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• W2032408313 abstract "Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-κB- and AP-1 reporter constructs indicated that an FasL-induced NF-κB and AP-1 activity are required for maximal promoter activity. FasL induced NF-κB activation with slower kinetics than did TNF-α, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-κB activation by FasL. A dominant-negative mutant of IKKγ inhibited the FasL-induced NF-κB activation. However, TRADD and RIP, which are essential for the TNF-α-induced NF-κB activation, were not involved in the FasL-induced NF-κB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-α-induced NF-κB activation. These results show that FasL induces NF-κB activation and IL-8 production by a novel mechanism, distinct from that of TNF-α. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-κB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-κB activation. Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-κB- and AP-1 reporter constructs indicated that an FasL-induced NF-κB and AP-1 activity are required for maximal promoter activity. FasL induced NF-κB activation with slower kinetics than did TNF-α, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-κB activation by FasL. A dominant-negative mutant of IKKγ inhibited the FasL-induced NF-κB activation. However, TRADD and RIP, which are essential for the TNF-α-induced NF-κB activation, were not involved in the FasL-induced NF-κB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-α-induced NF-κB activation. These results show that FasL induces NF-κB activation and IL-8 production by a novel mechanism, distinct from that of TNF-α. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-κB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-κB activation. The tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; TNFR1, TNF receptor type I; CTD, C-terminal domain; DD, death domain; DED, death effector domain; FasL, Fas ligand; fmk, fluoromethylketone; mAb, monoclonal antibody; RLU, relative luciferase units; Z, benzyloxycarbonyl; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; IL, interleukin; siRNA, short interfering RNA; RT-PCR, reverse transcription-PCR; GFP, green fluorescent protein; HEK, human embryonic kidney; CLARP, caspase-like apoptosis regulatory protein. receptor family is a still growing group of cytokine receptors that regulate cell proliferation, differentiation, and death. A subset of this family called death receptors possesses a characteristic cytoplasmic region named death domain (DD) (1Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar). Activation of these receptors induces recruitment of the death-inducing signaling complex, which consists of adaptor molecules (such as FADD and/or TRADD) and upstream caspases (such as caspase-8 and -10); this complex in turn initiates the activation cascade of caspases, which eventually results in apoptotic cell death. Like other members of the TNF receptor family, all the death receptors (TNF receptor type I (TNFR1), Fas, DR3, DR4, DR5, and DR6) have been reported to induce the activation of NF-κB (2Rensing-Ehl A. Hess S. Ziegler-Heitbrock H.W. Riethmuller G. Engelmann H. J. Inflamm. 1995; 45: 161-174PubMed Google Scholar, 3Ponton A. Clement M.V. Stamenkovic I. J. Biol. Chem. 1996; 271: 8991-8995Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 4Marsters S.A. Sheridan J.P. Donahue C.J. Pitti R.M. Gray C.L. Goddard A.D. Bauer K.D. Ashkenazi A. Curr. Biol. 1996; 6: 1669-1676Abstract Full Text Full Text PDF PubMed Google Scholar, 5Chaudhary P.M. Eby M. Jasmin A. Bookwalter A. Murray J. Hood L. Immunity. 1997; 7: 821-830Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, 6Schneider P. Thome M. Burns K. Bodmer J.L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 7Chinnaiyan A.M. O'Rourke K. Yu G.L. Lyons R.H. Garg M. Duan D.R. Xing L. Gentz R. Ni J. Dixit V.M. Science. 