The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m406620200
ISSN1083-351X
AutoresAitor G. Granja, Marı́a L. Nogal, Carolina Hurtado, Virginia Vila‐del Sol, Ángel L. Carrascosa, María Salas, Manuel Fresno, Yolanda Revilla,
Tópico(s)T-cell and Retrovirus Studies
ResumoCyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5′-terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calcium-dependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NFκB had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein down-regulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation. Cyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5′-terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calcium-dependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NFκB had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein down-regulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation. Viruses have been known for a long time to use a variety of strategies not only to alter the host metabolism via their signaling proteins but also to hijack cellular signaling pathways and transcription factors to control them to their own advantage. Both the nuclear factor-κB (NFκB) 1The abbreviations used are: NFκB, nuclear factor-κB; COX-2, cyclooxygenase-2; NFAT, nuclear factor of activated T cell; IκB, inhibitory proteins of the IκB family; Cot/TP12, serine/threonine kinase Cot; CsA, cyclosporin A; PMA, phorbol 12-myristate 13-acetate; Ion, calcium ionophore; PGE2, prostaglandin E2; RLU, relative luciferase unit(s); PBS, phosphate-buffered saline; ASFV, African swine fever virus; IL-2, interleukin-2; β-gus, β-glucuronidase. and the nuclear factor of activated T cells (NFAT) pathways appear to be attractive targets for common viral pathogens, probably due to their ability to promote the expression of numerous proteins involved in adaptative and innate immunity (1Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3321) Google Scholar, 2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2221) Google Scholar). Several viruses, including hepatitis C virus (3Bergqvist A. 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Cell Culture, Viruses, and Reagents—Vero (African green monkey kidney) cells were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (Invitrogen). Jurkat human leukemia T cell line was obtained from the American Type Culture Collection and cultured in RPMI 1640 (Invitrogen) medium supplemented with 10% fetal bovine serum. Both media were supplemented with 2 mm l-glutamine, 100 units of gentamicin per milliliter and non-essential amino acids. Cells were stimulated by phorbol 12-myristate 13-acetate (PMA; Sigma) at 15 ng/ml and A23187 calcium ionophore (Ion; Sigma) at 1 μm. Cyclosporin A (CsA, Sandoz, 100 ng/ml) was added 1 h before the addition of PMA and Ion. The Vero-adapted ASFV strain Ba71V was propagated and titrated by plaque assay on Vero cells as described previously (41Enjuanes L. Carrascosa A.L. Moreno M.A. Vinuela E. J. Gen. Virol. 1976; 32: 471-477Crossref PubMed Scopus (177) Google Scholar). ASFV A238L Deletion Mutant Construction—The A238L-defective mutant ΔA238L virus was obtained by insertion of the Escherichia coli β-glucuronidase (β-gus) gene into the A238L open reading frame. For this, a 473-bp left-flanking and a 480-bp right-flanking DNA fragments were generated by PCR amplification of the ASFV strain Ba71V genome using specific oligonucleotides and cloned into plasmid p72GUS10T (42Garcia R. Almazan F. Rodriguez J.M. Alonso M. Vinuela E. Rodriguez J.F. J. Biotechnol. 1995; 40: 121-131Crossref PubMed Scopus (26) Google Scholar). The recombinant ΔA238L virus was obtained as previously described (42Garcia R. Almazan F. Rodriguez J.M. Alonso M. Vinuela E. Rodriguez J.F. J. Biotechnol. 