Regulation of RelA/p65 and Endothelial Cell Inflammation by Proline-Rich Tyrosine Kinase 2.
ABSTRACT We investigated the role of proline-rich tyrosine kinase 2 (Pyk2) in the mechanism of NF-κB activation and endothelial cell (EC) inflammation induced by thrombin, a procoagulant serine protease released in high amounts during sepsis and other inflammatory conditions. Stimulation of EC with thrombin resulted in a time-dependent activation of Pyk2. RNAi knockdown of Pyk2 attenuated thrombin-induced activity of NF-κB and expression of its target genes, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). Depletion of Pyk2 also inhibited NF-κB activity induced by TNFα, suggesting that Pyk2 is a general regulator of NF-κB. Pyk2 knockdown impaired thrombin-induced activation of IKK and phosphorylation (Ser32 and Ser36) of IκBα, but surprisingly failed to prevent IκBα degradation. However, depletion of IKKα or IKKβ was effective in inhibiting IκBα phosphorylation/degradation, as expected. Intriguingly, Pyk2 knockdown impaired nuclear translocation and DNA binding of RelA/p65 despite the inability to prevent IκBα degradation. Additionally, Pyk2 knockdown was associated with inhibition of RelA/p65 phosphorylation at Ser536, which is important for transcriptional activity of RelA/p65. Depletion of IKKα or IKKβ, each impaired RelA/p65 phosphorylation. Taken together, these data identify Pyk2 as a critical regulator of EC inflammation by virtue of engaging IKK to promote the release and the transcriptional capacity of RelA/p65, and additionally, by its ability to facilitate the nuclear translocation of the released RelA/p65. Thus, specific targeting of Pyk2 may be an effective anti-inflammatory strategy in vascular diseases associated with EC inflammation and intravascular coagulation.
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ABSTRACT: The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.PLoS ONE 03/2013; 8(3):e59965. DOI:10.1371/journal.pone.0059965 · 3.53 Impact Factor
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ABSTRACT: Atherosclerosis initiates at predictable focal sites and develops to a spatially regional disease with limited distribution. There is compelling evidence that links hemodynamics to the localized origin of atherosclerotic lesions. Arterial flow in vivo is unsteady, dynamically complex, and regionally variable. Sites susceptible to atherosclerosis near arterial branches and curves are associated with regions of disturbed blood flow that contain repetitive phases of flow reversal resulting in steep multidirectional temporal and spatial gradients of wall shear stresses. Endothelium in atherosusceptible regions relative to protected sites show activation of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), the altered expression of pro-inflammatory Nuclear Factor kappa B (NFκB) and oxidant/anti-oxidant pathways, and low expression of major protective factors, notably endothelial nitric oxide synthase (eNOS) and Kruppel-like Factors KLF2 and KLF4. At some atherosusceptible locations, reactive oxygen species (ROS) levels are significantly elevated. Here we describe flow-related phenotypes identified in steady state in vivo and outline some of the molecular mechanisms that may contribute to pre-lesional atherosusceptibility as deduced from complementary cell experiments in vitro. We conclude that disturbed flow is a significant local risk factor for atherosclerosis that induces a chronic low-level inflammatory state, an adaptive response to ensure continued function at the expense of increased susceptibility to atherogenesis. Surprisingly, when challenged by short-term hypercholesterolemia in vivo, atherosusceptible endothelial phenotype was resistant to greater pro-inflammatory expression, suggesting that sustained hyperlipidemia is required to overcome these protective characteristics.Cardiovascular Research 04/2013; DOI:10.1093/cvr/cvt101 · 5.81 Impact Factor
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ABSTRACT: Background Human brain microvascular endothelial cells (hBMVECs) are integral part of the blood brain barrier. Post-translational modifications of adherens junction proteins regulate the permeability of human brain microvascular endothelial cells. Pro-inflammatory signals can induce tyrosine phosphorylation of adherens junction proteins. The primary objective of this work is to provide a molecular model; how the HIV-1 Tat protein can compromise the BBB integrity and eventually lead to neurological consequences. We exposed hBMVECs to recombinant HIV-1 clade C Tat protein to study the effect of HIV-1 Tat C on permeability of hBMVECs. Trans-endothelial electrical resistance and fluorescent dye migration assay have been used to check the permeability of hBMVECs. DCFDA staining has been used for intracellular reactive oxygen species (ROS) detection. Western blotting has been used to study the expression levels and co-immunoprecipitation has been used to study the interactions among adherens junction proteins. Results HIV-1 Tat C protein induced NOX2 and NOX4 expression level and increased intracellular ROS level. Redox-sensitive kinase; PYK2 activation led to increased tyrosine phosphorylation of VE-cadherin and β-catenin, leading to disruption of junctional assembly. The dissociation of tyrosine phosphatases VE-PTP and SHP2 from cadherin complex resulted into increased tyrosine phosphorylation of VE-cadherin and β-catenin in HIV-1 Tat C treated hBMVECs. Conclusion Unrestricted phosphorylation of junctional proteins in hBMVECs, in response to HIV-1 Tat C protein; leads to the disruption of junctional complexes and increased endothelial permeability.BMC Neuroscience 06/2014; 15(1):80. DOI:10.1186/1471-2202-15-80 · 2.85 Impact Factor