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The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling*

  • Bysani Chandrasekar
    Correspondence
    To whom correspondence should be addressed: Medicine/Cardiology, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-4598; Fax: 210-567-6960;
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Srinivas Mummidi
    Footnotes
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Anthony J. Valente
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Devang N. Patel
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Steven R. Bailey
    Footnotes
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Gregory L. Freeman
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Masahiko Hatano
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Takeshi Tokuhisa
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Liselotte E. Jensen
    Affiliations
    Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229
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  • Author Footnotes
    * This work was supported in part by the NHLBI, National Institutes of Health Grant HL68020 and by a pilot grant from the Executive Research Committee at the University of Texas Health Science Center at San Antonio. It was presented in part at the 77th Scientific Sessions of the American Heart Association, New Orleans, LA, November 7–10, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by the Merit Review Entry Program of the Department of Veterans Affairs.
    ¶ Supported by the Janey Briscoe Center of Excellence in Cardiovascular Disease.
Open AccessPublished:May 11, 2005DOI:https://doi.org/10.1074/jbc.M502586200
      We recently demonstrated that the chemokine CXCL16 is expressed in aortic smooth muscle cells (ASMC) and induces ASMC adhesion and proliferation (Chandrasekar, B., Bysani, S., and Mummidi, S. (2004) J. Biol. Chem. 279, 3188–3196). Here we reort that interleukin (IL)-18 positively regulates CXCL16 transcription in rat ASMC. We characterized the cis-regulatory region of CXCL16 and identified a functional activator protein-1 (AP-1) binding motif. Deletion or mutation of this site attenuated IL-18-mediated CXCL16 promoter activity. Gel shift, supershift, and chromatin immunoprecipitation assays confirmed AP-1-dependent CXCL16 expression. CXCL16 promoter-reporter activity was increased by constitutively active c-Fos and c-Jun and decreased by dominant negative or antisense c-Fos and c-Jun. Src kinase inhibitors PP1 and PP2, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, Akt inhibitor, the c-Jun N-terminal kinase (JNK) inhibitor SP600125, antisense JNK and dominant negative MyD88, interleukin-1 receptor-associated kinase (IRAK)-1, IRAK4, and phosphatidylinositol 3-kinase expression all attenuated IL-18-mediated AP-1 binding and reporter activity, CXCL16 promoter-reporter activity, and CXCL16 expression. Thus IL-18 induced CXCL16 expression via a MyD88 → IRAK1-IRAK4-TRAF6 (tumor necrosis factor receptor-associated factor 6) → c-Src→ PI3K → Akt → JNK → AP-1 pathway. Importantly, IL-18 stimulated ASMC proliferation in a CXCL16-dependent manner. These data provide for the first time a mechanism of IL-18-mediated CXCL16 gene transcription and CXCL16-dependent ASMC proliferation and suggest a role for IL-18-CXCL16 cross-talk in atherogenesis and restenosis following angioplasty.
      Atherosclerosis is an inflammatory disease responsible for considerable morbidity and mortality in Western societies. Both proinflammatory cytokines and chemokines play a central role in atherogenesis. The cross-talk between cytokines and chemokines amplifies the inflammatory cascade, resulting in the development and progression of atherosclerosis (
      • Libby P.
      ,
      • Steffens S.
      • Mach F.
      ,
      • Zernecke A.
      • Weber C.
      ).
      IL-18
      The abbreviations used are: IL-18, interleukin-18; IL-18R, IL-18 receptor; SMC, smooth muscle cells; ASMC, aortic smooth muscle cells; AP-1, activator protein-1; CXCL, CXC chemokine ligand; CXCR, CXC chemokine receptor; dn, dominant negative; EMSA, electrophoretic mobility shift assay; IRAK, interleukin-1 receptor-associated kinase; JNK, c-Jun N-terminal kinase; MyD88, myeloid differentiation primary response gene 88; NF-κB, nuclear factor κB; SNAP, S-nitroso-N-acetyl-dl-penicillamine; TNF, tumor necrosis factor; TRAF6, tumor necrosis factor receptor-associated factor 6; siRNA, small interfering RNA; contig, group of overlapping clones; CREB, cAMP-response element-binding protein; IFN, interferon; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; ODN, oligonucleotides; AS, antisense; rr, rat recombinant; RACE, rapid amplification of cDNA ends; UTR, untranslated region; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; p-, phospho-; PARP, poly(ADP-ribose) polymerase.
      1The abbreviations used are: IL-18, interleukin-18; IL-18R, IL-18 receptor; SMC, smooth muscle cells; ASMC, aortic smooth muscle cells; AP-1, activator protein-1; CXCL, CXC chemokine ligand; CXCR, CXC chemokine receptor; dn, dominant negative; EMSA, electrophoretic mobility shift assay; IRAK, interleukin-1 receptor-associated kinase; JNK, c-Jun N-terminal kinase; MyD88, myeloid differentiation primary response gene 88; NF-κB, nuclear factor κB; SNAP, S-nitroso-N-acetyl-dl-penicillamine; TNF, tumor necrosis factor; TRAF6, tumor necrosis factor receptor-associated factor 6; siRNA, small interfering RNA; contig, group of overlapping clones; CREB, cAMP-response element-binding protein; IFN, interferon; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; ODN, oligonucleotides; AS, antisense; rr, rat recombinant; RACE, rapid amplification of cDNA ends; UTR, untranslated region; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; p-, phospho-; PARP, poly(ADP-ribose) polymerase.
      is a proinflammatory and pro-atherogenic cytokine. Several lines of evidence suggest that it plays a critical role in the generation of atherosclerotic plaque. IL-18 expression is detected in human atherosclerotic lesions (
      • Mallat Z.
      • Corbaz A.
      • Scoazec A.
      • Besnard S.
      • Leseche G.
      • Chvatchko Y.
      • Tedgui A.
      ,
      • Gerdes N.
      • Sukhova G.K.
      • Libby P.
      • Reynolds R.S.
      • Young J.L.
      • Schonbeck U.
      ), and administration of IL-18 aggravates atherosclerosis in mice (
      • Mallat Z.
      • Corbaz A.
      • Scoazec A.
      • Besnard S.
      • Leseche G.
      • Chvatchko Y.
      • Tedgui A.
      ,
      • Gerdes N.
      • Sukhova G.K.
      • Libby P.
      • Reynolds R.S.
      • Young J.L.
      • Schonbeck U.
      ,
      • Tenger C.
      • Sundborger A.
      • Jawien J.
      • Zhou X.
      ,
      • Whitman S.C.
      • Ravisankar P.
      • Daugherty A.
      ,
      • Mallat Z.
      • Corbaz A.
      • Scoazec A.
      • Graber P.
      • Alouani S.
      • Esposito B.
      • Humbert Y.
      • Chvatchko Y.
      • Tedgui A.
      ). Furthermore, the generation of atherosclerotic lesions is reduced in IL-18-deficient apolipoprotein E null mice (
      • Elhage R.
      • Jawien J.
      • Rudling M.
      • Ljunggren H.G.
      • Takeda K.
      • Akira S.
      • Bayard F.
      • Hansson G.K.
      ), suggesting that IL-18 may play an important role in atherogenesis. Most of the IL-18 effects in atherogenesis are thought to be mediated via the induction of IFN-γ (
      • Tenger C.
      • Sundborger A.
      • Jawien J.
      • Zhou X.
      ,
      • Whitman S.C.
      • Ravisankar P.
      • Daugherty A.
      ,
      • Elhage R.
      • Jawien J.
      • Rudling M.
      • Ljunggren H.G.
      • Takeda K.
      • Akira S.
      • Bayard F.
      • Hansson G.K.
      ,
      • Wuttge D.M.
      • Zhou X.
      • Sheikine Y.
      • Wagsater D.
      • Stemme V.
      • Hedin U.
      • Stemme S.
      • Hansson G.K.
      • Sirsjo A.
      ).
      It was recently reported that intraperitoneal administration of IL-18 can induce the expression of the chemokine CXCL16 in atherosclerotic lesions and spleens of SCID (severe combined immunodeficiency)/apoE null mice (
      • Tenger C.
      • Sundborger A.
      • Jawien J.
      • Zhou X.
      ). CXCL16, a recently discovered transmembrane chemokine (
      • Matloubian M.
      • David A.
      • Engel S.
      • Ryan J.E.
      • Cyster J.G.
      ,
      • Wilbanks A.
      • Zondlo S.C.
      • Murphy K.
      • Mak S.
      • Soler D.
      • Langdon P.
      • Andrew D.P.
      • Wu L.
      • Briskin M.
      ), is a member of the non-ELR (absence of glutamic acid-leucine-arginine motif before the first conserved cysteine) CXC chemokine subfamily. Unlike other members of this subgroup, it is structurally similar to CX3CL1 (fractalkine (
      • Bazan J.F.
      • Bacon K.B.
      • Hardiman G.
      • Wang W.
      • Soo K.
      • Rossi D.
      • Greaves D.R.
      • Zlotnik A.
      • Schall T.J.
      )), containing four distinct domains; that is, a chemokine domain tethered to the cell surface via a mucin-like stack, which in turn is attached to transmembrane and cytoplasmic domains. As a transmembrane protein it acts as an adhesion molecule, and upon cleavage by ADAM10 (
      • Abel S.
      • Hundhausen C.
      • Mentlein R.
      • Schulte A.
      • Berkhout T.A.
      • Broadway N.
      • Hartmann D.
      • Sedlacek R.
      • Dietrich S.
      • Muetze B.
      • Schuster B.
      • Kallen K.J.
      • Saftig P.
      • Rose-John S.
      • Ludwig A.
      ,
      • Gough P.J.
      • Garton K.J.
      • Wille P.T.
      • Rychlewski M.
      • Dempsey P.J.
      • Raines E.W.
      ) it acts as a chemokine. It attracts CXC receptor 6 (CXCR6) expressing T, NK, and NKT cells to the sites of inflammation and injury (
      • Shashkin P.
      • Simpson D.
      • Mishin V.
      • Chesnutt B.
      • Ley K.
      ,
      • Heydtmann M.
      • Adams D.H.
      ,
      • Unutmaz D.
      • Xiang W.
      • Sunshine M.J.
      • Campbell J.
      • Butcher E.
      • Littman D.R.
      ,
      • Kim C.H.
      • Kunkel E.J.
      • Boisvert J.
      • Johnston B.
      • Campbell J.J.
      • Genovese M.C.
      • Greenberg H.B.
      • Butcher E.C.
      ,
      • Kim C.H.
      • Johnston B.
      • Butcher E.C.
      ). CXCL16 also functions as a scavenger receptor for phosphatidylserine and oxidized lipoprotein (hence, the name SR-PSOX (
      • Shimaoka T.
      • Kume N.
      • Minami M.
      • Hayashida K.
      • Kataoka H.
      • Kita T.
      • Yonehara S.
      )). By enhancing the uptake of oxidized low density lipoprotein, CXCL16 promotes foam cell formation. We recently demonstrated that CXCL16 induces SMC proliferation (
      • Chandrasekar B.
      • Bysani S.
      • Mummidi S.
      ). SMC proliferation is a hallmark of atherosclerosis and restenosis after angioplasty (
      • McBride W.
      • Lange R.
      • Hillis L.
      ,
      • Austin G.E.
      • Ratliff N.B.
      • Hollman J.
      • Tabei S.
      • Phillips D.F.
      ,
      • Ross R.
      ,
      • Ross R.
      ). Furthermore, a positive correlation was shown between mutations in CXCL16 gene and the severity of coronary artery stenosis (
      • Lundberg G.A.
      • Kellin A.
      • Samnegard A.
      • Lundman P.
      • Tornvall P.
      • Dimmeler S.
      • Zeiher A.M.
      • Hamsten A.
      • Hansson G.K.
      • Eriksson P.
      ), suggesting that CXCL16 may play a causal role in the development and progression of atherosclerosis.
