Vaccinia Virus Protein A52R Activates p38 Mitogen-activated Protein Kinase and Potentiates Lipopolysaccharide-induced Interleukin-10
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.m501917200
ISSN1083-351X
AutoresGeraldine Maloney, Martina Schröder, Andrew Bowie,
Tópico(s)Immune Response and Inflammation
ResumoVaccinia virus (VV) has many mechanisms to suppress and modulate the host immune response. The VV protein A52R was previously shown to act as an intracellular inhibitor of nuclear factor κB (NFκB) signaling by Toll-like receptors (TLRs). Co-immunoprecipitation studies revealed that A52R interacted with both tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 2 (IRAK2). The effect of A52R on signals other than NFκB was not determined. Here, we show that A52R does not inhibit TLR-induced p38 or c-Jun amino N-terminal kinase (JNK) mitogen activating protein (MAP) kinase activation. Rather, A52R could drive activation of these kinases. Two lines of evidence suggested that the A52R/TRAF6 interaction was critical for these effects. First, A52R-induced p38 MAP kinase activation was inhibited by overexpression of the TRAF domain of TRAF6, which sequestered A52R and inhibited its interaction with endogenous TRAF6. Second, a truncated version of A52R, which interacted with IRAK2 and not TRAF6, was unable to activate p38. Because interleukin 10 (IL-10) production is strongly p38-dependent, we examined the effect of A52R on IL-10 gene induction. A52R was found to be capable of inducing the IL-10 promoter through a TRAF6-dependent mechanism. Furthermore, A52R enhanced lipopolysaccharide/TLR4-induced IL-10 production, while inhibiting the TLR-induced NFκB-dependent genes IL-8 and RANTES. These results show that although A52R inhibits NFκB activation by multiple TLRs it can simultaneously activate MAP kinases. A52R-mediated enhancement of TLR-induced IL-10 may be important to virulence, given the role of IL-10 in immunoregulation. Vaccinia virus (VV) has many mechanisms to suppress and modulate the host immune response. The VV protein A52R was previously shown to act as an intracellular inhibitor of nuclear factor κB (NFκB) signaling by Toll-like receptors (TLRs). Co-immunoprecipitation studies revealed that A52R interacted with both tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 2 (IRAK2). The effect of A52R on signals other than NFκB was not determined. Here, we show that A52R does not inhibit TLR-induced p38 or c-Jun amino N-terminal kinase (JNK) mitogen activating protein (MAP) kinase activation. Rather, A52R could drive activation of these kinases. Two lines of evidence suggested that the A52R/TRAF6 interaction was critical for these effects. First, A52R-induced p38 MAP kinase activation was inhibited by overexpression of the TRAF domain of TRAF6, which sequestered A52R and inhibited its interaction with endogenous TRAF6. Second, a truncated version of A52R, which interacted with IRAK2 and not TRAF6, was unable to activate p38. Because interleukin 10 (IL-10) production is strongly p38-dependent, we examined the effect of A52R on IL-10 gene induction. A52R was found to be capable of inducing the IL-10 promoter through a TRAF6-dependent mechanism. Furthermore, A52R enhanced lipopolysaccharide/TLR4-induced IL-10 production, while inhibiting the TLR-induced NFκB-dependent genes IL-8 and RANTES. These results show that although A52R inhibits NFκB activation by multiple TLRs it can simultaneously activate MAP kinases. A52R-mediated enhancement of TLR-induced IL-10 may be important to virulence, given the role of IL-10 in immunoregulation. The recently described Toll-like receptor (TLR) 1The abbreviations used are: TLR, Toll-like receptor; IL-1, interleukin 1; IRAK, interleukin-1 receptor-associated kinase; JNK, c-Jun amino N-terminal kinase; LPS, lipopolysaccharide; MAP kinase, mitogen-activated protein