Abstract
Bacterial endotoxin (lipopolysaccharide or LPS) has potent pro-inflammatory properties and acts on many cell types including endothelial cells. Secretion of the CC chemokine, MCP-1 (CCL2) by LPS-activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in LPS-induced MCP-1 production in endothelial cells is not well understood. Using human microvascular endothelial cells (HMVEC), we analyzed the involvement of the non-receptor tyrosine kinase, Pyk2, in LPS-mediated MCP-1 production. There was a marked activation of the non-receptor tyrosine kinase, Pyk2, in response to LPS. Inhibition of Pyk2 activity using a pharmacological inhibitor, Tyrphostin A9 significantly attenuated LPS-induced Pyk2 tyrosine phosphorylation, p38 MAP kinase (MAPK) activation, NF-kappaB activation, and MCP-1 expression. Furthermore, specific inactivation of Pyk2 activity by transducing microvascular endothelial cells with catalytically inactive Pyk2 mutant (AAV-Pyk2MT) or Pyk2-specific siRNA significantly blocked LPS-induced MCP-1 production. The supernatants of these LPS-stimulated cells with attenuated Pyk2 activity demonstrated decreased trans-endothelial monocyte migration in comparison to LPS-treated controls, thus confirming the inhibition of functional MCP-1 production. In summary, our data suggest a critical role for the Pyk2 mediated pathway involving p38 MAP kinase and NF-kappaB in LPS-induced MCP-1 production in human microvascular endothelial cells.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Cell Movement / drug effects
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Cell Movement / genetics
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Cell Movement / immunology
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Cells, Cultured
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Chemokine CCL2 / biosynthesis
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Chemokine CCL2 / genetics
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Chemokine CCL2 / immunology*
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Endothelial Cells / immunology*
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Endothelial Cells / metabolism
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Enzyme Activation / drug effects
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Enzyme Activation / genetics
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Enzyme Activation / immunology
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Enzyme Inhibitors / pharmacology
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Focal Adhesion Kinase 2 / antagonists & inhibitors
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Focal Adhesion Kinase 2 / genetics
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Focal Adhesion Kinase 2 / immunology*
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Focal Adhesion Kinase 2 / metabolism
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Gene Expression Regulation / drug effects*
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Gene Expression Regulation / genetics
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Gene Expression Regulation / immunology
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Humans
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Lipopolysaccharides / pharmacology*
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MAP Kinase Signaling System / drug effects*
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MAP Kinase Signaling System / genetics
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MAP Kinase Signaling System / immunology
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Monocytes / immunology
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Monocytes / metabolism
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Mutation
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NF-kappa B / genetics
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NF-kappa B / immunology*
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NF-kappa B / metabolism
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Phosphorylation / drug effects
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Phosphorylation / genetics
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Phosphorylation / immunology
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Sepsis / genetics
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Sepsis / immunology
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Sepsis / metabolism
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Tyrphostins / pharmacology
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p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
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p38 Mitogen-Activated Protein Kinases / genetics
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p38 Mitogen-Activated Protein Kinases / immunology*
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p38 Mitogen-Activated Protein Kinases / metabolism
Substances
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CCL2 protein, human
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Chemokine CCL2
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Enzyme Inhibitors
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Lipopolysaccharides
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NF-kappa B
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Tyrphostins
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tyrphostin A9
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Focal Adhesion Kinase 2
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p38 Mitogen-Activated Protein Kinases