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A Novel Role of the Interferon-inducible Protein IFI16 as Inducer of Proinflammatory Molecules in Endothelial Cells

2007; Elsevier BV; Volume: 282; Issue: 46 Linguagem: Inglês

10.1074/jbc.m701846200

ISSN

1083-351X

Autores

Patrizia Caposio, Francesca Gugliesi, Claudia Zannetti, Simone Sponza, Michele Mondini, Enzo Médico, John Hiscott, Howard A. Young, Giorgio Gribaudo, Marisa Gariglio, Santo Landolfo,

Tópico(s)

Atherosclerosis and Cardiovascular Diseases

Resumo

The human IFI16 gene is an interferon-inducible gene implicated in the regulation of endothelial cell proliferation and tube morphogenesis. Immunohistochemical analysis has demonstrated that this gene is highly expressed in endothelial cells in addition to hematopoietic tissues. In this study, gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 revealed an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. Consistent with these observations, IFI16 triggered expression of adhesion molecules such as ICAM-1 and E-selectin or chemokines such as interleukin-8 or MCP-1. Treatment of cells with short hairpin RNA targeting IFI16 significantly inhibited ICAM-1 induction by interferon (IFN)-γ demonstrating that IFI16 is required for proinflammatory gene stimulation. Moreover, functional analysis of the ICAM-1 promoter by deletion- or site-specific mutation demonstrated that NF-κB is the main mediator of IFI16-driven gene induction. NF-κB activation appears to be triggered by IFI16 through a novel mechanism involving suppression of IκBα mRNA and protein expression. Support for this finding comes from the observation that IFI16 targeting with specific short hairpin RNA down-regulates NF-κB binding activity to its cognate DNA and inhibits ICAM-1 expression induced by IFN-γ. Using transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrate indeed that activation of the NF-κB response is mediated by IFI16-induced block of Sp1-like factor recruitment to the promoter of the IκBα gene, encoding the main NF-κB inhibitor. Activation of NF-κB accompanied by induction of proinflammatory molecules was also observed when IκBα expression was down-regulated by specific small interfering RNA, resulting in an outcome similar to that observed with IFI16 overexpression. Taken together, these data implicate IFI16 as a novel regulator of endothelial proinflammatory activity and provide new insights into the physiological functions of the IFN-inducible gene IFI16. The human IFI16 gene is an interferon-inducible gene implicated in the regulation of endothelial cell proliferation and tube morphogenesis. Immunohistochemical analysis has demonstrated that this gene is highly expressed in endothelial cells in addition to hematopoietic tissues. In this study, gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 revealed an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. Consistent with these observations, IFI16 triggered expression of adhesion molecules such as ICAM-1 and E-selectin or chemokines such as interleukin-8 or MCP-1. Treatment of cells with short hairpin RNA targeting IFI16 significantly inhibited ICAM-1 induction by interferon (IFN)-γ demonstrating that IFI16 is required for proinflammatory gene stimulation. Moreover, functional analysis of the ICAM-1 promoter by deletion- or site-specific mutation demonstrated that NF-κB is the main mediator of IFI16-driven gene induction. NF-κB activation appears to be triggered by IFI16 through a novel mechanism involving suppression of IκBα mRNA and protein expression. Support for this finding comes from the observation that IFI16 targeting with specific short hairpin RNA down-regulates NF-κB binding activity to its cognate DNA and inhibits