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• W2108733091 abstract "Toll-like receptor 4 (TLR4) and TLR2 agonists from bacterial origin require acylated saturated fatty acids in their molecules. Previously, we reported that TLR4 activation is reciprocally modulated by saturated and polyunsaturated fatty acids in macrophages. However, it is not known whether fatty acids can modulate the activation of TLR2 or other TLRs for which respective ligands do not require acylated fatty acids. A saturated fatty acid, lauric acid, induced NFκB activation when TLR2 was co-transfected with TLR1 or TLR6 in 293T cells, but not when TLR1, 2, 3, 5, 6, or 9 was transfected individually. An n-3 polyunsaturated fatty acid (docosahexaenoic acid (DHA)) suppressed NFκB activation and cyclooxygenase-2 expression induced by the agonist for TLR2, 3, 4, 5, or 9 in a macrophage cell line (RAW264.7). Because dimerization is considered one of the potential mechanisms for the activation of TLR2 and TLR4, we determined whether the fatty acids modulate the dimerization. However, neither lauric acid nor DHA affected the heterodimerization of TLR2 with TLR6 as well as the homodimerization of TLR4 as determined by co-immunoprecipitation assays in 293T cells in which these TLRs were transiently overexpressed. Together, these results demonstrate that lauric acid activates TLR2 dimers as well as TLR4 for which respective bacterial agonists require acylated fatty acids, whereas DHA inhibits the activation of all TLRs tested. Thus, responsiveness of different cell types and tissues to saturated fatty acids would depend on the expression of TLR4 or TLR2 with either TLR1 or TLR6. These results also suggest that inflammatory responses induced by the activation of TLRs can be differentially modulated by types of dietary fatty acids. Toll-like receptor 4 (TLR4) and TLR2 agonists from bacterial origin require acylated saturated fatty acids in their molecules. Previously, we reported that TLR4 activation is reciprocally modulated by saturated and polyunsaturated fatty acids in macrophages. However, it is not known whether fatty acids can modulate the activation of TLR2 or other TLRs for which respective ligands do not require acylated fatty acids. A saturated fatty acid, lauric acid, induced NFκB activation when TLR2 was co-transfected with TLR1 or TLR6 in 293T cells, but not when TLR1, 2, 3, 5, 6, or 9 was transfected individually. An n-3 polyunsaturated fatty acid (docosahexaenoic acid (DHA)) suppressed NFκB activation and cyclooxygenase-2 expression induced by the agonist for TLR2, 3, 4, 5, or 9 in a macrophage cell line (RAW264.7). Because dimerization is considered one of the potential mechanisms for the activation of TLR2 and TLR4, we determined whether the fatty acids modulate the dimerization. However, neither lauric acid nor DHA affected the heterodimerization of TLR2 with TLR6 as well as the homodimerization of TLR4 as determined by co-immunoprecipitation assays in 293T cells in which these TLRs were transiently overexpressed. Together, these results demonstrate that lauric acid activates TLR2 dimers as well as TLR4 for which respective bacterial agonists require acylated fatty acids, whereas DHA inhibits the activation of all TLRs tested. Thus, responsiveness of different cell types and tissues to saturated fatty acids would depend on the expression of TLR4 or TLR2 with either TLR1 or TLR6. These results also suggest that inflammatory responses induced by the activation of TLRs can be differentially modulated by types of dietary fatty acids. Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptor; COX, cyclooxygenase; LPS, lipopolysaccharide; DHA, docosahexaenoic acid; NFκB, nuclear factor κB; MyD88, myeloid differentiation factor 88; IRAK-1, interleukin-1 receptor-associated kinase-1; TRAF6, tumor necrosis factor receptor-associated factor 6; IL-1, interleukin-1; TRIF, TIR domaincontaining adapter inducing IFN-β; TICAM, Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule; IFN-β, interferon β; IKK, IκB kinase; IRF3, IFN-regulatory factor 3; ISRE, interferon-stimulated regulatory element; PamCAG, palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)propyl)-Ala-Gly-OH: MALP-2, Macrophage-activating lipopeptide, 2 kDa; HA, hemagglutinin; FBS, fetal bovine serum; PGE2, prostaglandin E2; RT, reverse transcription; DN, dominant-negative; RLA, relative luciferase activity; CMV, cytomegalovirus. 