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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-?B 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 ECs with thrombin resulted in a time-dependent activation of Pyk2. RNA interference knockdown of Pyk2 attenuated thrombin-induced activity of NF-?B and expression of its target genes, vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1. Pyk2 knockdown impaired thrombin-induced activation of I?B kinase (IKK) and phosphorylation (Ser32 and Ser36) of IkappaB?, but, surprisingly, failed to prevent I?B? degradation. However, depletion of IKK? or IKK? was effective in inhibiting I?B? phosphorylation/degradation, as expected. Intriguingly, Pyk2 knockdown impaired nuclear translocation and DNA binding of RelA/p65, despite the inability to prevent I?B? degradation. In addition, Pyk2 knockdown was associated with inhibition of RelA/p65 phosphorylation at Ser536, which is important for transcriptional activity of RelA/p65. Depletion of IKK? or IKK? 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.

SUBMITTER: Bijli KM

PROVIDER: S-EPMC3547104 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Regulation of Rela/p65 and endothelial cell inflammation by proline-rich tyrosine kinase 2.

Bijli Kaiser M KM Fazal Fabeha F Rahman Arshad A

American journal of respiratory cell and molecular biology 20120727 5

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 ECs with thrombin resulted in a time-dependent activation of Pyk2. RNA interference knockdown of Pyk2 attenuated thrombin-induced activity of NF-κB and expression of its target genes, vascular cell adhesion molecule-1 ...[more]

PMID: 22842493

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Project description:BackgroundAnti-angiogenic therapy inhibits tumor growth and is considered as a potential clinical therapy for malignant glioma. However, inevitable recurrences and unexpected tumor resistance, particularly increased invasion ability of glioma cell, were observed after anti-angiogenic treatment. The underlying mechanism remains undetermined. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration; therefore, we investigated the possible role of these kinases in rat C6 glioma cell invasion induced by bevacizumab, a recombinant monoclonal antibody against vascular endothelial growth factor (VEGF).MethodsThe effects of bevacizumab on migration and invasion of C6 glioma cells were investigated in vitro and in vivo. The cells proliferation, migration, and invasion were determined by MTT assay, wound healing, and transwell assay, respectively. Invasive potential of glioma cells in vivo was assessed by counting vimentin-positive cells crossing the solid tumor rim by immunohistochemical staining. The total and phosphorylated protein levels of FAK and Pyk2 were detected by Western blotting.ResultsBevacizumab exposure increased migration and invasion of cultured C6 cells in a concentration-dependent manner. In addition, the continuous bevacizumab treatment also promoted tumor invasion in rat C6 intracranial glioma models. Bevacizumab treatment enhanced Pyk2 phosphorylation at Tyr402, but no effect on FAK phosphorylation at Tyr397 both in vitro and in vivo. Knockdown of Pyk2 by siRNA or inhibition of Pyk2 phosphorylation by Src kinase specific inhibitor PP1 partially inhibited bevacizumab-induced cell invasion in cultured C6 glioma cells. Furthermore, the combined administration of bevacizumab and PP1 significantly suppressed glioma cell invasion into surrounding brain tissues compared to bevacizumab treatment alone in experimental rats.ConclusionsThese results suggest that anti-VEGF treatment promotes glioma cell invasion via activation of Pyk2. Inhibition of Pyk2 phosphorylation might be a potential target to ameliorate the therapeutic efficiency of anti-VEGF treatment.

Dataset Information

Regulation of Rela/p65 and endothelial cell inflammation by proline-rich tyrosine kinase 2.

Publications

Regulation of Rela/p65 and endothelial cell inflammation by proline-rich tyrosine kinase 2.

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