays a role in CCL2 3.22 1.27 the inflammatory response to infection and in inflammatory diseases including arthritis, multiple sclerosis and atherosclerosis CD83 antigen (activated B lymphocytes immunoglobulin superfamily), may play a role in antigen presentation and CD83 3.15 1.02 lymphocyte activation, expressed on dendritic cells at final stage of maturation Fibrinogen (B beta polypeptide), an acute phase protein involved in cell adhesion and probably inflammation; gene mutation FGB 3.06 1.35 causes increased risks of myocardial infarction, coronary artery disease and autosomal recessive congenital afibrinogenemia Piwi-like homolog 1 (Drosophila), involved in the control of cell PIWIL1 proliferation and apoptosis, may be involved in hemopoiesis and 3.06 1.21 spermatogenesis Integrin beta-like 1, contains ten beta integrin epidermal growth ITGBL1 factor (EGF) like repeat domains similar to those found in the 2.91 0.69 cysteine rich stalk like structure of integrin beta subunits Cytokine receptor family class 1 member 1, a type 1 cytokine receptor that may be involved in regulation of the immune WSX1 2.87 1.31 response; mouse Tccr is involved in the response to Leishmania major and mycobacterial infection Protein with moderate similarity to spinocerebellar ataxia 2 (human SCA2), which functions in vision, may play roles in A2LP 2.83 1.33 neurogenesis and pathogensis, and is associated with spinocerebellar ataxia type 2 Protein of unknown function, has strong similarity to MGC4734 2.60 1.20 uncharacterized mouse 5830442J12Rik Protein containing a pleckstrin homology (PH) domain, which I_1002182 mediate protein-protein and protein-lipid interactions, has very 2.60 1.15 strong similarity to uncharacterized human KIAA0638 NM_145260.1 Homo sapiens odd-skipped related 1 (OSR1), mRNA 2.55 0.96 Retired, was Thrombospondin 2, an adhesive molecule that binds heparin and plays roles in cell adhesion and inhibition of I_964991 2.55 1.18 angiogenesis, may have roles in development and response to viruses; lack of expression is important in the enhancement of ... Melanoma growth stimulating activity gamma, a chemokine and mitogenic factor, activates neutrophils and induces chemotaxis, CXCL3 may be involved in the inflammatory response; increased 2.55 1.08 expression may correlate with inflammation in hemolytic-uremic syndrome Homo sapiens procollagen, type XXIII, alpha 1 (COL23A1), NM_173465.2 2.54 1.49 mRNA Member of the rhodopsin family of G protein-coupled receptors (GPCR), has moderate similarity to olfactory receptor family 51 I_930084 2.50 1.22 subfamily E member 2 (human OR51E2), which is a prostate specific G protein-coupled receptor that interacts with GNA12

Protein containing an RNA recognition motif (RRM, RBD, or RNP), has a region of moderate similarity to a region of S. FLJ10290 2.50 1.22 cerevisiae Ecm2p, which is a putative pre-mRNA splicing factor that is also involved in cell wall structure or biosynthesis IFN-gamma-inducible protein 10, member of the C-X-C chemokine family that is involved in chemotaxis, responses to CXCL10 hypoxia and pathogens, tumor necrosis, inhibition of 2.47 0.79 angiogenesis, and cAMP-mediated signaling, overexpressed in most colorectal cancers TNF receptor-associated factor 3, part of heteromeric complex that interacts with the cytoplasmic domain of TNF receptors TRAF3 2.47 1.27 (TNFRSF5) and (TNFRSF4) and activates intracellular signaling cascades, involved in the acquired immune defense Desmocollin 3 (desmocollin 4), a member of the cadherin family of adhesive glycoproteins found in desmosomes, downregulated DSC3 2.47 1.21 in breast cancer, associated with autoantibody formation in pemphigus vulgaris Fatty acid amide hydrolase, degrades neuromodulatory fatty acid amides, may be involved in pregnancy; inhibition may be FAAH 2.46 1.42 therapeutic in breast cancer and sleep disorders; missense mutation of