1996; 274: 990-992Crossref PubMed Scopus (532) Google Scholar, 8Pan G. Bauer J.H. Haridas V. Wang S. Liu D. Yu G. Vincenz C. Aggarwal B.B. Ni J. Dixit V.M. FEBS Lett. 1998; 431: 351-356Crossref PubMed Scopus (227) Google Scholar), one of the most important transcription factors for the activation and regulation of the immune system. However, how these receptors activate NF-κB is still obscure. Fas (Apo1/CD95), a prototype of the death receptors, directly recruits FADD and strongly induces apoptosis in a variety of cell types upon its ligation by FasL (1Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar). The Fas-FasL system plays pivotal roles in various aspects of immune regulation and function, such as self-tolerance, and cell-mediated cytotoxicity (9Suda T. Nagata S. J. Allergy Clin. Immunol. 1997; 100: S97-S101Abstract Full Text Full Text PDF PubMed Google Scholar). It has been proposed that FasL is expressed in “immune privileged” organs (such as the eye and testis) and protects them from destructive inflammation by counterattacking inflammatory cells (10Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1879) Google Scholar, 11Bellgrau D. Gold D. Selawry H. Moore J. Franzusoff A. Duke R.C. Nature. 1995; 377: 630-632Crossref PubMed Scopus (1103) Google Scholar). In contrast, ectopic expression of FasL by genetic engineering induces inflammation accompanied by massive neutrophil infiltration in animals (12Seino K. Kayagaki N. Okumura K. Yagita H. Nat. Med. 1997; 3: 165-170Crossref PubMed Scopus (297) Google Scholar, 13Kang S.M. Schneider D.B. Lin Z. Hanahan D. Dichek D.A. Stock P.G. Baekkeskov S. Nat. Med. 1997; 3: 738-743Crossref PubMed Scopus (430) Google Scholar, 14Allison J. Georgiou H.M. Strasser A. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3943-3947Crossref PubMed Scopus (344) Google Scholar, 15Miwa K. Asano M. Horai R. Iwakura Y. Nagata S. Suda T. Nat. Med. 1998; 4: 1287-1292Crossref PubMed Scopus (346) Google Scholar). Furthermore, FasL seems to play detrimental roles in various inflammatory diseases such as hepatitis, graft-versus-host diseases, and pulmonary fibrosis (16Kondo T. Suda T. Fukuyama H. Adachi M. Nagata S. Nat. Med. 1997; 3: 409-413Crossref PubMed Scopus (461) Google Scholar, 17Miwa K. Hashimoto H. Yatomi T. Nakamura N. Nagata S. Suda T. Int. Immunol. 1999; 11: 925-931Crossref PubMed Scopus (54) Google Scholar, 18Kuwano K. Hagimoto N. Kawasaki M. Yatomi T. Nakamura N. Nagata S. Suda T. Kunitake R. Maeyama T. Miyazaki H. Hara N. J. Clin. Investig. 1999; 104: 13-19Crossref PubMed Scopus (320) Google Scholar). To clarify how FasL induces inflammation, we have been investigating the molecular mechanism of FasL-induced inflammation. We previously reported that FasL simultaneously induces apoptosis and the conversion of inactive pro-IL-1α into its active form; both of these processes are mediated by caspases (15Miwa K. Asano M. Horai R. Iwakura Y. Nagata S. Suda T. Nat. Med. 1998; 4: 1287-1292Crossref PubMed Scopus (346) Google Scholar). This active IL-1α plays an important role in the FasL-induced inflammation. On the other hand, several reports have shown that some normal and transformed cell lines produce IL-8, a chemokine for neutrophils, upon Fas ligation by an anti-Fas monoclonal antibody (mAb) or FasL (19Abreu-Martin M.T. Vidrich A. Lynch D.H. Targan S.R. J. Immunol. 1995; 155: 4147-4154PubMed Google Scholar, 20Sekine C. Yagita H. Kobata T. Hasunuma T. Nishioka K. Okumura K. Biochem. Biophys. Res. Commun. 1996; 228: 14-20Crossref PubMed Scopus (62) Google Scholar, 21Hagimoto N. Kuwano K. Kawasaki M. Yoshimi M. Kaneko Y. Kunitake R. Maeyama T. Tanaka T. Hara N. Am. J. Respir. Cell Mol. Biol. 1999; 21: 436-445Crossref PubMed Scopus (98) Google Scholar, 22Schaub F.J. Han D.K. Liles W.C. Adams L.D. Coats S.A. Ramachandran R.K. Seifert R.A. Schwartz S.M. Bowen-Pope D.F. Nat. Med. 2000; 6: 790-796Crossref PubMed Scopus (190) Google Scholar, 23Ahn J.H. Park S.M. Cho H.S. Lee M.S. Yoon J.B. Vilcek J. Lee T.H. J. Biol. Chem. 2001; 276: 47100-47106Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24Choi C. Xu X. Oh J.W. Lee S.J. Gillespie G.Y. Park H. Jo H. Benveniste E.N. Cancer Res. 2001; 61: 3084-3091PubMed Google Scholar, 25Manos E.J. Jones D.A. Cancer Res. 2001; 61: 433-438PubMed Google Scholar, 26Shimada M. Andoh A. Araki Y. Fujiyama Y. Bamba T. J. Gastroenterol. Hepatol. 