Thrombin inhibits nuclear factor kappaB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C.
The occupancy of endothelial protein C receptor (EPCR) by protein C switches the protease activated receptor 1 (PAR-1)-dependent signalling specificity of thrombin from a permeability enhancing to a barrier protective response in vascular endothelial cells. In this study, the modulatory effects of thrombin and thrombin receptor agonist peptides (TRAP) on tumour necrosis factor (TNF)-alpha-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A were evaluated by monitoring the expression of cell surface adhesion molecules (VCAM-1, ICAM-1 and E-selectin), adhesion of freshly isolated neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factor-kappaB (NF-kappaB) pathway. The analysis of results indicate that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither PAR-1 agonist influenced the proinflammatory effects of TNF-alpha in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin, thus leading to thrombin inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNF-alpha-activated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kappaB pathways in response to TNF-alpha in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is ligated by protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses, thus leading to activation of Rac1 and inhibition of RhoA and NF-kappaB signalling cascades in vascular endothelial cells.
Bae
Jong-Sup
JS
Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1100 S. Grand Blvd., St. Louis, MO 63104, USA.
Rezaie
Alireza R
AR
D015703:Antigens, CD / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000276:immunology; D000074283:Endothelial Protein C Receptor; D006801:Humans; D007962:Leukocytes / Q000276:immunology; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism
D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; D016328:NF-kappa B; C478370:PROCR protein, human; D011486:Protein C; D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; D013917:Thrombin; D020742:rhoA GTP-Binding Protein