Journal of Biological Chemistry · 2000 · 247 citations · 35 references
Life SciencesImmunologyMolecular BiologyCell SignalingInnate ImmunitySignal TransductionCytokine SignalingInflammatory Cell SignalingInflammatory SignalingInnate Immune Signaling PathwaysMacrophage BiologyToll-like ReceptorsCox ProcessesCyclooxygenase-2 PathwayMissense MutationsNuclear Factor Kappa B ActivationToll-like Receptor 4 GeneticsToll-like Receptor SignalingToll-like Receptor-mediated Innate Immune ResponseToll-like Receptors In Inflammation
Genetic evidence indicating that TOLL-like receptor 4 (Tlr4) is the lipopolysaccharide (LPS) receptor in mice was reported. However, biochemical evidence that murine Tlr4 confers LPS responsiveness has not been convincingly demonstrated. Inducible cyclooxygenase (COX-2) is selectively expressed in LPS-stimulated macrophages in part mediated through the activation of NFκB. Thus, we determined whether murine Tlr4 confers LPS responsiveness as evaluated by the activation of NFκB and COX-2 expression. Transfection of a murine macrophage-like cell line (RAW264.7) with the constitutively active form (ΔTlr4) of Tlr4 is sufficient to activate NFκB and COX-2 expression. However, the truncated form (ΔTlr4(P712H)) of the missense mutant Tlr4(P712H) found in LPS-hyporesponsive mouse strain (C3H/HeJ) inhibits LPS-induced NFκB activation and COX-2 expression. The inability of ΔTlr4(P712H) to activate NFκB and induce COX-2 expression is rescued by a constitutively active adapter protein myeloid differentiation factor 88 (MyD88), which interacts directly with the cytoplasmic domain of Tlr proteins. Furthermore, MyD88 is co-immunoprecipitated with the wild-type ΔTlr4 but not with the ΔTlr4(P712H) mutant. Together, these results indicate that Tlr4 confers LPS responsiveness in RAW264.7 cells and suggest that hyporesponsiveness of C3H/HeJ mice to LPS is attributed to the disruption of Tlr4-mediated signaling pathways that results from the inability of the mutant Tlr4(P712H) to interact with MyD88. Genetic evidence indicating that TOLL-like receptor 4 (Tlr4) is the lipopolysaccharide (LPS) receptor in mice was reported. However, biochemical evidence that murine Tlr4 confers LPS responsiveness has not been convincingly demonstrated. Inducible cyclooxygenase (COX-2) is selectively expressed in LPS-stimulated macrophages in part mediated through the activation of NFκB. Thus, we determined whether murine Tlr4 confers LPS responsiveness as evaluated by the activation of NFκB and COX-2 expression. Transfection of a murine macrophage-like cell line (RAW264.7) with the constitutively active form (ΔTlr4) of Tlr4 is sufficient to activate NFκB and COX-2 expression. However, the truncated form (ΔTlr4(P712H)) of the missense mutant Tlr4(P712H) found in LPS-hyporesponsive mouse strain (C3H/HeJ) inhibits LPS-induced NFκB activation and COX-2 expression. The inability of ΔTlr4(P712H) to activate NFκB and induce COX-2 expression is rescued by a constitutively active adapter protein myeloid differentiation factor 88 (MyD88), which interacts directly with the cytoplasmic domain of Tlr proteins. Furthermore, MyD88 is co-immunoprecipitated with the wild-type ΔTlr4 but not with the ΔTlr4(P712H) mutant. Together, these results indicate that Tlr4 confers LPS responsiveness in RAW264.7 cells and suggest that hyporesponsiveness of C3H/HeJ mice to LPS is attributed to the disruption of Tlr4-mediated signaling pathways that results from the inability of the mutant Tlr4(P712H) to interact with MyD88. lipopolysaccharide TOLL-like receptor nuclear factor κB interleukin myeloid differentiation factor 88 cyclooxygenase hemagglutinin polymerase chain reaction NFκB-inducing kinase inhibitor κBα TOLL/IL-1R homology The pathogenesis of Gram-negative septic shock is presumed to be because of excess stimulation of host cells by bacterial lipopolysaccharide (LPS)1endotoxin (1Morrison D.C. Ulevitch R.J. Am. J. Pathol. 