      Taken together, these studies suggest that IL-18 may induce SMC proliferation through a CXCL16-mediated mechanism. In this study we tested this hypothesis directly and delineated the mechanisms by which IL-18 induces CXCL16 expression in SMC. IL-18 induced CXCL16 expression through an AP-1-dependent mechanism, and this induction involves MyD88, IRAK, TRAF6, c-Src, PI3K, Akt, and JNK. More importantly, IL-18 induced SMC proliferation in a CXCL16-dependent manner. IL-18-mediated CXCL16 expression was independent of the classical inflammatory cytokines IL-1β, TNF-α, and IFN-γ. These results ascribe a previously unrecognized role for IL-18 in SMC proliferation and suggest that IL-18 and CXCL16 cross-talk may lead to the amplification of an inflammatory cascade in vessel wall-promoting atherosclerosis.

      MATERIALS AND METHODS

      Cell Culture—Non-transformed rat aortic smooth muscle cells (ASMC) were a generous gift from Dr. Sergei N. Orlov (University of Montreal, Montreal, Canada) and have been previously described (
      • Chandrasekar B.
      • Mummidi S.
      • Perla R.P.
      • Bysani S.
      • Dulin N.O.
      • Liu F.
      • Melby P.C.
      ). ASMC were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum and 100 units/ml each of penicillin and streptomycin. The cells were passaged when confluent. For cytokine stimulation experiments, ASMC were grown until 70–80% confluent, then the medium was replaced with Dulbecco's modified Eagle's medium plus 0.5% bovine serum albumin. After overnight culture recombinant rat IL-18 (rrIL-18; #521-RL-025/CF, R&D Systems, Minneapolis, MN) was added at the indicated doses and for the indicated time periods.
      Cytokine Stimulation—Recombinant IL-18 preparations contained <1.0 enzyme units/μg of endotoxin as determined by the LAL method (manufacturer's technical data sheet, R&D Systems). To determine any possible contribution of endotoxin to the IL-18-mediated effects, ASMC were treated simultaneously with 10 μg/ml polymyxin B (#P4932, Sigma). In the inhibition studies, before the addition of IL-18 ASMC were treated with either 100 nm wortmannin or 20 μm LY294002 for 1 h (PI3K inhibitors), 1 μm 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate for 1 h (Akt inhibitor), 1 μm 4-amino-1-tert-butyl-3-(1′-naphthyl)pyrazolo[3,4-d]pyrimidine (PP1), or 1 μm 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) for 30 min (selective inhibitors of the Src-family of tyrosine kinases), 1 μm 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3) for 30 min (negative control for PP1 and PP2), and 20 μm anthra[1,9-cd]pyrazol-6(2H)-one 1,9-pyrazoloanthrone (SP600125) for 30 min (JNK inhibitor). Control cells received the vehicle (Me2SO) alone. To determine whether p38 MAPK and ERK play a role in IL-18-mediated CXCL16 expression, ASMC were pretreated with 1 μm SB203580 for 30 min (p38 MAPK inhibitor) or 10 μm PD98059 for 1 h (ERK1/2 inhibitor) or Me2SO alone before the addition of IL-18 for 2 h. The above inhibitors were obtained from Calbiochem and Axxora, LLC. (San Diego, CA).
      Antisense Oligonucleotide and siRNA Treatment—To determine whether IL-18-induced CXCL16 expression is a direct mechanism or is mediated via induction of IL-1β, TNF-α, or IFN-γ, ASMC were transfected with 10 μm phosphorothioated-modified antisense (AS) oligonucleotides (ODN (
      • Schroeder R.A.
      • Gu J.S.
      • Kuo P.C.
      )) for TNF-α (5′-CTG ACT GCC TGG GCC AGA GGG CTG ATT AG-3′; phosphorothioated on three nucleotides on each end of the molecule), IL-1β (5′-CAG GAT GAG GAC ATG AGC ACC-3′), or IFN-γ (5′-TAC TGC CAC GGC ACA GTC ATT GAA-3′) before the addition of IL-18. Transfections were carried out using the Oligofectamine™ reagent, as recommended by the manufacturer (Invitrogen). After 16 h, cells were treated with IL-18 at the indicated times and doses. IFN-γ expression was also knocked down using 150 nm rat IFN-γ siRNAs (5′-GGC ACA CUC AUU GAA AGC Ctt; 5′-GGA CGG UAA CAC GAA AAU Att-3′; 5-GGA UGC AUU CAU GAG CAU Ctt-3′; Ambion, Inc., Austin, TX). To verify whether release of preformed cytokines mediates IL-18-induced CXCL16 expression, ASMC were incubated with 5 μg/ml neutralizing antibodies to rat TNF-α (#AF-510-NA), IL-1β (#AF-501-NA), or IFN-γ (#AF-585-NA) (R&D Systems) 1 h before the addition of IL-18. Normal goat IgG served as a control. Efficacy of these neutralizing antibodies was verified as previously described in transient transfection assays using NF-κB (pNF-κB-Luc) and IFN-γ-activating sequence (pGAS-Luc) reporter constructs (Stratagene, La Jolla, CA (
      • Chandrasekar B.
      • Vemula K.
      • Surabhi R.M.
      • Li-Weber M.
      • Owen-Schaub L.B.
      • Jensen L.E.
      • Mummidi S.
      )). The vector pEGFP-Luc served as a negative control. 24 h after transfection, cells were treated for 1 h with the neutralizing antibodies followed by the addition of respective recombinant proteins (100 pg/ml rrIL-1β, 100 pg/ml rrTNF-α, or 10 ng/ml rrIFN-γ). After 7 h of incubation, the cells were harvested for the dual-luciferase assay (Promega, Madison, WI).
      Akt1 and -2 expression was also targeted by siRNAs (Signal Silence™ Akt siRNA, #6211, Cell Signaling Technology, Beverly, MA (
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      )). Sense siRNA (5′-UUC UCC GAA CGU GUC ACG UdTdT-3′; #1022076, Qiagen, Inc., Valencia, CA) served as a negative control.
      Cell Death Assays—To investigate IL-18-induced apoptosis, ASMC were cultured in complete medium until ∼70% confluent. The medium was replaced with Dulbecco's modified Eagle's medium plus 0.5% bovine serum albumin, and the cells were incubated for 48 h. IL-18 was added at 25 ng/ml and incubated for 24 h. Cells were harvested, and the presence of mono- and oligonucleosomes in the cytoplasm was assayed by enzyme-linked immunoassay (Cell Death Detection ELISAplus kit, Roche Applied Science (
      • Chandrasekar B.
      • Vemula K.
      • Surabhi R.M.
      • Li-Weber M.
      • Owen-Schaub L.B.
      • Jensen L.E.
      • Mummidi S.
      ,
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      )). As a positive control, S-nitroso-N-acetylpenicillamine (SNAP, #487910, EMD Biosciences, Inc., La Jolla, CA) was added to a final concentration of 500 μm for 24 h (
      • Chandrasekar B.
      • Mummidi S.
      • Perla R.P.
      • Bysani S.
      • Dulin N.O.
      • Liu F.
      • Melby P.C.
      ). Apoptotic cell death was also analyzed using the annexin V-fluorescein isothiocyanate apoptosis detection kit (Oncogene Research Products, San Diego, CA). Floating cells were collected and added to the scraped adherent cells for the assay. Cells were counterstained with propidium iodide and analyzed by flow cytometry.
      Cell Proliferation Assays—Cell proliferation was determined by [3H]thymidine incorporation (
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      ). To confirm the role of CXCL16 in IL-18-mediated ASMC proliferation, ASMC were transfected as above with a mixture of CXCL16 siRNAs (5′-AGU UGC UAC UGU GAU CGU Att-3′, 5′-GUU UUC ACC ACC AAA AAC Att-3′, 5′-AAC CUC CAG A CA CAA GCA Ctt-3′) and incubated for 48 h before the addition of IL-18.
      5Rapid Amplification of cDNA Ends (5RACE)—The 5′ ends of rat CXCL16 mRNA transcripts were determined by 5′ RACE using ASMC total RNA. Total RNA was isolated using the TRIzol reagent (Invitrogen) and treated with RNase-free DNase to eliminate any genomic DNA contamination. We used the SMART RACE cDNA amplification kit (BD Biosciences, Clontech, Palo Alto, CA) to amplify complete CXCL16 transcripts following the manufacturer's instructions. Successive PCR was carried out using the rat CXCL16-specific antisense primers, 5′-TCA TCT GTC TGT CTG CTG GTT TGT-3′ and 5′-TTC CTC TGG CTC CAA GAT GCT TTC-3′, and the sense primers provided in the kit. The products of the second PCR amplification were gel-purified and cloned into the pCR2.1-TOPO vector (Invitrogen), and the nucleotide sequence of four independent clones was determined.
      Cloning of the 5-Flanking Sequence of Rat CXCL16 and Vector Construction—A blast search of the GenBank™ data base identified rat CXCL16 gene sequences in GenBank™ (NW_047334). This contained in tandem the open reading frame of CXCL16 and the 5′-untranslated region (UTR) sequences that we identified by 5′-RACE. Approximately ∼1.2 kilobases of the 5′-flanking region of the gene was amplified from rat genomic DNA using the sense primer 5′-CTG ACA TAA GGA CTC AGG TCT CT-3′ and the antisense primer 5′-TCA GTA GGA TCC AGT CAC ATG G-3′ and cloned into the pCR2.1-TOPO vector. A series of nested deletions was generated using the sense primers 5′-gag ctc GGA AAG TGG ATA TTT AGG GTT-3′, 5′-gag ctc AAA GGC TTG CTG AGA AAG GTA-3′, and 5′-gag ctc CAC AGC TCA TAA CTT CAG TGG-3′. All the sense primers contained a SacI restriction site at the 5′ end (lowercase). The antisense primer (5′-ctc gag TCA GTA GGA TCC AGT CAC ATG-3′) contained an XhoI restriction site. The PCR products were cloned into pCR2.1-TOPO and subcloned into the pGL3-Basic reporter vector (Promega). The deletion construct S5 was generated by annealing sense (5′-gag ctc CAG TCC TGG GGA TTT GTT TGA CTG CTT CAC CTT GGC CAT GTG ACT GGA TCC TAC TGA ctc gag) and complimentary antisense oligonucleotides, digesting with SacI and XhoI, and inserting into pGL3-Basic. The 5′-flanking sequence of the CXCL16 gene was analyzed by MatInspector Professional® software to identify the potential binding sites for various transcription factors. The nucleotide sequences of the rat CXCL16 5′-flanking region and the mRNA have been deposited with the GenBank™ under the accession numbers DQ025527 and DQ025528, respectively.
      Site-directed Mutagenesis—Mutation of the AP-1 binding site in the S2 reporter vector construct was performed by site-directed mutagenesis using the QuikChange kit (Stratagene), the sense primer 5′-ACAAAACTCAATGACTtgAATCCTAAAATGCG-3′, and the antisense primer 5′-CGCATTTTAGGATTcaAGTCATTAGTTTTGT-3′ (mutated nucleotides are in lowercase). Mutation was confirmed by nucleotide sequencing.
      Cell Transfection and Reporter Assays—ASMC were transfected with 3 μg of the CXCL16 reporter constructs and 100 ng of the control Renilla luciferase vector pRL-TK (Promega) using Lipofectamine® (
      • Chandrasekar B.
      • Bysani S.
      • Mummidi S.
      ,
      • Lundberg G.A.
      • Kellin A.
      • Samnegard A.
      • Lundman P.
      • Tornvall P.
      • Dimmeler S.
      • Zeiher A.M.
      • Hamsten A.
      • Hansson G.K.
      • Eriksson P.
      ,
      • Chandrasekar B.
      • Vemula K.
      • Surabhi R.M.
      • Li-Weber M.
      • Owen-Schaub L.B.
      • Jensen L.E.
      • Mummidi S.