corresponding gene is associated with drug abuse Retired, was Killer cell immunoglobulin-like receptor (KIR) two domains long cytoplasmic tail 5, a member of the natural killer I_962571 2.46 1.49 cell immunoglobulin-like receptor family, may inhibit NK cell activity Homo sapiens hypothetical protein FLJ20054 (FLJ20054), NM_019049.1 2.45 1.00 mRNA Homo sapiens spastic paraplegia 20, spartin (Troyer syndrome) SPG20 2.44 1.08 (SPG20), mRNA Protein (peptidyl-prolyl cis-trans isomerase) NIMA-interacting 4 (parvulin), involved in protein folding, preferentially isomerizes PIN4 2.42 1.06 arginine-proline bonds, may play a role in mitochondrial function CD8 antigen beta polypeptide 1 (p37), forms a heterodimer with CD8 alpha (CD8A), binds to class I major histocompatibity CD8B1 2.40 1.38 complex antigen, and involved in the development of cytotoxic T lymphocytes Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon (I kappa B epsilon), which interacts NFKBIE 2.39 1.26 with and inhibits the transcription factor NF-kappa B proteins by sequestering them to the cytoplasm Protein of unknown function, has a region of low similarity to a FLJ32954 region of C. elegans T05G5.7, which is involved in 2.38 0.94 embryogenesis and morphogenesis Solute carrier family 1 member 3, glutamate transporter, may be important for glutamate reuptake in excitatory neurotransmission SLC1A3 2.38 1.43 and synaptic transmission, expression is altered in cortical pyramidal neurons of Alzheimer disease patients Protein with strong similarity to transducin (beta)-like 1 (human TBL1X), which binds histone H3 and is part of the SMRT IRA1 2.37 0.81 corepressor complex and may be involved in vision and hearing, contains eight WD domains (WD-40 repeats) I_932272 Protein of unknown function 2.37 1.44 Homo sapiens hypothetical protein MGC39650 (MGC39650), NM_152465.1 2.36 1.45 mRNA Protein containing an RNA recognition motif (RRM, RBD, or RNP), has a region of low similarity to a region of S. cerevisiae HTF9C Trm2p, which is a uridine methyltransferase that catalyzes the 2.36 1.13 formation of ribothymidine in cytoplasmic and mitochondrial tRNA Protein with high similarity to mouse Ora16, which is an odorant I_959005 receptor, member of the rhodopsin family of G protein-coupled 2.35 1.03 receptors (GPCR) Retired, replaced by A_23_P354704, was Sialyltransferase 8A, an alpha-2,8-sialyltransferase which functions as ganglioside D3 I_943896 2.34 1.26 synthase, involved in cell proliferation, apoptosis, and probably neuron differentiation; altered expression is associated ... Protein containing nine ankyrin (Ank) repeats and two TPR (tetratrico peptide repeat) domains, which may mediate DKFZP564D166 2.33 0.72 protein-protein interactions, has a region of low similarity to a region of ankyrin (rat Ank3) Member of the rhodopsin family of G protein-coupled receptors (GPCR), has moderate similarity to olfactory receptor 17 (mouse I_935128 Olfr17), which may contribute to a sensory map in olfactory 2.32 1.41 bulb via axon guidance and may play a role in the olfactory response Retired, was Leukocyte immunoglobulin-like receptor subfamily A member 2, an activating receptor that associates with I_962563 monocyte Fc-epsilon-RI-gamma (FCER1G), involved in signal 2.31 0.99 transduction and in calcium homeostasis, may activate lymphoid cells in ... Protein containing a phorbol esters or diacylglycerol binding domain (C1 domain), which bind two zinc ions, has very strong I_959614 2.29 1.30 similarity to a region of beta isoform of protein kinase C (human PRKCB1), which is important for cellular signaling

