Activation of Syk by Protein Kinase C-δ Regulates Thrombin-induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells via Tyrosine Phosphorylation of RelA/p65

Protein kinase C-δ (PKC-δ) plays a pivotal role in mediating thrombin-induced NF-κB activation and ICAM-1 expression in endothelial cells. However, the downstream mechanisms mediating its function are unclear. In this study, we show that PKC-δ-mediated activation of protein-tyrosine kinase Syk plays an important role in thrombin signaling of NF-κB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Stimulation of human vascular endothelial cells with thrombin resulted in a time-dependent phosphorylation of Syk on tyrosine 525 and 526, an indication of Syk activation. Inhibition of PKC-δ by pharmacological and genetic approaches prevented Syk activation by thrombin. These results place Syk downstream of PKC-δ in transmitting thrombin-activated signaling in endothelial cells. Consistent with this, thrombin-induced NF-κB activity and ICAM-1 expression were prevented by the expression of a kinase-defective mutant or RNA interference knockdown of Syk. Similarly, inhibiting Syk also impaired NF-κB activity and ICAM-1 expression induced by a constitutively active mutant of PKC-δ. Analysis of the NF-κB pathway showed that Syk contributes to thrombin-induced NF-κB activation by controlling its transactivation potential and that this response is associated with tyrosine phosphorylation of RelA/p65. Thus, these data unveil a novel pathway in which Syk signals downstream of PKC-δ to mediate thrombininduced ICAM-1 expression in endothelial cells by increasing transcriptional capacity of NF-κB via a mechanism that relies on tyrosine phosphorylation of RelA/p65.