Department of Gastroenterology and Endocrinology, Center of Internal Medicine, University of Göttingen, Göttingen, Germany.
Lab Invest. 2010 Sep;90(9):1306-24. doi: 10.1038/labinvest.2010.85. Epub 2010 May 10.
The source of circulating erythropoietin (EPO), the mediators and the mechanisms involved in the upregulation of EPO gene expression during acute-phase reaction are still poorly understood. Acute-phase reaction was induced by either intramuscular turpentine oil (TO) or intraperitoneal lipopolysaccharide (LPS) administration into wild-type and interleukin (IL)-6 knockout (KO) mice. Animals were killed at different time points and blood, liver and muscle tissue were collected. Serum levels of EPO were measured by enzyme-linked immunoadsorbent assay; liver and injured muscle samples were processed for RNA isolation and for protein analysis. EPO, hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) mRNA were analyzed by RT-PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1alpha and HIF-2alpha localization was performed through immunofluorescence staining. EPO, HIF-1 and HIF-2 gene and protein expression levels were also analyzed in isolated mouse hepatocytes after stimulation with IL-6. In the wild-type animals, EPO serum levels increased dramatically at 12 h after the insults together with the hepatic gene expression. In TO-treated animals, the EPO gene expression reached an 8.2-fold increase at 12 h, and in LPS-treated mice a similar induction was recorded at 6 h (about 4.5-fold increase). In the IL-6KO strain, the upregulation after the inflammatory stimuli was much lower (only 2.0-fold increase). A progressive upregulation of HIF-1alpha and HIF-2alpha was detectable until 6 h after the insults, but only HIF-1alpha upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1alpha, in parallel with an increase of EPO mRNA. No effect on HIF-2alpha expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1alpha induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2alpha, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.
循环红细胞生成素 (EPO) 的来源、急性期反应中 EPO 基因表达上调的介质和机制仍知之甚少。通过肌肉内松节油油 (TO) 或腹腔内脂多糖 (LPS) 给药诱导野生型和白细胞介素 (IL)-6 敲除 (KO) 小鼠发生急性期反应。在不同时间点处死动物，并采集血液、肝脏和肌肉组织。通过酶联免疫吸附测定法测量血清 EPO 水平；处理肝和受损肌肉样本以进行 RNA 分离和蛋白质分析。通过 RT-PCR 分析 EPO、缺氧诱导因子 1α 和 2α (HIF-1α 和 HIF-2α) mRNA，通过 Western blot 和电泳迁移率变动分析测定蛋白质水平。通过免疫荧光染色进行 HIF-1α 和 HIF-2α 定位。还分析了刺激后分离的小鼠肝细胞中 EPO、HIF-1 和 HIF-2 基因和蛋白质表达水平。在野生型动物中，损伤后 12 小时 EPO 血清水平显著增加，同时肝基因表达增加。在 TO 处理的动物中，EPO 基因表达在 12 小时达到 8.2 倍的增加，在 LPS 处理的小鼠中，在 6 小时记录到类似的诱导（增加约 4.5 倍）。在 IL-6KO 株中，炎症刺激后的上调幅度要低得多（仅增加 2.0 倍）。在损伤后 6 小时内可检测到 HIF-1α 和 HIF-2α 的逐渐上调，但仅在 IL-6KO 小鼠中观察到 HIF-1α 上调减少。在分离的肝细胞中，单次剂量的 IL-6 刺激诱导 HIF-1α 的核积累，同时 EPO mRNA 增加。未发现 HIF-2α 表达的影响。IL-6 似乎是 EPO 基因表达的主要调节剂，也是急性期反应中肝细胞和枯否细胞中 HIF-1α 诱导的主要贡献者。主要表达在内皮细胞和成纤维样细胞中的 HIF-2α 的增加似乎不受缺乏 IL-6 的影响。