ICAM-1 expression induced by IFN-γ. Using transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrate indeed that activation of the NF-κB response is mediated by IFI16-induced block of Sp1-like factor recruitment to the promoter of the IκBα gene, encoding the main NF-κB inhibitor. Activation of NF-κB accompanied by induction of proinflammatory molecules was also observed when IκBα expression was down-regulated by specific small interfering RNA, resulting in an outcome similar to that observed with IFI16 overexpression. Taken together, these data implicate IFI16 as a novel regulator of endothelial proinflammatory activity and provide new insights into the physiological functions of the IFN-inducible gene IFI16. Interferons (IFNs) 2The abbreviations used are: IFN, interferon; HUVEC, human umbilical vein endothelial cell; siRNA, small interfering RNA; shRNA, short hairpin RNA; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; TNF, tumor necrosis factor; m.o.i., multiplicity of infection; RT, reverse transcription; ChIP, chromatin immunoprecipitation; IL, interleukin; PFU, plaque-forming unit; PBS, phosphate-buffered saline; EGM, endothelial growth medium; EMSA, electrophoretic mobility shift assay; IKK, IκB kinase; Ab, antibody; p.i., post-infection; h.p.i., hours p.i.; FACS, fluorescence-activated cell sorter. are important regulators of viral replication, cell growth, immunomodulation, and inflammation (1Bekisz J. Schmeisser H. Hernandez J. Goldman N.D. Zoon K.C. Growth Factors. 2004; 22: 243-251Crossref PubMed Scopus (171) Google Scholar, 2Parmar S. Platanias L.C. Cancer Treat. Res. 2005; 126: 45-68Crossref PubMed Scopus (12) Google Scholar). Moreover, it is now well accepted that IFNs play a critical role in the pathogenesis and perpetuation of specific autoimmune diseases, including systemic lupus erythematosus, autoimmune thyroid disease, and type 1 diabetes (3Kong J.S. Teuber S.S. Gershwin M.E. Autoimmun. Rev. 2006; 5: 471-485Crossref PubMed Scopus (28) Google Scholar). The interferon-inducible p200 family of proteins (Ifi200 in the mouse and HIN200 in the humans) is among the numerous gene products induced by IFNs (4Asefa B. Klarman K.D. Copeland N.G. Gilbert D.J. Jenkins N.A. Keller J.R. Blood Cells Mol. Dis. 2004; 32: 155-167Crossref PubMed Scopus (114) Google Scholar, 5Landolfo S. Gariglio M. Gribaudo G. Lembo D. Biochimie (Paris). 1998; 80: 721-728Crossref PubMed Scopus (89) Google Scholar, 6Ludlow L.E.A. Johnstone R.W. Clarke C.J.P. Exp. Cell Res. 2005; 308: 1-17Crossref PubMed Scopus (137) Google Scholar). Recently, the Pyrin domain, commonly found among cell death-associated proteins such as Pyrin, ASC, and zebrafish caspase, and also referred to as the PAAD/DAPIN domain (7Inohara N. Nunez G. Nat. Rev. Immunol. 2003; 3: 371-382Crossref PubMed Scopus (870) Google Scholar, 8Tschopp J. Martinon F. Burns K. Nat. Rev. Mol. Cell Biol. 2003; 4: 95-104Crossref PubMed Scopus (601) Google Scholar), has been found in the N terminus of most Ifi200/HIN200 proteins, suggesting a role of these proteins in inflammation and apoptosis (9Aglipay J.A. Lee S.W. Okada S. Fujiuchi N. Ohtsuka T. Kwak J.C. Wang Y. Johnstone R.W. Deng C. Qin J. Ouchi T. Oncogene. 2003; 22: 8931-8938Crossref PubMed Scopus (106) Google Scholar, 10Albrecht M. Choubey D. Lengauer T. Biochem. Biophys. Res. Commun. 2005; 327: 679-687Crossref PubMed Scopus (61) Google Scholar). The IFI16 gene, a member of the HIN200 family (4Asefa B. Klarman K.D. Copeland N.G. Gilbert D.J. Jenkins N.A. Keller J.R. Blood Cells Mol. Dis. 2004; 32: 155-167Crossref PubMed Scopus (114) Google Scholar, 5Landolfo S. Gariglio M. Gribaudo G. Lembo D. Biochimie (Paris). 