Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: Initiated by hypoxia inducible factor 1α, presented by exosomes
Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: Initiated by hypoxia inducible factor 1α, presented by exosomes
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DOI：
10.1016/j.yjmcc.2012.10.002
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发表时间：
2012-12-01
影响因子：
5
通讯作者：
Liao, Yuhua
中科院分区：
文献类型：
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作者：
Yu, Xian;Deng, Lingyan;Liao, Yuhua
肿瘤坏死因子-α（TNF-α）的过度表达越来越多地被认为是有害的急性心肌梗死心肌细胞。在心肌缺血期间，TNF-α主要从巨噬细胞释放，但在持续性缺血时，它可以起源于心肌细胞并有助于心脏重塑。心肌细胞释放TNF-α的起始因子和确切的分子机制目前尚不清楚。在这项研究中，我们研究了缺氧对心肌细胞TNF-α表达的直接影响，缺氧诱导因子-1 α（HIF-1 α）在TNF-α调节中的作用以及TNF-α的潜在分泌途径。采用实时荧光定量PCR、Western blotting和免疫荧光法检测缺氧条件下原代培养心肌细胞中TNF-α表达和HIF-1 α活化的升高。HIF-1 α小干扰RNA（siRNA）核转染和HIF-1 α蛋白抑制剂2-甲氧基乙烯醇处理可明显抑制TNF-α mRNA的升高和蛋白的分泌。在HEK 293和HepG 2细胞中观察到类似的结果。在人TNF-α基因启动子中鉴定了推定的缺氧反应元件。缺失分析和定点突变表明，HIF共识结合位点跨越bp-1295 bp-1292相对于转录起始位点的激活TNF-α启动子，这是由电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）分析证实的功能。在Western印迹中通过抗CD 63抗体鉴定缺氧心肌细胞上清液中的外泌体（介导非经典蛋白质分泌途径的囊泡），并通过电子显微镜观察。通过免疫电子显微镜和免疫印迹验证了从缺氧心肌细胞上清液沉淀的外来体内TNF-α的存在。这项研究的结果表明，在缺氧条件下，HIF-1 α启动TNF-α的表达，由心肌细胞中的外来体介导。(C)2012爱思唯尔有限公司保留所有权利。
Excessive tumor necrosis factor-alpha (TNF-alpha) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-alpha is mainly released from macrophages, but with persistent ischemia, it can originate from cardiomyocytes and contribute to cardiac remodeling. The initiating factor and exact molecular mechanism of TNF-alpha release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-alpha expression of cardiomyocytes, the role of hypoxia inducible factor-1 alpha (HIF-1 alpha) in TNF-alpha regulation and potential secretory pathway of TNF-alpha. Elevated TNF-alpha expression and HIF-1 alpha activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofiuorescence. TNF-alpha mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1 alpha small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1 alpha protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-alpha gene promoter. Deletion analysis and site-directed mutagenesis demonstrated that HIF consensus binding sites spanning bp-1295 to bp-1292 relative to the transcription start site were functional for activation of the TNF-alpha promoter which was confirmed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Exosomes (vesicles mediating a non-classical route of protein secretion) in supernatants from hypoxic cardiomyocytes were identified by an anti-CD63 antibody in Western blot and observed by electron microscopy. The presence of TNF-alpha within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1 alpha initiates expression of TNF-alpha, mediated by exosomes in cardiomyocytes. (C) 2012 Elsevier Ltd. All rights reserved.