Lung Cell Genomic Threats from Physiological Signals

描述（由申请人提供）：在缺氧信号传导中用作第二信使的活性氧物质（ROS）氧化修饰位于肺动脉内皮细胞（PAEC）VEGF基因中HIF-1 DNA识别序列3'末端的鸟嘌呤。 当在包含VEGF基因的缺氧反应元件的寡核苷酸中的缺氧修饰的鸟嘌呤处引入脱碱基位点时，该序列结合更多的HIF-1并产生更强的缺氧诱导的报告基因表达。 这些发现支持ROS参与低氧信号传导的新模型，其中ROS介导的关键DNA调控序列中的碱基氧化影响转录复合物的形成和伴随的基因表达。 如果这个模型具有普遍意义，那么由非缺氧刺激产生的ROS应该引起类似的氧化DNA修饰模式，并且这些应该导致转录复合物和基因表达的组成的可预测的改变。 因此，我们现在提出使用受体介导的激动剂凝血酶和PDGF的实验，它们在ROS依赖性信号通路方面不同，但共同参与HIF-1诱导VEGF表达。 我们将测试工作假设的关键要素，即在凝血酶和PDGF信号传导的背景下产生的ROS氧化修饰功能相关DNA序列内的特定核苷酸，从而改变转录复合物的组成和伴随的基因表达。 在PAEC中进行的研究将：（1）确定凝血酶和PDGF对诱导型VEGF基因以及非诱导型肌动蛋白基因和静止胰岛素基因的启动子和编码区中的氧化修饰的平衡密度的影响的动力学;（二）在诱导型VEGF基因的缺氧反应元件和已知的VEGF基因中，以单核苷酸分辨率绘制凝血酶和PDGF诱导的修饰图。（3）检验在DNA反应元件内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形成的转录复合物的组成的假设;和（4）确定在DNA应答元件内ROS修饰的核苷酸处引入模型氧化碱基产物是否改变了应答凝血酶和PDGF的报告基因表达。 这项研究将提供一个以前未被重视的机制，通过该机制，在生理信号中产生的ROS调节基因表达的概念验证。 此外，这些研究将证实特定核基因的完整性受到生理信号背景下发生的氧化碱基修饰的威胁。 这一发现可能指向导致体细胞突变的新途径，从而更好地理解癌症，衰老和其他疾病，其中ROS被认为发挥致病作用。

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.