1998; 80: 721-728Crossref PubMed Scopus (89) Google Scholar, 6Ludlow L.E.A. Johnstone R.W. Clarke C.J.P. Exp. Cell Res. 2005; 308: 1-17Crossref PubMed Scopus (137) Google Scholar), was originally identified as a target of interferons (IFN-α/β and -γ). Recently, however, it has become clear that oxidative stress, cell density, and various proinflammatory cytokines also trigger IFI16 expression (11Gugliesi F. Mondini M. Ravera R. Robotti A. De Andrea M. Gribaudo G. Gariglio M. Landolfo S. J. Leukocyte Biol. 2005; 77: 820-829Crossref PubMed Scopus (51) Google Scholar, 12Mondini M. Vidali M. Airò P. De Andrea M. Riboldi P.S. Meroni P.L. Gariglio M. Landolfo S. Ann. N. Y. Acad. Sci. 2007; 1110: 47-56Crossref PubMed Scopus (68) Google Scholar). IFI16 expression is seen in vascular endothelial cells from blood and lymph vessels in addition to hematopoietic cells, suggesting a possible link to angiogenesis and inflammation (13Gariglio M. Azzimonti B. Pagano M. Palestro G. De Andrea M. Valente G. Voglino G. Navino L. Landolfo S. J. Interferon Cytokine Res. 2002; 22: 815-821Crossref PubMed Scopus (61) Google Scholar, 14Wei W. Clarke C.J. Somers G.R. Gresswell K.S. Loveland K.A. Trapani J.A. Johnstone R.W. Histochem. Cell Biol. 2003; 119: 45-54Crossref PubMed Scopus (55) Google Scholar). Consistent with this hypothesis, expression of IFI16 in HUVEC efficiently suppressed endothelial migration, invasion, and formation of capillary-like structures in vitro accompanied by inhibition of cycle progression and arrest in the G1 phase of the cell cycle (15Raffaella R. Gioia D. De Andrea M. Cappello P. Giovarelli M. Marconi P. Manservigi R. Gariglio M. Landolfo S. Exp. Cell Res. 2004; 293: 331-345Crossref PubMed Scopus (53) Google Scholar). Additionally, anti-IFI16 autoantibody titers are significantly elevated in patients with autoimmune diseases such as cutaneous systemic sclerosis, systemic lupus erythematosus, and Sjogren syndrome, but not in those with other autoimmune diseases when compared with controls (16Mondini M. Vidali M. De Andrea M. Azzimanti B. Airò P. Riboldi P.S. Meroni P.L. Albano E. Shoenfeld Y. Gariglio M. Landolfo S. Arthritis Rheum. 2006; 54: 3939-3944Crossref PubMed Scopus (58) Google Scholar). Taken together, these results indicate that IFI16 may be involved in the early steps of inflammation by modulating endothelial cell functions. One of the initial key events in the endothelial response to inflammatory stimuli is the expression of adhesion molecules such as intercellular cell adhesion molecule (ICAM-1, VCAM-1, and E-selectin) and production of chemokines, including IL-8 and MCP-1 (17Cook-Mills J.M. Deem T.L. J. Leukocyte Biol. 2005; 77: 487-495Crossref PubMed Scopus (219) Google Scholar, 18Millan J. Hewlett L. Glyn M. Toome D. Clark P. Ridley A.J. Nat. Cell Biol. 2006; 8: 113-123Crossref PubMed Scopus (327) Google Scholar, 19Muller W.A. Trends Immunol. 2003; 24: 327-334PubMed Scopus (0) Google Scholar). The majority of genes expressed in endothelial cells in response to inflammatory mediators such as lipopolysaccharide, IL-1β, or TNF-α contain functionally important NF-κB-binding sites in their promoter regions (20Denk A. Goebeler M. Schmid S. Berberich I. Ritz O. Lindemann D. Ludwig S. Wirth T. J. Biol. Chem. 2001; 276: 28451-28458Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 21Kempe S. Kestler H. Lasar A. Wirth T. Nucleic Acids Res. 2005; 33: 5308-5319Crossref PubMed Scopus (228) Google Scholar). Moreover, inhibition of NF-κB activity resulted in efficient inhibition of endothelial cell activation (22Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar, 23Li X. Stark G.R. Exp. Hematol. 2002; 4: 285-296Abstract Full Text Full Text PDF Scopus (313) Google Scholar, 24Karin M. Greten F.R. Nat. Rev. Immunol. 