1The abbreviations used are: TLR, Toll-like receptor; COX, cyclooxygenase; LPS, lipopolysaccharide; DHA, docosahexaenoic acid; NFκB, nuclear factor κB; MyD88, myeloid differentiation factor 88; IRAK-1, interleukin-1 receptor-associated kinase-1; TRAF6, tumor necrosis factor receptor-associated factor 6; IL-1, interleukin-1; TRIF, TIR domaincontaining adapter inducing IFN-β; TICAM, Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule; IFN-β, interferon β; IKK, IκB kinase; IRF3, IFN-regulatory factor 3; ISRE, interferon-stimulated regulatory element; PamCAG, palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)propyl)-Ala-Gly-OH: MALP-2, Macrophage-activating lipopeptide, 2 kDa; HA, hemagglutinin; FBS, fetal bovine serum; PGE2, prostaglandin E2; RT, reverse transcription; DN, dominant-negative; RLA, relative luciferase activity; CMV, cytomegalovirus. play a critical role in inducing innate immune responses by recognizing invading microbial pathogens (1Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4416) Google Scholar, 2Barton G.M. Medzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1054) Google Scholar, 3Medzhitov R. Janeway Jr., C. Trends Microbiol. 2000; 8: 452-456Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 4Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4731) Google Scholar). The activation of TLRs by agonists recruits an adaptor molecule, MyD88, and initiates the activation of downstream signaling cascades leading to the activation of NFκB and mitogen-activated protein kinase and the expression of inflammatory gene products, including cyclooxygenase-2 (COX-2), cytokines, and chemokines (2Barton G.M. Medzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1054) Google Scholar). Currently, eleven TLRs in mammalian cells are identified, and each TLR responds to different types of agonists: viral double-stranded RNA for TLR3, flagellin and single-stranded RNA for TLR5 and TLR7, respectively, and unmethylated CpG DNA for TLR9 (3Medzhitov R. Janeway Jr., C. Trends Microbiol. 2000; 8: 452-456Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 4Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4731) Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2617) Google Scholar, 53Zhang D. Zhang G. Hayden M.S. Greenblatt M.B. Bussey C. Flavell R.A. Ghosh S. Science. 2004; 303: 1522-1526Crossref PubMed Scopus (877) Google Scholar, 54Heil F. Hemmi H. Hochrein H. Ampenberger F. Kirschning C. Akira S. Lipford G. Wagner H. Bauer S. Science. 2004; 303: 1526-1529Crossref PubMed Scopus (3036) Google Scholar, 55Diebold S.S. Kaisho T. Hemmi H. Akira S. Reis E.S.C. Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2725) Google Scholar). TLR4 recognizes lipopolysaccharide (LPS) derived from Gram-negative bacteria (6Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6422) Google Scholar, 7Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1352) Google Scholar, 8Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). TLR4 can be also activated by non-bacterial agonists such as heat shock protein 60, fibronectin, taxol, respiratory syncytial virus coat protein, and saturated fatty acids (9Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1364) Google Scholar, 10Okamura Y. Watari M. Jerud E.S. Young D.W. Ishizaka S.T. Rose J. Chow J.C. Strauss 3rd, J.F. J. Biol. Chem. 2001; 276: 10229-10233Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 11Kurt-Jones E.A. Popova L. Kwinn L. Haynes L.M. Jones L.P. Tripp R.A. Walsh E.E. Freeman M.W. Golenbock D.T. Anderson L.J. Finberg R.W. Nat. Immunol. 