1995; 40: 121-131Crossref PubMed Scopus (26) Google Scholar). Briefly, Vero cells were transfected by lipofection with pΔA238L, using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions, mixed in Opti-MEM (Invitrogen), and then infected with virus strain Ba71V. After 48 h of infection, the cells were harvested, and diluted samples were used to infect Vero cell monolayers. The infected cells were covered with agar (Invitrogen), and, 4 days later, the β-gus substrate 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc, Biomol), was added to the culture medium. The blue-stained plaques were selected and used to infect fresh monolayers of Vero cells. The recombinant virus was purified after successive rounds of plaque isolation. The lack of gene A238L in the recombinant virus was assessed by Southern blot hybridization. Briefly, DNA samples from the genome of previously purified virus BA71V and ΔA238L were digested with the restriction endonuclease EcoRI, subjected to electrophoresis in agarose gels, and transferred to nylon membranes following standard procedures. The DNA probes, specific for the β-gus and A238L genes, and for the SalI I′ fragment of Ba71V genome were labeled with a DIG DNA labeling kit (Roche Applied Science). Plasmid Constructs—Human COX-2 promoter constructs P2-1900 (-1796, +104), P2-1102 (-998, +104), P2-431 (-327, +104), P2-274 (-170, +104), and P2-150 (-46, +104) were generated as described previously (40Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The COX-2 promoter mutants P2-274 dNFAT MUT, P2-274 pNFAT MUT, and P2-274 d,pNFAT MUT were generated as described (40Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The AP-1-Luc plasmid includes the AP-1-responsive (-73 to +63 bp) region of the human collagenase promoter fused to the luciferase gene (43Deng T. Karin M. Genes Dev. 1993; 7: 479-490Crossref PubMed Scopus (291) Google Scholar). NFAT-luc, containing three tandem copies of the NFAT binding site of the IL-2 promoter, and the full-length human NFATc (p1SH107c) expression plasmid (14Northrop J.P. Ho S.N. Chen L. Thomas D.J. Timmerman L.A. Nolan G.P. Admon A. Crabtree G.R. Nature. 1994; 369: 497-502Crossref PubMed Scopus (524) Google Scholar), were a generous gift from Dr. G. Crabtree. The pNFκB-luc contains three tandem copies of the NF-κB site of the conalbumin promoter driving the luciferase reporter gene. The pcDNA-A238L expression plasmid was generated by cloning the A238L open reading frame from Ba71V viral strain of ASFV into the pcDNA3.1 mammalian expression vector (Invitrogen). The ΔCAM-AI expression plasmid encoded a deletion mutant of murine calcineurin catalytic subunit. The p65 expression plasmid pcDNA3-p65 was a gift from Dr. J. Alcamí. The GAL4 luciferase constructs (GAL4-Luc) contain five GAL4 DNA consensus binding sites derived from the yeast GAL4 gene fused to luciferase reporter gene (44Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1446) Google Scholar), and the pGAL4-hNFAT1 constructs containing the first 1–451 amino acids of human NFAT1 fused to the DNA-binding domain of yeast GAL4 transcription factor were originated as described previously (21de Gregorio R. Iniguez M.A. Fresno M. Alemany S. J. Biol. Chem. 2001; 276: 27003-27009Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Transfection and Luciferase Assays—Generation of A238L stably expressing Jurkat cells was done by transfecting 0.5 μg of empty plasmid pcDNA3.1 or pcDNA3.1-A238L using the LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions and mixing in Opti-MEM (Invitrogen). Two days later, G418 antibiotic selection was applied (0.5 mg of G418 (Invitrogen) per milliliter). Cells were refed with fresh medium every 3 days until colonies were apparent (2–3 weeks). These cellular lines were named Jurkat-pcDNA and Jurkat-A238L. Vero or Jurkat-pcDNA and Jurkat-A238L cells were transfected with 250 ng of specific plasmids per 106 cells as described above. In cotransfection assays, 0.05–0.5 μg of the corresponding expression plasmid per 106 cells was added. 