      ,
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      ). After incubation for the indicated time periods, the cells were harvested for the dual-luciferase assay. Data were normalized by dividing firefly luciferase activity with that of the corresponding Renilla luciferase. The transfection efficiency of rat ASMC (∼32%) was determined using pEGFP-N1 vector (BD Biosciences Clontech (
      • Chandrasekar B.
      • Mummidi S.
      • Perla R.P.
      • Bysani S.
      • Dulin N.O.
      • Liu F.
      • Melby P.C.
      )). All plasmids were purified using EndoFree Plasmid Maxi kit (Qiagen).
      To determine whether IL-18 induces AP-1 activation in ASMC, we used pAP-1-Luc (PathDetect® AP-1 Cis-reporting System, Stratagene), an inducible AP-1 reporter vector containing seven repeats of the AP-1 enhancer element. ASMC were co-transfected with pAP-1-Luc and pRL-TK and after 24 h of incubation were treated with the indicated dose of IL-18. pEGFP-Luc, a vector that encodes a fusion of enhanced green fluorescent protein and luciferase under the regulation of the CMV promoter (Clontech) served as the control.
      In addition to the pharmacological inhibitors described above, ASMCs were transfected with rat MyD88 siRNA (siGENOME SMARTpool siRNA, #M-099508-00, Dharmacon, Lafayette, CO), dominant negative (dn) IRAK1 in pCI-Neo, dnIRAK4 (1–191) in pcDNA4/HisMaxC (Invitrogen), dnTRAF6 in pRK5 (TRAF6-(289–522)-FLAG), and dnPI3K in pcDNA3 as described previously (
      • Chandrasekar B.
      • Vemula K.
      • Surabhi R.M.
      • Li-Weber M.
      • Owen-Schaub L.B.
      • Jensen L.E.
      • Mummidi S.
      ,
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      ). The corresponding empty vectors served as controls. Inhibition of JNK activation was also achieved by treating ASMC with antisense oligonucleotides against JNK1 (5′-CTC TCT GTA GGC CCG CTT GG-3′) and JNK2 (5′-GTC CGG GCC AGG CCA AAG TC-3′). Scrambled sequences (JNK1, 5′-CTT TCC GTT GAA CCC CTG GG-3′) and JNK2 (5′-GTG CGC GCG AGC CCG AAA TC-3′) served as controls (
      • Potapova O.
      • Gorospe M.
      • Bost F.
      • Dean N.M.
      • Gaarde W.A.
      • Mercola D.
      ). Ectopic expression of c-Fos and c-Jun was achieved by transfecting cells with constitutively active c-Fos or c-Jun expression vectors (
      • Okada S.
      • Obata S.
      • Hatano M.
      • Tokuhisa T.
      ). pcDNA3 served as a control. Expression of c-Fos was inhibited by transfecting the cells with dn-c-Fos in CMV500 (a kind gift from Dr. C. Vinson, Laboratory of Metabolism, National Institutes of Health, Bethesda, MD). CMV500 served as a control. Expression of c-Fos was also inhibited by treating the cells with phosphorothioated c-Fos antisense oligodeoxynucleotides (5′-GAA CAT CAT GGT CGT-3′ (
      • Quercia S.
      • Chang S.L.
      )). Sense (5′-ACG ACC ATG ATG TTC-3′) as well as scrambled mismatched oligonucleotides (5′-TGC TGG TAC TAC AAG-3′) served as controls. c-Jun expression was also targeted using phosphorothioated c-jun-specific antisense oligonucleotide (5′-CGT TTC CAT CTT TGC AGT-3′ (
      • Zhang S.
      • Liu J.
      • MacGibbon G.
      • Dragunow M.
      • Cooper G.J.
      )). Sense oligonucleotides (5′-ACT GCA AAG ATG GAA ACG-3′) served as controls.
      Electrophoretic Mobility Shift Assay (EMSA)—AP-1 DNA binding activity was analyzed by EMSA (
      • Chandrasekar B.
      • Melby P.C.
      • Sarau H.M.
      • Raveendran M.
      • Perla R.P.
      • Marelli-Berg F.M.
      • Dulin N.O.
      • Singh I.S.
      ) using ASMC nuclear extracts and 32P-labeled double-stranded DNA probes for the AP-1 site of the CXCL16 promoter (5′-ACA AAA CTC AAT GAC Tca AAT CCT AAA ATG CG-3′) and mutated CXCL16 AP-1 site (5′-ACA AAA CTC AAT GAC Tgt AAT CCT AAA ATG CG-3′). Supershift EMSA was performed as above, except that the nuclear extracts were preincubated for 1 h with 1 μg of rabbit antibodies to either c-Jun (sc-45X), c-Fos (sc-52X), JunB (sc-46X), or JunD (sc-74X) (TransCruz Gel Supershift reagents; Santa Cruz Biotechnology, Inc.). Isotype-matched rabbit polyclonal antibodies served as controls.
      Chromatin Immunoprecipitation Assay—ASMC were cultured in complete medium in 100-mm dishes until ∼70% confluent. The medium was replaced with Dulbecco's modified Eagle's medium plus 0.5% bovine serum albumin and incubated overnight. IL-18 was added at the indicated dose and incubated for 1 h. The cells were then fixed by the addition of 280 μl of 37% formaldehyde (Sigma) to 10 ml of culture medium for 10 min at 37 °C, harvested, and processed for immunoprecipitation using a commercially available kit (Chromatin Immunoprecipitation assay kit, #17-295; Upstate Biotechnology Inc., Lake Placid, NY) and following the manufacturer's protocol. Immune complexes were eluted, reverse cross-linked using 5 m NaCl, and purified by phenol/chloroform extraction. Ethanol-precipitated DNA pellets were redissolved in Tris-EDTA buffer. The supernatant of an immunoprecipitation reaction carried out in the absence of c-Fos antibody was purified and used for the total input DNA control. The supernatant DNA was diluted 1:100 before PCR analysis. PCR was carried out on 1 μl of each sample using sense primer, 5′-CTT GAG GTA AGA TCT ACA TAG GAA-3′, and antisense primer, 5′-CAA ATT TAC CTT TCT CAG CAA-3′, which amplified a 239-bp segment of the rat CXCL16 gene from –1094 to –845 relative to the transcription start site. PCR products were analyzed on 2% agarose gels. Primers from the CXCL16 open reading frame (sense, 5′-TGT GGA ATC GGT CAT GGG CAG-3′; antisense, 5′-TGC CAG CAC CAG CTC CTG GTT-3′) that would amplify a 294-bp fragment were used in a control PCR.
      Analysis of RNA Expression—IL-18Rα and IL-18Rβ mRNA expression were determined by Northern blot analysis using 2 μg of ASMC poly(A)+ RNA (
      • Chandrasekar B.
      • Melby P.C.
      • Sarau H.M.
      • Raveendran M.
      • Perla R.P.
      • Marelli-Berg F.M.
      • Dulin N.O.
      • Singh I.S.
      ) (TRIzol® reagent (Invitrogen) and the PolyATtract® mRNA isolation system (Promega). IL-18Rα and IL-18Rβ cDNA were amplified by reverse transcription-PCR as previously described (
      • Chandrasekar B.
      • Colston J.T.
      • de la Rosa S.D.
      • Rao P.P.
      • Freeman G.L.
      ). Northern blotting, autoradiography, and densitometry were performed as described previously (
      • Chandrasekar B.
      • Colston J.T.
      • de la Rosa S.D.
      • Rao P.P.
      • Freeman G.L.
      ). Rat CXCL16 cDNA was amplified from reverse-transcribed ASMC poly(A)+ RNA using the following primers: sense, 5′-CTT CTT TGC GCT GCT GAC TC-3′; antisense, 5′-GGT GCT CTG AAA CTG TAC AG-3′. Rat CXCR6 cDNA was amplified using the following primers derived from the mouse sequence (GenBank™ accession number NM_030712.1): sense, 5′-ATG GAT GAT GGG CAT CAA GAG-3′; antisense, 5′-TCA TAG TGA GCA ATG GCA GG-3′. The amplified PCR products were subcloned into pCR2.1-TOPO (Invitrogen), and sequenced on both strands. The rate of CXCL16 gene transcription was analyzed by nuclear run-on assays. Glyceraldehyde-3-phosphate dehydrogenase served as an internal control. CXCL16 mRNA stability after IL-18 or vehicle (control) treatment was determined after actinomycin D treatment (
      • Chandrasekar B.
      • Melby P.C.
      • Sarau H.M.
      • Raveendran M.
      • Perla R.P.
      • Marelli-Berg F.M.
      • Dulin N.O.
      • Singh I.S.
      ).
      Analysis of Protein Expression—Protein extraction, Western blotting, autoradiography, and densitometry were performed as described previously (
      • Chandrasekar B.
      • Melby P.C.
      • Sarau H.M.
      • Raveendran M.
      • Perla R.P.
      • Marelli-Berg F.M.
      • Dulin N.O.
      • Singh I.S.
      ). Goat anti-mouse CXCL16 antibodies (AF503) and anti-IFN-γ antibodies (AF-585-NA) were obtained from R&D Systems. Cross-reactivity of the anti-CXCL18 antibody between rat and mouse CXCL16 was confirmed in pilot studies. Antibodies to c-Src (sc-5266), p-c-Src Tyr530 (sc-16846), p-c-Src Tyr139 sc-12928-R), p-c-Jun Ser63 (sc-7980-R), p-c-Jun Ser73 (sc-16311-R), MyD88 (sc-8197), and actin were all obtained from Santa Cruz Biotechnology. Rabbit antibody to p85 (06-497) was obtained from Upstate Biotechnology, Inc. Antibodies to Akt, phospho-Akt (Thr308), ERKl/2 (9102), phospho-ERK1/2 (9101S), p38 MAPK (9212), phospho-JNK (9251S), Bad (9292), phospho-Bad Ser136 (9295), caspase-3 (9662), and PARP (9542) were purchased from Cell Signaling Technology, Inc. The anti-caspase-3 monoclonal antibodies detect both the full-length (35 kDa) and cleaved active forms (17/19 kDa). The anti-PARP antibodies detect both full-length (116 kDa) and the cleaved active form (89 kDa). Activation-specific antibody against p38 MAPK (Thr180 and Tyr182; anti-ACTIVE® p38 polyclonal antibody, V1211) was from Promega. Actin was used as an internal control.
      Measurement of Src Tyrosine Kinase, PI3K, Akt Kinase, and JNK Activities—Src kinase activity was determined as previously described (
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      ) using as substrate the C-terminal domain of Sam 68, corresponding to amino acids 331–443 of the mouse sequence (Santa Cruz Biotechnology). PI3K lipid kinase assays were performed as described before using p85 immunoprecipitates (
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      ). Akt kinase activity was performed using a commercially available kit (Cell Signaling Technology (
      • Chandrasekar B.
      • Mummidi S.
      • Perla R.P.
      • Bysani S.
      • Dulin N.O.
      • Liu F.
      • Melby P.C.
      )). JNK activity in cell extracts was measured using a commercially available kit (stress-activated protein kinase/JNK assay kit, 9810, Cell Signaling Technology). A c-Jun fusion protein (glutathione S-transferase fused to the N terminus of c-Jun, codons 1–89) was used to pull down JNK enzyme from cell extracts, which in the presence of kinase buffer and ATP phosphorylates c-Jun (1–89). Phosphorylated c-Jun was detected by Western blot analysis using anti-phospho-c-Jun (Ser63) antibody.
      Statistical Analyses—Comparison between experimental groups was made using the unpaired t test with the Bonferroni correction for multiple comparisons if needed. If three comparisons were made, a p value of <0.025 was considered significant. For two comparisons, a p value of <0.05 was considered significant. Each experiment was performed at least three times, and group data were expressed as mean ± S.E.

      RESULTS

      The pluripotent effects of IL-18 are mediated through a heterodimeric receptor IL-18R comprising a ligand binding subunit IL-18Rα and a signal transducing subunit IL-18Rβ. We confirmed that ASMC express both of these receptors at basal conditions (Fig. 1A), and these results are in agreement with the observations of Gerdes et al. (
      • Gerdes N.
