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

We investigated the role of proline-rich tyrosine kinase 2 (Pyk2) in the mechanism of NF-kappa B activation and endothelial cell (EC) inflammationinducedby thrombin, aprocoagulant 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-kappa 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 kappa B kinase (IKK) and phosphorylation (Ser32 and Ser36) of IkappaB alpha, but, surprisingly, failed to prevent I kappa B alpha degradation. However, depletion of IKK alpha or IKK beta was effective in inhibiting I kappa B alpha phosphorylation/degradation, as expected. Intriguingly, Pyk2 knockdown impaired nuclear translocation and DNA binding of RelA/p65, despite the inability to prevent I kappa B alpha 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 alpha or IKK beta each impaired RelA/p65 phosphorylation. Taken together, these data identify Pyk2 as a critical regulatorofECinflammationby virtueofengagingIKKtopromotethe 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.