kinase; MEF, murine embryonic fibroblasts; TIR, Toll-interleukin-1 receptor-resistance domain; TNF, tumor necrosis factor; TRAF6, tumor necrosis factor-associated factor 6; VV, vaccinia virus; TAK1, transforming growth factor-β activated kinase 1; NFκB, nuclear factor κB. family are critical in initiating an appropriate innate immune response to infectious agents, and in directing the later adaptive response. To date, 13 members of the TLR family have been identified in mammals. The TLRs belong to a superfamily that includes the interleukin 1 (IL-1) receptors. This family share significant homology in their cytoplasmic regions, which is defined by the presence of a Toll-IL-1 receptor-resistance domain (TIR) (1Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6751) Google Scholar). Similar to the IL-1R, engagement of the TLRs with their ligands leads to activation of several intracellular signal transduction pathways, culminating in the induction of proinflammatory cytokines such as IL-1 and tumor necrosis factor (TNF), of chemokines such as IL-8 and RANTES (2Yamamoto M. Takeda K. Akira S. Mol. Immunol. 2004; 40: 861-868Crossref PubMed Scopus (304) Google Scholar), and of the immunoregulatory cytokine IL-10 (3Moore K.W. de Waal Malefyt R. Coffman R.L. O'Garra A. Annu. Rev. Immunol. 2001; 19: 683-765Crossref PubMed Scopus (5364) Google Scholar). Among the most prominent and best characterized of these intracellular signaling pathways are those leading to the activation of mitogen-activated protein (MAP) kinases and the transcription factor NFκB. Triggering of the IL-1R or of TLRs causes TIR adaptor molecules to be recruited to the receptor complex such as MyD88 (4Janssens S. Beyaert R. Trends Biochem. Sci. 2002; 27: 474-482Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and TIR domain containing adaptor inducing interferon-β (TRIF) (5Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1025) Google Scholar, 6Hoebe K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar). Subsequently, the IL-1 receptor-associated kinases (IRAKs) such as IRAK1, IRAK2, and IRAK4 are activated, which then engage with TRAF6, ultimately activating the IκB kinase complex. This complex phosphorylates the inhibitory molecule IκB, which leads to NFκB entering the nucleus and inducing target gene expression (7Akira S. Yamamoto M. Takeda K. Biochem. Soc. Trans. 2003; 31: 637-642Crossref PubMed Scopus (168) Google Scholar). Activation of TRAF6 also results in the activation of TAK1 and subsequent activation of MAP kinases (p42/p44, p38, and JNK). The TLR family is now known to be important in sensing and responding to viruses. Double-stranded RNA is a molecular pattern associated with viral infection, and TLR3 has been shown to sensitize cells to activation by poly(I:C) a synthetic double-stranded RNA analogue (8Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar). Other TLRs involved in sensing viral infection include TLR7 and TLR8, which detect single-stranded RNA from influenza, human immunodeficiency virus, and vesicular stomatitis virus (9Diebold S.S. Kaisho T. Hemmi H. Akira S. Reis e Sousa C. Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2757) Google Scholar, 10Lund J.M. Alexopoulou L. Sato A. Karow M. Adams N.C. 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A. 2000; 97: 10162-10167Crossref PubMed Scopus (391) Google Scholar, 14DiPerna G. Stack J. Bowie A.G. Boyd A. Kotwal G. Zhang Z. Arvikar S. Latz E. Fitzgerald K.A. Marshall W.L. J. Biol. Chem. 2004; 279: 36570-36578Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar, 16Stack J. Haga I.R. Schroder M. Bartlett N.W. Maloney G. Reading P.C. Fitzgerald K.A. Smith G.L. Bowie A.G. J. Exp. Med. 2005; 201: 1007-1018Crossref PubMed Scopus (307) Google Scholar). The VV genome contains numerous genes encoding proteins involved in immunomodulation and immunoevasion. For example, the virus encodes proteins that act as decoy receptors for IL-1, IL-18, and TNF (17Tortorella D. Gewurz B.E. Furman M.H. Schust D.J. Ploegh H.L. Annu. Rev. Immunol. 