GLIPR1 Homo sapiens GLI pathogenesis-related 1 (glioma) (GLIPR1), 2.28 1.29 mRNA Protein containing three ankyrin (Ank) repeats, which may ASB14 mediate protein-protein interactions, has low similarity to 2.27 0.93 uncharacterized human ASB1 Protein containing three TPR (tetratrico peptide repeat) domains, EKN1 2.27 1.42 which may mediate protein-protein interactions Myotubularin related protein 3, contains a double zinc finger FYVE domain that binds phosphatidylinositol 3-phosphate, MTMR3 2.25 1.38 dephosphorylates inositol lipid 3-phosphates and proteins at Ser, Thr and Tyr residues, involved in membrane lipid metabolism PRO2086 Protein of unknown function 2.23 1.42 Protein phosphatase 1D magnesium-dependent delta isoform, a type 2C Ser/Thr phosphatase, inhibits TP53-mediated apoptosis, PPM1D UV-induced p53 (TP53) and p38 MAP kinase phosphorylation; 2.23 1.46 overexpression in breast cancer cells inhibits p53 (TP53) activation Cyclin-dependent kinase like 1 (CDC2-related kinase 1), member of the proline-directed Ser/Thr kinase family, contains a CDKL1 MAP kinase phosphorylation motif, activated by epidermal 2.23 1.46 growth factor (EGF), phosphorylates histones, upregulated in gliosis Retired, replaced by A_23_P360302, was Guanylyl cyclase F, a membrane-bound guanylyl cyclase that converts GTP to cGMP I_959396 2.22 1.42 in the retina probably for photoreceptor recovery or adaptation, and is regulated intracellularly by Ca2+ binding proteins MGC4677 Protein of unknown function 2.21 1.10 Homo sapiens tumor necrosis factor (TNF superfamily, member TNF 2.19 1.00 2) (TNF), mRNA Homo sapiens transition protein 2 (during histone to protamine TNP2 2.19 1.38 replacement) (TNP2), mRNA Diubiquitin, ubiquitin-like protein that interacts with MAD2 (MAD2L1), induces apoptosis, may be involved in B-cell and UBD 2.18 0.89 dendritic cell-cycle regulation and antigen processing, induced by IFN-gamma (IFNG), TNF-alpha (TNF) Protein with high similarity to mouse Or83, which is an odorant I_962026 receptor, member of the rhodopsin family of G protein-coupled 2.16 1.18 receptors (GPCR) Adaptor-related protein complex 1 mu 2 subunit, involved in AP1M2 2.15 1.21 protein sorting Protein phosphatase 4 regulatory subunit 2, interacts with protein phosphatase 4 catalytic subunit (PPP4C), may target PPP4C to PPP4R2 2.14 1.45 the centrosome and regulate its activity at centrosomal microtubule organizing centers cGMP-dependent protein kinase type 1, relaxes vascular smooth muscle and inhibits platelet aggregation, may be involved PRKG1 cardiac contractility, may be associated with hypertension and 2.14 1.35 atherosclerosis; mouse Prkg1 is associated with erectile dysfunction Immediate early response 3, a radiation-inducible protein that may regulate cell growth, apoptosis, and T-cell homeostasis, IER3 2.13 1.20 may also play roles in inflammation and keratinocyte growth and differentiation Member of the ABC transporter family, which are involved in translocation of a variety of compounds across biological ABCA5 2.12 1.25 membranes, has moderate similarity to a region of rat Abca2, which is a putative ATP-binding cassette transporter Protein containing six Kelch motifs and a BTB (BR-C, ttk and babor) or POZ (Pox virus and zinc finger) domain, which are KLHL5 found in some DNA and actin binding proteins, has moderate 2.11 1.48 similarity to Kelch-like 2 (Mayven, human KLHL2), which binds actin Protein with high similarity to olfactory receptor family 51 subfamily E member 2 (human OR51E2), which interacts with I_1100410 human GNA12 and may be associated with prostate cancer, 2.11 0.76 member of the rhodopsin family of G protein-coupled receptors (GPCR) GBA2 Homo sapiens glucosidase, beta (bile acid) 2 (GBA2), mRNA 2.10 1.26 Cis-retinol-androgen dehydrogenase 7, converts 3 alpha-adiol to RODH 2.10 1.47 dihydrotestosterone MGC21854 Protein of unknown function 2.09 1.06 Nephrocystin, SH3 domain-containing protein that associates with Pyk2 (PTK2B) and induces its tyrosine phosphorylation, NPHP1 2.09 1.20 appears to be important to renal function; gene mutations result in the cystic kidney disease familial juvenile nephronophthisis