2000; 1: 398-401Crossref PubMed Scopus (1339) Google Scholar, 12Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, 13Byrd-Leifer C.A. Block E.F. Takeda K. Akira S. Ding A. Eur. J. Immunol. 2001; 31: 2448-2457Crossref PubMed Scopus (235) Google Scholar). TLR2 detects a variety of microbial components such as bacterial lipopeptides, peptidoglycan, and lipoteichoic acids (4Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4731) Google Scholar). TLR2 forms a heterodimer with TLR1 or TLR6 to respond to and discriminate different types of agonists (4Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4731) Google Scholar, 14Ozinsky A. Underhill D.M. Fontenot J.D. Hajjar A.M. Smith K.D. Wilson C.B. Schroeder L. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13766-13771Crossref PubMed Scopus (1675) Google Scholar). The activation of diacylated mycoplasmal lipopeptides, macrophage-activating lipopeptide 2-kDa (MALP-2), requires TLR2 dimerized with TLR6 to induce cytokine production, whereas the activation of TLR2 by triacylated bacterial lipopeptides requires the dimerization with TLR1 (15Takeuchi O. Kawai T. Muhlradt P.F. Morr M. Radolf J.D. Zychlinsky A. Takeda K. Akira S. Int. Immunol. 2001; 13: 933-940Crossref PubMed Scopus (1006) Google Scholar, 16Takeuchi O. Sato S. Horiuchi T. Hoshino K. Takeda K. Dong Z. Modlin R.L. Akira S. J. Immunol. 2002; 169: 10-14Crossref PubMed Scopus (1075) Google Scholar). The saturated fatty acid moieties acylated in LPS and lipopeptides are critical for ligand recognition and receptor activation for TLR4 and TLR2. Deacylated LPS loses its endotoxic activity (17Munford R.S. Hall C.L. Science. 1986; 234: 203-205Crossref PubMed Scopus (195) Google Scholar, 18Kitchens R.L. Ulevitch R.J. Munford R.S. J. Exp. Med. 1992; 176: 485-494Crossref PubMed Scopus (232) Google Scholar), and the deacylated bacterial lipoproteins are unable to activate TLR2 and to induce cytokine expression in monocytes (19Brightbill H.D. Libraty D.H. Krutzik S.R. Yang R.B. Belisle J.T. Bleharski J.R. Maitland M. Norgard M.V. Plevy S.E. Smale S.T. Brennan P.J. Bloom B.R. Godowski P.J. Modlin R.L. Science. 1999; 285: 732-736Crossref PubMed Scopus (1402) Google Scholar). Furthermore, LPS-containing unsaturated fatty acids is also inactive and acts as an antagonist against native LPS (20Krauss J.H. Seydel U. Weckesser J. Mayer H. Eur. J. Biochem. 1989; 180: 519-526Crossref PubMed Scopus (77) Google Scholar, 21Qureshi N. Takayama K. Kurtz R. Infect. Immun. 1991; 59: 441-444Crossref PubMed Google Scholar). Broadly, TLR agonists can activate two different downstream signaling pathways that are MyD88-dependent and -independent, leading to differential target gene expression and cellular responses. TLR2 and TLR9 induce NFκB activation and cytokine production through MyD88-dependent signaling pathways (22Takeuchi O. Kaufmann A. Grote K. Kawai T. Hoshino K. Morr M. Muhlradt P.F. Akira S. J. Immunol. 2000; 164: 554-557Crossref PubMed Scopus (504) Google Scholar, 23Hacker H. Vabulas R.M. Takeuchi O. Hoshino K. Akira S. Wagner H. J. Exp. Med. 2000; 192: 595-600Crossref PubMed Scopus (418) Google Scholar, 24Schnare M. Holt A.C. Takeda K. Akira S. Medzhitov R. Curr. Biol. 2000; 10: 1139-1142Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The stimulation of TLR4 triggers the activation of both MyD88-dependent and -independent signaling pathways. The activation of TLR3 is known to stimulate primarily MyD88-independent signaling pathway. Toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF)/TIR-domain-containing adaptor molecule (TI-CAM)-1 is an adaptor molecule responsible for activation of MyD88-independent pathways leading to the activation of IRF3 and the expression of IFNβ (25Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2068) Google Scholar, 26Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat Immunol. 