2000; 18: 861-926Crossref PubMed Scopus (710) Google Scholar). VV is a member of the Poxviridae, a family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells. The most notorious member, variola virus, causes smallpox. This disease was eradicated using prophylactic inoculations with the antigenically related VV (18Moss B. Knipe D.M. Griffin D.E. Chanock R.M. Lamb R.A. Lamb M.A. Roizman B. Straus S.E. Fields Virology. 2. Lippincott-Raven, New York2001: 2849-2883Google Scholar). One VV protein implicated in the evasion of the host TLR response is A52R. A role for A52R in VV virulence has been clearly established in that deletion of a52r from VV led to an attenuated virus in a murine intranasal infection (15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). A52R was shown to be capable of interacting with IRAK2 and TRAF6 and to block every IL-1R-TLR pathway to NFκB activation tested (13Bowie A. Kiss-Toth E. Symons J.A. Smith G.L. Dower S.K. O'Neill L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10162-10167Crossref PubMed Scopus (391) Google Scholar, 15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). However, the effect of A52R on signals other than NFκB has not been determined. Given that many viral proteins have multiple activities and that other intracellular effects of A52R might contribute to its role in virulence, we tested the effect of A52R on signals other than NFκB. Here we show that A52R can activate the MAP kinases p38 and JNK in a TRAF6-dependent manner. In addition, A52R leads to enhancement of the TLR-induced p38-dependent gene IL-10. In contrast, inhibition of the TLR-induced NFκB-dependent genes IL-8 and RANTES is observed. These results highlight the ability of A52R to differentially modulate TLR signaling. This ability of A52R to activate p38 and potentiate TLR-induced IL-10 may be important to its role in virulence. Expression and Reporter Plasmids—CD4-TLR4 was a kind gift from R. Medzhitov (Yale University, New Haven, CT), and TLR3 was kindly provided by D. Golenbock (University of Massachusetts Medical School, Worcester, MA). Myc-IRAK2 was a gift from M. Muzio (19Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google Scholar). The TRAF6 expression plasmids, FLAG-TRAF6 and FLAG-TRAF domain (ΔTRAF6, amino acids 289–522), were provided by Tularik Inc. (San Francisco, CA). A52R expression plasmid was previously described (13Bowie A. Kiss-Toth E. Symons J.A. Smith G.L. Dower S.K. O'Neill L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10162-10167Crossref PubMed Scopus (391) Google Scholar). ΔA52R was generated by PCR of the A52R plasmid and comprised amino acids 1–144 of the wild type protein (which is 190 amino acids in length), plus an extra 27 amino acids derived from the vector sequence. The NFκB luciferase reporter construct was a gift from R. Hofmeister (University of Regensburg, Germany). The human IL-10 promoter reporter plasmid was a kind gift from L. Ziegler-Heitbrock (20Benkhart E.M. Siedlar M. Wedel A. Werner T. Ziegler-Heitbrock H.W. J. Immunol. 2000; 165: 1612-1617Crossref PubMed Scopus (218) Google Scholar). Antibodies and Reagents—Anti-FLAG M2 monoclonal antibody and anti-Myc monoclonal antibody clone 9E10 were purchased from Sigma. Anti-A52R antibody was previously described (15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). Anti-IκBα antibody was provided by R. Hay (University of St. Andrews, Scotland). Other antibodies used were anti-TRAF6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-phospho-p38 MAP kinase (Thr180/Tyr182) antibody and anti-p38 MAP kinase antibody (both from Cell Signaling Technology, Beverly, MA), and anti-rabbit IgG antibody-F(ab)2 fragment (Abcam, Cambridge, United Kingdom). Human rIL-1α was from the National Cancer Institute (Frederick, MD). The synthetic double-stranded RNA analogue, poly(I:C), was purchased from Amersham Biosciences (Bucks, UK). Lipopolysaccharide from Escherichia coli serotype EH100(Ra) and the p38 MAP kinase inhibitor SB203580 were purchased from Alexis Biochemicals (Bingham, Nottingham, UK). Reporter Gene Assays—HEK 293 cells (2 × 104 cells per well) were seeded into 96-well plates and transfected 24 h later with expression vectors and the indicated luciferase reporter genes using GeneJuice™ (Novagen) according to the manufacturer's instructions. In all cases, 20 ng/well of phRL-TK reporter gene (Promega) was co-transfected to normalize the data for transfection efficiency. The total amount of DNA per transfection was kept constant at 230 ng by addition of pcDNA3.1 (Stratagene). For NFκB assays, 60 ng of a NFκB luciferase reporter gene was used (15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). For IL-10 promoter assays, 60 ng of the IL-10 promoter luciferase reporter gene was used. For MAP kinase reporter assays the PathDetect System™ (Stratagene) was used, whereby either 0.25 ng of a c-Jun-Gal4 (to assay JNK) or a CHOP-Gal4 (to assay p38 MAP kinase) fusion vector was used in combination with 60 ng of pFR-luciferase reporter. Cells were stimulated with 100 ng/ml IL-1, 1 μg/ml LPS, or 25 μg/ml poly(I:C), where indicated 6 h prior to harvesting. After 24 h, reporter gene activity was measured (15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). Data are expressed as mean -fold induction ± S.D. relative to control levels, for a representative experiment from a minimum of three separate experiments, each preformed in triplicate. Immunoprecipitation and Immunoblotting—HEK 293T cells (1.5 × 106) were seeded into 10-cm dishes 24 h prior to transfection. Transfections were carried out using GeneJuice™. For coimmunoprecipitations, 4 μg of each construct were transfected. Where only one construct was transfected the total amount of DNA (8 μg) was kept constant by supplementation with pcDNA3.1. Cells were harvested 24 h post-transfection, washed twice in phosphate-buffered saline, and lysed in 850 μl of lysis buffer (50 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm EDTA, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40 containing 1 mm phenylmethylsulfonyl fluoride, 0.01% (v/v) aprotinin, and 1 mm sodium orthovanadate). For immunoprecipitation, the indicated antibodies were precoupled to either protein A-Sepharose (polyclonal and FLAG monoclonal antibodies) or protein G-Sepharose beads (all other monoclonal antibodies) overnight at 4 °C. The beads were then washed twice in lysis buffer and incubated with the cell lysates overnight at 4 °C. The immune complexes were washed, boiled with 30 μl of 3× sample buffer (62.5 mm Tris, 2% (w/v) SDS, 10% v/v glycerol, 0.1% (w/v) bromphenol blue), and analyzed using standard SDS-PAGE and Western blotting techniques. For analysis of p38 MAP kinase activation by Western blot, a specific antibody raised against phosphorylated p38 (Thr180/Tyr182) was employed. Total levels of p38 MAP kinase protein were also analyzed using anti-p38 MAP kinase antibody. HEK 293 cells (1 × 105 cells per well) were seeded into 6-well plates and transfected 24 h later with ΔTRAF6 or A52R encoding plasmids as indicated, using GeneJuice™. The total amount of DNA (2.3 μg) was kept constant by supplementation with pcDNA3.1. 