Lo Huey-ming, Lai Tsung-hsuan, Li Chih-hung, Wu Wen-bin
1] School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan, China [2] Section of Cardiology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, China.
1] School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan, China [2] Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan, China [3] Institute of Systems Biology and Bioinformatics, National Central University, Jhongli City, Taoyuan, Taiwan, China.
Acta Pharmacol Sin. 2014 Mar;35(3):339-50. doi: 10.1038/aps.2013.182. Epub 2014 Feb 3.
Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-α has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro.
Human umbilical vein endothelial cells (HUVECs) were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-α-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTT assay. Monocyte migration was measured with transwell migration assay.
Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α (2, 5 ng/mL) induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α (5 ng/mL) caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor (SP600125), p38 MAPK inhibitor (SB202190) or PI-3K inhibitor (LY294002) significantly suppressed TNF-α-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-α-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone (up to 500 nmol/L) failed to affect TNF-α-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-α-induced CXCL1 from HUVECs attracted human monocyte migration.
TNF-α stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.
趋化因子通常引导循环白细胞向炎症或损伤部位移动。CXCL1/GRO-α已被证明在动脉粥样硬化病变和各种癌症中上调。本研究的目的是探讨体外肿瘤坏死因子-α（TNF-α）诱导人血管内皮细胞释放CXCL1的潜在机制。
用人脐静脉内皮细胞（HUVECs）分别用不同的促炎介质和生长因子处理。分别用逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）法检测CXCL1的表达和分泌。用蛋白质免疫印迹法检测TNF-α诱导的细胞信号传导。用MTT法检测细胞活力/生长情况。用transwell迁移试验检测单核细胞迁移。
在测试的17种介质和生长因子中，TNF-α、脂多糖（LPS）和凝血酶可显著诱导HUVEC细胞释放CXCL1增加。TNF-α（2、5 ng/mL）以浓度和时间依赖性方式诱导细胞释放CXCL1并使其信使核糖核酸（mRNA）表达增加。TNF-α（5 ng/mL）可导致应激活化蛋白激酶（JNK）、p38丝裂原活化蛋白激酶（p38 MAPK）、磷脂酰肌醇-3激酶（PI3K）和蛋白激酶B（Akt）激活，而用JNK抑制剂（SP600125）、p38 MAPK抑制剂（SB202190）或PI-3激酶抑制剂（LY294002）预处理可显著抑制TNF-α诱导的细胞释放CXCL1。但只有SP600125能显著降低TNF-α诱导的细胞CXCL1 mRNA表达。此外，地塞米松（高达500 nmol/L）未能影响TNF-α诱导的细胞释放CXCL1。在功能研究中，重组CXCL1可增强HUVEC增殖，并且重组CXCL1和TNF-α诱导的HUVEC细胞CXCL1均能吸引人类单核细胞迁移。
TNF-α通过JNK介导的CXCL1 mRNA表达以及p38 MAPK和PI-3K介导的CXCL1分泌过程刺激人内皮细胞释放CXCL1。