2003; 4: 161-167Crossref PubMed Scopus (1009) Google Scholar, 27Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1018) Google Scholar). Recently, it was reported that a new adaptor, TRIF-related adaptor molecule, interacts specifically with TRIF in an MyD88-independent pathway derived from the activation of TLR4, but not TLR3 (28Fitzgerald 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 (928) Google Scholar, 29Yamamoto M. Sato S. Hemmi H. Uematsu S. Hoshino K. Kaisho T. Takeuchi O. Takeda K. Akira S. Nat. Immunol. 2003; 4: 1144-1150Crossref PubMed Scopus (823) Google Scholar). Results from our previous studies demonstrated that saturated and unsaturated fatty acids reciprocally modulate MyD88-dependent signaling pathways and target gene expression, including COX-2 derived from TLR4 activation (12Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, 30Lee J.Y. Plakidas A. Lee W.H. Heikkinen A. Chanmugam P. Bray G. Hwang D.H. J. Lipid Res. 2003; 44: 479-486Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 31Lee J.Y. Ye J. Gao Z. Youn H.S. Lee W.H. Zhao L. Sizemore N. Hwang D.H. J. Biol. Chem. 2003; 278: 37041-37051Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). However, it is not known whether fatty acids also modulated MyD88-independent signaling pathways. The results from our previous studies showed that unsaturated fatty acids also suppressed NFκB activation and COX-2 expression induced by TLR2 agonist, a synthetic lipopeptide (PamCAG), in macrophages (30Lee J.Y. Plakidas A. Lee W.H. Heikkinen A. Chanmugam P. Bray G. Hwang D.H. J. Lipid Res. 2003; 44: 479-486Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). In addition, saturated fatty acid potentiated TLR2 agonist-induced NFκB activation and COX-2 expression in macrophages (30Lee J.Y. Plakidas A. Lee W.H. Heikkinen A. Chanmugam P. Bray G. Hwang D.H. J. Lipid Res. 2003; 44: 479-486Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). However, it remained to be determined whether the potentiation by the saturated fatty acid was mediated through the direct activation of TLR2. Moreover, it is not known whether fatty acids modulate the activation of other TLRs for which cognate ligands are not acylated by fatty acids. TLR4 homodimerizes whereas TLR2 heterodimerizes with TLR6 or TLR1 (14Ozinsky A. Underhill D.M. Fontenot J.D. Hajjar A.M. Smith K.D. Wilson C.B. Schroeder L. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13766-13771Crossref PubMed Scopus (1675) Google Scholar, 15Takeuchi O. Kawai T. Muhlradt P.F. Morr M. Radolf J.D. Zychlinsky A. Takeda K. Akira S. Int. Immunol. 2001; 13: 933-940Crossref PubMed Scopus (1006) Google Scholar, 16Takeuchi O. Sato S. Horiuchi T. Hoshino K. Takeda K. Dong Z. Modlin R.L. Akira S. J. Immunol. 2002; 169: 10-14Crossref PubMed Scopus (1075) Google Scholar, 32Zhang H. Tay P.N. Cao W. Li W. Lu J. FEBS Lett. 2002; 532: 171-176Crossref PubMed Scopus (94) Google Scholar). Forced dimerization of TLR4 or TLR2 can lead to the activation of the downstream signaling pathways and target gene expression (1Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4416) Google Scholar, 14Ozinsky A. Underhill D.M. Fontenot J.D. Hajjar A.M. Smith K.D. Wilson C.B. Schroeder L. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13766-13771Crossref PubMed Scopus (1675) Google Scholar, 32Zhang H. Tay P.N. Cao W. Li W. Lu J. FEBS Lett. 2002; 532: 171-176Crossref PubMed Scopus (94) Google Scholar). These results suggest that the dimerization may be one of the initial steps for the activation of TLR2 or TLR4. Therefore, in this study, we investigated whether the activation of TLRs other than TLR4 and MyD88-independent signaling pathways are modulated by fatty acids and whether the dimerization of TLR4 or TLR2 is affected by different types of fatty acids. Reagents—Sodium salts of saturated and unsaturated fatty acids were purchased from Nu-Chek (Eslyan, MN) and were dissolved in endotoxin-free water. Purified LPS was obtained from List Biological Laboratory Inc. A synthetic bacterial lipoprotein (PamCAG: palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)propyl)-Ala-Gly-OH) was purchased from Bachem (King of Prussia, PA). Poly(I:C) was purchased from Amersham Biosciences (Piscataway, NJ). Unmethylated CpG