2001; 16: 1060-1067Crossref PubMed Scopus (16) Google Scholar). Activation of NF-κB is correlated with the FasL-induced IL-8 production; however, there is no direct evidence that NF-κB activation is essential for this response. Furthermore, it has not been investigated whether FasL induces NF-κB activation directly or indirectly through the induction of another cytokine, such as IL-1. In this study, we have found that FasL induces cell-autonomous NF-κB activation and IL-8 production without inducing detectable apoptosis in HEK293 cells. Subsequent experiments using this cell line revealed that FasL induces NF-κB activation by a mechanism distinct from that of TNF-α. Reagents—Recombinant mouse (m)TNF-α was purchased from Genzyme (Cambridge, MA). Recombinant soluble mFasL (previously termed WX1) (27Suda T. Tanaka M. Miwa K. Nagata S. J. Immunol. 1996; 157: 3918-3924PubMed Google Scholar) and the neutralizing mAb against mFasL (FLIM58) were prepared and purified as previously described (17Miwa K. Hashimoto H. Yatomi T. Nakamura N. Nagata S. Suda T. Int. Immunol. 1999; 11: 925-931Crossref PubMed Scopus (54) Google Scholar). Z-VAD-fluoromethylketone (fmk) Z-YVAD-fmk, Z-DEVD-fmk, Z-IETD-fmk, and Z-AAD-fmk were purchased from Calbiochem (La Jolla, CA). Lactacystin was purchased from Sigma. Cell Lines—The caspase-8-deficient Jurkat cell lines (JB-6) and a stable transfectant of human (h)caspase-8 derived from JB-6 (BC22) was kindly provided by Dr. Shigekazu Nagata (Osaka University Medical School, Osaka, Japan) (28Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar). Plasmids—The firefly luciferase reporter constructs driven by the –133/–44 hIL-8 promoter (–133-Luc), or its mutants were described previously (29Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The construct driven by the 12-O-tetradecanoylphorbol 13-acetate responsive element (2xTRE-Luc (30Yoshioka K. Deng T. Cavigelli M. Karin M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4972-4976Crossref PubMed Scopus (210) Google Scholar)) was kindly provided by Dr. Katsuji Yoshioka (Kanazawa University, Kanazawa, Japan). The plasmid pNF-κB-Luc carrying a firefly luciferase cDNA driven by 5xNF-κB-binding sites was purchased from Stratagene (La Jolla, CA), and pRL-TK carrying Renilla luciferase cDNA driven by HSV-TK promoter was purchased from Promega (Madison, WI). The pEF-BOS expression vector carrying a cDNA for FasLDC, FasLDC2, FasLS was described previously (31Shudo K. Kinoshita K. Imamura R. Fan H. Hasumoto K. Tanaka M. Nagata S. Suda T. Eur. J. Immunol. 2001; 31: 2504-2511Crossref PubMed Scopus (72) Google Scholar), while that with a cDNA for hFas, its deletion or point mutants (FD2, FD5, FD7, and FP1) (32Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Abstract Full Text PDF PubMed Google Scholar), hFADD (33Matsumura H. Shimizu Y. Ohsawa Y. Kawahara A. Uchiyama Y. Nagata S. J. Cell Biol. 2000; 151: 1247-1256Crossref PubMed Scopus (207) Google Scholar), or hcaspase-8 (28Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar), and IκBα-S32A/S36A mutant was kindly provided by Dr. Shigekazu Nagata and Dr. Ken-ichi Yamamoto (Kanazawa University, Kanazawa, Japan), respectively. cDNAs for mFADD and hp84 were obtained by RT-PCR and cloned into pEF-BOS. The cDNAs for hIKKγ (IMAGE:2820134) was purchased from Invitrogen (Carlsbad, CA). To generate cDNAs encoding FLAG-tagged proteins, full-length and truncated cDNAs were cloned into pCMV-Tag2 (Stratagene). The cDNAs encoding FLAG-tagged proteins were then subcloned into pEF-BOS to generate pEF-FLAG-hFADD (full-length), pEF-Flag-hFADD-DD (amino acids 80–208), pEF-Flag-hFADDΔC (amino acids 1–181), pEF-Flag-mFADD (full-length), pEF-Flag-p84-DD (amino acids 465–657), and pEF-Flag-IKKγ-DN (amino acids 135–419), respectively. To generate fusion molecules of hFADD and mFADD, recombinant PCR was performed as described previously (31Shudo K. Kinoshita K. Imamura R. Fan H. Hasumoto K. Tanaka M. Nagata S. Suda T. Eur. J. Immunol. 