2005; 5: 749-759Crossref PubMed Scopus (2546) Google Scholar). The five members of the mammalian NF-κB family, p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2), are present in unstimulated cells as homo- or heterodimers bound to IκB family proteins (22Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar, 23Li X. Stark G.R. Exp. Hematol. 2002; 4: 285-296Abstract Full Text Full Text PDF Scopus (313) Google Scholar, 24Karin M. Greten F.R. Nat. Rev. Immunol. 2005; 5: 749-759Crossref PubMed Scopus (2546) Google Scholar). NF-κB proteins are characterized by the presence of a conserved 300-amino acid Rel homology domain that is located toward the N terminus of the protein and is responsible for dimerization, interaction with IκBs, and binding to DNA. Binding to IκB prevents the NF-κB-IκB complex from translocating to the nucleus, thereby maintaining NF-κB in an inactive state. Three distinct NF-κB-activating pathways have emerged, and all of them rely on sequentially activated kinases (25Janssens S. Tschopp J. Cell Death Differ. 2006; 13: 1-12Crossref PubMed Scopus (254) Google Scholar, 26Kato Jr., T. Delhase M. Hoffman A. Karin M. Mol. Cell. 2003; 12: 829-839Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 27Viatour P. Merville M.-P. Bours V. Chariot A. Trends Biochem. Sci. 2005; 30: 43-52Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar). The first pathway, i.e. the classical pathway, is triggered by proinflammatory cytokines such as TNF-α and leads to the sequential recruitment of various adaptors, including TNF receptor-associated death domain protein, receptor interacting protein, and TNF receptor-associated factor 2 (TRAF2) to the cytoplasmic membrane. This is followed by the recruitment and activation of the IκB kinase (IKK) complex, which includes the scaffold protein NF-κB essential modulator (NEMO; also named IKKγ), IKKα, and IKKβ kinases. Once activated, the IKK complex phosphorylates on Ser-32 and Ser-36 IκBα, which is subsequently ubiquitinated and then degraded via the proteosome pathway. A second pathway, i.e. the alternative pathway, is NEMO-independent and is triggered by cytokines, including lymphotoxin b, B-cell activating factor or the CD40 ligand, and by viruses (e.g. human T-cell leukemia virus and the Epstein-Barr virus). This pathway relies on the recruitment of TRAF proteins to the membrane and on the NF-κB-inducing kinase (NIK), which activates an IKKα homodimer. IKKα phosphorylates the ankyrin-containing and inhibitory molecule p100 on specific serine residues located in both the N- and C-terminal regions. p100 is then ubiquitinated and cleaved to generate the NF-κB protein p52 that moves as a heterodimer with RelB into the nucleus. The third signaling pathway is classified as "atypical" because it is independent of IKK but still requires the proteosome and is triggered by DNA-damaging agents such as UV radiation or doxorubicin. UV radiation induces IκBα degradation via the proteosome, but the targeted serine residues are located within a C-terminal cluster that is recognized by the p38-activated casein kinase 2 (CK2). To add new information to the physiological role of IFI16 in the modulation of the immune response and inflammation, we initiated microarray analysis of IFI16-overexpressing HUVEC. The results obtained indicate that genes involved in inflammation, cell proliferation, and apoptosis are affected by IFI16 overexpression. In particular we demonstrate that IFI16 stimulates the expression of proinflammatory genes, including ICAM-1, E-selectin, IL-8, and MCP-1. ICAM-1 stimulation by IFNs appears to depend on functional IFI16, because inhibition of its expression by RNA interference blocks the IFN capability to increase ICAM-1 expression. ICAM-1 induction takes place