DNA (ODN2006 and ODN1668) was purchased from TIB MolBiol (Berlin, Germany). Flagellin was obtained from Calbiochem (San Diego, CA). Macrophage-activating lipopeptide, 2 kDa (MALP-2) was purchased from Alexis Biochemical (San Diego, CA). Mouse monoclonal HA-antibody was obtained from Roche Applied Science (Indianapolis, IN). Mouse monoclonal FLAG-antibody was obtained from Sigma (St. Louis, MO). All other reagents were purchased from Sigma unless otherwise described. Cell Culture—RAW264.7 cells (a murine monocytic cell line, ATCC TIB-71) and 293T (human embryonic kidney cells; provided by Sam Lee, Beth Israel Hospital, Boston, MA) were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, Intergen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). SW620 epithelial cells (ATCC CCL-227) were cultured in RPMI containing 10% FBS, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a 5% CO2/air environment. RAW264.7 cells stably transfected with murine COX-2 promoter (–3.2 kb) luciferase plasmid were prepared as described in our previous study (30Lee J.Y. Plakidas A. Lee W.H. Heikkinen A. Chanmugam P. Bray G. Hwang D.H. J. Lipid Res. 2003; 44: 479-486Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Plasmids—pDisplay-FLAG-tagged TLR6 was prepared as follows: First, pDisplay-FLAG-tagged vector was obtained by the modification of pDisplay-HA-tagged vector (Invitrogen). A HindIII/BglII fragment in pDisplay-HA-tagged vector was replaced with a PCR product containing the coding sequence for FLAG peptide (KDDDDKYD), generated by the primers as follows: forward primer, 5′-CTATAGGGAGACCCAAGCTTGG-3′; reverse primer, 5′-GCGAGATCTCTTATCGTCGTCATCCTTGTAATCGTCACCAGTGGAACCTGGAAC-3′. The PCR product was digested with HindIII and BglII and ligated into pDisplay-HAtagged vector generating pDisplay-FLAG-tagged vector. Next, PCR for mouse TLR6 was performed with the deletion of the endogenous leader sequence and the addition of BglII and XhoI sites at the two ends. The primers used for the PCR of TLR6 were as follows: forward primer, 5′-GCGAGATCTAATGAACTTGAGTCTATGGTAGAC-3′; reverse primer, 5′-GCGCTCGAGTCAAGTTTTCACATCATCCTCATTG-3′. The resulting product was ligated into BglII and SalI sites in pDisplay-FLAG-tagged vector. The integrity of the sequences was confirmed by DNA sequencing. pcDNA-HA-TLR4 and pcDNA-FLAG-TLR4 were prepared as described in our previous studies (8Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). NFκB(2×)-luciferase reporter construct was provided by Frank Mercurio (Signal Pharmaceuticals, San Diego, CA). The luciferase reporter plasmid (pGL2) containing the promoter region of the murine COX-2 gene (–3.2 kb) was a kind gift from David Dewitt (Michigan State University, East Lansing, MI). The luciferase reporter plasmid containing the promoter of inducible nitricoxide synthase was from Christopher Glass (University of California, San Diego). Heat shock protein 70 (HSP70)-β-galactosidase reporter plasmid was from Robert Modlin (University of California, Los Angeles, CA). Mouse pDisplay-HA-TLR1, 2, 4, 6, and pDisplay-HA-TLR2(P>H) were obtained from Lynn Hajjar (University of Washington). Human pEF6-TLR5 was from Andrew Gewirtz (Emory University). Human pcDNA-TLR3 and a dominant-negative mutant of tumor necrosis factor receptor-associated kinase 6 (TRAF6) (pCMV4-TRAF6-(300–524)) were provided by Ruslan Medzhitov (Yale University School of Medicine). A dominant-negative mutant form of MyD88 (MyD88(ΔDD)) was kindly provided by Jurg Tschopp (University of Lausanne, Lausanne, Switzerland). A dominant-negative IL-1 receptor-associated kinase (IRAK)-1 (pCMV4-IRAK-1-(1–211)) was a kind gift from Sankar Ghosh (Yale University School of Medicine). A dominant-negative mutant of IKK (IKK(K44M)) was obtained from Michael Karin (University of California, San Diego). A dominant-negative mutant of inhibitor κB (pCMV4-IκBα(ΔN)) was provided by Dean Ballard (Vanderbilt University, Nashville, TN). A dominant-negative mutant of AKT (pSRα-AKT-T308A/S473A) was obtained from Bing-Hua Jiang (West Virginia University). A dominant-negative mutant of TRIF (TRIF ΔNΔC) was obtained from Shizuo Akira (Osaka University, Japan). A dominant-negative mutant of IRF3 (IRF3-DBD) was obtained from Genhong Cheng (University of California, Los Angeles, CA). All DNA constructs were prepared in large scale using the EndoFree Plasmid Maxi kit (Qiagen, Chatsworth, CA) for transfection. Preparation of Bone Marrow-derived Macrophages and Measurement of Prostaglandin E2 Production—Wild type (C3H/HeOUJ) and TLR4-mutant (C3H/HeJ) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Bone marrow cells isolated from femur were cultured in Dulbecco's modified Eagle's medium containing 10% FBS, 2 mm l-glutamine, 1 mm Na+ pyruvate, 10 mm Hepes buffer, and 20% L929 cell-conditioned medium for 6 days, and adherent cells were used as macrophages. After treatment with lauric acid or docosahexaenoic acid in the absence or presence of LPS to bone marrow-derived macrophages, the levels of PGE2 in the culture medium were determined by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Real-time RT-PCR Analysis of COX-2 Expression in Mouse Bone Marrow-derived Macrophages—Bone marrow-derived macrophages from wild type (C3H/HeOUJ) and TLR4-mutant (C3H/HeJ) mice were treated with lauric acid, docosahexaenoic acid, or LPS for 4 h. Total RNAs were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instruction. Five micrograms of total RNAs were used for cDNA synthesis with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative real-time PCR was performed with a LightCycler (Roche Molecular Biochemicals) using the LightCycler FastStart DNA Master SYBR Green I kit. The primers used to detect mouse COX-2 are as follows: forward primer, 5′-ACACTCTATCACTGGCACCC-3′; reverse primer, 5′-GAAGGGACACCCCTTCACAT-3′ generating the amplified PCR product of 585 bp in length. The primers for mouse β-actin (used as an internal control) are as follows: forward primer, 5′-TCATGAAGTGTGACGTTGACATCCGT-3′; reverse primer, 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′ generating the PCR product of 285 bp in length. The following program was used: denaturation at 95 °C for 10 min and 40 cycles consisting of denaturation at 95 °C for 1 s, annealing at 60 °C for 5 s, and extension at 72 °C for 25 s. The quality and specificity of the amplified PCR products were assessed by performing a melting curve analysis and a conventional RT-PCR followed by agarose gel analysis. Samples were compared using the relative crossing-point value (Cp) method. The Cp value, which is inversely proportional to the initial template copy number, was determined by the LightCycler software program provided by the manufacturer (Roche Molecular Biochemicals). The -fold induction of COX-2 expression by real-time PCR was measured three times in duplicate relative to vehicle control and calculated after adjusting for β-actin using 2ΔΔCp, where ΔCp = Cpβ-actin – CpCOX-2, and ΔΔCp =ΔCp treatment – ΔCp control. Transient Transfection and Luciferase Assays—These were performed as described in our previous studies (8Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 12Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, 30Lee J.Y. Plakidas A. Lee W.H. Heikkinen A. Chanmugam P. Bray G. Hwang D.H. J. Lipid Res. 2003; 44: 479-486Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 31Lee J.Y. Ye J. Gao Z. Youn H.S. Lee W.H. Zhao L. Sizemore N. Hwang D.H. J. Biol. Chem. 2003; 278: 37041-37051Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Briefly, RAW264.7 or 293T cells were co-transfected with a luciferase plasmid containing NFκB(2×)-binding site, murine COX-2 promoter (–3.2 kb), or inducible nitric-oxide synthase promoter, and HSP70-β-galactosidase plasmid as an internal control using SuperFect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. SW620 cells were transfected with an IL-8 promoter-luciferase plasmid. Various expression plasmids or corresponding empty vector plasmids for signaling components were co-transfected. The total amount of