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The Tumor Suppressor Cylindromatosis (CYLD) Acts as a Negative Regulator for Toll-like Receptor 2 Signaling via Negative Cross-talk with TRAF6 and TRAF7

2005; Elsevier BV; Volume: 280; Issue: 49 Linguagem: Inglês

10.1074/jbc.m509526200

ISSN

1083-351X

Autores

Hiroki Yoshida, Hirofumi Jono, Hirofumi Kai, Jian‐Dong Li,

Tópico(s)

Ocular Infections and Treatments

Resumo

Toll-like receptor 2 (TLR2) plays an important role in host defense against bacterial pathogens. Activation of TLR2 signaling not only induces the activation of innate immunity and instructs the development of the acquired immunity but also leads to the detrimental inflammatory responses in inflammatory and infectious diseases. To avoid detrimental inflammatory responses, TLR2 signaling must be tightly regulated. In contrast to the relative known positive regulation of TLR2 signaling, its negative regulation, however, is largely unknown. In addition the distal signaling components that link TLR2 to its downstream signaling pathways have yet to be further defined. In the present study we have provided direct evidence for the negative regulation of TLR2 signaling by the tumor suppressor cylindromatosis (CYLD). We showed that activation of TLR2 signaling by TLR2 ligands including peptidoglycan (PGN), MALP-2, and Pam3CSK4 induces activation of IKKs-IκBα and MKK3/6-p38 pathways not only by TRAF6 but also by TRAF7, a recently identified TRAF family member. The activation of both pathways leads to the transcription of TNF-α, IL-1β, and IL-8 as well as CYLD. CYLD in turn leads to the inhibition of TRAF6 and TRAF7 likely via a deubiquitination-dependent mechanism. The present studies thus unveil a novel autoregulatory feedback mechanism that negatively controls TLR2-IKKs-IκBα/MKK3/6-p38-NF-κB-dependent induction of immune and inflammatory responses via negatively cross-talking with both TRAF6 and TRAF7. These findings provide novel insights into autoregulation and negative regulation of TLR signaling. Toll-like receptor 2 (TLR2) plays an important role in host defense against bacterial pathogens. Activation of TLR2 signaling not only induces the activation of innate immunity and instructs the development of the acquired immunity but also leads to the detrimental inflammatory responses in inflammatory and infectious diseases. To avoid detrimental inflammatory responses, TLR2 signaling must be tightly regulated. In contrast to the relative known positive regulation of TLR2 signaling, its negative regulation, however, is largely unknown. In addition the distal signaling components that link TLR2 to its downstream signaling pathways have yet to be further defined. In the present study we have provided direct evidence for the negative regulation of TLR2 signaling by the tumor suppressor cylindromatosis (CYLD). We showed that activation of TLR2 signaling by TLR2 ligands including peptidoglycan (PGN), MALP-2, and Pam3CSK4 induces activation of IKKs-IκBα and MKK3/6-p38 pathways not only by TRAF6 but also by TRAF7, a recently identified TRAF family member. The activation of both pathways leads to the transcription of TNF-α, IL-1β, and IL-8 as well as CYLD. CYLD in turn leads to the inhibition of TRAF6 and TRAF7 likely via a deubiquitination-dependent mechanism. The present studies thus unveil a novel autoregulatory feedback mechanism that negatively controls TLR2-IKKs-IκBα/MKK3/6-p38-NF-κB-dependent induction of immune and inflammatory responses via negatively cross-talking with both TRAF6 and TRAF7. These findings provide novel insights into autoregulation and negative regulation of TLR signaling. Toll-like receptors (TLRs) 2The abbreviations used are: TLRToll-like receptorCYLDcylindromatosisILinterleukinMAPKmitogen-activated protein kinaseMKKMAPK kinasePGNpeptidoglycansiRNAsmall interfering RNATNFtumor necrosis factorTRAFthe tumor necrosis factor receptor-associated factorCAPEcaffeic acid phenethyl esterQ-PCRreal-time quantitative