Abstract
Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDA-containing fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Anti-Bacterial Agents / pharmacology
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Antigens, Surface / metabolism
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Blotting, Western
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Cell Line
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Dose-Response Relationship, Drug
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Drosophila Proteins*
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Enzyme Activation
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Exons
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Fibronectins / chemistry*
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Fibronectins / metabolism*
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Hot Temperature
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Humans
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Inflammation
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Interleukin-10 / biosynthesis
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Lipid A / analogs & derivatives*
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Lipid A / pharmacology
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Lipopolysaccharides / antagonists & inhibitors
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Lipopolysaccharides / pharmacology
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Lymphocyte Antigen 96
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Matrix Metalloproteinase 9 / metabolism
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Membrane Glycoproteins / metabolism*
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Mice
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Mice, Inbred C3H
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Plasmids / metabolism
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Polymyxin B / pharmacology
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Protein Structure, Tertiary
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Receptors, Cell Surface / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Signal Transduction
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Spleen / cytology
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Time Factors
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Toll-Like Receptor 4
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Toll-Like Receptors
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Transfection
Substances
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Anti-Bacterial Agents
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Antigens, Surface
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Drosophila Proteins
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E5564
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Fibronectins
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LY96 protein, human
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Lipid A
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Lipopolysaccharides
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Lymphocyte Antigen 96
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Membrane Glycoproteins
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Receptors, Cell Surface
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Recombinant Proteins
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TLR4 protein, human
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Toll-Like Receptor 4
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Toll-Like Receptors
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Interleukin-10
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Matrix Metalloproteinase 9
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Polymyxin B