c-Src 依赖性 EGF 受体激活促进内皮素-1 诱导的脑微血管内皮细胞 COX-2 表达。
c-Src-dependent EGF receptor transactivation contributes to ET-1-induced COX-2 expression in brain microvascular endothelial cells.
机构信息
Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan.
出版信息
J Neuroinflammation. 2012 Jul 2;9:152. doi: 10.1186/1742-2094-9-152.
BACKGROUND
Endothelin-1 (ET-1) is elevated and participates in the regulation of several brain inflammatory disorders. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2) gene expression. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear.
OBJECTIVE
The goal of this study was to examine whether ET-1-induced COX-2 expression and prostaglandin E2 (PGE2) release were mediated through a c-Src-dependent transactivation of epidermal growth factor receptor (EGFR) pathway in brain microvascular endothelial cells (bEnd.3 cells).
METHODS
The expression of COX-2 induced by ET-1 was evaluated by Western blotting and RT-PCR analysis. The COX-2 regulatory signaling pathways were investigated by pretreatment with pharmacological inhibitors, short hairpin RNA (shRNA) or small interfering RNA (siRNA) transfection, chromatin immunoprecipitation (ChIP), and promoter activity reporter assays. Finally, we determined the PGE2 level as a marker of functional activity of COX-2 expression.
RESULTS
First, the data showed that ET-1-induced COX-2 expression was mediated through a c-Src-dependent transactivation of EGFR/PI3K/Akt cascade. Next, we demonstrated that ET-1 stimulated activation (phosphorylation) of c-Src/EGFR/Akt/MAPKs (ERK1/2, p38 MAPK, and JNK1/2) and then activated the c-Jun/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. The activated c-Jun/AP-1 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Ultimately, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in bEnd.3 cells.
CONCLUSIONS
These results demonstrate that in bEnd.3 cells, c-Src-dependent transactivation of EGFR/PI3K/Akt and MAPKs linking to c-Jun/AP-1 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.
背景
内皮素-1（ET-1）水平升高并参与多种脑部炎症性疾病的调节。ET-1 对血管内皮细胞的有害作用可能通过环氧合酶-2（COX-2）基因表达的上调加重脑炎症。然而，ET-1 诱导脑微血管内皮细胞中 COX-2 表达的信号机制仍不清楚。
目的
本研究旨在探讨 ET-1 是否通过 c-Src 依赖性表皮生长因子受体（EGFR）途径的转激活来介导 COX-2 表达和前列腺素 E2（PGE2）的释放。
方法
通过 Western blot 和 RT-PCR 分析评估 ET-1 诱导的 COX-2 表达。通过药理抑制剂预处理、短发夹 RNA（shRNA）或小干扰 RNA（siRNA）转染、染色质免疫沉淀（ChIP）和启动子活性报告基因分析研究 COX-2 调节信号通路。最后，我们测定 PGE2 水平作为 COX-2 表达功能活性的标志物。
结果
首先，数据表明 ET-1 诱导的 COX-2 表达是通过 c-Src 依赖性 EGFR/PI3K/Akt 级联的转激活介导的。接下来，我们证明 ET-1 刺激 c-Src/EGFR/Akt/MAPKs（ERK1/2、p38 MAPK 和 JNK1/2）的激活，然后通过 Gq/i 蛋白偶联的 ETB 受体激活 c-Jun/激活蛋白 1（AP-1）。激活的 c-Jun/AP-1 与 COX-2 启动子内的相应结合位点结合，从而开启 COX-2 基因转录。最终，ET-1 上调 COX-2 促进 bEnd.3 细胞中 PGE2 的生物合成和释放。
结论
这些结果表明，在 bEnd.3 细胞中，c-Src 依赖性 EGFR/PI3K/Akt 的转激活和连接到 c-Jun/AP-1 级联的 MAPKs 对于 ET-1 诱导的 COX-2 上调是必不可少的。了解 ET-1/ETB 系统调节脑微血管内皮细胞中 COX-2 表达和 PGE2 释放的机制可为脑损伤和炎症性疾病提供合理的治疗干预。
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