reverse transcription-PCRPBSphosphate-buffered salineWTwild-typeHAhemagglutinin. play critical roles in host defense against invading pathogens by recognizing microbial components and activating complex signaling networks that in turn induce distinct patterns of gene expression, which not only lead to the activation of innate immunity but also instructs the development of antigen-specific acquired immunity (1Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar, 2Dunne A. O'Neill L.A. Science's STKE. 2003; 2003: re3Crossref PubMed Scopus (514) Google Scholar, 3Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1261) Google Scholar, 4Liew F.Y. Xu D. Brint E.K. O'Neill L.A. Nat. Rev. Immunol. 2005; 5: 446-458Crossref PubMed Scopus (1257) Google Scholar). However, TLRs have also been shown to be involved in the pathogenesis of autoimmune, chronic inflammatory, and infectious diseases (4Liew F.Y. Xu D. Brint E.K. O'Neill L.A. Nat. Rev. Immunol. 2005; 5: 446-458Crossref PubMed Scopus (1257) Google Scholar). Therefore, to avoid detrimental and overactive inflammatory responses, TLR signaling must be tightly regulated. In contrast to the known positive regulation of TLR signaling, the negative regulation of TLR signaling remains largely unknown (4Liew F.Y. Xu D. Brint E.K. O'Neill L.A. Nat. Rev. Immunol. 2005; 5: 446-458Crossref PubMed Scopus (1257) Google Scholar, 5Jono H. Lim J.H. Chen L.F. Xu H. Trompouki E. Pan Z.K. Mosialos G. Li J.D. J. Biol. Chem. 2004; 279: 36171-36174Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Mikami F. Gu H. Jono H. Andalibi A. Kai H. Li J.D. J. Biol. Chem. 2005; 280: 36185-36194Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). TLRs are type I transmembrane receptors with leucine-rich repeats in the extracellular domains and cytoplasmic domains that resemble the mammalian IL-1 receptor (IL-1R) (1Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar, 2Dunne A. O'Neill L.A. Science's STKE. 2003; 2003: re3Crossref PubMed Scopus (514) Google Scholar, 3Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1261) Google Scholar). To date, 11 members of the human TLR family have been cloned (1Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar, 2Dunne A. O'Neill L.A. Science's STKE. 2003; 2003: re3Crossref PubMed Scopus (514) Google Scholar, 3Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1261) Google Scholar). Of these, TLR2 and TLR4 have been well studied. While TLR4 seems to be mainly involved in Gram-negative bacteria lipopolysaccharide signaling, TLR2 can respond to a variety of bacterial products, including peptidoglycan (PGN), lipoprotein, lipoteichoic acid, and lipoarabinomannan (1Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar, 2Dunne A. O'Neill L.A. Science's STKE. 2003; 2003: re3Crossref PubMed Scopus (514) Google Scholar, 3Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1261) Google Scholar, 4Liew F.Y. Xu D. Brint E.K. O'Neill L.A. Nat. Rev. Immunol. 2005; 5: 446-458Crossref PubMed Scopus (1257) Google Scholar, 5Jono H. Lim J.H. Chen L.F. Xu H. Trompouki E. Pan Z.K. Mosialos G. Li J.D. J. Biol. Chem. 2004; 279: 36171-36174Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Mikami F. Gu H. Jono H. Andalibi A. Kai H. Li J.D. J. Biol. Chem. 2005; 280: 36185-36194Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7Wetzler L.M. Vaccine. 2003; 21: S55-S60Crossref PubMed Scopus (126) Google Scholar, 8Dziarski R. Gupta D. Infect. Immun. 2005; 73: 5212-5216Crossref PubMed Scopus (195) Google Scholar). The importance of TLR2 in host defense was further highlighted by the studies from knock-out mice showing decreased survival of TLR2-deficient mice after infection with Gram-positive Staphylococcus aureus (9Takeuchi O. Hoshino K. Akira S. J. Immunol. 2000; 165: 5392-5396Crossref PubMed Scopus (909) Google Scholar). Furthermore, our recent study demonstrated that TLR2 also plays a key role in activating host immune and inflammatory response by surface lipoprotein from the Gram-negative bacterium nontypeable Haemophilus influenzae, a major cause of otitis media and exacerbation of chronic obstructive pulmonary diseases (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T.F. