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

Department of Pathology, Ohio State University Medical Center, Columbus, OH 43210, USA.
The Journal of Immunology (Impact Factor: 4.92). 05/2008; 180(8):5636-44. DOI: 10.4049/jimmunol.180.8.5636
Source: PubMed


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.

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    • "PLC activation is required for the signal transduction events leading to inflammatory responses [16], [37]. In LPS-stimulated macrophages and human endothelial cells, proinflammatory cytokine secretion and expression are dependent on Src kinase [38], [39], [40]. Therefore, the role of Src kinase in immune response is rapidly emerging. "
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    ABSTRACT: Epidemiological evidence suggests that obesity is associated with inflammation of the respiratory tract and the pathogenesis of asthma. The purpose of this study was to examine the role of phospholipase D1 (PLD1) in leptin-induced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, and to suggest a molecular link between obesity and respiratory tract inflammation. We investigated whether leptin, a typical adipocytokine, plays a role in the expression of TNF-α through increased PLD1 activity in Raw 264.7. Leptin enhanced the activity of PLD1 through activation of PLCγ and Src, while PLD1 siRNA decreased the leptin-induced expression and production of TNF-α. Leptin-induced PLD activation was also inhibited by a PLCγ inhibitor (PAO) and Src kinase inhibitor (PP2), indicating that PLCγ and Src kinase are upstream activators of PLD1. Down-regulation of PLD1 also completely blocked activation of p70S6K, an activator of JNK. Leptin-induced expression of TNF-α was also prevented by inhibition of p70S6K and JNK. Taken together, these results indicate that PLD1 acts as an important regulator of leptin-induced expression of TNF-α by participating in the PLCγ/Src/PLD1/PA/p70S6K/JNK pathway.
    PLoS ONE 07/2014; 9(7):e102373. DOI:10.1371/journal.pone.0102373 · 3.23 Impact Factor
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    • "This may be explained by the negative feedback mechanism since TAT-Pyk2-CT treated and LPS challenged mice has less neutrophils in the lung. Anand AR et al. found that inhibition of Pyk2 activity in endothelial cells in vitro by transfection with the catalytically inactive C-terminal Pyk2 significantly blocked LPS-induced IL-8 production [34]. The inability of Pyk2 inhibition to affect IL-8 homolog secretion in mouse BAL in our study may suggest that lung resident cells (such as airway epithelial cells and alveolar macrophages) other than endothelial cells may be at least in part responsible for the secretion of IL-8 homologs found in the BAL. "
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    ABSTRACT: Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo. C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically. Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment. These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.
    Respiratory research 01/2012; 13(1):4. DOI:10.1186/1465-9921-13-4 · 3.09 Impact Factor
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    • "There is much evidence that central to the recognition of LPS expression in endothelial cells is a family of transmembrane proteins known as Toll-like receptors (TLRs).24–26 Most effector cells of the immune responses, especially the innate immune system, such as monocytes and endothelial cells express TLR2 and TLR4.27 When LPS binds to TLR4, multiple intracellular signaling pathways are activated, including transcription factor, NF-κB, pathway as well as MAPK.28 "
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    International Journal of COPD 09/2011; 6(1):439-48. DOI:10.2147/COPD.S23695 · 3.14 Impact Factor
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