transfected plasmids was equalized by supplementing with the corresponding empty vector to eliminate the experimental error from transfection itself. Luciferase and β-galactosidase enzyme activities were determined using the Luciferase Assay System and β-galactosidase Enzyme System (Promega, Madison, WI) according to the manufacturer's instructions. Luciferase activity was normalized by β-galactosidase activity to correct the transfection efficiency. Immunoprecipitation and Immunoblot Analysis—These were performed essentially the same as previously described (8Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 33Hwang D. Jang B.C. Yu G. Boudreau M. Biochem. Pharmacol. 1997; 54: 87-96Crossref PubMed Scopus (235) Google Scholar, 34Paik J.H. Ju J.H. Lee J.Y. Boudreau M.D. Hwang D.H. J. Biol. Chem. 2000; 275: 28173-28179Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Briefly, 293T cells were co-transfected with pDisplay-HA-TLR2 and pDisplay-FLAG-TLR6 (2 μg each). After 24 h, cells were washed with phosphate-buffered saline (pH 7.5) and lysed for 30 min on ice in lysis buffer (1% Nonidet P-40, 50 mm Hepes, pH 7.6, 250 mm NaCl, 10% glycerol, 1 mm EDTA, 20 mm β-glycerophosphate, 1 mm sodium orthovanadate, 1 mm sodium metabisulfite, 1 mm benzamidine hydrochloride, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 12,000 × g, 4 °C for 15 min. Supernatants were incubated with 1 μg of HA antibody (12CA5) for 4 h and further incubated with 70 μl of 50% (v/v) protein A-agarose (Amersham Biosciences, Arlington Heights, IL) for overnight at 4 °C with rocking. Immune complexes were solubilized with Laemmli sample buffer after five times of washing with lysis buffer. The samples were fractionated by 8% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with phosphate-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk and were blotted with the indicated antibodies. The reactive bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences). To reprobe with different antibodies, the membrane was stripped in the stripping buffer (35Chanmugam P. Feng L. Liou S. Jang B.C. Boudreau M. Yu G. Lee J.H. Kwon H.J. Beppu T. Yoshida M. Xia Y. Wilson C.B. Hwang D. J. Biol. Chem. 1995; 270: 5418-5426Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) at 56 °C for 1 h. Reciprocal Modulation of COX-2 Expression by Saturated and Polyunsaturated Fatty Acid in Bone Marrow-derived Macrophages Isolated from Wild-type and TLR4-mutant Mice—Results from our previous studies demonstrate that saturated fatty acid induces the activation of both endogenous and ectopically expressed TLR4 in macrophages (RAW264.7) and 293T cells, respectively, leading to NFκB activation and COX-2 expression (12Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, 30Lee J.Y. Plakidas A. Lee W.H. Heikkinen A. Chanmugam P. Bray G. Hwang D.H. J. Lipid Res. 2003; 44: 479-486Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 31Lee J.Y. Ye J. Gao Z. Youn H.S. Lee W.H. Zhao L. Sizemore N. Hwang D.H. J. Biol. Chem. 2003; 278: 37041-37051Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). To determine whether the saturated fatty acid can activate other TLRs in addition to TLR4, macrophages derived from TLR4-mutant mice (C3H/HeJ), which express non-functional TLR4 but express other wild-type TLRs, were treated with the saturated fatty acid. Macrophages were prepared by differentiating bone marrow cells isolated from wild-type (C3H/HeOUJ) and TLR4-mutant (C3H/HeJ) mice. Cells were further treated with saturated fatty acid (lauric acid, C12:0) or polyunsaturated fatty acid (docosahexaenoic acid, DHA) in the presence or absence o" @default.
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• W2108733091 title "Saturated Fatty Acid Activates but Polyunsaturated Fatty Acid Inhibits Toll-like Receptor 2 Dimerized with Toll-like Receptor 6 or 1" @default.
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• W2108733091 doi "https://doi.org/10.1074/jbc.m312990200" @default.
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