Hypoxia is an important stimulus for hypertensive pulmonary vascular remodeling. Like many stimuli promoting cell growth and differentiation, hypoxia seems to use reactive oxygen species (ROS) as second messengers. Among the observations supporting a role for ROS in hypoxic signaling is our discovery that hypoxia causes formation of oxidized bases and basic sites in the promoter of the VEGF gene, especially in the HIF-1 DNA recognition sequence required for hypoxia-induced gene expression. The long-term objective of this project is to understand the biological significance of the hypoxia-induced oxidative nuclear DNA modifications. During the initial award period, we determined that the hypoxia-related base modifications in the VEGF promoter might alter transcription factor binding to the hypoxic response element. Whereas a wild-type VEGF promoter sequence bound HIF-1, a sequence harboring an abasic site at the hypoxia-modified nucleotide in the HIF-1 DNA binding sequence bound not only HIF-1, but also Ref-l/Ape. Ref-l/Ape is a multifunctional protein exhibiting DNA binding and repair activity and, importantly, the ability to bind and redox activate HIF-1 along with other components of the hypoxia-inducible transcriptional complex. We also found that the hypoxia-related abasic site was functionally significant; hypoxic pulmonary artery endothelial cells (PAECs) transfected with an abasically modified VEGF promoter-reporter construct displayed more robust expression than cells transfected with the wild-type promoter-reporter construct. The proposed research will provide proof-of-concept for the novel hypothesis that the hypoxia-induced oxidative modification within the hypoxic response element serves to localize Ref-l/Ape binding, which then functions as a scaffold to optimize assembly and activation of the transcriptional complex leading to gene expression. Studies conducted in cultured rat PAECs and focusing on the VEGF gene as a model for other hypoxia-inducible genes will determine whether: (1) The position of the abasic site within the hypoxic response element is an important determinant of HIF-1 and Ref-l/Ape binding to DNA and assembly of other components of the transcriptional complex; (2) The DNA binding activity of Ref-l/Ape and its ability to redox activate HIF-1 as well as other co-activator integrator proteins are critical and independent factors governing assembly of the hypoxia-inducible transcriptional complex; and (3) The position of the hypoxia-related abasic site as well as the DNA binding, repair and redox-mediated activities of Ref-l/Ape are important determinants of hypoxia-induced VEGF gene expression. The outcome of these studies will provide new information about regulation of gene expression in hypoxia and may also point to new mechanisms of somatic mutation and thus lead to a better understanding of cancer, aging, and various pulmonary disorders where ROS are believed to play a pathogenic role.
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