24 h after transfection, cells were lysed in 100 μl of SDS sample buffer (62.5 mm Tris (pH 6.8), 2% (w/v) SDS, 50 mm dithiothreitol, 10% glycerol, 0.1% bromphenol blue). Lysates were then resolved by SDS-PAGE, transferred to poly(vinylidene difluoride) membranes, and probed with the indicated antibodies according to the manufacturer's instructions. Determination of Cytokine Concentrations—HEK 293 clonal cell lines stably expressing either TLR4/MD-2 (HEK-TLR4) or TLR3 (HEK-TLR3) (21Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (939) Google Scholar) or the murine macrophage cell line RAW 264.7 were used for determination of cytokine production. Cells (2 × 104 cell per well) were seeded into 96-well plates and transfected 24 h later with an expression plasmid encoding A52R, TRAF6, or ΔTRAF6 using GeneJuice™ where indicated. 24 h after transfection, cells were stimulated with 1 μg/ml LPS or 25 μg/ml poly(I:C). 2 h prior to stimulation the p38 MAP kinase inhibitor SB203580 was added were indicated. After 24 h supernatants were harvested, and IL-8, RANTES, or IL-10 concentrations were determined by enzyme-linked immunosorbent assay (R&D Biosystems). Experiments were performed three times in triplicate, and data are expressed as mean ± S.D. from one representative experiment. A52R Drives p38 MAP Kinase Activation—We previously showed that deletion of the vaccinia virus a52r gene reduced virus virulence in a murine intranasal infection model, that A52R acts as a potent inhibitor of NFκB activation induced by IL-1 and various TLRs, and that it was capable of interacting with both TRAF6 and IRAK2 (15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). However, the effect of A52R on signals other than NFκB was not determined and thus there might be other functions of A52R that also contribute to virulence. Therefore, here we examined the effect of A52R on p38 MAP kinase activation. For this we used the Stratagene Path-Detect™ System that is based on the ability of p38 MAP kinase to phosphorylate and activate the transcription factor CHOP. This is assayed by an increase in the ability of the Gal4-CHOP fusion protein to transactivate the pFR luciferase reporter, which contains Gal4 binding sites in its promoter. Fig. 1A shows that treatment of cells with IL-1, ectopic expression of CD4-TLR4, or ectopic expression of TLR3 together with poly(I:C) stimulation led to activation of p38 MAP kinase. Surprisingly, ectopic expression of increasing amounts of a plasmid encoding A52R enhanced IL-1-, CD4-TLR4-, and TLR3-mediated p38 MAP kinase activation (Fig. 1A). This was in contrast to the inhibitory effect of A52R on IL-1/TLR-induced NFκB activation (Fig. 1B and Refs. 13Bowie A. Kiss-Toth E. Symons J.A. Smith G.L. Dower S.K. O'Neill L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10162-10167Crossref PubMed Scopus (391) Google Scholar and 15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). In fact, expression of A52R alone in unstimulated cells led to both p38 MAP kinase and JNK activation (Fig. 1C), whereas A52R alone had no effect on basal levels of NFκB activity (not shown). Thus, A52R has opposite effects on IL-1/TLR-induced NFκB and MAP kinase activation and can in fact activate MAP kinases in the absence of any other stimulus. Activation of p38 by A52R Requires Interaction with TRAF6— Activation of p38 by A52R could conceivably be because of its ability to interact with either IRAK2 or TRAF6 (15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). To determine whether this was the case, we began to generate truncated versions of A52R to map the sites of interaction between A52R and TRAF6 and IRAK2. A truncated version of A52R lacking 46 amino acids at the C-terminal was constructed (Fig. 2A). This truncated A52R protein, here termed ΔA52R, was detectable by the anti-A52R antibody and was expressed at similar levels to A52R (Fig. 2, B and C). To determine whether ΔA52R was still capable of interacting with TRAF6 and/or IRAK2, co-immunoprecipitations were carried out. ΔA52R was unable to form a complex with TRAF6 but retained its ability to interact with IRAK2. A co-immunoprecipitation with anti-TRAF6 antibody pulled down A52R with both endogenous TRAF6 (Fig. 2B, top panel, lane 1) and overexpressed