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Jono H. Shuto T. Xu H. Kai H. Lim D.J. Gum Jr., J.R. Kim Y.S. Yamaoka S. Feng X.H. Li J.D. J. Biol. Chem. 2002; 277: 45547-45557Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Li J.D. J. Pharmacol. Sci. 2003; 91: 1-7Crossref PubMed Scopus (46) Google Scholar, 13Jono H. Xu H. Kai H. Lim D.J. Kim Y.S. Feng X.H. Li J.D. J. Biol. Chem. 2003; 278: 27811-27819Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Chen R. Lim J.H. Jono H. Gu X.X. Kim Y.S. Basbaum C.B. Murphy T.F. Li J.D. Biochem. Biophys. Res. Commun. 2004; 324: 1087-1094Crossref PubMed Scopus (118) Google Scholar). Thus, it is clear that TLR2 plays a crucial role in host defense against both Gram-positive and -negative bacteria. Similar to other TLRs, the negative regulation of TLR2 signaling also remains largely unclear. Despite recent studies showing that the tumor suppressor CYLD acts as a negative regulator for TNF receptor-induced activation of NF-κB and JNK via inhibition of the tumor necrosis factors receptor-associated factor 2 (TRAF2) (15Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (803) Google Scholar, 16Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (832) Google Scholar, 17Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. Nature. 2003; 424: 801-805Crossref PubMed Scopus (852) Google Scholar, 18Regamey A. Hohl D. Liu J.W. Roger T. Kogerman P. Toftgard R. Huber M. J. Exp. Med. 2003; 198: 1959-1964Crossref PubMed Scopus (101) Google Scholar, 19Reiley W. Zhang M. Sun S.C. J. Biol. Chem. 2004; 279: 55161-55167Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), its role in negatively regulation of TLR2 signaling has yet to be determined. Toll-like receptor cylindromatosis interleukin mitogen-activated protein kinase MAPK kinase peptidoglycan small interfering RNA tumor necrosis factor the tumor necrosis factor receptor-associated factor caffeic acid phenethyl ester real-time quantitative reverse transcription-PCR phosphate-buffered saline wild-type hemagglutinin. Although various proximal signaling components of TLR2 have been relatively well studied, the distal signaling components that link TLR2 to its downstream signaling pathways including NF-κB and MAPKs remain largely unknown (1Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar, 2Dunne A. O'Neill L.A. Science's STKE. 2003; 2003: re3Crossref PubMed Scopus (514) Google Scholar, 3Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1261) Google Scholar, 4Liew F.Y. Xu D. Brint E.K. O'Neill L.A. Nat. Rev. Immunol. 2005; 5: 446-458Crossref PubMed Scopus (1257) Google Scholar, 7Wetzler L.M. Vaccine. 2003; 21: S55-S60Crossref PubMed Scopus (126) Google Scholar). Among all known key signaling transducers downstream of TLR2, TRAF6 has been shown to be critically involved in activation of both NF-κB and p38 triggered by TLR family members (20Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1513) Google Scholar, 21Inoue J. Ishida T. Tsukamoto N. Kobayashi N. Naito A. Azuma S. Yamamoto T. Exp. Cell Res. 2000; 254: 14-24Crossref PubMed Scopus (371) Google Scholar, 22Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1257) Google Scholar, 23Ben-Neriah Y. Nat. Immunol. 2002; 3: 20-26Crossref PubMed Scopus (344) Google Scholar, 24Habelhah H. Takahashi S. Cho S.G. Kadoya T. Watanabe T. Ronai Z. EMBO J. 2004; 23: 322-332Crossref PubMed Scopus (189) Google Scholar). In addition to TRAF6, TRAF7 has been recently identified as a signaling transducer upstream of MEKK3-AP1 pathway (25Xu L.G. Li L.Y. Shu H.B. J. Biol. Chem. 2004; 279: 17278-17282Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 26Bouwmeester T. Bauch A. Ruffner H. Angrand P.O. Bergamini G. Croughton K. Cruciat C. Eberhard D. Gagneur J. Ghidelli S. Hopf C. Huhse B. Mangano R. Michon A.M. Schirle M. Schlegl