Thrombin and activated protein C inhibit the expression of secretory group IIA phospholipase A(2) in the TNF-alpha-activated endothelial cells by EPCR and PAR-1 dependent mechanisms.
BACKGROUND
Thrombin and tumor necrosis factor (TNF)-alpha up-regulate the expression of proinflammatory molecules in human umbilical vein endothelial cells (HUVECs). However, activated protein C (APC) down-regulates the expression of the same molecules. The expression level of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) is known to be elevated in inflammatory disorders including in sepsis. Here, we investigated the effects of APC and thrombin on the expression of sPLA(2)-IIA and extracellular signal-regulated kinase (ERK) in HUVECs.


METHODS
The expression level of sPLA(2)-IIA was quantitatively measured by an enzyme-linked-immunosorbent-assay following stimulation of HUVECs with either thrombin or TNF-alpha in the absence and presence of the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 and the cholesterol-depleting drug methyl-beta-cyclodextrin (MbetaCD).


CONCLUSIONS
Thrombin had no effect on the expression of sPLA(2)-IIA in HUVECs, however, TNF-alpha potently induced its expression. The prior treatment of cells with APC inhibited expression of sPLA(2)-IIA through the EPCR-dependent cleavage of PAR-1. Further studies revealed that if HUVECs were pretreated with the zymogen protein C to occupy EPCR, thrombin also inhibited the TNF-alpha-mediated expression of sPLA(2)-IIA through the cleavage of PAR-1. The EPCR-dependent cleavage of PAR-1 by both APC and thrombin increased the phosphorylation of ERK 1/2. Pretreatment of cells with either LY294002 or MbetaCD abolished the inhibitory activity of both APC and thrombin against sPLA(2)-IIA expression, suggesting that the protein C occupancy of EPCR confers a PI3-kinase dependent protective activity for thrombin such that its cleavage of the lipid-raft localized PAR-1 inhibits the TNF-alpha-mediated expression of sPLA(2)-IIA in HUVECs.
Bae
Jong-Sup
JS
Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-Industry, Daegu Haany University, Gyeongsan 712-715 Republic of Korea. BaeJS@dhu.ac.kr
Rezaie
Alireza R
AR
D015703:Antigens, CD / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D000074283:Endothelial Protein C Receptor; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D054501:Group II Phospholipases A2 / Q000037:antagonists & inhibitors*; D006801:Humans; D011486:Protein C / Q000378:metabolism* / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins / Q000378:metabolism
D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; C478370:PROCR protein, human; D011486:Protein C; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; D054501:Group II Phospholipases A2; D013917:Thrombin
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