16 h after transfection, Jurkat-pcDNA and Jurkat-A238L cells were stimulated with 15 ng/ml PMA plus 1 μm Ion during 4 h, and Vero cells were infected with Ba71V or Ba71VΔA238L at a multiplicity of infection of 5 plaque-forming units/cell at the indicated times. Then, Jurkat and Vero cells were lysed with 200 μl of cell culture lysis reagent (Promega) and microcentrifuged at full speed for 5 min at 4 °C, and 20 μl of each supernatant was used to determine firefly luciferase activity in a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Results were expressed as the luminescence units after normalization of protein concentration determined by the bicinchoninic acid spectrophotometric method (Pierce). Transfection experiments were performed in triplicate, and the data are presented as the mean of the relative luciferase units (RLUs) (mean ± S.D.). mRNA Analysis—Total RNA was prepared from Jurkat-pcDNA or Jurkat-A238L or ASFV-infected Vero cells by the TRIzol reagent RNA protocol (Invitrogen). Total RNA (1 μg) was reverse transcribed into cDNA by the RevertAid First Strand cDNA synthesis kit (MBI Fermentas) and used for PCR amplification with the addition of TaqDNA polymerase (Roche Applied Science) following the manufacturer's instructions. Specific primers used in PCR reactions were human COX-2 (forward: 5′-TTCAAATGAGATTGTGGGAAAATTGCT-3′ and reverse: 5′-AGATCATCTCTGCCTGAGTATCTT-3′), human β-actin (forward: 5′-GAGAAGATGACCCAGATCATG-3′ and reverse: 5′-TCAGGAGGAGCAATGATCTTG-3′), and A238L (forward: 5′-CGCGCGTCTAGATTACTTTCCATACTTGTT-3′ and reverse: 5′-GCGCGCAAGCTTATGGAACACATGTTTCCA-3′). The PCR reactions were performed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min. Amplified cDNAs were separated by agarose gel electrophoresis, and bands were visualized by ethidium bromide staining. Western Blot Analysis—Unstimulated or stimulated Jurkat-pcDNA and Jurkat-A238L cells were washed twice with PBS and lysed in radio immunolabeling protein assay (radioimmune precipitation assay) buffer supplemented with protease inhibitor mixture tablets (Roche Applied Science). Protein concentration was determined by the bicinchoninic acid spectrophotometric method. Cell lysates (50 μg of protein) were fractionated by SDS-8% polyacrylamide gel electrophoresis, electrophoretically transferred to an Immobilon extra membrane (Amersham Biosciences), and the separated proteins reacted with specific primary antibodies raised against COX-2 (Alexis Biochemicals, number 804-112-C050), β-actin (H-196, Santa Cruz Biotechnology), and NFAT (anti-NFAT1/c2 672 rabbit antiserum, previously described (45Lara-Pezzi E. Armesilla A.L. Majano P.L. Redondo J.M. Lopez-Cabrera M. EMBO J. 1998; 17: 7066-7077Crossref PubMed Scopus (88) Google Scholar)). Membranes were exposed to horseradish peroxidase-conjugated secondary antibodies (Dako), followed by chemiluminescence (ECL, Amersham Biosciences) detection by autoradiography. Determination of PGE2—PGE2 was determined in cell culture supernatants by a competitive enzyme-linked immunosorbent assay. The target (PGE2) competes with biotinylated PGE2 at the binding site of a specific monoclonal anti-PGE2 antibody. A streptavidin-peroxidase conjugate enables the detection of biotin via generation of a colored reagent. The detection limit was about 20 pg/ml PGE2. Jurkat pcDNA or Jurkat-A238L cells were stimulated with PMA/Ion, and supernatants were recovered at different times of stimulation. Concentrations of PGs were measured by a prostaglandin screen colorimetric assay kit, according to the