through activation of the NF-κB complex that appears to be triggered by transcriptional suppression of the IκBα gene expression. Consistent with these observations, reduction in IκBα expression by RNA interference results in the same outcome observed with IFI16 overexpression. Altogether, these results demonstrate that IFI16 modulates the response of proinflammatory cytokines in endothelial cells and may provide a molecular explanation for its role in the initial steps of the autoimmune response. Cells Lines and Reagents—HUVEC obtained by trypsin treatment of umbilical cord veins were cultured in endothelial growth medium (EGM-2, Clonetics, San Diego) containing 2% fetal bovine serum, human recombinant vascular endothelial growth factor, basic fibroblast growth factor, human epidermal growth factor, insulin growth factor (IGF-1), hydrocortisone, ascorbic acid, heparin, gentamycin, and amphotericin B (1 μg/ml each) and were seeded into 100-mm culture dishes coated with 0.2% gelatin. Experiments were performed with cells at passage 2–6. Human embryo kidney 293 cells (HEK-293) (Microbix Biosystems Inc.) were cultured in minimum Eagle'ns medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 2 mm glutamine, 100 units of penicillin per ml, and 100 μg per ml of streptomycin sulfate. Cells were kept in logarithmic growth phase by 1× citric saline detachment and reinoculation every 2–4 days. Interferon-γ and TNF-α were the generous gifts of Gianni Garotta, Geneva, Switzerland, and Tiziana Musso, Turin, Italy, respectively. Recombinant Adenovirus Preparations and HUVEC Infection—The pAC-CMV IFI16, containing the human IFI16 cDNA linked to a FLAG tag at the N terminus, was cotransfected together with pJM17 into human embryonic kidney 293 cells as described previously (11Gugliesi F. Mondini M. Ravera R. Robotti A. De Andrea M. Gribaudo G. Gariglio M. Landolfo S. J. Leukocyte Biol. 2005; 77: 820-829Crossref PubMed Scopus (51) Google Scholar). After several rounds of plaque purification, the adenovirus containing the IFI16 gene (AdVIFI16) was amplified on 293 cell monolayers and purified from cell lysates by banding twice on CsCl gradients. Desalting was performed using G-50 columns (Amersham Biosciences), and viruses were frozen in PBS, 10% glycerol at -80 °C. The infectious titers (PFU) were determined by a standard plaque assay on 293 cell monolayers (11Gugliesi F. Mondini M. Ravera R. Robotti A. De Andrea M. Gribaudo G. Gariglio M. Landolfo S. J. Leukocyte Biol. 2005; 77: 820-829Crossref PubMed Scopus (51) Google Scholar). The physical particles of the vector preparations were measured by spectrophotometry (28Mittereder N. March K.L. Trapnell B.C. J. Virol. 1996; 70: 7498-7509Crossref PubMed Google Scholar), and our viral preparations showed a 1:10–1:20 ratio between PFU and physical particles. Endotoxin contaminations were excluded by E-Toxate kit (Sigma) (sensitivity > 1.4 pg/ml). Recombinant AdVIFI16 was tested for IFI16 expression by Western blotting, using an anti-FLAG antibody (Sigma). For cell transduction, pre-confluent HUVEC were washed once with PBS and incubated with AdVIFI16 or AdVLacZ (used as a control) at a multiplicity of infection (m.o.i.) of 300 in EGM. After 60 min at 37 °C, the virus was washed off, and fresh medium was added. Cells were cultured for 36 h before use in the experiments. For IFI16 interference the pAC-CMV shRNA-IFI16, containing the short hairpin RNA sequence targeting the human IFI16 gene mRNA (shRNAIFI16) (5′-CCG TCA GAA GAC CAC AAT CTA CTT CAA GAG AGT AGA TTG TGG TCT TCT GAT TTT TTG GAA A-3′), or the pAC-CMV shRNA-SCRAMBLED (shRNASCR), containing a scrambled human IFI16 gene shRNA sequence (5′-CCG GCA CAG TCA CAA CAA ATC TTT CAA GAG AAG ATT TGT TGT GAC