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.
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