Department of Regenerative Medicine, and Stem Cell Research Center, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China.
Cytokine. 2013 Apr;62(1):115-22. doi: 10.1016/j.cyto.2013.01.021. Epub 2013 Mar 9.
We previously identified the tumor suppressor INT6/eIF3e as a novel down regulator of HIF2α. Small interfering RNA targeting Int6 (siRNA-Int6) in HeLa cells led to normoxic stabilization of HIF2α, with concomitant transcription of angiogenic factors, including angiopoietin, basic fibroblast growth factor, and vascular endothelial growth factor. Here we used HIF2α normoxic up-regulation via Int6 silencing to investigate the role of HIF2α in endothelial cells. As a result Int6 silencing in human umbilical vein endothelial cells (HUVECs) and in human aortic endothelial cells (HAECs) led to robust enhanced cord formation and the medium supernatant of Int6 silenced HUVECs enhanced migration of untreated HUVECs, indicating a HIF2α triggered secretory signaling. Within the responsible genes were the cytokines interleukin-6 (IL-6) and IL-8 and not unlike in HeLa cells vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF). In addition application of IL-6 and IL-8 antibodies to the medium of Int6 silenced HUVECs could reverse the enhanced migration effect and also abrogated their tube formation. Finally, a CHIP assay analysis confirmed hypoxia-responsive elements (HREs) in the IL-6 and IL-8 promoters. Our results demonstrate that expression of both IL-6 and IL-8 is regulated by HIF2α and we suggest that IL-6 and IL-8 are HIF2α controlled cytokines for angiogenesis particularly in endothelial cells.
我们之前发现肿瘤抑制因子 INT6/eIF3e 是 HIF2α 的新型下调因子。针对 Int6 的小干扰 RNA（siRNA-Int6）在 HeLa 细胞中导致 HIF2α 在常氧条件下稳定，同时伴有血管生成因子的转录，包括血管生成素、碱性成纤维细胞生长因子和血管内皮生长因子。在这里，我们使用通过 Int6 沉默实现的 HIF2α 常氧上调来研究 HIF2α 在血管内皮细胞中的作用。结果表明，Int6 沉默在人脐静脉内皮细胞（HUVEC）和人主动脉内皮细胞（HAEC）中导致强烈的增强的管形成，并且 Int6 沉默的 HUVEC 的培养基上清液增强了未经处理的 HUVEC 的迁移，表明 HIF2α 触发了分泌信号。在负责基因中包括细胞因子白细胞介素 6（IL-6）和白细胞介素 8（IL-8），与 HeLa 细胞中的情况类似，血管内皮生长因子（VEGF）、肝细胞生长因子（HGF）和表皮生长因子（EGF）。此外，将 IL-6 和 IL-8 抗体应用于 Int6 沉默的 HUVEC 的培养基中可以逆转增强的迁移作用，并且还消除了它们的管状形成。最后，CHIP 分析证实了 IL-6 和 IL-8 启动子中的缺氧反应元件（HREs）。我们的结果表明，IL-6 和 IL-8 的表达均受 HIF2α 调节，我们建议 IL-6 和 IL-8 是 HIF2α 控制的血管生成细胞因子，特别是在血管内皮细胞中。