      • Sukhova G.K.
      • Libby P.
      • Reynolds R.S.
      • Young J.L.
      • Schonbeck U.
      ). Although quiescent ASMC express low levels of CXCL16, IL-18 induced CXCL16 mRNA expression in a dose- and time-dependent manner. Peak levels of CXCL16 mRNA were detected at a concentration of 25 ng/ml (Fig. 1B). CXCL16 was detected after 30 min of treatment, peaked at 1 h, and persisted at these high levels throughout the 24-h study period (Fig. 1C), suggesting that IL-18 induces rapid and sustained expression of CXCL16 in ASMC. The increased CXCL16 mRNA levels also correlated with increased expression at the protein level (Fig. 1D). Because rat CXCL16-specific reagents are not available, we did not measure secreted CXCL16 in culture supernatants. The recombinant rat IL-18 used in the present study contained <1.0 enzyme units of endotoxin/μg of the cytokine as determined by the LAL method. Because lipopolysaccharide has previously been shown to induce CXCL16 expression in ASMC, we treated ASMC with IL-18 and polymyxin B (10 μg/ml) simultaneously. Treatment with polymyxin B failed to reduce or attenuate IL-18-mediated CXCL16 expression (Fig. 1E), indicating that it was the IL-18 and not the low contaminating levels of endotoxin that was responsible for the increased CXCL16 expression in ASMC. Furthermore, pretreatment of ASMC with anti-IL-18-neutralizing antibodies before IL-18 addition did not influence the induction of CXCL16 expression (data not shown), further supporting a specific role for the cytokine. Because an increase in mRNA expression can be the result of either increased gene transcription, mRNA stability, or both, we next investigated CXCL16 gene transcription by nuclear run-on assay and mRNA stability by actinomycin D pulse experiments. As shown in Fig. 1F, a marked increase in CXCL16 gene transcription resulted from the treatment of ASMC with IL-18. In contrast, no change was observed in CXCL16 mRNA stability after IL-18 treatment (Fig. 1G), indicating that IL-18-mediated CXCL16 expression was regulated predominantly at the transcriptional level. Together, these results demonstrate that IL-18 is a potent inducer of CXCL16 mRNA and protein expression in ASMC.
      Figure thumbnail gr1
      Fig. 1IL-18 induces CXCL16 expression in rat aortic smooth muscle cells. A, ASMC express IL-18Rα and -β mRNA. Northern blot analysis was performed using 2 μg of poly(A)+ RNA isolated from cultured ASMC. B, dose-dependent induction of CXCL16 mRNA by IL-18. Quiescent ASMC were treated with the indicated concentrations of IL-18 for 2 h, and total RNA was isolated and analyzed for CXCL16 mRNA and 28 S rRNA (loading control). C, time course studies. Quiescent ASMC were treated with 25 ng/ml IL-18 for up to 24 h, and total RNA was analyzed for CXCL16 mRNA expression. D, IL-18 induced CXCL16 protein expression. Quiescent ASMC were treated with IL-18 as in C, and cell extracts were analyzed by Western blotting for CXCL16 protein and β-actin. E, IL-18-induced CXCL16 expression was not mediated by endotoxin. Quiescent ASMC were either untreated or treated with polymyxin B vehicle (H2O) or 10 μg/ml polymyxin B (left three lanes) or with 25 ng/ml IL-18, 25 ng/ml IL-18 plus vehicle, or 25 ng/ml IL-18 plus 10 μg/ml polymyxin B (right three lanes) for 2 h. Total RNA was analyzed for CXCL16 expression as before. F, IL-18-induced CXCL16 expression was regulated at the transcriptional level. Quiescent ASMC were treated with 25 ng/ml IL-18 for 2 h, and the nuclear RNA was isolated and analyzed for CXCL16 transcription by the run-on assay. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the loading control, and pCR2.1-TOPO vector served as the internal control. A representative of three independent experiments is shown. G, IL-18-induced CXCL16 expression was not due to increased mRNA stability. Quiescent ASMC were treated with 25 ng/ml IL-18 for 2 h followed by the addition of 5 μg/ml actinomycin D for up to 6 h. Total RNA was isolated and analyzed for CXCL16 expression by Northern blotting as before. Band intensity was determined by densitometry and normalized to the value obtained at 2 h (100%). The results are the mean ± S.E. of four independent experiments.
      Although CXCL16 was strongly induced in the ASMC after IL-18 treatment, it is not evident from the above experiments whether this was dependent on the up-regulation of other proinflammatory cytokines such as IL-1β, TNF-α, or IFN-γ, all of which have been shown previously to induce CXCL16 expression in fibroblasts, endothelial cells, and SMC (
      • Abel S.
      • Hundhausen C.
      • Mentlein R.
      • Schulte A.
      • Berkhout T.A.
      • Broadway N.
      • Hartmann D.
      • Sedlacek R.
      • Dietrich S.
      • Muetze B.
      • Schuster B.
      • Kallen K.J.
      • Saftig P.
      • Rose-John S.
      • Ludwig A.
      ,
      • Gough P.J.
      • Garton K.J.
      • Wille P.T.
      • Rychlewski M.
      • Dempsey P.J.
      • Raines E.W.
      ,
      • Wagsater D.
      • Olofsson P.S.
      • Norgren L.
      • Stenberg B.
      • Sirsjo A.
      ,
      • Yamauchi R.
      • Tanaka M.
      • Kume N.
      • Minami M.
      • Kawamoto T.
      • Togi K.
      • Shimaoka T.
      • Takahashi S.
      • Yamaguchi J.
      • Nishina T.
      • Kitaichi M.
      • Komeda M.
      • Manabe T.
      • Yonehara S.
      • Kita T.
      ). To investigate whether IL-18 induces CXCL16 expression directly or its up-regulation is mediated via induction of IL-1β, TNF-α, or IFN-γ, we used antisense oligonucleotides (AS ODN) to suppress their expression. Our results indicate that treatment with AS ODN directed against IL-1β, TNF-α, or IFN-γ all failed to modulate either the low basal or the marked IL-18-induced, CXCL16 mRNA expression level (Fig. 2A). However, the AS ODN significantly inhibited lipopolysaccharide/IL-18-mediated corresponding cytokine secretion (data not shown). To rule out the role of preformed cytokines in IL-18-mediated CXCL16 induction, ASMC were incubated with neutralizing antibodies to either IL-1β, TNF-α, or IFN-γ for 1 h before IL-18 addition. Again, these treatments all failed to modulate IL-18-mediated CXCL16 mRNA expression (Fig. 2B). Normal goat IgG, used as a control, also failed to modulate IL-18-mediated CXCL16 expression (data not shown). The ability of these antibodies to neutralize their target cytokines was verified in transient transfection assays in which ASMC transfected with either NF-κB (pNF-κB-Luc) or IFN-γ-activating sequence (pGAS-Luc) reporter constructs were treated with the antibodies before incubation with their respective recombinant proteins (rrIL-1β, 100 pg/ml; rrTNF-α, 100 pg/ml; rrIFN-γ, 10 ng/ml) for an additional 7 h. The antibodies were highly effective, as they strongly inhibited reporter gene activity in ASMC resulting from the corresponding cytokine treatment (data not shown). To further confirm that IL-18-induced CXCL16 expression was not dependent on IFN-γ, we treated ASMC with IFN-γ siRNA, and knockdown of IFN-γ was confirmed by Western blotting (Fig. 2C). Once again, knockdown of IFN-γ failed to modulate IL-18-mediated CXCL16 expression (Fig. 2D). Together, these results demonstrate that IL-18 induces CXCL16 expression in ASMC independent of other cytokines.
      Figure thumbnail gr2
      Fig. 2IL-18-mediated CXCL16 expression is independent of IL-1β, TNF-α, and IFN-γ. A, quiescent ASMC were transfected with 10 μm IL-1β, TNF-α, or IFN-γ antisense oligonucleotides and incubated for 16 h before the addition of 25 ng/ml IL-18. Cells were harvested at 2 h, and total RNA was isolated and analyzed for CXCL16 expression by Northern blotting. A representative of three independent experiments is shown. B, to exclude the role of preformed cytokines in IL-18-mediated CXCL16 expression, quiescent ASMC were incubated with IL-1β-, TNF-α, or IFN-γ neutralizing antibodies (5 μg/ml) followed by the addition of 25 ng/ml IL-18 for 2 h, and CXCL16 expression was analyzed as in A. C and D, to confirm that the IL-18 induction of CXCL16 expression was independent of IFN-γ, ASMC were transfected with either IFN-γ or control siRNAs and incubated for 48 h. The cells were then untreated or treated with 25 ng/ml IL-18 for 3 h before isolation of total cell protein. Knock-down of IL-18-induced IFN-γ protein expression was confirmed by Western blotting (C, fifth lane). Knock-down of IFN-γ had no effect on the induction of CXCL16 mRNA by IL-18 (D, fifth lane).
      Because IL-18 strongly induced CXCL16 gene transcription (Fig. 1F), we sought to determine the cis-trans interactions that lead to its increased expression. To this end, we determined the genomic structure of rat CXCL16 gene and defined the cis-elements that are responsible for its induction by IL-18. As a preliminary step, we performed extensive blast searches to identify full-length mouse and human CXCL16 transcripts, for which cap-based libraries are available. Through this bioinformatics approach, we identified several mouse CXCL16 transcripts. The longest of these transcripts, the sequence of which was deposited under GenBank™ accession number AK028875, contained 537 nucleotides upstream of the ATG start codon. We also determined that this transcript showed a high degree of homology to the rat genomic sequence deposited under GenBank™ accession number NW_047334. This analysis suggested that the 5′-UTR sequences of the rat and mouse sequences are highly homologous and the 5′ end of the CXCL16 transcript from these two species may originate at the same relative position to the ATG start codon. To verify this experimentally, we performed 5′-RACE analysis with primers derived from rat genomic sequence that shared homology with the 5′-UTR of the mouse CXCL16 transcript. We analyzed four independent plasmid clones that were obtained after 5′-RACE, and this suggested that the rat CXCL16 transcript contained an additional 25 bases at the 5′ end compared with the longest known mouse transcript. Thus, the rat 5′-UTR sequence extends 562 nucleotides upstream of the CXCL16 start codon, and the 5′-most nucleotide of this novel sequence was designated as transcriptional start site. We also independently amplified CXCL16 5′-UTR from ASMC total RNA and found that it was completely co-linear with the genomic contig sequence. This further confirmed that the rat CXCL16 5′-UTR was not interrupted by introns. We then compared the genomic sequence of the rat CXCL16 locus with the mRNA sequence. This analysis suggested that the rat CXCL16 gene is organized in five exons and four introns. The genomic organization of the rat CXCL16 is shown in Fig. 3A.
      Figure thumbnail gr3
      Fig. 3Genomic organization and potential regulatory elements of rat CXCL16 gene. A, the CXCL16 gene is located on rat chromosome 10 and is composed of 5 exons (boxes, roman numerals) and 4 introns (straight lines, Arabic numbers and circled). The positions of the ATG start codon and the TAG stop codon are indicated. Regions of the exons encoding the open reading frame are shown in black. The lengths of the exonicregions that encode the open reading frame are shown in bold, and the lengths of the introns are in italics. Numbering is relative to the ATG start codon. The regions of the exons that encode the five domains (Sig, signal peptide; Chemokine, mucin stalk; TM, transmembrane; Cyt, cytoplasmic) of the CXCL16 protein are also shown. B, the 5′-flanking sequence of rat CXCL16 (lowercase) was analyzed by MatInspector Professional, and several potential transcription factor binding motifs that can mediate the inflammatory response are identified (in red and overlined with arrows; core sequences shown in large lowercase). The exon nucleotide sequence is shown in uppercase. Nucleotide residues in green encode the beginning of the open reading frame. The 5′-end of the transcript expressed in rat ASMC was verified by 5′-RACE. The nucleotide sequences of the forward primers (S1–S4) and the reverse primer (AS, in the 5′UTR region) used to prepare the deletion constructs are underlined. C, identification of the IL-18-responsive region in the 5′-flanking region of the rat CXCL16 gene. A deletion series of the 5′-flanking region of the rat CXCL16 gene in the pGL3-Basic reporter vector was co-transfected with the pRL-TK vector into rat ASMC, and firefly and Renilla luciferase activities were determined after 24 h of incubation. The results are the mean ± S.E. of six independent experiments.