2001; 31: 2504-2511Crossref PubMed Scopus (72) Google Scholar) using the following fusion primers: for m/hFADD1 consisting of amino acids 1–80 from mFADD and amino acids 81–208 from hFADD, sense 5′-CTGCAGCGCCTGGACGACTTCGAG-3′ and antisense 5′-CTAGAAGTCGTCCAGGCGCTGCAG-3′; for m/hFADD2 consisting of amino acids 1–137 from mFADD and amino acids 138–208 from hFADD, sense 5′-GTCTGACAGAGCGTGTGC-3′ and antisense 5′-TCTGTCAGACTTCGGGGGT-3′. The PCR products were cloned into pCMV-Tag2 and then subcloned into pEF-BOS. The expression vector (pEF-TRADD-296S) carrying a dominant negative mutant of hTRADD (amino acids 196–312 with alanine substitution at amino acids 296–299) was constructed by PCR-based mutagenesis as described previously (34Park A. Baichwal V.R. J. Biol. Chem. 1996; 271: 9858-9862Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The pcDNA3-HA-caspase8-mut (C377S), pcDNA3-Flag-CLARP(L), pcDNA3-Flag-CLARP(S), and pcDNA3-Myc-RIP-DD (amino acids 558–671) were kindly provided by Dr. N. Inohara (University of Michigan Medical School, Ann Arbor, MI). The pCAGGS-Bcl-XL was kindly provided by Dr. Y. Tsujimoto (Osaka University, Osaka, Japan). The CLARP-L and CLARP-S used in this study are identical with FLIP-L (GenBank™ accession number U97074) and FLIP-S (GenBank™ accession number U97075), respectively. To generate pNF-κB-GFP, the luciferase gene from pNF-κB-Luc was replaced with the GFP gene from pEGFP-1 (Clontech). Determination of the Cell Viability—Cells (5 × 104 cells/well) were cultured with recombinant mouse soluble FasL or TNF-α in flat-bottomed 96-well plates. Fifteen hours later, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and 1-methoxy-5-methylphenazinium methylsulfate (Dojin Laboratories, Kumamoto, Japan) were added to final concentrations of 5 and 0.2 mm, respectively, and further incubated for 4 h. The absorbance of the supernatant was measured at 450 nm using a microplate reader. Measurement of IL-8 —The amount of IL-8 in culture supernatant was determined using an ELISA kit (PharMingen, San Diego, CA). RT-PCR Analysis—Single-strand cDNAs were prepared from 2 μgof total RNA using an oligo(dT) primer. Specific cDNAs were amplified by PCR using the following primers: IL-8 sense, 5′-CAGTTTTGCCAAGGAGTGCTAA-3′, antisense, 5′-AACTTCTCCACAACCCTCTGC-3′; FADD sense, 5′-CTGCAGCGCCTGGACGACTTCGAG-3′, antisense, 5′-AAAGCAGCGGCCCATCAGGA3′; α-actin sense, 5′-TGCGTGACATTAAGGAGAAG-3′, antisense, 5′-CGGATGTCCACGTCACACTT-3′. Electrophoretic Mobility Shift Assays (EMSA)—EMSA were carried out as described previously (35Masuda E.S. Tokumitsu H. Tsuboi A. Shlomai J. Hung P. Arai K. Arai N. Mol. Cell. Biol. 1993; 13: 7399-7407Crossref PubMed Scopus (126) Google Scholar), except that the double-strand oligonucleotides containing the following NF-κB-binding sequence were used (sequence overhangs are lowercase): forward, 5′-gatcTGGGGACTTTCCGC-3′; reverse, 5′-gatcGCGGAAAGTCCCCA-3′. Competitive binding experiments were performed with a 50-fold excess of cold double-strand oligonucleotides with the wild-type or a mutated NF-κB-binding sequence (forward, 5′-gatcTGTTGACTTTTCGC-3′; reverse, 5′-gatcGCGAAAAGTCAACA-3′). Reporter Assays—HEK293 or 293T cells were transfected with one of the firefly luciferase reporter plasmids described above and pRL-TK using the TransIT-LT1 reagent (TAKARA, Otsu, Japan). Stable transfectants of HEK293 cells harboring the NF-κB-Luc construct and a hygromycin B resistance gene was also generated using the TransIT-LT1 reagent according to the manufacturer's protocol. Jurkat and Jurkat-derived cell lines were transfected with pNF-κB-Luc and pRL-TK using the LipofectAMINE PLUS reagent (Invitrogen). In some experiments, cells were cotransfected with one of the tester plasmids described above and/or an empty vector as a control. The total amount of transfected DNA per culture was kept constant