Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
J Mol Cell Cardiol. 2012 Dec;53(6):848-57. doi: 10.1016/j.yjmcc.2012.10.002. Epub 2012 Oct 16.
Excessive tumor necrosis factor-α (TNF-α) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-α 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-α release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-α expression of cardiomyocytes, the role of hypoxia inducible factor-1α (HIF-1α) in TNF-α regulation and potential secretory pathway of TNF-α. Elevated TNF-α expression and HIF-1α activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofluorescence. TNF-α mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1α small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1α protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-α 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-α 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-α within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1α initiates expression of TNF-α, mediated by exosomes in cardiomyocytes.
过度的肿瘤坏死因子-α（TNF-α）表达被认为对急性心肌梗死中的心肌细胞有害。在心肌缺血时，TNF-α主要由巨噬细胞释放，但持续缺血时，它可以来源于心肌细胞并导致心脏重构。目前尚不清楚TNF-α从心肌细胞释放的起始因子和确切分子机制。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-1α（HIF-1α）在 TNF-α调节中的作用以及 TNF-α的潜在分泌途径。通过实时 PCR、Western blot 和免疫荧光检测缺氧对原代培养心肌细胞中 TNF-α表达和 HIF-1α激活的影响。用 HIF-1α 小干扰 RNA（siRNA）转染和 2-甲氧基雌二醇（HIF-1α蛋白抑制剂）处理明显抑制 TNF-α mRNA 升高和蛋白分泌。在 HEK293 和 HepG2 细胞中也观察到类似的结果。在人类 TNF-α基因启动子中鉴定出潜在的缺氧反应元件。缺失分析和定点突变表明，相对于转录起始位点，跨越 bp-1295 到 bp-1292 的 HIF 共有结合位点对于 TNF-α启动子的激活是功能性的，这通过电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）分析得到证实。Western blot 中用抗 CD63 抗体鉴定出缺氧心肌细胞上清液中的外体（介导非经典途径蛋白分泌的囊泡），并通过电子显微镜观察。用免疫电子显微镜和免疫印迹法验证了从缺氧心肌细胞上清液沉淀的外体中存在 TNF-α。本研究结果表明，在缺氧下，HIF-1α通过心肌细胞中的外体启动 TNF-α的表达。
