title: Serum amyloid A3 binds MD-2 to activate p38 and NF-κB pathways in a MyD88-dependent manner.
abstract: Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20-86) (20-86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20-86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20-86) or SAA3 (43-57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 μM, respectively. FLAG-tagged SAA3 tightly bound to protein A-tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20-86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-β1. An i.v. injection of SAA3 (43-57) induced the lung recruitment of CD11b(+)Gr-1(+) cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.
authors.affiliation: Department of Pharmacology, Tokyo Women's Medical University, Tokyo 162-8666, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Deguchi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tomita
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Omori
authors.forename: Akiko
authors.initials: A
authors.lastname: Komatsu
authors.forename: Umeharu
authors.initials: U
authors.lastname: Ohto
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Natsuko
authors.initials: N
authors.lastname: Tanimura
authors.forename: Sachiko
authors.initials: S
authors.lastname: Akashi-Takamura
authors.forename: Kensuke
authors.initials: K
authors.lastname: Miyake
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Maru
mesh_terms: D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000473:pathology; D051218:Lymphocyte Antigen 96 / Q000172:deficiency / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D022423:Myeloid Cells / Q000502:physiology; D053594:Myeloid Differentiation Factor 88 / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D025941:Protein Interaction Mapping; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D000685:Serum Amyloid A Protein / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D008024:Ligands; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C076498:Saa3 protein, mouse; D000685:Serum Amyloid A Protein; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; C577686:interleukin-6, mouse; C482199:lipopolysaccharide, E coli O55-B5; C514862:Akt1 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020741:rho GTP-Binding Proteins