TGT GCT TTT TTG GA AA-3′), were cotransfected with pJM17 into 293 cells. After several rounds of plaque purification, recombinant adenoviruses were amplified on 293 cell monolayers and purified to homogeneity as before. PFU were assessed for virus titers on the 293 complementing cell line with agar overlay. Recombinant AdVshRNAIFI16 was tested for IFI16 knockdown by Western blotting. For cell transduction, HUVEC were washed once with PBS and incubated with AdVshRNAIFI16 or AdVshRNASCR at an m.o.i. of 300 in EGM. After 2 h at 37 °C, the virus was washed off, and cells were treated as indicated. For IκBα interference, the adenoviral human IκBα siRNA recombinant virus (AdVshRNAIκBα) containing the short hairpin RNA sequence targeting the human IκBα gene mRNA was purchased from Imgenex (San Diego, CA) and used according to the manufacturer'ns instruction at an m.o.i. of 1000. Microarray and Data Analysis—Total RNA was extracted from HUVEC cultured at subconfluence and infected with AdVIFI16 or AdVLacZ for 6 h or 24 h. RNA pooled from different cultures was used as a template for biotinylated probe synthesis. Total RNAs were quality-controlled and quantified on the Bioanalyzer 2100 (Agilent). For gene expression profiling on Beadchips from Illumina, reverse transcription, double-stranded cDNA, and biotinylated cRNA synthesis were carried out according to standard Illumina protocols, using the "Illumina RNA amplification kit" (Ambion) and biotin-16 UTP (Roche Applied Science). Briefly, 500 ng of total RNA were used for reverse transcription to synthesize double-stranded cDNA. The cDNA was used for in vitro transcription to obtain biotinylated cRNA, followed by a purification step. Subsequently, 550 ng of biotinylated cRNA, resuspended in Hyb E1 buffer, formamide, and RNase-free water, were denatured at 65 °C for 5 min and hybridized for 16 h at 55 °C on Ilumina "Human Sampler" bead arrays, exploring 516 genes. Hybridized bead arrays were then washed three times with E1BC buffer (Illumina), once with 100% EtOH, blocked using blocking E1 buffer (Illumina), and stained with streptavidin-Cy3 (2 μl per chip of 1 mg/ml stock, from Amersham Biosciences). After the final washing and drying, bead arrays were scanned on a GenePix Personal 4100A microarray reader (Axon). Images were processed with the Beadarray Studio software (Illumina) to extract signal intensities for each bead type in each experiment and export them to a tab-delimited text file. Statistical analysis and selection of regulated genes was carried out using Excel (Microsoft). We defined two criteria for selecting regulated genes as follows: (a) genes that were regulated more than 2-fold in both replicates (32 genes); (b) genes that were regulated by average at least 1.4-fold plus 2 standard deviations (45 genes). The first approach allowed capturing genes with greater response, and the second allowed identifying genes with smaller, but consistent, response. In total, we selected 55 genes, of which 22 passed both tests. Real Time RT-PCR Analysis—RT-PCR analysis was performed on an Mx 3000 P™ (Stratagene) using the SYBR Green I dye (Invitrogen) as a nonspecific PCR product fluorescence label. Total cellular RNA was isolated using the Eurozol reagent (Euroclone Ltd.). RNA (1 μg) was then retrotranscribed at 42 °C for 60 min in PCR buffer (1.5 mm MgCl2) containing 5 μm random primers, 0.5 mm dNTP, and 100 units of Moloney murine leukemia virus reverse transcriptase (Ambion) in a final volume of 20 μl. cDNAs (2 μl) or water as control was amplified in duplicate by real time RT-PCR using the Brilliant SYBR Green QPCR master mix (Stratagene) in a final volume of 25 μl. Primer sequences were as follows: IFI16 (sense, 5′-ACT GAG TAC AAC AAA GCC