1978; 93: 526-617PubMed Google Scholar, 2Bone R.C. Ann. Intern. Med. 1991; 115: 457-469Crossref PubMed Scopus (1238) Google Scholar, 3Morrison D.C. Ryan J.L. Annu. Rev. Med. 1987; 38: 417-432Crossref PubMed Google Scholar). Such stimulation leads to the expression and release of a plethora of pro-inflammatory marker gene products and lipid mediators, which in turn can initiate a chain of events leading to systemic toxicity (4Dinarello C.A. FASEB J. 1994; 8: 1314-1325Crossref PubMed Google Scholar, 5Tracey K.J. Lowry S.F. Adv. Surg. 1990; 23: 21-56PubMed Google Scholar). However, the initial recognition of LPS by cells of the innate immune system is required to defend the host from a Gram-negative infection before it becomes widely disseminated (6O'Brien A.D. Rosenstreich D.L. Scher I. Campbell G.H. MacDermott R.P. Formal S.B. J. Immunol. 1980; 124: 20-24PubMed Google Scholar,7Rosenstreich D.L. Weinblatt A.C. O'Brien A.D. Crit. Rev. Immunol. 1982; 3: 263-330PubMed Google Scholar). Identifying the downstream signaling pathways derived from LPS stimulation is of fundamental importance to understanding the cellular mechanism of Gram-negative septic shock. CD14, a glycosylphosphatidylinositol-linked membrane protein that is widely expressed in mononuclear cells, is considered a high affinity receptor for LPS (8Ingalls R.R. Heine H. Lien E. Yoshimura A. Golenbock D. Infect. Dis. Clin. North Am. 1999; 13: 341-353Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 9Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1325) Google Scholar). However, CD14 lacks a cytoplasmic domain, and there has been a puzzling question as to how CD14 transmits extracellular signals into downstream cytoplasmic signaling pathways. Recently, it was demonstrated that human TOLL-like receptor-2 (Tlr2) mediates the LPS-induced cellular signaling pathway (10Kirschning C.J. Wesche H. Merrill A.T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar, 11Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1102) Google Scholar). Human Tlr4 can constitutively activate NFκB but fails to confer LPS responsiveness (10Kirschning C.J. Wesche H. Merrill A.T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar). However, results from studies using Tlr2- or Tlr4-deficient mice and from in vitrotransfection studies suggest that Tlr4 recognizes LPS, but Tlr2 recognizes Gran-positive bacterial cell wall components (41Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2800) Google Scholar, 42Yoshimura A. Lien E. Ingalls R.R. Tuomanen E. Dziarski R. Golenbock D. J. Immunol. 1999; 163: 1-5Crossref PubMed Google Scholar). The human TOLL protein is a transmembrane protein with an extracellular domain consisting of leucine-rich repeats and a cytoplasmic domain homologous to that of the IL-1 receptor (12Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1458) Google Scholar). Genetic evidence indicating that murine Tlr4 is the LPS receptor was demonstrated using two mouse strains (C3H/HeJ and C57BL/10ScCr), which are hyporesponsive to LPS (13Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. 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 (6478) Google Scholar). The former strain has Tlr4 with a missense mutation to replace proline with histidine at position 712, whereas the latter strain is homozygous for a null mutation of Tlr4. This genetic evidence is confirmed by another independent investigation (14Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1356) Google Scholar). It is also demonstrated that the activation of NFκB and the expression of certain NFκB-induced gene products in LPS-stimulated macrophages derived from the C3H/HeJ mouse strain are impaired (15Vogel S.N. Johnson D. Perera P.Y. Medvedev A. Lariviere L. Qureshi S.T. Malo D. J. Immunol. 1999; 162: 5666-5670PubMed Google Scholar,16Beutler B. Krochin N. Milsark I.W. Luedke C. Cerami A. Science. 