Lin Chih-Chung, Hsieh Hsi-Lung, Chi Pei-Ling, Yang Chien-Chung, Hsiao Li-Der, Yang Chuen-Mao
Department of Anesthetics, Chang Gung Memorial Hospital at Linkuo, and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Mol Neurobiol. 2014 Jun;49(3):1256-69. doi: 10.1007/s12035-013-8597-1. Epub 2013 Nov 28.
Endothelin-1 (ET-1), a proinflammatory mediator, is elevated in the regions of several brain inflammatory disorders, implying that ET-1 may contribute to inflammatory responses. The deleterious effects of ET-1 on brain endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in mouse brain microvascular endothelial cells (bEnd.3 cells) remain unclear. Herein, we investigated the effects of Ca2+-dependent protein kinases on ET-1-induced COX-2 expression and PGE2 release in bEnd.3 cells. The data obtained with Western blotting, reverse transcription PCR, and intracellular Ca2+ analyses showed that ET-1-induced COX-2 expression was mediated through phosphatidylinositol-phospholipase C (PI-PLC) and phosphatidylcholine-phospholipase C (PC-PLC)/Ca2+-dependent activation of protein kinase C-alpha (PKC-α) and calmodulin kinase II (CaMKII) cascades. Next, we demonstrated that ET-1 stimulated intracellular Ca2+ increase, phoshorylation of PKC-α, CaMKII, and mitogen-activated protein kinases (MAPKs) (ERK1/2, p38 MAPK, and JNK1/2) and then activated the activating transcription factor 2 (ATF2)/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. Moreover, the data of chromatin immunoprecipitation and promoter reporter assay demonstrated that the activated ATF2/AP-1 and p300 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in these cells. Taken together, these results demonstrate that in bEnd.3 cells, Ca2+-dependent PKC-α and CaMKII linking to MAPKs, ATF2/AP-1, and p300 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2/PGE2 system upregulated by ET-1 on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.
内皮素 -1（ET -1）是一种促炎介质，在几种脑部炎症性疾病的区域中水平升高，这意味着ET -1可能参与炎症反应。ET -1对脑内皮细胞的有害作用可能会通过上调环氧化酶 -2（COX -2）/前列腺素E2（PGE2）系统来加重介导的脑部炎症。然而，ET -1诱导小鼠脑微血管内皮细胞（bEnd.3细胞）中COX -2表达的信号机制仍不清楚。在此，我们研究了钙依赖性蛋白激酶对ET -1诱导的bEnd.3细胞中COX -2表达和PGE2释放的影响。通过蛋白质印迹、逆转录PCR和细胞内钙分析获得的数据表明，ET -1诱导的COX -2表达是通过磷脂酰肌醇 -磷脂酶C（PI -PLC）和磷脂酰胆碱 -磷脂酶C（PC -PLC）/钙依赖性激活蛋白激酶C -α（PKC -α）和钙调蛋白激酶II（CaMKII）级联反应介导的。接下来，我们证明ET -1刺激细胞内钙增加、PKC -α、CaMKII和丝裂原活化蛋白激酶（MAPK）（ERK1/2、p38 MAPK和JNK1/2）的磷酸化，然后通过Gq/i蛋白偶联的ETB受体激活激活转录因子2（ATF2）/激活蛋白1（AP -1）。此外，染色质免疫沉淀和启动子报告基因分析的数据表明，活化的ATF2/AP -1和p300与COX -2启动子内其相应的结合位点结合，从而开启COX -2基因转录。最后，ET -1对COX -2的上调促进了这些细胞中PGE2的生物合成和释放。综上所述，这些结果表明，在bEnd.3细胞中，钙依赖性PKC -α和CaMKII与MAPK、ATF2/AP -1和p300级联反应对于ET -1诱导的COX -2上调至关重要。了解ET -1上调脑微血管内皮细胞中COX -2/PGE2系统的机制可能为脑损伤和炎症性疾病提供合理的治疗干预措施。