TRAF6 (Fig. 2B, top panel, lane 3). In contrast, under the same conditions ΔA52R was not detected in complex with either endogenous or overexpressed TRAF6 (Fig. 2B, top panel, lanes 4 and 6). However, a clear interaction between ΔA52R and IRAK2 was observed (Fig. 2C). The ability of ΔA52R and A52R to form a complex with IRAK2 appeared equal (Fig. 2C, top panels, compare lanes 3 and 6). Next, the ability of ΔA52R to induce MAP kinase activation was tested. Fig. 2D shows that in contrast to A52R, ΔA52R failed to drive p38 and JNK activation. Thus, the failure of ΔA52R to activate p38 and JNK correlated with its inability to interact with TRAF6, suggesting a role for TRAF6 in the stimulatory effect of A52R on MAP kinases. A52R Activates p38 via the TRAF Domain of TRAF6 —We next sought to examine more closely how the interaction between A52R and TRAF6 was responsible for the p38 MAP kinase activation. Fig. 3A shows that consistent with previous work (15Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar), A52R interacted with the TRAF domain of TRAF6 (amino acids 289–522). The TRAF domain of TRAF6, here termed ΔTRAF6, has been reported to act as a dominant negative, inhibiting IL-1-induced p38 and JNK activation (22Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Crossref PubMed Scopus (409) Google Scholar). We therefore hypothesized that if the A52R-TRAF domain interaction was important for A52R-induced p38 activation, overexpression of ΔTRAF6 would sequester A52R and prevent p38 MAP kinase activation. To test this we examined p38 activation by Western blot analysis in HEK 293 cells using a phospho-specific p38 antibody. Fig. 3B shows that transfection of cells with ΔTRAF6 alone had no effect on levels of phospho-p38 (upper panel, lane 2). Consistent with the reporter gene based assay (Fig. 1), overexpression of A52R led to an increase in phospho-p38 (Fig. 3B, upper panel, compare lane 3 to lane 1), whereas ΔA52R expression had no effect on phospho-p38 (not shown). Co-expression of ΔTRAF6 with A52R completely inhibited A52R-mediated p38 activation (Fig. 3B, upper panel, lane 4). As a control, A52R was shown to have no effect on IκBα protein levels (Fig. 3B, third panel), and similarly did not lead to an increase in IκBα phosphorylation (not shown). Inhibition of p38 MAP kinase activation by overexpression of ΔTRAF6 was likely because of ΔTRAF6 sequestering A52R and preventing its interaction with endogenous TRAF6. Fig. 3C shows this to be the case because the presence of overexpressed ΔTRAF6 inhibited the interaction of A52R with endogenous TRAF6 (compare lanes 4 and 2). Thus A52R activates p38 by engaging the TRAF domain of TRAF6. A52R Enhances TLR-induced IL-10 Induction While Inhibiting NFκB-dependent Genes—Previous studies have shown that LPS-induced IL-10 production is p38 dependent (23Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar, 24Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Therefore, we wondered whether A52R would have an effect on IL-10 induction. We first examined the effect of A52R on the IL-10 promoter using a reporter gene assay. Interestingly, A52R was capable of driving the IL-10 promoter in a dose-dependent manner (Fig. 4A). In contrast, A52R expression did not affect the basal activity of a range of NFκB-dependent promoters, including IL-8, RANTES, and interferon-β (not shown). To examine whether the induction of the IL-10 promoter was TRAF6-dependent, we next assessed the ability of ΔTRAF6 to inhibit A52R-mediated IL-10 induction. Fig. 4B shows that a single dose of ΔTRAF6 was capable of negating the stimulatory effect of A52R on the IL-10 promoter. In addition, A52R failed to activate the IL-10 reporter in TRAF6–/– murine embryonic fibroblasts (MEFs). In the absence of TRAF6, the ability of A52R to drive the IL-10 promoter was abolished compared with a 3-fold induction