      We analyzed the sequences upstream of the transcriptional start site for the presence of transcription factor binding sites that could be potentially involved in CXCL16 regulation. This analysis revealed the presence of strong consensus binding sites for several transcription factors that are known to be involved in inflammation. These include binding sites for AP-1 at position –952 to –933, CREB at positions –1339 to –1320 and –681 to –661, interferon regulatory factor at position –883 to –818, and an inverse NF-κB at position –587 to –574 (Fig. 3B). Based on this analysis, we designed oligonucleotide primers to generate a series of reporter constructs (S1-S5) to delineate the CXCL16 promoter and to determine the relative importance of the aforementioned cis binding sites in IL-18-mediated transcriptional regulation. After transfection into rat ASMC and treatment with IL-18, it was determined that the two longest constructs (S1 and S2) displayed the strongest IL-18-induced transcriptional activity (Fig. 3C). Deletion of sequences between –1016 and –882 led to a significant loss (∼60%) of activity (compare S2 and S3). This suggested that the S2 construct contained the most significant IL-18-responsive elements and that this loss of activity was associated with the deletion of binding sites for CREB, GATA4, and AP-1 (Fig. 3C).
      The induction of cellular genes by IL-18 is associated with the activation of both NF-κB and AP-1 transcription factors, often in a cell-type specific manner (
      • Chandrasekar B.
      • Vemula K.
      • Surabhi R.M.
      • Li-Weber M.
      • Owen-Schaub L.B.
      • Jensen L.E.
      • Mummidi S.
      ,
      • Morel J.C.
      • Park C.C.
      • Zhu K.
      • Kumar P.
      • Ruth J.H.
      • Koch A.E.
      ,
      • Lee J.K.
      • Kim S.H.
      • Lewis E.C.
      • Azam T.
      • Reznikov L.L.
      • Dinarello C.A.
      ). Because the induction of the CXCL16 promoter activity by IL-18 was significantly reduced after the deletion of the potential AP-1 binding site from construct S2 (Fig. 3C), we determined whether this site in fact binds AP-1 in vitro and in vivo after stimulation. We performed gel mobility shift assays with the putative AP-1 binding site from CXCL16 promoter using nuclear extracts obtained from ASMC that were either untreated (Fig. 4A, lane 4) or treated with IL-18 for different lengths of time (Fig. 4A, lanes 5–10). Treatment with IL-18 resulted in the formation of two bands that disappeared when unlabeled AP-1 consensus oligonucleotide was included in the reaction (Fig. 4A, lane 2). In contrast, inclusion of an oligonucleotide with a mutated AP1 binding site did not compete for the binding (Fig. 4A, lane 1), suggesting that the induced complexes were highly specific. In separate EMSA experiments, we confirmed that IL-18 can induce AP-1 activation in ASMC using a consensus AP-1 gel shift DNA probe (data not shown). Mutant consensus AP-1 probes served as controls. Furthermore, to confirm that the induction of the AP-1 DNA-protein complexes was due to IL-18 treatment, we pretreated ASMC with anti-IL-18 neutralizing antibodies before IL-18 addition. This completely attenuated the formation of the IL-18-induced AP-1 DNA binding activity (data not shown).
      Figure thumbnail gr4
      Fig. 4IL-18 induces AP-1 DNA binding activity and AP-1-dependent reporter activity. A, IL-18 induces AP-1 DNA binding activity in ASMC. EMSA for AP-1 DNA binding activity was carried out using the AP-1 DNA binding sequence from the CXCL16 promoter, and nuclear extracts from quiescent ASMC or ASMC treated with 25 ng/ml IL-18 for the indicated times. Competition experiments (lanes 1 and 2) were performed with nuclear protein extracts from ASMC treated with IL-18 for 1 h. Arrows denote specific DNA-protein complexes. B, supershift assays. Nuclear extracts from ASMC treated with IL-18 for 1 h were incubated with anti-c-Fos, -c-Jun, -JunD, -JunB, or isoform-specific control IgG antibodies, and EMSA was performed as in A. Supershifted complexes are indicated by the white arrow. C, chromatin immunoprecipitation analysis of the CXCL16 AP-1 binding site. Quiescent ASMC were untreated or treated with IL-18 for 1 h, then cross-linked chromatin was prepared and immunoprecipitated with or without antibodies (Ab) to c-Fos before amplification of the CXCL16 gene region containing the AP-1 site. No specific DNA was seen in the chromatin immunoprecipitated without specific antibody (lanes 1 and 3). Specific DNA sequences in the chromatin immunoprecipitated by c-Fos antibody are shown (lanes 2 and 4). Amplification of the input DNA is shown in lanes 5 and 6. D, IL-18 increases transactivation by AP-1. The pAP-1-Luc reporter plasmid was co-transfected with the control pRL-TK Renilla luciferase plasmid into ASMC, and after 24 h, the transfected cells were incubated with antibody to IL-18 or control IgG for 1 h followed by the addition of 25 ng/ml IL-18 for 7 h. Firefly and Renilla luciferase activities were determined, and the luciferase activity was normalized to the Renilla values. The data represent the mean ± S.E. of four independent experiments. *, p < 0.001 (versus untreated); †, p < 0.01 versus IL-18-treated transfected cells. E, the AP-1 site in the rat CXCL16 promoter mediates IL-18-induced transcription. The AP-1 site in the pCXCL16-S2 promoter construct was mutated by site-directed mutagenesis from TGACTcaAATCC to TGACTtgAATCC, and the wild-type and mutant constructs were transfected and assayed as in D. *, p < 0.001 versus S2-mut AP-1. IRF, interferon regulatory factor. F, IL-18 increases transactivation by NF-κB. The pNF-κB-Luc reporter plasmid was transfected and assayed as in D. The data represent the mean ± S.E. of four independent experiments. *, p < 0.001 (versus untreated); †, p < 0.01 versus IL-18-treated pNF-κB-1-Luc transfected cells.
      Because AP-1 occurs as a dimeric complex composed of Fos and Jun proteins, it was important to establish the identity of the nuclear proteins induced by IL-18 in ASMC. We used AP-1 subunit-specific antibodies to perform supershift assays, and these showed that both anti-c-Fos and -c-Jun antibodies supershifted the DNA-protein complexes (Fig. 4B), whereas no change in mobility was seen with anti-JunD, anti-JunB, or control IgG antibodies. This firmly established that the predicted CXCL16 AP-1 binding site bound c-Fos and c-Jun complexes after IL-18 stimulation of ASMC.
      Having established that CXCL16 promoter contains a functional AP-1 binding site, we sought to determine the nucleotides critical for binding of c-Fos/c-Jun complexes. Mutation of the CA residues in the CXCL16 AP-1 binding site (CA → TG) as well as in the consensus AP-1 oligomer led to a loss of binding activity (data not shown). Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated ASMC were used as a positive control in these experiments, and this led to the formation of identical bands on EMSA (data not shown).
      Binding of a nuclear factor in vitro to a cis-regulatory element, as demonstrated by EMSA, does not necessarily mean that this factor interacts with this element in vivo. To determine whether AP-1 complexes interact in vivo with CXCL16 promoter, we performed chromatin immunoprecipitation assays on ASMC that were either untreated or treated with IL-18. This analysis demonstrated that IL-18 treatment increased c-Fos binding to the AP-1 site in vivo (Fig. 4C).
      Binding of a transcription factor to a promoter does not unequivocally establish its involvement in its regulation. To establish the functional relevance of AP-1 induction in ASMC, we initially determined whether IL-18 induces AP-1 driven transcriptional events in ASMC. Treatment of ASMC with IL-18 resulted in an AP-1-driven luciferase activity (Fig. 4D), and anti-IL-18-neutralizing antibodies, but not control IgG, blocked this reporter activity.
      We then asked whether inhibiting AP-1 binding to the CXCL16 promoter would inhibit IL-18-mediated CXCL16 promoter activity. Mutation in the AP-1 binding site led to a significant decrease in the induction of the S2 construct with IL-18 (Fig. 4E). Together, these results indicate that the IL-18 induction of CXCL16 is strongly mediated by an AP-1-dependent pathway.
      IL-18 has also been demonstrated to activate the NF-κB pathway. We investigated IL-18 induced NF-κB promoter activity in ASMC using the reporter vector construct pNF-κB-Luc. Treatment of with IL-18 led to a marked increase in reporter gene activity in the pNF-κB-Luc-transfected cells, which was blocked by pretreatment of the cells with IL-18 antibody but not by control IgG (Fig. 4F). However, the transactivation studies with the CXCL16 promoter constructs (Figs. 3C and 4E) strongly suggested that the AP-1 site is the dominant IL-18 response element within this region. In addition, the CXCL16 promoter reporter constructs that included the putative NF-κB site at –587 to –574, but in which the AP-1 site was absent, demonstrated only weak transactivation in response to IL-18 treatment (compare S2, S3, and S4 with S2 in Fig. 3C). We investigated the binding of the NF-κB proteins to this putative NF-κB site and found the binding to be weak (data not shown). Thus, if NF-κB directly modulates transcription of the rat CXCL16 gene, the binding site responsible most likely lies outside the promoter region investigated here.
      Our experiments in this study indicate that AP-1 is a key regulator of CXCL16 expression in the rat. To further demonstrate the direct role of the AP-1 transcription factor in CXCL16 transcription, we blocked its expression using dominant negative and antisense oligonucleotide approaches. Expression of dn c-Fos, AS c-Fos, AS c-Jun oligonucleotides, or a combination of c-Fos and c-Jun AS oligonucleotides failed to modulate basal levels of CXCL16-dependent luciferase activity (data not shown). In contrast, dn-c-Fos, c-Fos AS, and c-Jun AS oligonucleotides all attenuated IL-18-mediated CXCL16-dependent luciferase activity (Fig. 5A). Treatment with c-Fos AS and c-Jun AS ODN together was more potent in inhibiting IL-18-mediated CXCL16 promoter-driven luciferase activity. Because dn-c-Fos and c-Fos/c-Jun AS ODN inhibited CXCL16 promoter activity, we next investigated whether ectopic expression of c-Fos or c-Jun increases CXCL16 promoter-reporter activity. Both c-Fos and c-Jun increased CXCL16 promoter activity individually, but these effects were more pronounced when c-Fos and c-Jun were coexpressed (Fig. 5B). Together, these results indicate that AP-1 plays an important role in CXCL16 induction in ASMC.
      Figure thumbnail gr5
      Fig. 5c-Jun and c-Fos transactivate the CXCL16 promoter. A, ASMC were co-transfected with CXCL16 promoter-reporter construct S2 and pRL-TK together with either dn-c-Fos, c-Fos antisense oligonucleotides, or c-Jun antisense oligonucleotides. Empty vector, sense, and scrambled oligonucleotides served as controls. After 48 h the cells were stimulated with 25 ng/ml IL-18 for 7 h before assay. B, ectopic expression of c-Fos and c-Jun activates CXCL16 promoter-reporter activity. ASMC were transfected with S2, and c-Fos, c-Jun, or c-Fos plus c-Jun expression vectors together with the pRL-TK control vector. After 48 h the transfected cells were incubated with 25 ng/ml IL-18 for 7 h. Cell lysates in both studies were analyzed for reporter gene activities as in . The data are the mean ± S.E. of 3–6 independent experiments. A, *, p < 0.001 versus untreated control; †, p < 0.05 versus IL-18. B:*, p < 0.05; **, p < 0.01 versus untreated and pcDNA3.
