LPS-induced MCP-1 expression in human microvascular endothelial cells is mediated by the tyrosine kinase, Pyk2 via the p38 MAPK/NF-kappaB-dependent pathway

Mol Immunol. 2009 Feb;46(5):962-8. doi: 10.1016/j.molimm.2008.09.022. Epub 2008 Oct 26.

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

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Movement / drug effects
  • Cell Movement / genetics
  • Cell Movement / immunology
  • Cells, Cultured
  • Chemokine CCL2 / biosynthesis
  • Chemokine CCL2 / genetics
  • Chemokine CCL2 / immunology*
  • Endothelial Cells / immunology*
  • Endothelial Cells / metabolism
  • Enzyme Activation / drug effects
  • Enzyme Activation / genetics
  • Enzyme Activation / immunology
  • Enzyme Inhibitors / pharmacology
  • Focal Adhesion Kinase 2 / antagonists & inhibitors
  • Focal Adhesion Kinase 2 / genetics
  • Focal Adhesion Kinase 2 / immunology*
  • Focal Adhesion Kinase 2 / metabolism
  • Gene Expression Regulation / drug effects*
  • Gene Expression Regulation / genetics
  • Gene Expression Regulation / immunology
  • Humans
  • Lipopolysaccharides / pharmacology*
  • MAP Kinase Signaling System / drug effects*
  • MAP Kinase Signaling System / genetics
  • MAP Kinase Signaling System / immunology
  • Monocytes / immunology
  • Monocytes / metabolism
  • Mutation
  • NF-kappa B / genetics
  • NF-kappa B / immunology*
  • NF-kappa B / metabolism
  • Phosphorylation / drug effects
  • Phosphorylation / genetics
  • Phosphorylation / immunology
  • Sepsis / genetics
  • Sepsis / immunology
  • Sepsis / metabolism
  • Tyrphostins / pharmacology
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / genetics
  • p38 Mitogen-Activated Protein Kinases / immunology*
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • CCL2 protein, human
  • Chemokine CCL2
  • Enzyme Inhibitors
  • Lipopolysaccharides
  • NF-kappa B
  • Tyrphostins
  • tyrphostin A9
  • Focal Adhesion Kinase 2
  • p38 Mitogen-Activated Protein Kinases