NRF2 Regulates Hyperoxia-induced NOX4 expression in Human Lung Endothelium: Identification of functional antioxidant response elements on NOX4 promoter

摘要

Reactive oxygen species (ROS) generated by vascular endothelial and smooth muscle cells contribute to the development and progression of vascular diseases. We have recently shown that hyperoxia enhances NADPH Oxidase 4 (NOX4) expression, which regulates lung endothelial cell migration and angiogenesis. Regulation of NOX4 is poorly understood in the vasculature. The objective of this study is to identify transcriptional factor(s) involved in regulation of endothelial NOX4. We found that hyperoxia induced NOX4 expression was markedly reduced in Nrf2^-/- mice, compared to Nrf2^+/+ mice. Exposure of human lung microvascular endothelial cells (HLMVECs) to hyperoxia stimulated NRF2 translocation from the cytoplasm to the nucleus and increased NOX4 expression. Knock down of NRF2 expression using a siRNA approach attenuated basal NOX4 expression; however, it enhanced superoxide/ROS generation under both normoxia and hyperoxia. In silico analysis revealed presence of at least three consensus sequences for the antioxidant response element (ARE) in the promoter region of NOX4. In transient transfections, hyperoxia stimulated NOX4promoter activity in HLMVECs, and deletion of -438 to -458 and -619 to -636 sequences markedly reduced hyperoxia-stimulated NOX4 promoter activation. ChIP analysis revealed an enhanced recruitment of NRF2 to endogenous NOX4 promoter spanning these two AREs following hyperoxic insult. Collectively, these results demonstrate, for the first time, a novel role of NRF2 in regulating hyperoxia-induced NOX4 transcription via AREs in lung endothelium.