      Diverse signaling pathways converge at AP-1 activation. Morel et al. (
      • Morel J.C.
      • Park C.C.
      • Zhu K.
      • Kumar P.
      • Ruth J.H.
      • Koch A.E.
      ) have demonstrated previously that IL-18 induces Src kinase activation and Src kinase-dependent AP-1 activation in rheumatoid arthritis synovial fibroblasts. Confirming their observations, our results demonstrated a time-dependent increase in Src kinase activity in ASMC after IL-18 treatment, with peak levels of activity detected at 30 min (Fig. 6A). Furthermore, treatment with PP2, a Src kinase inhibitor, blocked IL-18-mediated Src kinase activity (Fig. 6B). Because phosphorylation at Tyr139 indicates Src kinase activation and phosphorylation at Tyr530 indicates inhibition of its activity, we performed Western blot analysis using phosphorylation-specific antibodies. Our results demonstrated that treatment with IL-18 increased p-Src (Tyr139) levels (Fig. 6C, left panel) with a concomitant decrease in p-Src (Tyr530) levels (Fig. 6C, right panel). Together, these results indicate that IL-18 induces Src kinase activity in ASMC.
      Figure thumbnail gr6
      Fig. 6IL-18 induced Src kinase activity. A, IL-18 induced phosphorylation of Src substrate SAM68 (Src associated in mitosis, 68 kDa). Quiescent ASMC were treated with 25 ng/ml IL-18 for up to 2 h, and Src kinase activity was determined by autoradiography using Src immunoprecipitates (IP) and Sam 68 (331–445) in the presence of [γ-32P]ATP. C, control. Total Src levels were determined by Western blotting (bottom panel). B, IL-18-induced Src kinase activity was inhibited by Src kinase inhibitor PP2. Quiescent ASMC were treated with PP2 for 1 h followed by the addition of 25 ng/ml IL-18 for 30 min. Src kinase activity was determined as described above. C, phosphorylation at Tyr130 indicates activation, and phosphorylation at Tyr530 indicates inhibition of Src kinase activity. p-SrcTyr levels were determined by Western blot analyses using phosphorylation-specific antibodies. Levels of total Src served as a control.
      Induction of Src kinase activates several downstream second messenger molecules including PI3K. Therefore, we examined IL-18-mediated PI3K activation in ASMC. Quiescent ASMC were treated with IL-18 for 10 min. PI3K lipid kinase assays were performed in p85 immunoprecipitates (
      • Chandrasekar B.
      • Mummidi S.
      • Perla R.P.
      • Bysani S.
      • Dulin N.O.
      • Liu F.
      • Melby P.C.
      ,
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      ,
      • Chandrasekar B.
      • Marelli-Berg F.M.
      • Tone M.
      • Bysani S.
      • Prabhu S.D.
      • Murray D.R.
      ). Fig. 7A shows significantly increased levels of phosphatidylinositol 3-phosphate (PI3P) in ASMC treated with IL-18, and treatment with the PI3K-specific inhibitors wortmannin and LY294002 inhibited IL-18-mediated phosphatidylinositol 3-phosphate formation. This induction of PI3K activity could also be blocked specifically by pretreating the cells with antibody to IL-18 before the addition of IL-18 (data not shown). Akt is one of the downstream substrates for PI3K. Western blot analysis using anti-phospho Akt (Thr308) antibodies, which specifically recognize the activated form of Akt, revealed a rapid IL-18-dependent phosphorylation of Akt at Thr308. Furthermore, pretreatment with wortmannin attenuated IL-18-mediated Akt phosphorylation (Fig. 7B). In addition to wortmannin, LY294002, another PI3K-specific inhibitor and Akt inhibitor, attenuated IL-18-mediated Akt phosphorylation. IL-18 also increased Akt kinase activity (Fig. 7C), and this induction could be attenuated by pretreatment with wortmannin, LY294002, and Akt inhibitor (7C). Together, these results indicate that IL-18 induces PI3K-dependent Akt kinase activity in ASMC.
      Figure thumbnail gr7
      Fig. 7IL-18 induced PI3K-dependent Akt activation. A, IL-18 induced PI3K activity. Quiescent ASMC were treated with PI3K-specific inhibitors for 1 h followed by the addition of 25 ng/ml IL-18 for 10 min. PI3K lipid kinase assays were performed as described under “Materials and Methods.” The bottom panel shows immunoblot analysis of the same samples with anti-p85 antibody. DMSO, Me2SO. PI3P, phosphatidylinositol 3-phosphate. B, IL-18 induced PI3K-dependent Akt activation. Quiescent ASMC were treated with the PI3K-specific inhibitors wortmannin or LY294002 or the Akt inhibitor before the addition of 25 ng/ml IL-18. Total and phospho-Akt levels were determined by Western blotting. C, IL-18 induced PI3K-dependent Akt kinase activity. Quiescent ASMC were treated as in B, and phosphorylated glycogen synthase kinase (GSK)-3α/β was determined by Western blotting.
      IL-18 activates diverse signal transduction pathways including activation of stress- and mitogen-activated protein kinases p38 MAPK, p42/44 MAPK (ERK), and JNK (activated protein kinase (
      • Chandrasekar B.
      • Mummidi S.
      • Claycomb W.C.
      • Mestril R.
      • Nemer M.
      ,
      • Morel J.C.
      • Park C.C.
      • Zhu K.
      • Kumar P.
      • Ruth J.H.
      • Koch A.E.
      ,
      • Lee J.K.
      • Kim S.H.
      • Lewis E.C.
      • Azam T.
      • Reznikov L.L.
      • Dinarello C.A.
      ,
      • Wyman T.H.
      • Dinarello C.A.
      • Banerjee A.
      • Gamboni-Robertson F.
      • Hiester A.A.
      • England K.M.
      • Kelher M.
      • Silliman C.C.
      ). All three stress-activated protein kinase/MAPKs have been shown to play a role in AP-1 activation. Therefore, we examined the activation status of p38 MAPK, ERK, and JNK and investigated whether these might mediate IL-18-induced CXCL16 expression. Quiescent ASMC were treated with IL-18, and the levels of total and phosphorylated p38 MAPK, ERK, and JNK were semiquantitated by Western blotting using activation-specific antibodies. JNK kinase activity was also determined. The results show that IL-18 rapidly induced p38 MAPK activation, with increased levels of phospho-p38 MAPK seen at 15 min (Fig. 8A, upper panel). These levels remained high throughout the 120-min study period. However, total p38 MAPK levels remained unchanged after treatment (Fig. 8A, lower panel). Similarly, IL-18 increased phospho-ERK levels without affecting total ERK levels (Fig. 8B). IL-18 also increased phospho-JNK levels (Fig. 8C, upper panel) and JNK kinase activity (Fig. 8D, lower panel). Pretreatment with SB203580, PD98059, and SP600125 inhibited IL-18-induced p38 MAPK (Fig. 8E), ERK (Fig. 8F), and JNK (Fig. 8G) activation, respectively. However, inhibition of JNK by SP600125 was the only treatment to significantly attenuate IL-18-induced CXCL16 expression (Fig. 8H). Together, these results indicate that IL-18 induces p38 MAPK, ERK, and JNK activation in ASMC, but it is the activation of JNK and not p38 MAPK or ERK that plays a prominent role in IL-18-mediated CXCL16 expression in ASMC.
      Figure thumbnail gr8
      Fig. 8IL-18 induced p38 MAPK, ERK, and JNK activation in ASMC. A, IL-18 induced p38 MAPK activity. Quiescent ASMC were treated with 25 ng/ml IL-18 for the indicated time periods, and Western blot analysis was carried out on 40 μg of the whole cell homogenates using antibodies to p38 MAPK and phospho-p38 MAPK. C, control. B, IL-18 induced ERK activation. ASMC were treated with IL-18 as in A, and Western blot analysis was carried out using antibodies against ERK and phospho-ERK. C, IL-18 induced JNK activation. ASMC were treated with IL-18 as in A, and Western blot analysis was carried out using antibodies against JNK and phospho-JNK. D, IL-18 induced JNK kinase activity. ASMC were treated with IL-18 as in A and at the indicated time periods. Cell lysates were harvested and analyzed for JNK kinase activity as described under “Materials and Methods.” Phosphorylated c-Jun was detected by Western blot analysis using anti-phospho c-Jun (Ser63) antibody. E, SB203580 inhibited IL-18-induced p38 MAPK phosphorylation. ASMC were treated with 1 μm SB203580 for 30 min before the addition of 25 ng/ml IL-18. Total and phospho-p38 MAPK levels were determined by Western blotting. DMSO, Me2SO. F, PD98059 inhibited IL-18-induced ERK phosphorylation. ASMC were treated with 10 mm PD98059 for 1 h before the addition of 25 ng/ml IL-18. Total and phospho-ERK levels were determined by Western blotting. G, SP600125 inhibited IL-18-induced JNK phosphorylation. ASMC were treated with 20 mm SP600125 for 30 min before the addition of 25 ng/ml IL-18. Total and phospho-JNK levels were determined by Western blotting. H, however, JNK, but not p38 MAPK or ERK, mediated IL-18-induced CXCL16 mRNA expression. ASMC were treated with the p38 MAPK inhibitor SB203580, the ERK inhibitor PD98059, or the JNK inhibitor SP600125 (as above) followed by the addition of 25 ng/ml IL-18 for 2 h. Total RNA was isolated and analyzed for CXCL16 mRNA expression by Northern blotting. 28 S rRNA served as an internal control. A representative blot from three independent experiments is shown.
      MyD88, an adaptor molecule, links IL-18 receptor to IRAK and mediates IL-18 signal transduction (
      • Adachi O.
      • Kawai T.
      • Takeda K.
      • Matsumoto M.
      • Tsutsui H.
      • Sakagami M.
      • Nakanishi K.
      • Akira S.
      ). In fact, MyD88-deficient mice exhibited defective IL-18 signaling (
      • Adachi O.
      • Kawai T.
      • Takeda K.
      • Matsumoto M.
      • Tsutsui H.
      • Sakagami M.
      • Nakanishi K.
      • Akira S.
      ), and the MyD88-IRAK-TRAF6 module has been shown to be essential in IL-1β-mediated PI3K activation (
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      ). Therefore, we investigated whether IL-18 induces AP-1 activation via MyD88, IRAK, and TRAF6 in ASMC. ASMC were transiently transfected with MyD88 siRNA (knockdown of MyD88 was confirmed by Western blotting, data not shown), dominant negative IRAK1, IRAK4, or TRAF6. Cells transfected with control siRNA or the corresponding empty vectors served as controls. Cells were co-transfected with either AP-1 reporter or CXCL16 promoter-reporter vector. Knockdown of MyD88 or expression of dominant negative IRAK1, IRAK4, and TRAF6, but not the controls, all inhibited IL-18-mediated AP-1 reporter activity (Fig. 9A), CXCL16 promoter-reporter activity (Fig. 9B), and CXCL16 mRNA expression (Fig. 9C). Furthermore, the induction of AP-1 DNA binding activity was also inhibited (data not shown). These studies indicate that MyD88, IRAK1, IRAK4, and TRAF6 mediate IL-18-induced AP-1 activation and CXCL16 expression.