J. Schwab M. Stein M.A. Bauer A. Casari G. Drewes G. Gavin A.C. Jackson D.B. Joberty G. Neubauer G. Rick J. Kuster B. Superti-Furga G. Nat. Cell Biol. 2004; 6: 97-105Crossref PubMed Scopus (873) Google Scholar). Its role in mediating TLR-dependent activation of both NF-κB and p38 pathways still remains unclear. In the present study, we provided evidence for the first time that activation of TLR2 signaling by TLR2 ligands induces activation of IKKs-IκBα and MKK3/6-p38 signaling pathways not only by TRAF6 but also by TRAF7. The activation of both IKKs-IκBα and MKK3/6-p38 pathways will induce transcription of TNF-α, IL-1β, and IL-8 as well as CYLD. CYLD in turn leads to the inhibition of TRAF6 and TRAF7 likely via a deubiquitination-dependent mechanism. The present studies thus identified a novel autoregulatory feedback mechanism that negatively controls TLR2-IKKs-IκBα/MKK3/6-p38-NF-κB-dependent induction of immune and inflammatory responses via negatively cross-talking with both TRAF6 and TRAF7. These findings provide novel insights into autoregulation and negative regulation of TLR signaling. Reagents and Plasmids—Caffeic acid phenethyl ester (CAPE), MG-132, and SB203580 were purchased from Calbiochem (La Jolla, CA). PGN from S. aureus, Pam3CSK4 and R837 (Imiquimod) were purchased from InvivoGen (San Diego, CA). MALP-2 was purchased from ALEXIS Biochemicals (San Diego, CA). The plasmids IκBα (S32/36A), IKKα (K44M), IKKβ (K49A), NEMO DN, fp38α(AF), fp38β2 (AF), MKK3β(A), MKK6β(A), TRAF2 DN, TRAF6, TRAF6 DN, HA-TRAF7, HA-TRAF7 DN, HA-CYLD, FLAG-CYLD, and NF-κB-luciferase reporter were described previously (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T.F. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Jono H. Shuto T. Xu H. Kai H. Lim D.J. Gum Jr., J.R. Kim Y.S. Yamaoka S. Feng X.H. Li J.D. J. Biol. Chem. 2002; 277: 45547-45557Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Li J.D. J. Pharmacol. Sci. 2003; 91: 1-7Crossref PubMed Scopus (46) Google Scholar, 13Jono H. Xu H. Kai H. Lim D.J. Kim Y.S. Feng X.H. Li J.D. J. Biol. Chem. 2003; 278: 27811-27819Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Chen R. Lim J.H. Jono H. Gu X.X. Kim Y.S. Basbaum C.B. Murphy T.F. Li J.D. Biochem. Biophys. Res. Commun. 2004; 324: 1087-1094Crossref PubMed Scopus (118) Google Scholar, 15Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (803) Google Scholar, 25Xu L.G. Li L.Y. Shu H.B. J. Biol. Chem. 2004; 279: 17278-17282Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 27Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar, 28Latz E. Visintin A. Lien E. Fitzgerald K.A. Monks B.G. Kurt-Jones E.A. Golenbock D.T. Espevik T. J. Biol. Chem. 2002; 277: 47834-47843Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Cell Culture—Human lung epithelial cell line A549 and human cervix epithelial cell line HeLa were maintained as described (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T.F. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 33Shuto T. Imasato A. Jono H. Sakai A. Xu H. Watanabe T. Rixter D.D. Kai H. Andalibi A. Linthicum F. Guan Y.L. Han J. Cato A.C. Lim D.J. Akira S. Li J.D. J. Biol. Chem. 2002; 277: 17263-17270Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 34Imasato A. Desbois-Mouthon C. Han J. Kai H. Cato A.C. Akira S. Li J.D. J. Biol. Chem. 2002; 277: 47444-47450Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Stable cell lines of HEK293-pcDNA, HEK293-TLR2, and HEK293-TLR4 were kindly provided by Dr. Douglas T. Golenbock (28Latz E. Visintin A. Lien E. Fitzgerald K.A. Monks B.G. Kurt-Jones E.A. Golenbock D.T. Espevik T. J. Biol. Chem. 2002; 277: 47834-47843Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 29Latz E. Franko J. Golenbock D.T. Schreiber J.R. J. Immunol. 