in normal MEFs (Fig. 4C), thus implicating TRAF6 in the activation of the IL-10 promoter by A52R. We next used the murine macrophage cell line RAW 264.7 to analyze the effect of A52R-induced IL-10 protein production. The p38 inhibitor SB203580 has previously been shown to inhibit LPS/TLR4-induced IL-10 production in human peripheral blood mononuclear cells (23Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar) and in the human monocyte cell line THP-1 (24Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Consistent with this, here SB203580 inhibited LPS-induced IL-10 production (Fig. 5A, right panel). Expression of A52R alone did not lead to IL-10 production in unstimulated cells (Fig. 5A, left panel). However, expression of A52R strongly enhanced LPS-induced IL-10 production (Fig. 5A, left panel). This is suggestive of a potent effect on TLR-induced IL-10 production given that only a small fraction (3% on average, not shown) of the RAW 264.7 cells stimulated by LPS to release IL-10 would be expected to be expressing A52R in this transient transfection system. The fact that A52R failed to drive the IL-10 promoter in TRAF6–/– MEFs suggested that the TRAF6-A52R interaction was critical for the effect of A52R on IL-10 production. This conclusion prompted us to examine the role of TRAF6 on LPS-induced IL-10 production. Consistent with the effect of A52R on LPS-induced IL-10, ectopic expression of TRAF6 enhanced (Fig. 5B, left panel), whereas ΔTRAF6 inhibited (Fig. 5B, right panel), LPS-induced IL-10 production. Next we sought to compare the effect of A52R on IL-10 induction to its effects on two NFκB-dependent genes, the chemokines IL-8 (25Jung Y.D. Fan F. McConkey D.J. Jean M.E. Liu W. Reinmuth N. Stoeltzing O. Ahmad S.A. Parikh A.A. Mukaida N. Ellis L.M. Cytokine. 2002; 18: 206-213Crossref PubMed Scopus (84) Google Scholar) and RANTES (26Genin P. Algarte M. Roof P. Lin R. Hiscott J. J. Immunol. 2000; 164: 5352-5361Crossref PubMed Scopus (189) Google Scholar). Stimulation of HEK-TLR4 cells with LPS led to IL-8 production, whereas stimulation of HEK-TLR3 cells with poly(I:C) led to RANTES production (Fig. 5C). In both cases transient transfection with A52R inhibited chemokine production in a dose-dependent manner (Fig. 5C). Thus A52R differentially modulates TLR-induced gene expression, by potentiating the p38-dependent gene IL-10, while inhibiting the NFκB-dependent genes IL-8 and RANTES.Fig. 5Differential effects of A52R on TLR-dependent gene induction. A, murine macrophage RAW 264.7 cells were transfected with 180 ng of plasmid encoding A52R, 24 h prior to stimulation with 1 μg/ml LPS (left panel). Two hours after stimulation with LPS, 10 μm SB203580 was added (right panel). Twenty-four hours after stimulation supernatants were harvested and assayed for IL-10 by enzyme-linked immunosorbent assay. Data are expressed as picograms/ml ± S.D. relative to control levels, for a representative experiment from a minimum of three separate experiments, each performed in triplicate. B, RAW 264.7 cells were transfected with 180 ng of plasmid encoding TRAF6 (left panel) or ΔTRAF 6 (right panel) 24 h prior to stimulation with 1 μg/ml LPS. Twenty-four hours after stimulation, supernatants were harvested and assayed for IL-10 by enzyme-linked immunosorbent assay. C, HEK-TLR4 (left panel) or HEK-TLR3 (right panel) cells were transfected with the indicated amounts of a plasmid encoding A52R 24 h prior to stimulation with 1 μg/ml LPS (left panel) or 25 μg/ml poly(I:C) (right panel). Twenty-four hours after stimulation, supernatants were harvested and assayed for IL-8 and RANTES by enzyme-linked immunosorbent assay. Data are expressed as picograms/ml ± S.D. relative to control levels, for a representative experiment from two experiments, each performed in triplicate.View Lar
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