Protein with high similarity to RNase 8 placental (human RNASE8), which exhibits relatively low ribonucleolytic activity RNASE7 2.08 1.27 and is expressed only in placenta, member of the pancreatic ribonuclease family of pyrimidine-specific endoribonucleases Cadherin 13 (H cadherin), a GPI-linked member of the cadherin family of calcium dependent adhesion molecules, mediates CDH13 cell-cell interactions, may act as a tumor suppressor; decreased 2.07 1.18 expression may be indicative of breast and lung cancer progression LIM homeobox protein 5, a member of the LIM-homeodomain LHX5 family of transcription factors, may regulate development of the 2.07 0.68 central nervous system Member of the rhodopsin family of G protein-coupled receptors I_931434 (GPCR), has moderate similarity to mouse Ora16, which is an 2.07 1.02 odorant receptor Protein with high similarity to olfactory receptor family 51 subfamily E member 2 (human OR51E2), which is a I_1002493 prostate-specific GPCR that is overexpressed in prostate cancer, 2.06 1.37 member of the rhodopsin family of G protein coupled-receptors (GPCR) Homo sapiens hypothetical protein FLJ25965 (FLJ25965), NM_173598.1 2.05 1.14 mRNA NM_001956.1 Homo sapiens endothelin 2 (EDN2), mRNA 2.05 1.31 Homo sapiens ELK3, ETS-domain protein (SRF accessory ELK3 2.04 1.47 protein 2) (ELK3), mRNA Neurolysin (metallopeptidase M3 family), a zinc metalloendopeptidase that cleaves a variety of regulatory NLN peptides including neurotensin (NTS), bradykinin (KNG), 2.03 1.30 angiotensin 1 (SERPINA8), and the dynorphins 1-8 and 1-9 (PDYN) WDR17 Homo sapiens WD repeat domain 17 (WDR17), mRNA 2.03 1.25 Protein with very strong similarity to synaptotagmin 6 (mouse Syt6), which is a putative calcium sensing protein that forms I_1999239.FL2 homodimers and heterodimers with other synaptotagmins to 2.03 1.11 regulate neurotransmitter release, contains two C2 domains [425-aa form] Ninein, GSK3B binding and putative GTP-binding protein that plays a role in microtubule organization and may be involved in NIN 2.02 1.38 centrosome matrix formation, contains a large coiled coil domain and four leucine zipper domains Member of the DEAD or DEAH box ATP-dependent RNA helicase family, contains two helicase conserved C-terminal RIG-I domains, has low similarity to melanoma differentiation 2.02 1.44 associated protein 5 (human MDA5), which is a putative ATP-dependent RNA helicase Plasminogen, a fibrinolytic protein that is cleaved to form angiostatin and plasmin, which inhibit angiogenesis and function PLG 2.02 1.35 in cell migration, proliferation, and extracellular-matrix degradation; deficiency leads to thrombosis and ligneous ... Member of the M20, M25, or M40 peptidase family of zinc metalloproteases, has a region of moderate similarity to a region FLJ32569 2.01 0.98 of gly-X carboxypeptidase yscS (S. cerevisiae Cps1p), which is involved in nitrogen metabolism Protein containing a ubiquitin-like protein-specific protease (Ulp1) family C-terminal catalytic domain, has a region of high MGC27076 2.00 0.89 similarity to a region of smt3-specific isopeptidase 1 (mouse Smt3ip1), which cleaves SUMO-1 conjugates from proteins

Suppressed genes Homo sapiens methyl-CpG binding domain protein 3-like 1 MBD3L1 0.50 0.85 (MBD3L1), mRNA HHLA3 Protein of unknown function 0.50 0.77 Member of the cyclophilin type peptidyl-prolyl cis-trans isomerase family, has a region of high similarity to a region of MGC41939 matrin cyclophilin (rat Ppig), which is a peptidyl-prolyl 0.49 0.88 isomerase that may act in coordinating transcription and pre-mRNA splicing Homo sapiens ATPase, H+/K+ exchanging, alpha polypeptide NM_000704.1 0.49 0.88 (ATP4A), mRNA Kazal type serine protease inhibitor 2 (acrosin-trypsin inhibitor), SPINK2 a putative Kazal-type protease inhibitor expressed in testis, 0.49 1.12 epididymis, and seminal vesicles but not in prostate Retired, replaced by A_23_P422809, was