Mechanism of leptin expression in breast cancer cells:: role of hypoxia-inducible factor-1α
Mechanism of leptin expression in breast cancer cells:: role of hypoxia-inducible factor-1α
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DOI：
10.1038/sj.onc.1210660
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发表时间：
2008-01-17
期刊：
影响因子：
8
通讯作者：
Surmacz, E.
中科院分区：
文献类型：
--
作者：
Cascio, S.;Bartella, V.;Surmacz, E.
我们先前报道过，肥胖激素瘦素在乳腺癌活检组织中过度表达。在此，我们研究了这一过程所涉及的分子机制，重点关注与肥胖相关的状况，即高胰岛素血症和缺氧诱导。通过使用实时定量聚合酶链反应、蛋白质的免疫荧光检测以及酶联免疫吸附测定，我们发现用高剂量胰岛素或缺氧模拟剂氯化钴（CoCl₂）处理MCF - 7乳腺癌细胞，或者在缺氧条件下培养细胞，会显著增加瘦素mRNA和蛋白质的表达。值得注意的是，在高胰岛素血症和缺氧联合或缺氧模拟处理下，观察到瘦素mRNA和蛋白质表达量最高。荧光素酶报告基因检测表明，瘦素合成增加可能与瘦素基因启动子的激活有关。DNA亲和沉淀和染色质免疫沉淀实验显示，胰岛素、CoCl₂和/或缺氧处理增加了缺氧诱导因子 - 1α（HIF - 1α）在细胞核内的积累，并增强了其与几个上游瘦素调控序列的相互作用，尤其是与包含四个缺氧反应元件和三个富含GC区域的近端启动子的相互作用。通过使用反向染色质沉淀，我们确定HIF - 1α在近端瘦素启动子上的结合与主要的HIF共激活因子p300的募集同时发生，这表明HIF / p300复合物参与瘦素转录。利用RNA干扰证实了HIF - 1α在胰岛素和CoCl₂激活的瘦素mRNA和蛋白质表达中的重要性。
We reported previously that the obesity hormone leptin is overexpressed in breast cancer biopsies. Here, we investigated molecular mechanisms involved in this process, focusing on conditions that are associated with obesity, that is, hyperinsulinemia and induction of hypoxia. By using quantitative real-time PCR, immunofluorescent detection of proteins and enzyme-linked immunosorbent assays, we found that treatment of MCF-7 breast cancer cells with high doses of insulin or the hypoxia-mimetic agent CoCl2, or culturing the cells under hypoxic conditions significantly increased the expression of leptin mRNA and protein. Notably, the greatest leptin mRNA and protein expression were observed under combined hyperinsulinemia and hypoxia or hypoxia-mimetic treatments. Luciferase reporter assays suggested that increased leptin synthesis could be related to the activation of the leptin gene promoter. DNA affinity precipitation and chromatin immunoprecipitation experiments revealed that insulin, CoCl2 and/or hypoxia treatments augmented nuclear accumulation of hypoxia-inducible factor-1 alpha ( HIF-1 alpha) and increased its interaction with several upstream leptin regulatory sequences, especially with the proximal promoter containing four hypoxia-response elements and three GC-rich regions. By using reverse chromatin precipitation, we determined that loading of HIF-1 alpha on the proximal leptin promoter concurred with the recruitment of p300, the major HIF coactivator, suggesting that the HIF/p300 complex is involved in leptin transcription. The importance of HIF-1 alpha in insulin- and CoCl2-activated leptin mRNA and protein expression was confirmed using RNA interference.