2004; 172: 2431-2438Crossref PubMed Scopus (112) Google Scholar). All stable cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen), 0.5 mg/ml G418, and 10 μg/ml ciprofloxacin (Cellgro, Herndon, VA) in a 5% saturated CO2 atmosphere at 37 °C. Transfection and Luciferase Assay—Cells were cultured on 24-well plates. After 24 h, cells were co-transfected with NF-κB-luciferase reporter plasmid and various expression plasmids as indicated in the legends to Figs. 1, 2, 3, 4, 5. Empty vector was used as a control and was added where necessary to ensure a constant amount of input DNA. All transient transfections were carried out in triplicate using a TransIT-LT1 reagent (Mirus, Madison, WI) following the manufacturer's instructions. At 40 h after the start of transfection, cells were pretreated with or without chemical inhibitors including 10 μg/ml CAPE, 10 μm MG-132, and 1 μm SB203580 for 1 h. PGN (5 μg/ml), MALP-2 (10 ng/ml), Pam3CSK4 (1 μg/ml), or R837 (10 μg/ml) were then added to the cells for 5 h before cell lysis for luciferase assay. Luciferase activity was normalized with respect to β-galactosidase activity.FIGURE 2The tumor suppressor CYLD negatively regulates TLR2-dependent activation of both NF-κB and p38 MAPK pathways. A, overexpression of WT CYLD inhibited PGN-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. B, CYLD knockdown by siRNA-CYLD markedly reduced both exogenous and endogenous expression of CYLD and enhanced PGN-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. C, CYLD knockdown by siRNA-CYLD enhanced MALP-2- and Pam3CSK4-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. D, overexpressing WT CYLD inhibited, whereas CYLD knockdown by siRNA-CYLD enhanced, PGN-induced phosphorylation of IκBα, MKK3/6, and p38 in HEK293-TLR2 cells. E, overexpressing WT CYLD inhibited, whereas CYLD knockdown by siRNA-CYLD enhanced, PGN-induced TNF-α, IL-1β, and IL-8 expression at mRNA level in HEK293-TLR2 cells. Values are the means ± S.D. (n = 3). CON, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Both TRAF6 and TRAF7 are required for mediating TLR2-dependent activation of NF-κB and p38 MAPK pathways. A, overexpression of dominant-negative mutant forms of TRAF6 and TRAF7 but not TRAF2 inhibited PGN-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. B, overexpression of a dominant-negative mutant form of TRAF7 inhibited MALP-2- and Pam3CSK4-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. C, TRAF7 knockdown by siRNA-TRAF7 reduced exogenous expression of TRAF7 in HEK293-TLR2 cells. D, TRAF7 knockdown by siRNA-TRAF7 reduced endogenous expression of TRAF7 at mRNA level in HEK293-TLR2 cells. E, TRAF7 knockdown by siRNA-TRAF7 inhibited PGN-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. F, TRAF7 knockdown by siRNA-TRAF7 inhibited MALP-2- and Pam3CSK4-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. G, TRAF7 DN and siRNA-TRAF7 did not affect IKKβ-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. H, TRAF7 DN and siRNA-TRAF7 inhibited R837-induced NF-κB-dependent promoter activity in A549 cell line. A549 cells were co-transfected with NF-κB-luciferase reporter plasmid, wild-type TLR7 and TRAF7 DN (left panel), or siRNA-TRAF7 (right panel). At 40 h after the transfection, R837 was added to the cells for 5 h before cell lysis for luciferase assay. I and J, overexpression of dominant-negative mutant forms of TRAF6 and TRAF7 inhibited PGN-induced phosphorylation of IκBα and p38 in HEK293-TLR2 cells. K, TRAF7 knockdown by siRNA-TRAF7 inhibited PGN-induced phosphorylation of IκBα and p38 in HEK293-TLR2 cells. L, overexpression of dominant-negative mutant forms of TRAF6 and TRAF7 inhibited PGN-induced TNF-α, IL-1β, and IL-8 expression at mRNA level in HEK293-TLR2 cells. Values are the means ± S.D. (n = 3). CON, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4TRAF7 synergizes with TRAF6 in mediating NF-κB activation. A, TRAF7 synergistically enhanced TRAF6-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. B, overexpression of a dominant-negative mutant form of TRAF7 inhibited TRAF6-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. C, TRAF7 knockdown by siRNA-TRAF7 inhibited TRAF6-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. D, TRAF7 