title: Protease-activated receptor-1 activation of endothelial cells induces protein kinase Calpha-dependent phosphorylation of syntaxin 4 and Munc18c: role in signaling p-selectin expression.
abstract: Endothelial cells exhibit regulated exocytosis in response to inflammatory mediators such as thrombin and histamine. The exocytosis of Weibel-Palade bodies (WPBs) containing von Willebrand factor, P-selectin, and interleukin-8 within minutes after stimulation is important for vascular homeostasis. SNARE proteins are key components of the exocytic machinery in neurons and some secretory cells, but their role in regulating exocytosis in endothelial cells is not well understood. We examined the function of SNARE proteins in mediating exocytosis of WPBs in endothelial cells. We identified the presence of syntaxin 4, syntaxin 3, and the high affinity syntaxin 4-regulatory protein Munc18c in human lung microvascular endothelial cells. Small interfering RNA-induced knockdown of syntaxin 4 (but not of syntaxin 3) inhibited exocytosis of WPBs as detected by the reduction in thrombin-induced cell surface P-selectin expression. Thrombin ligation of protease-activated receptor-1 activated the phosphorylation of syntaxin 4 and Munc18c, which, in turn, disrupted the interaction between syntaxin 4 and Munc18. Protein kinase Calpha activation was required for the phosphorylation of syntaxin 4 and Munc18c as well as the cell surface expression of P-selectin. We also observed that syntaxin 4 knockdown inhibited the adhesion of neutrophils to thrombin-activated endothelial cells, demonstrating the functional role of syntaxin 4 in promoting endothelial adhesivity. Thus, protease-activated receptor-1-induced protein kinase Calpha activation and phosphorylation of syntaxin 4 and Munc18c are required for the cell surface expression of P-selectin and the consequent binding of neutrophils to endothelial cells.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Fu
authors.forename: Anjaparavanda P
authors.initials: AP
authors.lastname: Naren
authors.forename: Xiaopei
authors.initials: X
authors.lastname: Gao
authors.forename: Gias U
authors.initials: GU
authors.lastname: Ahmmed
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D005089:Exocytosis / Q000502:physiology; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D008168:Lung / Q000098:blood supply; D008565:Membrane Proteins / Q000378:metabolism*; D008833:Microcirculation; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology; D019007:P-Selectin / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D050765:Qa-SNARE Proteins; D011817:Rabbits; D044463:Receptor, PAR-1 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D006490:Hemostatics; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D019007:P-Selectin; D050765:Qa-SNARE Proteins; D044463:Receptor, PAR-1; D033921:Vesicular Transport Proteins; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013917:Thrombin; D002118:Calcium