Division of Cardiovascular Sciences, Center for Applied Medical Research, University of Navarra, Avda. Pío XII 55, 31008 Pamplona, Spain.
Cardiovasc Res. 2011 Nov 1;92(2):247-55. doi: 10.1093/cvr/cvr202. Epub 2011 Jul 19.
Cardiotrophin-1 (CT-1) is a cytokine of the interleukin-6 superfamily which is up-regulated in cardiac diseases, in part via hypoxia-dependent mechanisms. However, no evidence for a direct regulation of CT-1 gene (CTF1) promoter by hypoxia inducible factor-1 (HIF-1) has been provided.
Hypoxia increased CT-1 mRNA levels in the murine adult cardiomyocyte cell line HL-1 in a time-dependent manner. Interestingly, in a murine model (C57BL/6), we show that systemic hypoxia also significantly up-regulated CT-1 in myocardial tissue. The effect of hypoxia on CT-1 expression was mediated through a transcriptional mechanism, since hypoxia increased luciferase activity of constructs containing CTF1 promoter sequences. The increase in CT-1 levels was significantly reduced by drugs that prevent calcium mobilization, such as lercanidipine, or that inhibit the activation of the PI3K/Akt pathway (wortmannin) or mammalian target of rapamycin (rapamycin). The CT-1 elevation was similarly induced by HIF-1α over-expression in co-transfection experiments and prevented by HIF-1α silencing. The direct interaction of HIF-1α with the CTF1 promoter was confirmed through site-directed mutagenesis of hypoxia response elements, electrophoreric mobility shift, and ChIP assays. Hypoxia induced HL-1 apoptosis (measured as annexin-V binding or caspase 3/7 activity) which was increased when CT-1 was silenced in knocked-down cells by lentiviral vectors.
Hypoxia increased CT-1 levels in cardiac cells (in vitro and in vivo) through a direct regulation of CTF1 promoter by HIF-1α. This CT-1 activation by hypoxia may protect cells from apoptosis, thus supporting a protective role for CT-1 as a survival factor for cardiomyocytes.
心肌营养素 1（CT-1）是白介素 6 超家族的细胞因子，在部分心脏疾病中上调，部分原因是通过缺氧依赖机制。然而，没有证据表明缺氧诱导因子 1（HIF-1）直接调节 CT-1 基因（CTF1）启动子。
缺氧以时间依赖的方式增加了鼠成年心肌细胞系 HL-1 中的 CT-1 mRNA 水平。有趣的是，在鼠模型（C57BL/6）中，我们发现全身缺氧也显著上调心肌组织中的 CT-1。缺氧对 CT-1 表达的影响是通过转录机制介导的，因为缺氧增加了包含 CTF1 启动子序列的构建体的荧光素酶活性。通过阻止钙动员的药物（如乐卡地平）或抑制 PI3K/Akt 途径（wortmannin）或哺乳动物雷帕霉素靶蛋白（rapamycin）的激活来减少 CT-1 水平的增加。在共转染实验中，通过过表达 HIF-1α 也可以诱导 CT-1 水平升高，并通过 HIF-1α 沉默来阻止。通过对缺氧反应元件进行定点突变、电泳迁移率改变和 ChIP 测定，证实了 HIF-1α 与 CTF1 启动子的直接相互作用。缺氧诱导 HL-1 凋亡（通过 annexin-V 结合或 caspase 3/7 活性测量），当 CT-1 在敲低细胞中通过慢病毒载体沉默时，凋亡增加。
缺氧通过 HIF-1α 直接调节 CTF1 启动子增加心脏细胞（体外和体内）中的 CT-1 水平。这种 CT-1 激活可能通过防止细胞凋亡来保护细胞，从而支持 CT-1 作为心肌细胞存活因子的保护作用。
