The Tyrosine Kinase, Pyk2/RAFTK, Mediates LPS-Induced IL-8 and MCP-1 Expression in Human Endothelial Cells.

Abstract

Lipopolysaccharide (LPS), an essential component of the surface of Gram-negative bacteria, has been shown to induce various inflammatory cytokines, including IL-8 and MCP-1, in endothelial cells. However, the mechanism by which LPS mediates the production of these inflammatory cytokines is not well known. In the present study, we investigated the role of proline-rich tyrosine kinase 2 (Pyk2), also known as RAFTK, in LPS-induced IL-8 and MCP-1 expression in endothelial cells. We observed the concentration-dependent and time-dependent tyrosine phosphorylation of Pyk2 following LPS treatment in human umbilical vein endothelial cells (HUVEC). We next analyzed the site of Pyk2 phosphorylation using antibodies specific to the individual phosphorylated tyrosine residues in Pyk2: pY402 (Src binding site), pY881 (Grb2 binding site) and pY580 (autophosphorylation site). LPS stimulation significantly enhanced phosphorylation of the pY402 and pY580 residues, but not of the pY881 site. The role of Pyk2 in LPS-induced IL-8 and MCP-1 expression was further investigated using a recombinant adeno-associated virus (AAV) expressing kinase-defective mutant Pyk2. Endothelial cells infected with the catalytically inactive Pyk2 mutant significantly attenuated LPS-induced IL-8 and MCP-1 production in HUVEC, compared to cells infected with the control AAV vector. Treatment of endothelial cells with a specific inhibitor of Pyk2, Tyrphostin A9, also blocked the expression of IL-8 and MCP-1 in a dose-dependent manner. Both p42/p44 and p38 MAP kinase pathways have been implicated in the production of inflammatory cytokines in endothelial cells. We found that blocking of Pyk2 using Tyrphostin A9 inhibited the phosphorylation of p38 MAP kinase but merely altered the kinetics of p42/p44 MAPK phosphorylation, indicating that p38 MAP kinase is an important downstream molecule of Pyk2 in LPS-induced IL-8 production. The involvement of the p38 MAP kinase pathway was confirmed by the dose-dependent blocking of IL-8 production upon treatment with a specific inhibitor of p38 MAP kinase. In summary, our data illustrate a key role for Pyk2 in LPS-mediated signaling, resulting in IL-8 and MCP-1 secretion in human endothelial cells. These findings may have important implications for the treatment of sepsis and other inflammatory disorders.