manufacturer's protocol, (Cayman Chemical). Immunofluorescence and Confocal Microscopy—ASFV-infected Vero cells were grown on coverslips to 2 × 105 cells/cm2. Cultures were rinsed three times with PBS and fixed with cold 99.8% methanol (Merck) for 15 min at -20 °C, before rehydrating twice with PBS and blocking with 1% bovine serum albumin in PBS for 10 min at room temperature. The cells were incubated during 2 h with the specific antibody against NFAT (G1-D10, Santa Cruz Biotechnologies), rinsed extensively with PBS, and then incubated with the secondary antibody (Alexa, Molecular Probes) for 1 h at room temperature in the dark. Finally, the cells were rinsed successively with PBS, distilled water, and ethanol, and mounted with a drop of Mowiol on a micro slide. Visualization of stained cultures was performed under a fluorescence Axioskop2 plus (Zeiss) microscope coupled to a color charge-coupled device camera or to a Confocal Microradiance (Bio-Rad) equipment. Images were digitalized, processed, and organized with Metamorph, Lasershap2000 version 4, Adobe Photoshop 7.0, Adobe Illustrator 10, and Microsoft PowerPoint SP-2 software. Electrophoretic Mobility Shift Assay—Nuclear extracts from Jurkat-pcDNA and Jurkat-A238L cells unstimulated or stimulated with 15 ng/ml PMA plus 1 μm Ion treated or not with 100 ng/ml CsA were prepared. Cells were harvested by centrifugation, washed twice with PBS, and resuspended in 500 μl of Buffer A (10 mm HEPES, pH 7.6; 10 mm KCl; 0.1 mm EDTA; 0.1 mm EGTA; 0.75 mm spermidine; 0.15 mm spermine; 1 mm dithiothreitol; 0.5 mm phenylmethylsulfonyl fluoride; 10 mm Na2MoO4; and 2 μg/ml each of inhibitors leupeptin, aprotinin, and pepstatin A). After 15 min at 4 °C, 5 μl of a 10% Nonidet P-40 solution were added. Samples were vortexed for 10 s and centrifuged for 20 min at 3000 rpm and 4 °C. The supernatants were used as cytosolic extracts. To avoid cytosolic contamination, nuclei were washed twice with 200 μl of buffer A. For nuclear protein extraction, 50 μl of Buffer C (20 mm HEPES, pH 7.6; 0.4 m NaCl; 1 mm EDTA; 1 mm EGTA; 1 mm dithiothreitol; 0.5 mm phenylmethylsulfonyl fluoride; 10 mm Na2MoO4; and 2 μg/ml each of inhibitors leupeptin, aprotinin, and pepstatin A) were added, and nuclear pellets were incubated for 30 min at 4 °C with gentle agitation. Samples were centrifuged for 10 min at 14,000 rpm and 4 °C, and supernatants were used as nuclear extracts. Protein concentration was determined by Bradford assay (Bio-Rad). Electrophoretic mobility shift assays were performed basically as described previously (40Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). For binding reaction 5 μg of nuclear extract was incubated with 1 μg of poly(dI-dC) in DNA Binding Buffer (10% (w/v) polyvinyl ethanol, 12.5% (v/v) glycerol, 50 mm Tris-HCl, pH 8; 2.5 mm dithiothreitol; 2.5 mm EDTA) on ice for 15 min. Then, 32P-labeled double-stranded oligonucleotide probe (0.5 ng) was added, and samples were incubated for additional 45 min at room temperature. In competition experiments, a 50-fold molar excess of unlabeled oligonucleotide was added to the binding reaction mixture prior to the probe. Supershift assays were performed by incubating nuclear extracts with either preimmune serum or anti-NFAT antiserum prior to the addition of the probe. DNA-protein complexes were resolved by polyacrylamide gel electrophoresis on a 4% non-denaturing gel. The sequences of the oligonucleotides used were: 5′-TCGACAAGGGGAGAGGAGGGAAAAATTTGTGGC-3′ (nucleotides -117 to -91 containing the NFAT distal site of the human COX-2 promoter), 5′-TCGACAAAAGGCGGAAAGAAACAGTCATTTC-3′ (nucleotides -82 to -58, including the NFAT/AP-1 proximal site of the human COX-2 promoter), and 5′-GATCGGAGGAAAAACTGTTTCA
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