      Figure thumbnail gr9
      Fig. 9IL-18 induced CXCL16 expression in ASMC through MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling pathways. A, IL-18 increased AP-1-dependent reporter gene activity via MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling. ASMC were co-transfected with pAP-1-Luc and the pRL-TK transfection control vector together with dnMyD88, dnIRAK1, dnIRAK4, or dnTRAF6 expression vectors. After 48 h the cells were treated with 25 ng/ml IL-18 for 7 h, and the reporter gene activity was assayed as before. Results are the mean ± S.E. of four independent experiments. *, p < 0.001 versus untreated cells; †, p < 0.05 versus IL-18. B, IL-18 induced CXCL16 gene transcription activity via MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling. ASMC were transfected with the S2 CXCL16 promoter-reporter vector and either dnMyD88, dnIRAK1, dnIRAK4, or dnTRAF6 expression vectors or their vector controls. Luciferase activity was assayed as before. The results are the mean ± S.E. of six experiments. *, p < 0.001 (versus untreated); †, p < 0.05 versus IL-18. C, IL-18 induced CXCL16 mRNA expression via MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling. ASMC were transfected with dnMyD88, dnIRAK1, dnIRAK4, or dnTRAF6 and after 24 h treated with 25 ng/ml IL-18 for 2 h. Total RNA was isolated and analyzed for CXCL16 mRNA expression by Northern blotting as before.
      We have demonstrated that IL-18 induces PI3K-dependent Akt kinase activity in ASMC (Fig. 7C). However, it is not known whether PI3K and Akt play a role in IL-18-mediated AP-1 activation. Therefore, quiescent ASMC were treated with PI3K- and Akt-specific inhibitors before the addition of IL-18. In addition to the use of pharmacological inhibitors, we also transfected cells with dnPI3K. Wortmannin, LY294002, dnPI3K, Akt inhibitor, and Akt knockdown significantly attenuated IL-18-mediated AP-1-driven luciferase activity (Fig. 10A), CXCL16 promoter-reporter activity (Fig. 10B) and CXCL16 mRNA expression (Fig. 10C). Together, these results indicate that PI3K and Akt both play a role in IL-18-mediated AP-1 activation and CXCL16 expression.
      Figure thumbnail gr10
      Fig. 10IL-18 induced CXCL16 expression via PI3K and Akt. A, IL-18 increased AP-1-dependent reporter gene activity via PI3K and Akt. ASMC were transfected with an AP-1 reporter vector (pAP-1-Luc) and pRL-TK transfection control vector. In some cases the cells were also co-transfected with a dominant negative PI3K expression vector, the empty vector (pcDNA3.1), or Akt siRNA. After 48 h some cells were treated with PI3K inhibitor, Akt inhibitor, or vehicle (Me2SO (DMSO)) for 1 h then treated with 25 ng/ml IL-18 or vehicle for 7 h. Reporter genes were assayed as before. Results are the mean ± S.E. of four independent experiments. *, p < 0.001 (versus untreated); †, p < 0.05 versus IL-18. B, IL-18 induced CXCL16 promoter-reporter activity via PI3K and Akt. The experiments were carried out as in A but using the S2 construct. Results are the mean ± S.E. of six experiments. *, p < 0.001 (versus untreated); †, p < 0.05 versus IL-18. C, IL-18 induced CXCL16 mRNA expression via PI3K and Akt. Experimental conditions were as described for panel B, except that the cells were treated with 25 ng/ml IL-18 for 2 h before Northern blot analysis. A representative blot from three experiments is shown.
      IL-1β has been shown to activate AP-1 in a TRAF6- and c-Src-dependent manner (
      • Morel J.C.
      • Park C.C.
      • Zhu K.
      • Kumar P.
      • Ruth J.H.
      • Koch A.E.
      ). We demonstrated above that IL-18 induced AP-1 activation via TRAF6 (Fig. 9, A and B). We also showed that IL-18 induces c-Src activation (Fig. 6). However, it is not known whether IL-18-mediated AP-1 activation is dependent on Src activation. Therefore, we treated quiescent ASMC with the Src kinase family-specific inhibitors PP1 and PP2. Pretreatment with PP1 and PP2 attenuated IL-18-mediated AP-1 reporter activity (Fig. 11A), CXCL16 promoter-reporter activity (Fig. 11B), and CXCL16 mRNA expression (Fig. 11C). Together, these results indicate that IL-18 induces AP-1 activation and CXCL16 expression via Src activation.
      Figure thumbnail gr11
      Fig. 11IL-18 induced CXCL16 expression through Src and JNK. A, IL-18 increased AP-1-dependent reporter gene activity via Src and JNK. ASMC were transfected with pAP-1-Luc and pRL-TK and after 24 h treated with the Src kinase inhibitors PP1 and PP2, the JNK inhibitor SP600125, or antisense JNK oligonucleotides followed by 25 ng/ml IL-18 for 7 h. Reporter gene assays were carried out as before. The results are the mean ± S.E. of four experiments. *, p < 0.001 (versus untreated); †, p < 0.05 versus IL-18. DMSO, Me2SO. B, IL-18 induced CXCL16 promoter-reporter activity via Src and JNK. Experimental conditions were as in A, except that the S2 construct was used instead of pAP-1-Luc. The results are the mean ± S.E. of six experiments. *, p < 0.001 (versus untreated); †, p < 0.05 versus IL-18. C, IL-18 induced CXCL16 mRNA expression via Src and JNK. Experimental conditions were as in A, except that the cells were treated with 20 ng/ml IL-18 for 2 h. Northern analysis was carried out as before. D, SP600125 inhibited the IL-18-induced phosphorylation of c-Jun Ser63 and c-Jun Ser73 in ASMC. To confirm that the dose of JNK inhibitor used in A inhibited the IL-18 activation of c-Jun, ASMC were treated with 20 μm SP600125 for 30 min, then 25 ng/ml IL-18 for 1 h, and whole cell lysates were assayed for total c-Jun, c-Jun Ser63, and c-Jun Ser73 by Western blot. β-Actin was used as the cell loading control.
      Activation of the serine/threonine protein kinase JNK has been shown to induce AP-1 activation (
      • Karin M.
      • Liu Z.
      • Zandi E.
      ). JNK phosphorylates c-Jun at N-terminal serine 63 and 73, resulting in AP-1 transactivation (Fig. 11D (
      • Karin M.
      • Liu Z.
      • Zandi E.
      )). Because our results identified c-Jun in the IL-18-induced AP-1 DNA protein complex (Fig. 4C), we investigated whether JNK plays a role in IL-18-mediated AP-1 activation. JNK was targeted by AS ODN or the JNK-specific inhibitor SP600125. Treatment with JNK AS ODN, but not scrambled ODN, attenuated IL-18-mediated AP-1 DNA binding activity (Fig 11A). Furthermore, SP600125 and JNK AS ODN inhibited IL-18-mediated AP-1 reporter activity (Fig. 11B), CXCL16 promoter-reporter activity (Fig. 11C), CXCL16 mRNA expression (Fig. 11C), and phosphorylation of c-Jun at serine 63 and 73 (Fig. 11D). Together, these results indicate that IL-18 induces AP-1 activation and CXCL16 expression via JNK activation.
      IL-18 is a pluripotent cytokine. It exerts both proinflammatory and pro-apoptotic effects. We have previously demonstrated that IL-18 induces death in cardiac microvascular endothelial cells (
      • Chandrasekar B.
      • Vemula K.
      • Surabhi R.M.
      • Li-Weber M.
      • Owen-Schaub L.B.
      • Jensen L.E.
      • Mummidi S.
      ). Therefore, we investigated whether IL-18 induces ASMC death. Our results demonstrate that IL-18 does not induce ASMC apoptosis as evidenced by low levels of mono- and oligonucleosomal fragmented DNA in the cytoplasmic extracts of ASMC (Fig. 12A). Further supporting this observation, IL-18 treatment did not lead to an increase in annexin V-positive cells (Fig. 12B). IL-18 also induced phosphorylation of the pro-apoptotic Bad at Ser136 (Fig. 12C) and failed to activate the pro-apoptotic caspase-3 (Fig. 12D) and PARP (Fig. 12E) proteins. In contrast, treatment with SNAP, a nitric oxide donor (
      • Lundberg G.A.
      • Kellin A.
      • Samnegard A.
      • Lundman P.
      • Tornvall P.
      • Dimmeler S.
      • Zeiher A.M.
      • Hamsten A.
      • Hansson G.K.
      • Eriksson P.
      ), induced ASMC death as expected (Fig. 12A). SNAP induced activation of pro-apoptotic caspase-3 and PARP as seen by the cleaved active products (Fig. 12, D and E). Together, these results confirm that IL-18 does not exert pro-apoptotic effects on ASMC.
      Figure thumbnail gr12
      Fig. 12IL-18 induced ASMC proliferation in a CXCL16-dependent manner. A, IL-18 failed to induce ASMC death. Quiescent ASMC were treated with 25 ng/ml IL-18 for 24 h. The levels of mono- and oligonucleosomal fragmented DNA in the cytoplasmic extracts were quantified by enzyme-linked immunosorbent assay (ELISA). SNAP (500 mm), a nitric oxide donor, was used as a positive apoptosis-inducing control. The data represent the mean ± S.E. of four experiments. *, p < 0.001 versus untreated and IL-18-treated cells. B, cells treated in the identical manner were also analyzed for annexin V/PI staining. The data shown are the percentage of annexin V-positive and phosphatidylinositol-negative cells after the treatment and represent the mean ± S.E. of three experiments. C, IL-18 induced phosphorylation of Bad. Quiescent ASMC were treated with 25 ng/ml IL-18 for 4 h. Cell lysates were analyzed by Western blotting for total Bad and for phospho-Bad using an antibody that specifically recognizes the phosphorylated Ser136 residue. β-Actin was used as the internal loading control. D, IL-18 failed to induce caspase-3 activation. Quiescent ASMC were treated with 25 ng/ml IL-18 for 16 h. Activation of caspase-3 was analyzed by Western blotting. SNAP was used as a positive control. E, IL-18 failed to activate PARP. Quiescent ASMC were treated with 25 ng/ml IL-18 for 16 h, and activation of PARP was analyzed in nuclear protein extracts by Western blotting. SNAP was used as a positive control. These experiments were repeated three times with similar results. F, ASMC expressed CXCR6. 1 mg of poly(A)+ RNA from quiescent ASMC was analyzed for CXCR6 mRNA by reverse transcription-PCR. Lane 1, 100-bp ladder; lane 2, CXCR6 cDNA (indicated by an arrow); lane 3, reverse transcription negative control. G, IL-18 induced ASMC proliferation. Quiescent ASMC were treated with or without 100 μm CXCL16 siRNA or scrambled control RNA for 48 h before the addition of 25 ng/ml IL-18. Cell proliferation was determined by [3H]thymidine incorporation. The data shown are the mean ± S.E. of six experiments. *, p < 0.01 versus control siRNA; †, p < 0.025 versus IL-18. H, CXCL16 siRNAs specifically inhibited IL-18-induced CXCL16 expression. To confirm that the CXCL16 siRNA in G specifically inhibited IL-18-induced CXCL16 expression, ASMC treated exactly as in G were assayed for CXCL16 protein by Western blotting.
      Because IL-18 induced CXCL16 expression (Fig. 1), and CXCL16 exerts pro-mitogenic effects (
      • Chandrasekar B.
      • Bysani S.
      • Mummidi S.
      ), we investigated whether IL-18 induces ASMC proliferation and whether IL-18-mediated ASMC proliferation was dependent on CXCL16 expression. Because CXCL16 signals via CXCR6, at first we determined CXCR6 expression in ASMC by reverse transcription-PCR. Confirming our earlier results in human ASMC (
      • Chandrasekar B.
      • Bysani S.
      • Mummidi S.
      ), Fig. 12F shows that rat ASMC expressed CXCR6 mRNA under normal basal conditions. We then targeted CXCL16 expression using siRNA. ASMC were transfected with CXCL16 siRNA and then treated with IL-18 for an additional 3 days. Cell proliferation was assessed by [3H]thymidine incorporation. Our results indicate that IL-18 indeed induced ASMC proliferation (Fig. 12G), and knockdown of CXCL16 (confirmed by Western blotting, Fig. 12H) attenuated IL-18-mediated ASMC proliferation by 27% (Fig. 12G). Together these results indicate that IL-18 is a pro-mitogenic cytokine and induces ASMC proliferation in part via CXCL16 induction.