within an experiment. Cells were harvested 24 h after the transfection, and then assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) in a LB9501 Lumat luminometer (Berthold, Bad Wildbad, Germany). Firefly luciferase activity was normalized to the Renilla luciferase activity. In some experiments, HEK293 cells were transfected with pNF-κB-GFP and the internal control plasmid pCMV-DsRed-Express (Clontech) using the LipofectAMINE PLUS reagent. Twenty-four hours after the transfection, cells were subjected to flow cytometry analyses using a FACSCalibur® (BD Biosciences) equipped with a 488-nm argon laser. The DsRed-positive cells were gated and their mean fluorescent intensity of GFP and DsRed was calculated using CELLQuest software. Relative NF-κB activity was assessed as follows; mean fluorescent intensity of GFP/mean fluorescent intensity of DsRed × 100. Western Blotting—Western blotting was carried out as previously described (36Suda T. Nagata S. J. Exp. Med. 1994; 179: 873-878Crossref PubMed Scopus (501) Google Scholar) using the M2 mAb against FLAG peptide (Sigma), the 5F7 mAb against hcaspase-8 fragment (MBL, Nagoya, Japan), or a mAb against mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Chemicon, Temecula CA). Small Interfering RNA (siRNA)—The siRNA targeting hFADD (sense, 5′-GAUU GGAGAAGGCUGGCUCTT-3′; antisense, 5′-GAGCCAGCCUUCUCCAAUCTT-3′) and control siRNA (sense, 5′-CUCGGUCGGAAGAGGUUAGTT-3′; antisense, 5′-CUAACCUCUUCCGACCGAGTT-3′) were custom-synthesized (JBioS, Osaka, Japan). The siRNA targeting hcaspase-8 (sense, 5′-GGAACAACUGGACAGUGAAGAUCUGAG-3′; antisense, 5′-CAGAUCUUCACUGUCCAGUUGUUCCAU-3′) was custom-synthesized (iGENE, Tsukuba, Japan). HEK293 cells were transfected with double-stranded siRNA with or without various plasmids using LipofectAMINE PLUS reagent according to the manufacturer's protocol. 24 or 48 hours after the transfection, the cells were harvested and subjected to a flow cytometry, Western blotting, or RT-PCR analysis as described above. FasL Induces Delayed IL-8 Expression at the mRNA Level—We tested various human cell lines for their potential to produce IL-8 in response to FasL stimulation, and found that HEK293 cells exhibit this response without showing any evidence of apoptosis (Fig. 1, A and B). In addition, U937 and HeLa cells produced IL-8 upon FasL stimulation only when apoptosis was inhibited by a pan-caspase inhibitor z-VAD-fmk (data not shown). A neutralizing mAb against FasL inhibited the FasL-induced but not the TNF-α-induced IL-8 production (Fig. 1A), confirming that FasL was responsible for this response. The artificial soluble mFasL (27Suda T. Tanaka M. Miwa K. Nagata S. J. Immunol. 1996; 157: 3918-3924PubMed Google Scholar) used in these experiments has strong cytotoxic activity, resembling the membrane-bound form rather than the soluble form of FasL. To investigated which form of FasL is responsible for its IL-8-inducing activity, we transfected HEK293 cells with expression vectors carrying a FasL cDNA producing both membrane-bound and soluble forms (FasLDC), the membrane-bound form only (FasLDC2), or a soluble form only (FasLS) (31Shudo K. Kinoshita K. Imamura R. Fan H. Hasumoto K. Tanaka M. Nagata S. Suda T. Eur. J. Immunol. 2001; 31: 2504-2511Crossref PubMed Scopus (72) Google Scholar) (Fig. 1C, left panel). As expected, the cytotoxicity of FasL was detected in culture supernatant of the cells expressing FasLDC and FasLS but not FasLDC2, which is not released into the culture medium (Fig. 1C, right panel). Transfection of FasLDC and FasLDC2 but not FasLS induced IL-8 production, indicating that the membrane-bound form mediates the IL-8-inducing activity. TNF-α induced significant IL-8 production within 1.5 h, which lasted more than 12 h; in contrast, significant IL-8 production was detected starting 6 h after the FasL addition (Fig. 1D). Consistent with this, strong IL-8 mRNA expression was detected as early as 1.5 h after TNF-α stimulation, whereas weaker IL-8 mRNA expression was detected beginning 3 h after the FasL