1986; 232: 977-980Crossref PubMed Scopus (1014) Google Scholar). In addition, the overexpression of mutant Tlr4 derived from C3H/HeJ mice into human dermal endothelial cells results in the inhibition of LPS-induced NFκB activation (17Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). However, biochemical evidence that murine Tlr4 indeed confers LPS responsiveness has not been conclusively demonstrated. Activation of TOLL proteins and IL-1 receptor induces recruitment of the adapter molecule, myeloid differentiation factor 88 (MyD88) (18Medzhitov R. Janeway Jr., C.A. Cell. 1997; 91: 295-298Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar, 19Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (527) Google Scholar, 20O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (499) Google Scholar), which in turn leads to the activation of NFκB and the expression of NFκB-induced gene products (10Kirschning C.J. Wesche H. Merrill A.T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar, 11Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1102) Google Scholar, 21Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4458) Google Scholar). Results from our previous studies indicate that LPS induces selective expression of the mitogen-induced cyclooxygenase (COX-2) in murine macrophages (22Lee S.H. Soyoola E. Chanmugam P. Hart S. Sun W. Zhong H. Liou S. Simmons D. Hwang D. J. Biol. Chem. 1992; 267: 25934-25938Abstract Full Text PDF PubMed Google Scholar). LPS activates NFκB through TOLL-like receptors in macrophages (23Qureshi S.T. Gros P. Malo D. Trends Genet. 1999; 15: 291-294Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 24Yang R.B. Mark M.R. Gurney A.L. Godowski P.J. J. Immunol. 1999; 163: 639-643PubMed Google Scholar, 25Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar, 26Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). However, the role of NFκB in LPS-induced COX-2 expression in macrophages is not clearly established; there were contradicting reports regarding the role of NFκB in LPS-induced COX-2 expression in macrophages (27Hwang D. Jang B.C., Yu, G. Boudreau M. Biochem. Pharmacol. 1997; 54: 87-96Crossref PubMed Scopus (235) Google Scholar, 28Wadleigh D.J. Reddy S.T. Kopp E. Ghosh S. Herschman H.R. J. Biol. Chem. 2000; 275: 6259-6266Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). COX-2 is shown to be overexpressed in sites of inflammation and in tissues of many types of tumors (29Crofford L.J. J. Rheumatol. 1997; 24: 15-19Google Scholar, 30Kutchera W. Jones D.A. Matsunami N. Groden J. McIntyre T.M. Zimmerman G.A. White R.L. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4816-4820Crossref PubMed Scopus (445) Google Scholar, 31Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrenbach S. DuBois R.N. Gastroenterology. 1994; 107: 1183-1188Abstract Full Text PDF PubMed Google Scholar, 32Sano H. Kawahito Y. Wilder R.L. Hashiramoto A. Mukai S. Asai K. Kimura S. Kato H. Kondo M. Hla T. Cancer Res. 1995; 55: 3785-3789PubMed Google Scholar, 33Kargman S.L. O'Neill G.P. Vickers P.J. Evans J.F. Mancini J.A. Jothy S. Cancer Res. 1995; 55: 2556-2559PubMed Google Scholar). Elucidating the signaling pathways is the key to understanding why COX-2 is overexpressed in such pathological states and can provide crucial information for identifying the potential targets for pharmacological and dietary modulation. In this study, we have addressed three important issues in elucidating LPS-stimulated signaling pathways in murine macrophages. First, we determined whether the activation of NFκB is sufficient and required for LPS-induced COX-2 expression. Second, we determined whether the activation of Tlr4 confers LPS responsiveness as evaluated by the activation of NFκB and the expression of COX-2. Third, we determined why the Tlr4 mutant with a missense mutation at position 712 fails to transmit the LPS-induced signal to downstream signaling pathways. RAW264.7 cells (murine macrophage-like cell line, ATCC TIB-71) were cultured in LPS-free Dulbecco's modified Eagle's medium containing 10% (v/v) heat-inactivated fetal bovine serum (Intergen) and 10 units/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Inc.) at 37 °C in a 5% CO2 air environment. Human embryonic kidney cells (293T cells) were provided by Sam Lee (Beth Israel Hospital, Boston, MA) and cultured in the same medium used for the RAW264.7 cells. Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-tagged murine Tlr4 cDNA constructs (both wild-type and mutant pFlag-CMV1-Tlr4(wt) and pFlag-CMV1-Tlr4(P712H), respectively) were kindly provided by Bruce Beutler (Southwestern Medical Center, Dallas, TX). These constructs lack DNA sequences for the first 20 amino acids representing the signal peptide. The mutant Tlr4 found in LPS-hyporesponsive mouse strain (C3H/HeJ) has a missense mutation at position 712 to replace proline with histidine. The Flag-Tlr4 constructs were subcloned into pcDNA3.1/zeo(−) vector (Invitrogen, San Diego, CA), and the Flag epitope tag was replaced with the HA tag sequence (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) by polymerase chain reaction (PCR) cloning. The first PCR reaction was performed with the primers named Xho-SS-FOR and SS-HA-REV. The second PCR reaction was performed with the primers named Xho-SS-FOR and Not-HA-REV using the first PCR product as a template. The final PCR product was inserted in the XhoI and NotI sites of pcDNA3.1/zeo(−)-Flag-Tlr4 (wt) and pcDNA3.1/zeo(−)-Flag-Tlr4(P712H) plasmids resulting in pcDNA3.1/zeo(−)-HA-Tlr4(wt) and pcDNA3.1/zeo(−)-HA-Tlr4(P712H) constructs, respectively. The primers used are as follows: Xho-SS-FOR, 5′-GTTAAACTCGAGCCACCATGTCTGCACTTCTGATCC-3′; SS-HA-REV, 5′-AGCGTAGTCTGGGACGTCGTATGGGTAAGCAACTGCAGCTCCAACAAG-3′; Not-HA-REV, 5′-GGGATTCGCGGCCGCAGCGTAGTCTGGGACGTCGTATGGG-3′. For truncated Tlr4 constructs (Flag/HA epitope-tagged ΔTlr4(wt) and ΔTlr4(P712H)) lacking the extracellular leucine-rich repeat domain, DNA sequences spanning the epitope tag plus N-terminal 21–78 amino acids and transmembranes plus the cytoplasmic region (amino acids 629–835) were amplified by PCR using Flag/HA-Tlr4(wt) and Flag/HA-Tlr4(P712H) plasmids, respectively, as templates. The primers used for the PCR cloning are as follows: Xho-Tlr-F208, 5′-GTTAAACTCGAGCCAGGATGATGCCTCCCTGGCTCC-3′; TlrR441Bam, 5′-ACACCGGATCCTTCTGAAAAATTGGAGAAGCTATAGC-3′ for amplifying the N-terminal region; BmTlrF2092, 5′-GAAGGGGATCCAAGACAATCATCAGTGTGTCAGTG3′; and Tlr-REV, for the region spanning amino acids respectively. The PCR products were subcloned into the XhoI and sites in pcDNA3.1/zeo(−) expression vector for and The of the sequences was confirmed by DNA of Tlr4 cDNA constructs that were used in these studies is shown in The expression of was into cells by using Flag HA and immune were at the Medical using a containing amino acids the of human Tlr4. plasmids for the wild-type NFκB-inducing kinase and a mutant were from Rothe San murine MyD88 mutant and the constitutively active mutant were kindly provided by of K. F. C. H. P. J.L. F. L. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The constructs were subcloned into expression vector plasmids for wild-type inhibitor κB and a mutant were provided by was a from San Diego, was from of R.B. M. S.T. P.J. Godowski P.J. R.L. Science. 