Hypoxia upregulates osteopontin expression in NIH-3T3 cells via a ras-activated enhancer
Hypoxia upregulates osteopontin expression in NIH-3T3 cells via a ras-activated enhancer
复制标题
DOI：
10.1038/sj.onc.1208800
复制
发表时间：
2005-09-01
期刊：
影响因子：
8
通讯作者：
Le, QT
中科院分区：
文献类型：
--
作者：
Zhu, YH;Denhardt, DT;Le, QT
骨桥蛋白(OPN)是一种分泌的磷酸糖蛋白，与多种实体肿瘤的进展和生存有关，包括头颈癌。以往的研究表明，OPN的表达是由肿瘤缺氧诱导的，其血浆水平可作为头颈癌患者肿瘤缺氧和治疗结果的替代标志物。在这项研究中，我们研究了低氧促进OPN表达的转录机制。我们发现，在头颈部鳞状细胞癌(HNSCC)细胞系和NIH3T3细胞中，低氧以时间依赖的方式在mRNA和蛋白质水平上诱导OPN。放线菌素D-Chase实验表明，低氧诱导OPN不是由于增加了mRNA的稳定性。对小鼠OPN启动子区域的缺失分析表明，ras激活的增强子(RAE)位于转录起始点的-731到-712之间，是低氧增强OPN转录所必需的。对RAE DNA序列进行凝胶迁移率分析，我们发现缺氧诱导了序列特异的DNA结合复合体。此外，低氧和ras暴露导致OPN蛋白和mRNA水平的相加诱导，这似乎是由RAE介导的。低氧通过RAE元件诱导OPN是由Akt-激酶信号通路介导的，因为具有显性负结构的Akt水平降低导致低氧抑制OPN的诱导。综上所述，这些结果已经确定了一种新的受Akt信号调控的低氧反应转录增强子。
Osteopontin (OPN) is a secreted phosphoglycoprotein that has been linked to tumor progression and survival in several solid tumors, including head and neck cancers. Previous studies showed that OPN expression is induced by tumor hypoxia, and its plasma levels can serve as a surrogate marker for tumor hypoxia and treatment outcome in head and neck cancer patients. In this study, we investigate the transcriptional mechanism by which hypoxia enhances OPN expression. We found that OPN is induced in head and neck squamous cell carcinoma (HNSCC) cell lines and in NIH3T3 cells by hypoxia at both mRNA and protein levels in a time-dependent manner. Actinomycin D chase experiments showed that hypoxic induction of OPN was not due to increased mRNA stability. Deletion analyses of the mouse OPN promoter regions indicated that a ras-activated enhancer (RAE) located at -731 to -712 relative to the transcription start site was essential for hypoxia-enhanced OPN transcription. Using electrophoretic mobility shift assays with the RAE DNA sequence, we found that hypoxia induced sequence-specific DNA-binding complexes. Furthermore, hypoxia and ras exposure resulted in an additive induction of OPN protein and mRNA levels that appeared to be mediated by the RAE. Induction of OPN through the RAE element by hypoxia is mediated by an Akt-kinase signaled pathway as decreasing Akt levels with dominant negative constructs resulted in inhibition of OPN induction by hypoxia. Taken together, these results have identified a new hypoxia responsive transcriptional enhancer that is regulated by Akt signaling.