凝血酶和活化蛋白 C 通过 EPCR 和 PAR-1 依赖性机制抑制 TNF-α 激活的内皮细胞中分泌型 IIA 磷脂酶 A(2)的表达。
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.
机构信息
Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-Industry, Daegu Haany University, Gyeongsan 712-715 Republic of Korea.
出版信息
Thromb Res. 2010 Jan;125(1):e9-e15. doi: 10.1016/j.thromres.2009.07.015. Epub 2009 Aug 15.
INTRODUCTION
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.
MATERIALS AND 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).
RESULTS AND 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.
简介
凝血酶和肿瘤坏死因子 (TNF)-α上调人脐静脉内皮细胞 (HUVEC) 中促炎分子的表达。然而，活化蛋白 C (APC) 下调相同分子的表达。已知分泌型 IIA 组磷酯酶 A2 (sPLA2-IIA) 的表达水平在包括脓毒症在内的炎症性疾病中升高。在这里，我们研究了 APC 和凝血酶对 HUVECs 中 sPLA2-IIA 和细胞外信号调节激酶 (ERK) 表达的影响。
材料和方法
在不存在和存在磷脂酰肌醇 3-激酶 (PI3-kinase) 抑制剂 LY294002 和胆固醇耗竭药物甲基-β-环糊精 (MbetaCD) 的情况下，通过酶联免疫吸附试验定量测量 HUVECs 用凝血酶或 TNF-α刺激后 sPLA2-IIA 的表达水平。
结果和结论
凝血酶对 HUVECs 中 sPLA2-IIA 的表达没有影响，但 TNF-α 强烈诱导其表达。APC 的预先处理通过 PAR-1 的 EPCR 依赖性裂解抑制 sPLA2-IIA 的表达。进一步的研究表明，如果 HUVECs 用酶原蛋白 C 预处理以占据 EPCR，则凝血酶也通过 PAR-1 的裂解抑制 TNF-α 介导的 sPLA2-IIA 表达。APC 和凝血酶对 PAR-1 的 EPCR 依赖性裂解增加 ERK 1/2 的磷酸化。细胞用 LY294002 或 MbetaCD 预处理可消除 APC 和凝血酶对 sPLA2-IIA 表达的抑制活性，表明蛋白 C 对 EPCR 的占据赋予了 PI3-kinase 依赖的保护活性，使得其对脂质筏定位的 PAR-1 的裂解抑制了 HUVECs 中 TNF-α 介导的 sPLA2-IIA 表达。
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