      DISCUSSION

      Results from the present study demonstrated that 1) the proinflammatory and pro-atherogenic cytokine IL-18 induced CXCL16 expression in ASMC, 2) IL-18-mediated CXCL16 expression was independent of IL-1β, TNF-α, and IFN-γ, 3) CXCL16 expression was regulated mainly at the transcriptional level, 4) IL-18 induced CXCL16 expression in AP-1-dependent manner, 5) JNK, but not p38 MAPK or ERK, played a role in IL-18-mediated CXCL16 induction, 6) IL-18 mediates CXCL16 expression via a MyD88 → IRAK → TRAF6 → c-Src → PI3K → Akt → JNK → AP-1-signaling pathway, and 7) IL-18 was mitogenic for ASMC, inducing their proliferation in a CXCL16-dependent manner.
      A number of proinflammatory cytokines has been shown to induce CXCL16 expression in human vascular cells, including IFN-γ, TNF-α, IL-12, and IL-15 (
      • Matloubian M.
      • David A.
      • Engel S.
      • Ryan J.E.
      • Cyster J.G.
      ,
      • Wilbanks A.
      • Zondlo S.C.
      • Murphy K.
      • Mak S.
      • Soler D.
      • Langdon P.
      • Andrew D.P.
      • Wu L.
      • Briskin M.
      ,
      • Abel S.
      • Hundhausen C.
      • Mentlein R.
      • Schulte A.
      • Berkhout T.A.
      • Broadway N.
      • Hartmann D.
      • Sedlacek R.
      • Dietrich S.
      • Muetze B.
      • Schuster B.
      • Kallen K.J.
      • Saftig P.
      • Rose-John S.
      • Ludwig A.
      ,
      • Gough P.J.
      • Garton K.J.
      • Wille P.T.
      • Rychlewski M.
      • Dempsey P.J.
      • Raines E.W.
      ,
      • Potapova O.
      • Gorospe M.
      • Bost F.
      • Dean N.M.
      • Gaarde W.A.
      • Mercola D.
      ,
      • Yamauchi R.
      • Tanaka M.
      • Kume N.
      • Minami M.
      • Kawamoto T.
      • Togi K.
      • Shimaoka T.
      • Takahashi S.
      • Yamaguchi J.
      • Nishina T.
      • Kitaichi M.
      • Komeda M.
      • Manabe T.
      • Yonehara S.
      • Kita T.
      ). Among these cytokines, IFN-γ has been reported to be the most potent inducer of CXCL16 in cultured ASMC, increasing CXCL16 mRNA expression and both the secretion and surface expression of the protein (
      • Wagsater D.
      • Olofsson P.S.
      • Norgren L.
      • Stenberg B.
      • Sirsjo A.
      ). TNF-α and IFN-γ synergistically induce CXCL16 expression in endothelial cells (
      • Abel S.
      • Hundhausen C.
      • Mentlein R.
      • Schulte A.
      • Berkhout T.A.
      • Broadway N.
      • Hartmann D.
      • Sedlacek R.
      • Dietrich S.
      • Muetze B.
      • Schuster B.
      • Kallen K.J.
      • Saftig P.
      • Rose-John S.
      • Ludwig A.
      ). IFN-γ also induces CXCL16 expression in both primary non-transformed human monocytes and the monocytic cell line THP-1 in vitro (
      • Wuttge D.M.
      • Zhou X.
      • Sheikine Y.
      • Wagsater D.
      • Stemme V.
      • Hedin U.
      • Stemme S.
      • Hansson G.K.
      • Sirsjo A.
      ). Furthermore, in vivo administration of IFN-γ increases CXCL16 expression in the atherosclerotic lesions in normal chow-fed apoE-null mice (
      • Wuttge D.M.
      • Zhou X.
      • Sheikine Y.
      • Wagsater D.
      • Stemme V.
      • Hedin U.
      • Stemme S.
      • Hansson G.K.
      • Sirsjo A.
      ). Thus, IFN-γ is a potent inducer of CXCL16 both in vivo and in vitro. However, the molecular mechanisms involved in IFN-γ-mediated CXCL16 expression are not known. Within the 1636-bp fragment of the 5′-flanking region of the rat CXC16 gene (Fig. 3), we identified several potential transcription factor binding sites, including those for interferon regulatory factor and AP-1. Thus, we speculate that IFN-γ, which is known to regulate gene transcription through these factors (
      • Pestka S.
      • Krause C.D.
      • Walter M.R.
      ), may also regulate CXCL16 expression by interferon regulatory factor and AP-1-dependent mechanisms. Studies are in progress to identify the roles of interferon regulatory factor and AP-1 in IFN-γ mediated CXCL16 induction in ASMC.
      IFN-γ has been indirectly implicated in IL-18-mediated CXCL16 induction in vivo (
      • Wuttge D.M.
      • Zhou X.
      • Sheikine Y.
      • Wagsater D.
      • Stemme V.
      • Hedin U.
      • Stemme S.
      • Hansson G.K.
      • Sirsjo A.
      ). IL-18 induces the maturation of naïve CD4+ cells into IFN-γ-producing Th1 effector cells (
      • Nakanishi K.
      • Yoshimoto T.
      • Tsutsui H.
      • Okamura H.
      ), and IFN-γ is a potent inducer of CXCL16 in a variety of cell types (
      • Abel S.
      • Hundhausen C.
      • Mentlein R.
      • Schulte A.
      • Berkhout T.A.
      • Broadway N.
      • Hartmann D.
      • Sedlacek R.
      • Dietrich S.
      • Muetze B.
      • Schuster B.
      • Kallen K.J.
      • Saftig P.
      • Rose-John S.
      • Ludwig A.
      ,
      • Wagsater D.
      • Olofsson P.S.
      • Norgren L.
      • Stenberg B.
      • Sirsjo A.
      ). Sustained administration of IL-18 to apoE-null mice was shown to substantially increase the size of the spontaneously occurring atherosclerotic lesions compared with mice receiving vehicle alone (
      • Whitman S.C.
      • Ravisankar P.
      • Daugherty A.
      ). These pro-atherogenic effects were ablated in mice null for apoE and IFN-γ (
      • Whitman S.C.
      • Ravisankar P.
      • Daugherty A.
      ), suggesting that IFN-γ may mediate IL-18 effects. However, the results from the present study clearly demonstrate that IFN-γ is not necessary for IL-18-mediated CXCL16 induction in ASMC. Using neutralizing antibodies, antisense oligonucleotide, and siRNA-mediated knockdown, we have demonstrated that IL-18 induces CXCL16 expression in ASMC in an IFN-γ independent manner. IFN-γ independent effects of IL-18 have also been shown in other cell systems. Udagawa et al. (
      • Udagawa N.
      • Horwood N.J.
      • Elliott J.
      • Mackay A.
      • Owens J.
      • Okamura H.
      • Kurimoto M.
      • Chambers T.J.
      • Martin T.J.
      • Gillespie M.T.
      ) have shown that IL-18 inhibits osteoclast formation in vivo, and neutralization of IFN-γ failed to attenuate these inhibitory effects. Similarly, IL-18 induced activation and proliferation of natural killer cells in both wild type and IFN-γR knock-out mice (
      • Lauwerys B.R.
      • Renauld J.C.
      • Houssiau F.A.
      ), demonstrating the IFN-γ-independent effects of IL-18. Furthermore, our results also showed that IL-18 induces CXCL16 expression independent of IL-1β and TNF-γ, two other proinflammatory cytokines known to induce CXCL16 expression (
      • Gough P.J.
      • Garton K.J.
      • Wille P.T.
      • Rychlewski M.
      • Dempsey P.J.
      • Raines E.W.
      ,
      • Wagsater D.
      • Olofsson P.S.
      • Norgren L.
      • Stenberg B.
      • Sirsjo A.
      ), indicating that IL-18 is potent and direct inducer of CXCL16 expression in ASMC.
      Our results further show that IL-18 induces AP-1 activation and CXCL16 expression via c-Src, PI3K, Akt, and JNK activation. Morel et al. (
      • Morel J.C.
      • Park C.C.
      • Zhu K.
      • Kumar P.
      • Ruth J.H.
      • Koch A.E.
      ) have previously demonstrated that IL-18 induces vascular cell adhesion molecule expression in rheumatoid arthritis synovial fibroblasts via direct activation of Src. Using Src antisense oligonucleotides, these authors found that Src lies upstream of PI3K and Akt in the signaling pathway. In fact, Beraud et al. (
      • Beraud C.
      • Henzel W.J.
      • Baeuerle P.A.
      ) have shown recruitment and activation of PI3K by Src. In ASMC, IL-18 induced PI3K-dependent Akt activation, and inhibition of Src, PI3K, and Akt attenuated IL-18-mediated AP-1 activation and CXCL16 expression, indicating that IL-18 induces CXCL16 expression in c-Src, PI3K, and Akt-dependent signaling.
      Proinflammatory cytokines induce AP-1 activation via p38 MAPK and JNK. IL-18 is a potent activator of p38 MAPK, ERK, and JNK activation, and all three stress-activated protein kinase/MAPKs have been shown to play a role in AP-1 activation (
      • Chang L.
      • Karin M.
      ). Using pharmacological inhibitors, we show here that IL-18 induced phosphorylation and activation of all three stress-activated protein kinase/MAPKs in ASMC. However, JNK, but not p38 MAPK or ERK, appears to be involved in IL-18-mediated CXCL16 induction. Although the inhibition of p38 MAPK and ERK lowers CXCL16 expression by 18 and 20%, respectively, inhibition of JNK attenuated CXCL16 expression by 46%. Furthermore, IL-18 induced c-Jun phosphorylation at Ser63 and Ser73, and inhibition of JNK attenuated IL-18-mediated AP-1 activation and CXCL16 expression. Together, these results indicate that IL-18 induces AP-1 activation and CXCL16 expression via JNK and to a limited extent via p38 MAPK and ERK.
      Unlike in endothelial cells (
      • Chandrasekar B.
      • Vemula K.
      • Surabhi R.M.
      • Li-Weber M.
      • Owen-Schaub L.B.
      • Jensen L.E.
      • Mummidi S.
      ), IL-18 failed to induce ASMC death. In fact, IL-18 induces ASMC proliferation. This is the first report demonstrating the pro-mitogenic effects of IL-18 on ASMC. Effects of IL-18 on cell survival and proliferation are cell type-dependent. IL-18 promotes endothelial cell migration and angiogenesis in vivo independent of TNF-α (
      • Park C.C.
      • Morel J.C.
      • Amin M.A.
      • Connors M.A.
      • Harlow L.A.
      • Koch A.E.
      ). In contrast, IL-18 inhibits tumor growth by suppressing angiogenesis (
      • Cao R.
      • Farnebo J.
      • Kurimoto M.
      • Cao Y.
      ). IL-18 induces melanoma cell proliferation via induction of stem cell factor (
      • Hue J.
      • Kim A.
      • Song H.
      • Choi I.
      • Park H.
      • Kim T.
      • Lee W.J.
      • Kang H.
      • Cho D.
      ). In the present study we demonstrated that IL-18 induces SMC proliferation in a CXCL16-dependent manner. IL-18-mediated SMC proliferation is significantly inhibited by CXCL16 knockdown. However, knockdown of CXCL16 did not completely block IL-18-mediated ASMC proliferation, suggesting that IL-18 might also induce other pro-survival and pro-mitogenic factors in ASMC.
      Together, our results indicate that IL-18 induces CXCL16 expression in aortic smooth muscle cells via MyD88 → IRAK → TRAF6 → c-Src → PI3K → Akt → JNK → AP-1 signaling. Our results also demonstrated for the first time that IL-18 is mitogenic for ASMC in a CXCL16-dependent manner. These results suggest a role for IL-18-CXCL16 cross-talk in atherosclerosis and in restenosis after angioplasty and provide novel targets to reduce/attenuate atherosclerosis

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