Homeodomain interacting protein kinase 3, a serine/threonine protein kinase I_929690 that interacts with homeodomain transcription factors, inhibits 0.49 0.86 JNK activation, may be involved in apoptosis and cell growth regulation, ... Protein containing a DnaJ domain, which are part of chaperone (protein folding) system that mediates interaction with heat C21orf55 0.49 0.76 shock proteins, has high similarity to uncharacterized mouse BC020175 Tankyrase, an ankyrin-repeat-containing protein with TNKS poly(ADP-ribose) polymerase activity, functions in telomere 0.49 0.85 maintenance, binds to telomeric protein TERF1 Myeloblastosis oncogene-like 1, member of the myb family of transcription factors, expression is cell-cycle regulated, MYBL1 stimulates B-cell proliferation and likely regulates B-cell 0.47 0.91 differentiation; may play a role in the pathogenesis of Burkitts lymphoma I_963857 Retired, was Protein of unknown function 0.47 0.70 Protein of unknown function, has strong similarity to PCNX 0.47 0.82 uncharacterized mouse Pcnx Thymosin beta 4 (X chromosome), a thymic hormone that sequesters actin monomers thereby inhibiting actin TMSB4X polymerization, may be involved in neurogenesis, induction of 0.46 0.74 apoptosis, and the wounding response; expression may be upregulated in pancreatic ... Gastrulation brain homeo box 2, a putative transcription factor of GBX2 the homeobox family, may play a role in neurogenesis and cell 0.46 0.74 proliferation, induces malignant growth of prostate cancer cells Receptor-like protein tyrosine phosphatase type B, a receptor tyrosine phosphatase, functions as the receptor for Helicobacter PTPRB 0.45 1.00 pylori VacA toxin, may be involved in density-dependent growth arrest and may be a tumor suppressor Homo sapiens hypothetical protein FLJ25027 (FLJ25027), NM_152643.1 0.45 1.14 mRNA I_958498 Retired, was Protein of unknown function 0.45 0.72 Member of the sulfatase family, which hydrolyze sulfate esters, has moderate similarity to arylsulfatase A (human ARSA), KIAA1001 0.45 1.06 which is a lysosomal enzyme which hydrolyzes cerebroside sulfate and is associated with metachromatic leukodystrophy Homo sapiens hypothetical protein MGC20470 (MGC20470), NM_145053.1 0.45 1.33 mRNA Tumor specific protein HAGE, a member of the DEAD-box HAGE family of ATP-dependent RNA helicases, expressed highly in 0.45 1.21 testis and tumors ODF4 Homo sapiens outer dense fiber of sperm tails 4 (ODF4), mRNA 0.44 0.94 Protein containing a C3HC4 type (RING) zinc finger and two PDZ, DHR, or GLGF domains, which are found in signaling I_963094 proteins, has a region of low similarity to a region of human 0.44 0.80 MPDZ, which interacts with the C-terminus of the 5-HT2C receptor Member of the amidohydrolase family, which hydrolyze adenine MGC35366 to form hypoxanthine and ammonia, has high similarity to 0.44 0.74 uncharacterized C. elegans T12A2.1 C14orf132 Protein of unknown function 0.44 0.69 Hyaluronoglucosaminidase 4, member of a family of HYAL4 0.43 1.03 hyaluronidases that function in glycosaminoglycan catabolism Selectin E, a lectin and EGF-domain containing adhesion SELE molecule that mediates adhesion of neutrophils to the blood 0.43 1.17 vessel lining, important for responses to injury and inflammation; genetic variants may be risk factors for early severe atherosclerosis