1999; PubMed Scopus Google Scholar). COX-2 was from DNA constructs were in a using for RAW264.7 cells were in and with of DNA plasmids as an using to the was determined by with as in our previous Lee Boudreau Hwang J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in and for were with a of of or and expression cells were with and for in of 10 μg/ml 10 μg/ml were by at 4 °C for at were with of HA for 4 and of (v/v) protein was for at 4 °C with were with Nature. PubMed Scopus Google three with The were by and to The were with 20 containing 5% and were with the The were with the system as in our previous studies (22Lee S.H. Soyoola E. Chanmugam P. Hart S. Sun W. Zhong H. Liou S. Simmons D. Hwang D. J. Biol. Chem. 1992; 267: 25934-25938Abstract Full Text PDF PubMed Google Scholar, Lee Boudreau Hwang J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, P. L. Liou S. Jang Boudreau Yu, G. Lee T. M. Y. Hwang D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). whether LPS-induced expression of COX-2 is mediated through the activation of we whether or not the inhibition of LPS-induced activation of NFκB leads to the of COX-2 expression. were with the for NFκB or COX-2 and with an expression containing a mutant of or The results that LPS-induced COX-2 expression is by the of cells with a mutant of or These results indicate that the activation of NFκB is required for the expression of COX-2 in LPS-stimulated RAW264.7 cells. activation of NFκB of LPS leads to the expression of this that NFκB is required for the expression of COX-2 by The results that activation of NFκB by of cells with the wild-type leads to the expression of COX-2. Furthermore, the of cells with and a mutant of the COX-2 expression Together, these results indicate that the activation of NFκB is required for the expression of COX-2 in LPS-stimulated macrophages. were two reports regarding the role of NFκB in LPS-induced COX-2 expression in RAW264.7 cells. The for NFκB was demonstrated in our previous using the pharmacological of or nuclear of NFκB (27Hwang D. Jang B.C., Yu, G. Boudreau M. Biochem. Pharmacol. 1997; 54: 87-96Crossref PubMed Scopus (235) Google Scholar). However, the results from another using the gene that NFκB not be required for the expression of COX-2 in LPS-stimulated RAW264.7 cells D.J. Reddy S.T. Kopp E. Ghosh S. Herschman H.R. J. Biol. Chem. 2000; 275: 6259-6266Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). It is not the these two studies (27Hwang D. Jang B.C., Yu, G. Boudreau M. Biochem. Pharmacol. 1997; 54: 87-96Crossref PubMed Scopus (235) Google Scholar, 28Wadleigh D.J. Reddy S.T. Kopp E. Ghosh S. Herschman H.R. J. Biol. Chem. 2000; 275: 6259-6266Abstract Full Text Full Text PDF PubMed Scopus (319) Google in of NFκB for the expression of COX-2. used the murine COX-2 containing whereas the COX-2 used in another the results that were from our sequences D.J. Reddy S.T. Kopp E. Ghosh S. Herschman H.R. J. Biol. Chem. 2000; 275: 6259-6266Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). the κB in the murine COX-2 is it is that of the region of COX-2 gene are required for the expression of COX-2 in LPS-stimulated RAW264.7 cells. whether or not the activation of Tlr4 is sufficient to activate NFκB and induce the expression of cells were with the truncated mouse Tlr4 (ΔTlr4) lacking the leucine-rich repeat extracellular domain and the or gene The of by the truncated TOLL proteins lacking the extracellular domain was demonstrated in and in human cells R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4458) Google Scholar, C. Biol. Cell. 1995; PubMed Scopus Google Scholar). The results that of cells with the constitutively active ΔTlr4 leads to the activation of NFκB and COX-2 expression The same truncated ΔTlr4(P712H) with the missense mutation at position 712 is to activate NFκB and COX-2 expression. Thus, these results indicate that the activation of Tlr4 is sufficient to activate NFκB and induce COX-2 expression in RAW264.7 cells. Kirschning (10Kirschning C.J. Wesche H. Merrill A.T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google that human with CD14 into cells, LPS of NFκB. However, the overexpression of human Tlr4 constitutively the NFκB and the of these cells with LPS not the gene these that human Tlr2 but not Tlr4 confers LPS results that of RAW264.7 cells with the truncated ΔTlr4 but not the wild-type murine Tlr4 a