Murine TOLL-like receptor 4 confers lipopolysaccharide responsiveness as determined by activation of NF kappa B and expression of the inducible cyclooxygenase.
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 NF kappa B. Thus, we determined whether murine Tlr4 confers LPS responsiveness as evaluated by the activation of NF kappa B and COX-2 expression. Transfection of a murine macrophage-like cell line (RAW264.7) with the constitutively active form (delta Tlr4) of Tlr4 is sufficient to activate NF kappa B and COX-2 expression. However, the truncated form (delta Tlr4(P712H)) of the missense mutant Tlr4(P712H) found in LPS-hyporesponsive mouse strain (C3H/HeJ) inhibits LPS-induced NF kappa B activation and COX-2 expression. The inability of delta Tlr4(P712H) to activate NF kappa B 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 delta Tlr4 but not with the delta Tlr4(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.
Rhee
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Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808, USA.
Hwang
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D051546:Cyclooxygenase 2; D017931:DNA Primers; D029721:Drosophila Proteins; D004790:Enzyme Induction; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D020125:Mutation, Missense; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D000097782:NF-kappaB-Inducing Kinase
D051546:Cyclooxygenase 2; D017931:DNA Primers; D029721:Drosophila Proteins; D007527:Isoenzymes; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D016328:NF-kappa B; D011451:Prostaglandin-Endoperoxide Synthases; D017346:Protein Serine-Threonine Kinases; D011956:Receptors, Cell Surface; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D000097782:NF-kappaB-Inducing Kinase; C495345:TLR4 protein, human; C496540:PTGS2 protein, human