addition by RT-PCR analyses (Fig. 1E). These results indicate that FasL, compared with TNF-α, induces IL-8 expression more slowly at the mRNA level. NF-κB and AP-1 Binding Elements Are Required for the IL-8 Promoter Activation by FasL and NF-κB Activation Is Required for the FasL-induced IL-8 Production—The IL-8 promoter region contains three important cis-acting elements for IL-8 gene transcription, namely NF-κB, AP-1, and NF-IL-6 binding sites (29Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and IL-8 transcription required either the combination of NF-κB and NF-IL-6 or that of NF-κB and AP-1 sites, depending on the type of cells or stimulation (37Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Abstract Full Text PDF PubMed Google Scholar, 38Mahe Y. Mukaida N. Kuno K. Akiyama M. Ikeda N. Matsushima K. Murakami S. J. Biol. Chem. 1991; 266: 13759-13763Abstract Full Text PDF PubMed Google Scholar, 39Yasumoto K. Okamoto S. Mukaida N. Murakami S. Mai M. Matsushima K. J. Biol. Chem. 1992; 267: 22506-22511Abstract Full Text PDF PubMed Google Scholar). The contribution of these individual elements to the FasL-induced IL-8 promoter activity was examined using a luciferase reporter gene construct controlled under the minimal essential promoter region of the IL-8 gene (–133 to –44 bp) containing all three elements (40Okamoto S. Mukaida N. Yasumoto K. Rice N. Ishikawa Y. Horiguchi H. Murakami S. Matsushima K. J. Biol. Chem. 1994; 269: 8582-8589Abstract Full Text PDF PubMed Google Scholar) and a series of constructs with mutation in each of the elements (Fig. 2A). FasL induced strong luciferase activity in HEK293 cells transiently transfected with the construct containing the wild-type IL-8 promoter. The mutation in the NF-κB-binding site completely abolished the FasL-induced luciferase activity. The mutation in the AP-1 binding site inhibited it strongly but not completely. In contrast, the mutation in the NF-IL-6 binding site had no effect on it. These results strongly suggest that the NF-κB site is essential, and the AP-1 site is partially required for the FasL-induced IL-8 promoter activation. Consistent with these results, FasL induced the expression of a luciferase gene controlled under the NF-κB or AP-1 elements in HEK293 cells (Fig. 2, B and C). In agreement with the results shown in Fig. 1, addition of an anti-FasL mAb completely abolished the FasL-induced and NF-κB-driven expression of luciferase (Fig. 2B), and FasL induced slow NF-κB activation compared with TNF-α (data not shown). Furthermore, proteasome inhibitor lactacystin and expression of proteasome-resistant IκBα mutant (IκBα super-repressor) inhibited both NF-κB activation and IL-8 production induced by FasL or TNF-α (Fig. 2, D–G and data not shown). Considering the transfection efficiency in these experimental conditions (about 70%), the inhibition of IL-8 production by IκBα superrepressor is almost completely. These results indicate that NF-κB activation is essential for the FasL-induced IL-8 production. Induction of NF-κB Activity and IL-8 Promoter Activity by FasL Is a Cell-autonomous Response and Neither Transcriptional nor Translational Events Are Required—The delayed time course of FasL-induced IL-8 production and NF-κB activation raised the possibility that the effect of FasL might be indirect. To determine whether some secretory factor is involved in the FasL-induced NF-κB activation in a paracrine manner, we examined the potential of culture supernatant from FasL-treated HEK293 cells to activate transcription driven by NF-κB sites after the neutralization of FasL (Fig. 3A). We detected no induction of the NF-κB activity, indicating that there was no stable secondary mediator in the supernatant. TNF-α-treated supernatant induced NF-κB activity because of the carryover of TNF-α, serving as a positive control. However, these results do not rule out the possibility that the secondary mediator was unstable or a membrane-bound. To exclude this possibility, we mixed two HEK293 cell cultures that had been