ATT TGA-3′; antisense, 5′-TTG TGA CAT TGT CCT GTC CCC AC-3′); ICAM-1 (sense, 5′-CAA CCG GAA GGT GTA TGA AC-3′; antisense, 5′-CAG CGT AGG GTA AGG TTC-3′); V-CAM1 (sense, 5′-CAT GGA ATT CGA ACC ACA-3′; antisense, 5′-GAC CAA GAC GGT TGT ATC GG-3′); BAD (sense, 5′-CAT TGA GCC GAG TGA GCA GG-3′; antisense, 5′-TCG TCA CTC ATC CTC CGG AG-3′); UCP3 (sense, 5′-CGT GGT GAT GTT CGT AAC CTA TG-3′; antisense, 5′-CGG TGA TTC CCG TAA CAT CTG-3′); CDC25a (sense, 5′-CTC CTC CGA GTC AAC AGA TT-3′; antisense, 5′-CAG AGT TCT GCC TCT GTG TG-3′); SSTR1 (sense, 5′-GGC GAA ATG CGT CCC AG-3′; antisense, 5′-CGG AGT AGA TGA AAG AGA TCA GGA-3′); CCR4 (sense, 5′-CCT CAG AGC CGC TTT CAG A-3′; antisense, 5′-GCC TTG ATG CCT TCT TTG GT-3′); E-Selectin (sense, 5′-CTC TGA CAG AAG AAG CCA AG-3′; antisense, 5′-ACT TGA GTC CAC TGA AGT CA-3′); MCP-1 (sense, 5′-TCC TGT GCC TGC TGC TGA TAG C-3′; antisense, 5′-TTC TGA ACC CAC TTC TGC TTG G-3′); IL-8 (sense, 5′-ATG ACT TCC AAG CTG GCC GTG GCT-3′; antisense, 5′-TCT CAG CCC TCT TCA AAA ACT TCT C-3′); PAI-1 (sense, 5′-TGC TGG TGA ATG CCC TCT ACT-3′; antisense, 5′-CGG TCA TTC CCA GGT TCT CTA-3′); PTX-3 (sense, 5′-TGG CTG CCG GCA GGT; antisense, 5′-TCC ACC CAC CAC AAA CAC TAT-3′); and β-actin (sense, 5′-GTT GCT ATC CAG GCT GTG-3′; antisense, 5′-TGT CCA CGT CAC ACT TCA-3′). Following an initial denaturing step at 95 °C for 2 min to activate 0.75 units of Platinum TaqDNA polymerase (Invitrogen), the cDNAs were amplified for 30 cycles (95 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min). For quantitative analysis, semi-logarithmic plots were constructed of Δ fluorescence versus cycle number, and a threshold was set for the changes in fluorescence at a point in the linear PCR amplification phase (Ct). The Ct values for each gene were normalized to the Ct values for β-actin with the ΔCt equation. The level of target RNA, normalized to the endogenous reference and relative to the mock-infected and untreated cells, was calculated by the comparative Ct method with the 2-ΔΔCt equation. Plasmids and Transfection Assays—Reporter luciferase vectors for human full-length ICAM-1 promoter pIC1352 (-1352/+1) and the relevant mutant constructs pIC339 (-339/+1), pIC135 (-135/+1), and pIC135 (ΔAP2) have been described previously (29Van de Stolpe A. Caldenhoven E. Stade B.G. Koenderman L. Raaijmakers J.A. Johnson J.P. van der Saag P.T. J. Biol. Chem. 1994; 269: 6185-6192Abstract Full Text PDF PubMed Google Scholar). To derive ICAM-1 promoter with mutated NF-κB-binding sites, the sequence of these sites was modified by two consecutive rounds of site-directed mutagenesis (QuikChange XL site-directed mutagenesis kit; Stratagene). The two NF-κB recognition sites were abrogated and changed to unique restriction sites (-187 BamHI, and -491 KpnI) in the plasmid pIC1352 (mut2NF-κB). In the plasmid pIC339 (mutNF-κB) the -187 NF-κB-binding site was mutated and changed with BamHI restriction site. The correctness of the introduced mutations was confirmed by sequencing the derived constructs. The pGL2-ICAM-1 construct, spanning 137 nucleotides before the translation start site and carrying two Ets-responsive sites, pGL2-ICAM-1/Mut117, pGL2-ICAM-1/Mut97, and pGL2-ICAM-1/Mut117–97, the specific mutants for one or both Ets sites, respectively, have been described previously (30de Launoit Y. Audette M. Pelczar H. Plaza S. Baert J.L. Oncogene. 