interacted with TRAF6 in HEK293-TLR2 cells. Cells were co-transfected with TRAF6 and HA-TRAF7. Whole cell extracts were analyzed by immunoblotting (IB) with anti-TRAF6 or anti-HA antibodies either directly or after co-immunoprecipitation (IP) with control (Con) IgG, anti-TRAF6, or anti-HA antibodies. Values are the means ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5CYLD inhibits TLR2-dependent NF-κB activation via negative cross-talk with both TRAF6 and TRAF7 likely in a deubiquitination-dependent manner. A, CYLD knockdown by siRNA-CYLD enhanced wild-type TRAF6- and TRAF7-induced NF-κB-dependent promoter activity in HEK293-TLR2 cells. B, CYLD interacted with TRAF6 and TRAF7 in HEK293-TLR2 cells. Cells were co-transfected with HA-CYLD and TRAF6 or FLAG-CYLD and HA-TRAF7. Whole cell extracts were analyzed by immunoblotting (IB) with anti-TRAF6, anti-HA, or anti-FLAG antibodies either directly or after co-immunoprecipitation (IP) with control IgG, anti-TRAF6, anti-HA, or anti-FLAG antibodies. C, CYLD co-localized with TRAF6 and TRAF7 in HEK293-TLR2 cells. Cells were co-transfected with FLAG-CYLD and TRAF6 (upper) or FLAG-CYLD and HA-TRAF7 (lower), and their cellular localization was analyzed by staining with anti-FLAG (green), anti-TRAF6 (red), and anti-HA (red) antibody. D, PGN induced ubiquitination of TRAF6 and TRAF7. Cells were transfected with TRAF6 or HA-TRAF7. At40 h after transfection, cells were treated with PGN for the indicated times. Whole cell extracts were subjected to co-immunoprecipitatoin (IP) with control IgG, anti-TRAF6, or anti-HA antibodies and immunoblotting with anti-ubiquitin antibody. The same blots were reprobed with anti-TRAF6 or anti-HA antibodies. E, co-expressing WT CYLD inhibited, whereas CYLD knockdown by siRNA-CYLD enhanced the ubiquitination of TRAF6 and TRAF7 in HEK293-TLR2 cells. Cells were co-transfected with TRAF6, FLAG-CYLD, and siRNA-CYLD or HA-TRAF7, FLAG-CYLD, and siRNA-TRAF7. Whole cell extracts were subjected to co-immunoprecipitatoin (IP) with control IgG, anti-TRAF6, or anti-HA antibodies and immunoblotting with anti-ubiquitin antibody. The same blots were reprobed with anti-TRAF6 or anti-HA antibodies. Values are the means ± S.D. (n = 3). Con, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Real-time Quantitative Reverse Transcription-PCR (Q-PCR) Analysis—Total RNA was isolated by using TRIzol reagent (Invitrogen) by following the manufacturer's instructions. For the reverse transcription reaction, TaqMan reverse transcription reagents (Applied Biosystems) were used. Briefly, the reverse transcription reaction was performed for 60 min at 37 °C, followed by 60 min at 42 °C by using oligo(dT) and random hexamers. PCR amplification was performed by using TaqMan universal master mix for human TNF-α, IL-1β, IL-8, and CYLD, or by using SYBR Green universal master mix for human TRAF7. In brief, reactions were performed in duplicate containing 2× universal master mix, 1 μl of template cDNA, 100 nm primers, and 100 nm probe in a final volume of 12.5 μl, and they were analyzed in a 96-well optical reaction plate (Applied Biosystems). Probes for TaqMan include a fluorescent reporter dye, 6-carboxyfluorescein, on the 5′ end and labeled with a fluorescent quencher dye, 6-carboxytetramethylrhodamine, on the 3′ end to allow direct detection of the PCR product. Reactions were amplified and quantified by using as ABI 7700 sequemce detector and the manufacturer's corresponding software (Applied Biosystems). Relative quantity of mRNAs were obtained by using the comparative Ct method (for details, see User Bulletin 2 for the Applied Biosystems PRISM 7700 sequence-detection system) and was normalized by using TaqMan predeveloped assay reagent human cyclophilin as an endogenous control (Applied Biosystems). Universal master mix and TaqMan predeveloped assay reagents (primer and probe mixture of human TNF-α, IL-1β, and IL-8) were purchased from Applied Biosystems. The primers and probe for human