The Tyrosine Kinase Pyk2 Mediates Lipopolysaccharide-Induced IL-8 Expression in Human Endothelial Cells

Abstract

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells.

Regulation of Rela/p65 and Endothelial Cell Inflammation by Proline-Rich Tyrosine Kinase 2

“…Pyk2 activation was also observed in LPS-stimulated human umbilical vein ECs and human dermal microvascular ECs (26,27), as well as in cyclic strain-exposed bovine aortic ECs (22). Thus, Pyk2 appears to be responsive to the proinflammatory environment in ECs from different vascular beds.…”

“…Recently, it has also been implicated in proinflammatory gene expression and leukocyte TEM (26,27); however, the mechanistic basis of these responses is only partially understood. Because NF-kB is an essential regulator of genes involved in TEM of leukocytes, the present study was undertaken to address the role of Pyk2 in the mechanism of NF-kB activation in ECs.…”

“…Pyk2 is also present in different types of ECs, and has been implicated in various signaling pathways (21,22). It is an important regulator of endothelial permeability and angiogenesis (23,24), and has been shown to promote TEM of leukocytes via intercellular adhesion molecule-1 (ICAM-1)-dependent tyrosine phosphorylation of vascular endothelial cadherins and expression of IL-8 and MCP-1 (25)(26)(27). Thrombin has been shown to activate Pyk2 in ECs in a Ca 21 -dependent manner (21); however, the role of Pyk2 in thrombin-induced NF-kB activation and proinflammatory gene expression remains unclear.…”

Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function

“…Increased expression of IL-8 by endothelial cells can be induced in culture by endotoxin [22], and IL-8 production is increased by Escherichia coli in Crohn's disease [23], suggesting that increased IL-8 observed at the transcriptional and protein levels in cirrhosis, is indeed in response to bacterial products. Similarly, Trem-1 + macrophages and intestinal epithelial cells have been shown to be major sources of IL-6 in Crohn's disease, and elevated IL-6 levels have been demonstrated in serum and tissue of IBD patients.…”

SHP-1–Pyk2–Src Protein Complex and p38 MAPK Pathways Independently Regulate IL-10 Production in Lipopolysaccharide-Stimulated Macrophages

“…Lyn associates with CD14 and is activated upon LPS stimulation in macrophages (21). The Src kinase was shown to phosphorylate and activate the focal adhesion proline-rich tyrosine kinase 2 (Pyk2), leading to actin reorganization and IL-8 production in LPS-stimulated macrophages and human endothelial cells, respectively (22,23) The role of Pyk2 in immune response is rapidly emerging. Pyk2 is expressed in hematopoietic cells and is rapidly phosphorylated following stimulation with extracellular ligands, including inflammatory cytokines.…”