separately transfected with mFas cDNA and either the NF-κB reporter or IL-8 promoter reporter construct, and then cultured them with or without Jo2, the agonistic mAb specific for mFas (Fig. 3B). Jo2 did not induce luciferase activity when the reporter constructs were transfected alone, because Jo2 cannot activate the endogenous hFas of HEK293 cells (lane 2). However, the cotransfection of mFas and reporter constructs induced luciferase activity (lanes 5 and 7) and Jo2 enhanced it (lanes 6 and 8). In contrast, significant luciferase activity was not detected when mFas cDNA and a reporter construct were separately transfected, even after Jo2 stimulation (lanes 9 and 10). Consistent with reporter assays shown in Fig. 2B, FasL as well as TNF-α induced NF-κB-specific DNA binding activity in the nuclear extract of HEK293 cells, as revealed by the electrophoretic mobility shift assay (Fig. 3C). Importantly, pretreatment with actinomycin D or cycloheximide had no effect on NF-κB DNA binding activity induced either FasL or TNF-α. Therefore, neither transcriptional nor translational events are required for FasL- and TNF-α-induced NF-κB activation. These results indicate that no secondary mediator is essential for the FasL-induced NF-κB activation. Consistent with a previous report (25Manos E.J. Jones D.A. Cancer Res. 2001; 61: 433-438PubMed Google Scholar), addition of anti-p65 and -p50 antibodies resulted in the supershift of the nuclear NF-κB induced by either FasL or TNF-α (data not shown), suggesting that FasL and TNF-α induce a similar NF-κB complex. The DD of Fas and FADD Play a Critical Role for the FasL-induced NF-κB Activation—To clarify which cytoplasmic region of the Fas receptor was responsible for the FasL-induced NF-κB activation, we expressed a set of mutants of hFas in HEK293 cells. Comparable expression of the wild-type and mutant Fas on the cell surface of transfectants was confirmed by FACS analysis (see Supplemental Fig. S1). Consistent with previous reports, overexpression of hFas activated NF-κB (Fig. 4A). Deletion of the C-terminal 15 amino acids from hFas up-regulates its ability to induce apoptosis (32Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Abstract Full Text PDF PubMed Google Scholar). However, this deletion did not affect its capacity to induce NF-κB activation. On the other hand, the further deletion of Fas up to a part of the DD (FD7 and FD2) or the lprcg-type point mutation (Val238 to Asn) in the DD (FP1), which abolishes its apoptosis-inducing capacity (32Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Abstract Full Text PDF PubMed Google Scholar), also abrogated its ability to activate NF-κB. These results indicate that the C-terminal 15 amino acids of hFas are dispensable, but the DD of Fas is indispensable for its ability to activate NF-κB. We next addressed the role of FADD, which is essential to recruit caspase-8 and to induce apoptosis upon Fas ligation (28Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar, 41Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), in the FasL-induced NF-κB activation. As shown in Fig .4B, overexpression of a dominant-negative mutant of FADD (FADD-DD) efficiently blocked the FasL- but not TNF-α-induced NF-κB activation. Because overexpression of FADD-DD might interfere with Fas to recruit signaling molecules other than FADD, we sought to suppress FADD expression specifically using siRNA. The effect of siRNA to endogenous FADD was validated by semi-quantitative RT-PCR (Fig. 4C). FADD-targeting siRNA but not a control siRNA with the reverse sequence of the FADD-targeting siRNA reduced the expression of FADD gene. In addition, the specificity of our FADD-targeting siRNA was confirmed by its ability to diminish the" @default.
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• W2032408313 title "Fas Ligand Induces Cell-autonomous NF-κB Activation and Interleukin-8 Production by a Mechanism Distinct from That of Tumor Necrosis Factor-α" @default.
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