activation of the NFκB gene whether the expression of COX-2 is mediated through activation of we whether the inhibition of NFκB by a mutant of or results in the of COX-2 expression. The results that inhibition of NFκB activation by the mutant of or leads to of COX-2 expression indicating that COX-2 expression is at in part mediated through NFκB. whether activation of Tlr4 confers LPS we determined whether the expression of the mutant Tlr4(P712H) found in C3H/HeJ mouse strain inhibits LPS-induced activation of NFκB and COX-2 expression. The results that the truncated mutant ΔTlr4(P712H) inhibits whereas the truncated wild-type ΔTlr4 LPS-induced activation of NFκB and COX-2 expression LPS responsiveness is in cells with the Tlr4 as with the cells. These results suggest that ΔTlr4 as a mutant. Together, these results indicate that the activation of Tlr4 confers LPS responsiveness in RAW264.7 cells. However, our results not the that TOLL-like receptors also LPS responsiveness in RAW264.7 cells. we why the missense mutation to replace histidine with proline at position 712 of Tlr4 in of It was demonstrated that MyD88 is an adapter protein directly with the cytoplasmic TOLL/IL-1R homology domain of human Tlr4 R. Preston-Hurlburt P. Kopp E. A. C. Ghosh S. Janeway Jr., C.A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google and considered as of the components of the human Tlr4-mediated signaling This domain is also the N-terminal domain of MyD88. Such a sequence homology is also murine at position 712 this domain of murine which is for the MyD88 adapter Thus, we determined whether the mutation at position 712 with the of MyD88 to resulting in of the signal Human embryonic kidney cells (293T cells) were with an epitope MyD88 and wild-type ΔTlr4 or the mutant ΔTlr4(P712H) cell from these cells were with and using or Tlr4 MyD88 was co-immunoprecipitated with the wild-type but not with the mutant These results suggest that the mutant Tlr4(P712H) is to interact with MyD88 and fails to activate downstream signaling pathways. The adapter MyD88 is to be an downstream signaling directly with the domain of Tlr4 (18Medzhitov R. Janeway Jr., C.A. Cell. 1997; 91: 295-298Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar, 19Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (527) Google Scholar, 20O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (499) Google Scholar). at position 712 is in this it be to whether the of proline with histidine resulting from the missense mutation with the of MyD88 to the domain of Tlr4. the of the mutant Tlr4 to activate downstream signaling pathways is because of inability to the adapter cells with a constitutively active form of MyD88 signal the of RAW264.7 cells with the mutant (ΔTlr4(P712H)) and a constitutively active form of MyD88 lacking TOLL/IL-1R domain results in the of NFκB activation and COX-2 expression these results suggest that hyporesponsiveness of the mouse strain (C3H/HeJ) to LPS is because of the disruption of Tlr4-mediated signaling pathways resulting from the inability of the mutant to the downstream signaling MyD88. In the results indicate that activation of Tlr4 confers LPS responsiveness and that disruption of Tlr4-mediated signaling pathways leads to hyporesponsiveness to LPS in murine macrophage-like cell line (RAW264.7) as determined by the activation of NFκB and the expression of COX-2. factor NFκB the expression of a of inflammation marker gene products that are in innate immune and pathogenesis of Gram-negative septic shock. our results the importance of signaling pathways in these A. and for this
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Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
Ulrich K. Laemmli · Nature · 1970 · 251.6K citations
Defective LPS Signaling in C3H/HeJ and C57BL/10ScCr Mice: Mutations in <i>Tlr4</i> Gene
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