CYLD were as follows: 5′-ACG CCA CAA TCT TCA TCA CAC T-3′ (forward primer) and 5′-AGG TCG TGG TCA AGG TTT CAC T-3′ (reverse primer); TaqMan probe, 5′-6-carboxyfluorescein-AAA AAG CTG TTT CCC TTG GTA CAC CCC C-6-carboxytetramethylrhodamine-3′. The primers for human TRAF7 were as follows: 5′-TGG AGT TCC GGC GGG-3′ (forward primer) and 5′-AGC CGC GCG TTG ATG T-3′ (reverse primer). Immunofluorescence—Cells were cultured on four-chamber slides. After treatment with PGN, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min. Fixed cells were subsequently blocked with 1.5% bovine serum albumin in PBS for 20 min and incubated with 1:250 dilution of mouse anti-p65 NF-κB antibody for 1 h (Santa Cruz Biotechnology). Primary antibody was detected with 1:200 dilution of fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology). Samples were examined and photographed by using an Axiophot microscope (Zeiss). For analyzing the co-localization of CYLD with TRAF6 and TRAF7, cells were co-transfected with the indicated combinations of TRAF6, HA-TRAF7, and FLAG-CYLD plasmids. After fixing, permeabilizing, and blocking, the cells were incubated with rabbit anti-TRAF6 antibody (Santa Cruz Biotechnology), rabbit anti-HA antibody (Santa Cruz Biotechnology), or mouse anti-FLAG antibody (Sigma). Primary antibodies were detected with fluorescein isothiocyanate-conjugated anti-mouse (Santa Cruz Biotechnology) or rhodamine-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology). Small Interfering RNA (siRNA)—RNA-mediated interference for down-regulating CYLD expression was done using small interfering siRNA-CYLD (pSUPER-CYLD) as described previously (5Jono H. Lim J.H. Chen L.F. Xu H. Trompouki E. Pan Z.K. Mosialos G. Li J.D. J. Biol. Chem. 2004; 279: 36171-36174Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 15Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (803) Google Scholar). For down-regulating TRAF7 expression, the siRNA-TRAF7 was purchased from Dharmacon. HEK293-TLR2 cells were cultured on 48-well plates. A final concentration of 200 nm siRNA-TRAF7 was co-transfected with NF-κB-luciferase reporter plasmid into 40-50% confluent cells using Lipofectamine 2000 (Invitrogen). At 40 h after the start of transfection, cells were treated with PGN, MALP-2, Pam3CSK4, or R837 for 5 h before being harvested for Luciferase assay. In contrast, for measuring siRNA effectiveness, cells were cultured on 12-well plates before cotransfection of siRNA-TRAF7 with HA-TRAF7, and Western blot analysis was performed. For confirming the down-regulation of the endogenous TRAF7, cells were transfected with siRNA-TRAF7, and Q-PCR was performed. Western Blot Analysis and Immunoprecipitation—Antibodies against phospho-IκBα (Ser-32), phospho-p38 (Thr-180/Tyr-182), p38, and phospho-MKK3/6 (Ser-189/207) were purchased from Cell Signaling Technology (Beverly, MA); HA-probe (Y-11), and TRAF6 (H-274) were from Santa Cruz Biotechnology; TRAF6 (IMG-536) was from IMGENEX (San Diego, CA), CYLD was from ALEXIS Biochemicals (San Diego, CA), FLAG and β-actin were from Sigma. Western blots were performed as described (11Jono H. Shuto T. Xu H. Kai H. Lim D.J. Gum Jr., J.R. Kim Y.S. Yamaoka S. Feng X.H. Li J.D. J. Biol. Chem. 2002; 277: 45547-45557Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 27Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar) and following the manufacturer's instructions. Briefly, Western blots were performed using whole cell extracts, separated on 6-10% SDS-PAGE gels, and transferred to polyvinylidine difluoride membranes (Pall Life Sciences, Pensacola, FL). The membrane was blocked with a solution of PBS containing 0.1% Tween 20 (PBS-T) and 5% nonfat milk. After three washes in PBS-T, the membrane was incubated in a 1:2000 dilution of a primary antibody. After another three washes in PBS-T, the membrane was incubated with 1:2000 dilution of the corresponding secondary antibody. The membrane was react

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