1998; 16: 2065-2073Crossref PubMed Scopus (57) Google Scholar). The 0.4SK-pGL3 Luc, obtained by subcloning the 0.4-kb fragment of the IκBα promoter, and the relevant mutant constructs, mutkB1, mutSp1, and mutkB1/Sp1, have been described previously (31Algarté M. Kwon H. Génin P. Hiscott J. Mol. Cell. Biol. 1999; 19: 6140-6153Crossref PubMed Google Scholar). For transfection experiments, HUVEC cells grown to subconfluence were transfected with 2 μg of the reporter vector of interest that was added to serum-free EGM-2, mixed with 6 μl of Reagent Plus and 3 μl of Lipofectamine (Invitrogen), and incubated for 2 h at 37 °C. After 4 h, the medium was replaced with EGM-2 medium containing 2% fetal bovine serum. Twenty four hours later, cells were infected with AdVIFI16 or AdVLacz (at an m.o.i. of 300) or mock-infected. After 24 h of incubation, chemiluminescence was measured using the Lumino luminometer (Stratec Biomedical Systems, Birkenfeld, Germany). Reporter gene activity was normalized to the amount of plasmid DNA introduced into the recipient cells measured by real time PCR with appropriate luciferase-actin primers. Nuclear Extract Isolation and Electrophoretic Mobility Shift Assay (EMSA)—HUVEC were plated at a density of 3 × 105 cells/100-mm diameter dish and after 72 h infected with AdVIFI16, AdVLacz, or mock-infected. At the indicated times post-infection (p.i.), cells were washed in cold PBS, harvested, and centrifuged to collect the pellet. The pellets were then incubated for 15 min on ice with a cytoplasmic isolation buffer (10 mm HEPES (pH 7.6), 60 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, protease and phosphatase inhibitor mixture (Sigma)). The samples were centrifuged, and the nuclear pellets were then collected by removing the supernatant containing the cytoplasmic extract, washed in cytoplasmic isolation buffer without Non-idet P-40, centrifuged, and incubated for 10 min on ice with a nuclear isolation buffer (20 mm Tris-HCl (pH 8.0), 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 25% glycerol, protease inhibitor mixture (Sigma)). After centrifugation, supernatants containing the nuclear extracts were collected and stored at -70 °C. EMSA was performed as described previously (32Caposio P. Dreano M. Garotta G. Gribaudo G. Landolfo S. J. Virol. 2004; 78: 3190-3195Crossref PubMed Scopus (36) Google Scholar). Briefly, nuclear extracts (15 μg of proteins) were incubated in a binding buffer (10 mm Tris-HCl (pH 7.9), 50 mm NaCl, 0.5 mm EDTA, 1 mm dithiothreitol, 7.5 mm MgCl2) with 1 μg of poly(dI-dC) (GE Healthcare) and the 32P-labeled double-stranded NF-κB, AP-1, and Sp1 consensus oligonucleotides (Promega). The oligonucleotide probe was labeled with [γ-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase according to the manufacturer'ns protocol, and finally column-purified on G-25 Sephadex (Bio-Rad). Complexes were analyzed by nondenaturing 4% PAGE, dried, and detected by autoradiography. Immunoblotting—Whole-cell protein extracts were prepared by resuspending pelleted cells in lysis buffer containing 125 mm Tris-Cl (pH 6.8), 1% SDS, 20 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, 4 μg/ml aprotinin, 1 μg/ml pepstatin. After a brief sonication, soluble proteins were collected by centrifugation at 15,000 × g. Supernatants were analyzed for protein concentration with a Dc protein assay kit (Bio-Rad) and stored at -70 °C in 10% glycerol. Proteins were separated by SDS-PAGE and then transferred to Immobilon-P membranes (Millipore). Filters were blocked in 5% nonfat dry milk in 10 mm Tris-Cl (pH 7.5), 100 mm NaCl, 0.1% Tween 20 and immunostained with the rabbit anti-IFI16 Ab (diluted 1:2000), the rabbit anti-IκBα polyclonal antibody (Santa Cruz Biotechnology) (diluted 1:500), the rabbit anti-p50 polyclonal antibody (Santa Cruz Biotechnology) (diluted 1:300), the mouse anti-p65 mAb (Santa Cruz Biotechnology) (diluted 1:500), the mouse anti-IKK2 mAb (Pharmingen) (diluted 1:1000), or the mouse anti-actin mAb (Chemicon) (diluted 1:4000). Immunocomplexes were detected

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