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• W1994985348 abstract "In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse ΔTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IκB-α degradation, and activation of NF-κB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-κB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes. In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse ΔTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IκB-α degradation, and activation of NF-κB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-κB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes. Activation of innate immune responses is critical for the early host defense against microbial infections and for subsequent development of adaptive immunity (1.Medzhitov R. Janeway Jr., C. N. Engl. J. Med. 2000; 343: 338-344Crossref PubMed Scopus (1721) Google Scholar, 2.Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Crossref PubMed Scopus (335) Google Scholar, 3.Iwasaki A. Medzhitov R. Nature Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3300) Google Scholar, 4.Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1351) Google Scholar). Toll-like receptors (TLRs) 4The abbreviations used are: TLR, Toll-like receptor; TIR, Toll-IL-1R resistance; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; DC, dendritic cells; STF, soluble tuberculosis factor; HEK, human embryonic kidney cells; PAMPs, pathogen-associated molecular patterns; TRIF, TIR domain containing adapter-inducing interferon; MAL, MyD88-like adapter; IRAK, IL-1R-associated kinase; IFN, interferon; Ab, antibody; p, phospho; hu, human; HA, hemagglutinin; HRP, horseradish peroxidase; WT, wild-type; LPS, lipopolysaccharide; IL, interleukin; ELISA, enzyme-linked immunosorbent assay.4The abbreviations used are: TLR, Toll-like receptor; TIR, Toll-IL-1R resistance; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; DC, dendritic cells; STF, soluble tuberculosis factor; HEK, human embryonic kidney cells; PAMPs, pathogen-associated molecular patterns; TRIF, TIR domain containing adapter-inducing interferon; MAL, MyD88-like adapter; IRAK, IL-1R-associated kinase; IFN, interferon; Ab, antibody; p, phospho; hu, human; HA, hemagglutinin; HRP, horseradish peroxidase; WT, wild-type; LPS, lipopolysaccharide; IL, interleukin; ELISA, enzyme-linked immunosorbent assay. play a central role in these processes by sensing conserved pathogen-associated molecular patterns (PAMPs) from bacteria (TLR2, TLR4, TLR5, TLR9) (4.Qureshi S.T. 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In this report, we suggest that impaired recruitment of MyD88 to TLR4 and activation of IRAK-1 in endotoxin-tolerant cells is secondary to a decrease in TLR4 tyrosine phosphorylation. Our findings support the hypothesis that tyrosine phosphorylation of the TLR4 TIR domain is required for TLR4 signaling, and is blocked in endotoxin-tolerant cells. Tyrosine-deficient TLR4 species exhibited constitutive associations with MyD88, suppressed LPS-inducible MyD88 recruitment to TLR4, increased interactions with a short isoform of IRAK-1, and impaired activation of IRAK-1 at the receptor. Furthermore, endotoxin-tolerant cells were found to show suppressed LPS-inducible TLR4 tyrosine phosphorylation, which correlates with impaired MyD88 recruitment to TLR4 and inhibited IRAK-1 activation. Moreover, the ability of protein tyrosine kinase and Src kinase inhibitors to block TLR4 tyrosine phosphorylation and signaling, coupled with the observation of LPS-inducible recruitment of Lyn kinase to TLR4 and its impairment in LPS-tolerant cells, suggest involvement of Lyn kinase in TLR4 signaling. Reagents and Cell Culture−Polyclonal antibody (Ab) to human TLR4 (H80), Abs to MyD88, IκB-α, tubulin, β-actin, and IRAK-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-phospho (p)-p38 and anti-pJNK Abs were from Promega (Madison, WI). Anti-FLAG monoclonal Ab (M2), M2-horseradish peroxidase (HRP) conjugate, and anti-hemagglutinin (HA) polyclonal Ab were obtained from Sigma, and anti-phosphotyrosine Ab PY20 was from BD Biosciences (San Jose, CA). Human HEK293T cells were maintained as described previously (64.Medvedev A.E. Vogel S.N. J. Endotoxin Res. 2003; 9: 60-64Crossref PubMed Google Scholar). Protein-free, phenol/water-extracted Escherichia coli LPS and mycobacterial soluble tuberculosis factor (STF) were prepared as described (65.McIntire F.C. Sievert H.W. Barlow G.H. Finley R.A. Lee A.Y. Biochemistry. 1967; 6: 2363-2372Crossref PubMed Scopus (182) Google Scholar, 66.Medvedev A.E. Henneke P. Schromm A. Lien E. Ingalls R. Fenton M.J. Golenbock D.T. Vogel S.N. J. Immunol. 2001; 167: 2257-2267Crossref PubMed Scopus (140) Google Scholar), and the synthetic TLR2 agonist, Pam3Cys (S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride) was purchased from EMC Microcollections GmbH (Tubingen, Germany). An HEK293 cell line stably transfected with untagged human TLR4 and FLAG-tagged human MD-2 (HEK/TLR4/MD-2) was kindly provided by Dr. Douglas Golenbock (University of Massachusetts Medical School, Worchester, MA). Protein tyrosine kinase inhibitors herbimycin A, genistein, and Src kinase inhibitors PP1 and PP2 were purchased from Calbiochem. Human monocytes were prepared by counterflow elutriation and resuspended in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 5% fetal bovine serum (HyClone, Logan, UT), 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA). Recombinant Plasmids and Transient Transfection−pCDNA3-YFP-human (hu)TLR4, pCDNA3-huCD14, pCMV1-FLAG-huTLR2, pCMV-β-galactosidase, and pELAM-luciferase, were obtained from Dr. Douglas T. Golenbock (University of Massachusetts Medical School, Worcester, MA). pEFBOS-His/FLAG-huMD-2 was provided by Dr. Kensuke Miyake (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan), and pCDNA3-YFP-MyD88 was obtained from Dr. Katherine A. Fitzgerald (University of Massachusetts Medical School). pGL3-RANTES-luciferase reporter plasmid was kindly provided by Dr. John Hiscott (McGill University, Montreal, Canada). Expression vectors encoding HA-ΔTLR4 WT and HA-ΔTLR4 P712H have been described (67.Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), and expression plasmids pFLAG-CMV-1 encoding WT or PGV714–716 CD4-TLR4 were kindly provided by Dr. Stephen T. Smale (Howard Hughes Medical Institute, UCLA, Los Angeles, CA). P714H, Y674A, and Y680A mutations were introduced into the TIR domain of CD4-TLR4 or YFP-TLR4 by site-directed mutagenesis, using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). HEK293T cells were cultured overnight in 150-mm TC dishes (5 × 106 cells per dish) and cotransfected for 3 h with expression vectors as described in the figure legends (25 μg of total plasmid DNA per dish) using Superfect transfection reagent (Qiagen, Valencia, CA). After 48 h, cellular extracts were prepared as described (68.Medvedev A.E. Thomas K. Awomoyi A. Kuhns D.B. Gallin J.I. Li X. Vogel S.N. J. Immunol. 2005; 174: 6587-6591Crossref PubMed Scopus (42) Google Scholar). For real-time PCR analyses and determination of cytokine levels by ELISA, transfections were carried out in 6-well plates according to the manufacturer’s protocol. Isolation of RNA, Real-time PCR Analysis, and IL-8 Secretion−Total RNA was isolated with RNeasy kits (Qiagen), followed by DNase digestion and re-purification as recommended by the manufacturer. cDNA was prepared from 1 μg of RNA using the Reverse Transcription System (Promega), and subjected to real-time PCR with gene-specific primers for human hypoxanthine phosphoribosyltransferase (HPRT), IL-8, TNF-α, and IFN-β on a MyIQ minicycler with Optical Detection Module (Bio-Rad). Real-time PCR data were processed using 2–ΔΔCT method (69.Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (121290) Google Scholar). IL-8 levels in supernatants were measured by ELISA in the Cytokine Core Laboratory (University of Maryland, Baltimore). Co-immunoprecipitation and Immunoblotting−Cell extracts (1–3 mg of total protein) were precleared with protein G-agarose beads (20 μl per sample, Roche Applied Science, Indianapolis, IN) and incubated overnight with 1 μg of respective Ab in lysis buffer containing 20 mm HEPES (pH 7.4), 0.5% Triton X-100, 150 mm NaCl, 12.5 mm β-glycerophosphate, 50 mm NaF, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Roche Applied Science). Thereafter, protein G-agarose beads were added (45 μl per sample) and incubation continued for 4 h. Beads were washed five times with lysis buffer, and proteins were separated by SDS-PAGE on 4–20% minigels (Invitrogen), transferred to Immobilon-P membranes (Millipore, Billerica, MA), and subjected to Western analysis as described previously (68.Medvedev A.E. Thomas K. Awomoyi A. Kuhns D.B. Gallin J.I. Li X. Vogel S.N. J. Immunol. 2005; 174: 6587-6591Crossref PubMed Scopus (42) Google Scholar). Reporter Assays−Expression vectors pELAM-luciferase (NF-κB reporter assays) and pGL3-RANTES-luciferase (RANTES reporter assays) were added (0.3 μg/well each) to corresponding transfection mixtures, pCMV-β-galactosidase reporter (0.2 μg/well) was co-transfected for normalization (NF-κB reporter assays), and total plasmid DNA amount was adjusted to 1.5 μg/well with pCDNA3. Following transfections, cells were recovered for 24 h, lysed in a passive lysis buffer (Promega) and firefly luciferase versus β-galactosidase activities were measured using Reporter Assay Systems (Promega) and β-galactosidase assay kit (Tropix, Galacto-Light System) on a Berthold LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). For RANTES reporter assays, luciferase activity was normalized to total protein concentrations measured in cell extracts. Statistical Analysis−Statistical analysis was performed using the GraphPad Prism 4 program for Windows. Statistical differences among experimental groups were evaluated by the Student’s t test with the level of significance set at p < 0.05. Values are expressed as mean ± S.D. The P712H Mutation in the TIR Domain of Murine HA-ΔTLR4 Abolishes TLR4 Signaling and Tyrosine Phosphorylation−Positional cloning of TLR4 identified a P712H mutation within the TIR domain as the molecular basis for the LPS-hyporesponsive phenotype of C3H/HeJ mice (4.Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1351) Google Scholar, 5.Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Aleojos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6401) Google Scholar). However, the molecular mechanisms responsible for the signaling deficiency of the mutant P712H TLR4 still remain obscure. In this study, we hypothesized that the P712H mutation alters the secondary structure of the TIR domain, resulting in the failure of TLR4 to undergo agonist-induced post-translational modifications that may be necessary for initiation of downstream signaling. Because an earlier report correlated LPS-induced TLR4 tyrosine phosphorylation with TLR4 signaling (70.Chen L.Y. Zuraw B.L. Zhao M. Liu F.T. Huang S. Pan Z.K. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003; 284: L607-L613Crossref PubMed Scopus (22) Google Scholar), we initially sought to determine whether signaling incompetence of P712H" @default.
• W1994985348 created "2016-06-24" @default.
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• W1994985348 date "2007-06-01" @default.
• W1994985348 modified "2023-10-11" @default.
• W1994985348 title "Role of TLR4 Tyrosine Phosphorylation in Signal Transduction and Endotoxin Tolerance" @default.
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