C-type (calcium dependent carbohydrate-recognition domain) lectin superfamily member 6, contains immunoreceptor CLECSF6 tyrosine-based inhibitory motif (ITIM), upregulated upon 0.43 0.74 dendritic cell differentiation, and downregulated upon dendritic cell maturation Ovary testis epididymis expressed, a putative transcription factor OTEX of the paired-like homeodomain family, highly expressed in 0.43 0.74 reproductive tissues and upregulated by androgen treatment Protein with weak similarity to cysteinyl leukotriene receptor 1 (human CYSLTR1), which is a G protein-coupled receptor I_929972 0.41 0.70 which mobilizes calcium upon activation by leukotrienes C4, D4 or E4 and is involved in bronchial asthma Protein with low similarity to melanoma inhibitory activity (cartilage derived retinoic acid sensitive protein, human MIA), MIA2 0.41 0.96 which is a marker for malignant melanoma that also inhibits the growth of melanoma cells Retired, replaced by A_23_P389465, was Protein containing a phorbol ester or diacylglycerol binding domain (C1 domain), I_931680 which bind two zinc ions, has high similarity to a region of 0.41 0.75 kinase suppressor of Ras (human KSR), which is a protein kinase that ... Homo sapiens hypothetical protein FLJ31819 (FLJ31819), NM_152529.2 0.40 0.73 mRNA Calcium/calmodulin-dependent protein kinase II delta, member of the multifunctional CAMKII family involved in Ca2+ CAMK2D 0.39 0.94 regulated processes; alternative form delta 3 is specifically upregulated in the myocardium of patients with heart failure Retired, was Bruno-like 4 RNA binding protein, a member of a I_963263 family of RNA-binding proteins related to the Drosophila Bruno 0.39 1.35 translational regulator, regulates splice site selection Protein with high similarity to dnaJ (Hsp40) homolog subfamily B member 1 (human DNAJB1), which physically interacts with I_1219826 0.39 1.26 and stimulates the ATPase activity of HSP70, contains a DnaJ C-terminal domain and a DnaJ domain Megakaryocyte-associated tyrosine kinase, non-receptor protein kinase, signals through phosphorylation of Src family kinases, MATK 0.39 0.76 may have roles cell migration and proliferation, and may act in megakaryopoesis and neurogenesis Calpain 11, a member of the calpain superfamily of calcium-dependent cysteine proteinases, contains putative CAPN11 0.38 0.67 protease and calcium-binding domains, exhibits restricted tissue distribution with highest abundance in testis Protein with high similarity to small glutamine-rich tetratricopeptide repeat-containing protein (human SGT), which LOC54557 interacts with the parvovirus H-1 NS1 protein and may act in 0.38 0.68 parvoviral replication, contains three TPR (tetratrico peptide repeat) ... I_961804 Protein of unknown function 0.37 1.13 Calcium channel (voltage-dependent) beta 4 subunit, a regulatory subunit that modulates channel activity and plays a CACNB4 0.36 0.78 role in synaptic transmission; gene mutations cause epilepsy, focal myoclonus and ataxia Carboxypeptidase A6, precursor of a putative zinc exopeptidase CPA6 with predicted specificity for large hydrophobic C-terminal 0.36 1.44 amino acids PTPRF interacting protein binding protein 1 (liprin beta 1), a beta liprin that contains an N-terminal coiled-coil region and a PPFIBP1 0.35 1.16 C-terminal liprin homology domain, which binds to alpha liprins, also binds metastasis-associated protein (mouse S100A4) Alpha 3 subunit of type IV collagen, basement membrane component involved in cell adhesion and apoptosis, tumstatin COL4A3 peptide has antitumor function; mutations and autoantibodies are 0.34 0.90 associated with Alport syndrome and Goodpasture syndrome, respectively Zona pellucida glycoprotein 2, one of three major proteins of oocyte zona pellucida, acts in fertilization as a secondary sperm ZP2 receptor to ZP3, allows species-specific sperm binding to zona 0.34 0.80 pellucida, can be used to generate immunocontraceptive vaccines KIAA0157 Protein of unknown function 0.33 1.29 Member of the ubiquitin family, has moderate similarity to a region of homocysteine-inducible endoplasmic reticulum FLJ22313 stress-inducible ubiquitin-like domain member 1 (human 0.29 0.82 HERPUD1), which is involved in the unfolded protein stress response Homo sapiens WAP four-disulfide core domain 10A WFDC10A 0.15 0.67 (WFDC10A), mRNA 1,2) Fold-induction of gene expression in KBG cells after stimulation with 10 ng/ml TNF-α (1) or MDP (2) for 16 hr.