Lung Cell Genomic Threats from Physiological Signals

描述(由申请人提供)：在低氧信号中用作第二信使的活性氧物种(ROS)氧化修饰位于肺动脉内皮细胞(PAEC)血管内皮细胞(PAEC)中HIF-1 DNA识别序列末端3‘端的鸟嘌呤。当在低氧修饰的鸟嘌呤上引入一个包含血管内皮生长因子基因低氧反应元件的寡核苷酸的碱性位点时，该序列结合了更多的HIF-1，并产生了更强劲的低氧诱导报告基因表达。这些发现支持一种新的ROS参与低氧信号传递的模型，在该模型中，ROS介导的关键DNA调控序列的碱基氧化影响转录复合体的形成和伴随的基因表达。如果这个模型具有普遍意义，那么非低氧刺激产生的ROS应该引起类似的DNA氧化修饰模式，这些应该导致转录复合体的组成和基因表达的可预见的变化。因此，我们现在建议使用受体介导的激动剂凝血酶和PDGF进行实验，这两种药物在ROS依赖的信号通路方面有所不同，但共同参与了HIF-1对血管内皮生长因子表达的诱导。我们将测试工作假说的关键元素，即在凝血酶和PDGF信号转导的背景下产生的ROS氧化修饰功能相关DNA序列中的特定核苷酸，从而改变转录复合体的组成和伴随的基因表达。在PAECs中进行的研究将：(1)确定凝血酶和PDGF对可诱导的VEGF基因启动子和编码区以及非诱导的肌动蛋白基因和静止的胰岛素基因的氧化修饰平衡密度的影响的动力学；(2)在可诱导的VEGF基因的缺氧反应元件和已知的非诱导肌动蛋白启动子和静止的胰岛素启动子的转录因子结合序列中，以单核苷酸分辨率定位凝血酶和PDGF诱导的修饰；(3)测试假设，即在DNA反应元件内的ROS修饰的核苷酸处引入模型氧化碱基产物改变了响应凝血酶和PDGF而形成的转录复合体的组成；以及(4)确定在DNA反应元件内的ROS修饰的核苷酸处引入模型氧化碱基产物是否改变了报告基因的表达以响应凝血酶和PDGF。这项研究将为生理信号中产生的ROS调节基因表达的一种以前未被认识的机制提供概念证明。此外，这些研究将证实，特定核基因的完整性受到发生在生理信号背景下的氧化碱基修饰的威胁。这一发现可能会指出导致体细胞突变的新途径，从而更好地理解癌症、衰老和其他被认为在其中起致病作用的疾病。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) used as second messengers in hypoxic signaling oxidatively modify the guanine located at the extreme 3' end of the HIF-1 DNA recognition sequence in the pulmonary artery endothelial cell (PAEC) VEGF gene. When an abasic site was introduced at the hypoxia-modified guanine in an oligonucleotide encompassing the VEGF gene's hypoxic response element, the sequence bound more HIF-1 and engendered more robust hypoxia-induced reporter gene expression. These findings support a new model for ROS involvement in hypoxic signaling in which ROS-mediated base oxidation in key DNA regulatory sequences impacts on formation of the transcriptional complex and attendant gene expression. If this model is of general significance, then ROS generated by non-hypoxic stimuli should cause similar patterns of oxidative DNA modifications and these should result in predictable alterations in the composition of transcriptional complexes and gene expression. Accordingly, we now propose experiments using the receptor-mediated agonists, thrombin and PDGF, which differ in terms of their ROS-dependent signaling pathways but have in common the involvement of HIF-1 in induction of VEGF expression. We will test key elements of the working hypothesis that ROS generated in the context of thrombin and PDGF signaling oxidatively modify specific nucleotides within functionally-relevant DNA sequences and thereby alter the composition of the transcriptional complex and attendant gene expression. Studies performed in PAECs will: (1) Define kinetics by which thrombin and PDGF impact on the equilibrium density of oxidative modifications in the promoter and coding regions of the inducible VEGF gene as well as the non-inducible actin gene and the quiescent insulin gene; (2) Map modifications induced by thrombin and PDGF at single nucleotide resolution in the hypoxic response element of the inducible VEGF gene and in known transcription factor binding sequences of the non-inducible actin promoter and the quiescent insulin promoter; (3) Test the hypothesis that introduction of a model oxidized base product at ROS-modified nucleotides within DNA response elements alters composition of the transcriptional complex forming in response to thrombin and PDGF; and (4) Determine whether introduction of a model oxidized base product at ROS-modified nucleotides within DNA response elements alters reporter gene expression in response to thrombin and PDGF. This research will provide proof-of-concept for a previously unappreciated mechanism by which ROS generated in physiological signaling regulate gene expression. In addition, these studies will confirm that integrity of specific nuclear genes is threatened by oxidative base modifications occurring in the context of physiological signaling. Such a finding could point to new pathways leading to somatic mutation and thus lead to a better understanding of cancer, aging, and other disorders wherein ROS are believed to play pathogenic roles.