Abstract 11900: The Main Mediator of Cellular Adaptation to Redox Stress NRF2 is Regulated by NADPH Oxidase NOX4 in Endothelial Cells: A Novel Feedback Mechanism
The NADPH oxidase NOX4 is a major source of reactive oxygen species (ROS) in endothelial cells. ROS formation is downregulated by long-term exposure to hemodynamic forces. A main mediator of cellular adaptation to oxidative stress is the transcription factor NRF2. We could recently show that downregulation of NRF2 induces NOX4. However, the underlying molecular mechanism is not well understood. In this study, we analyzed the cross-talk between NOX4 and NRF2 in human endothelial cells (HUVEC). Transduction of HUVEC with lentiviral particles containing scrambled shRNA, shNRF2, or shNOX4 did not alter cell viability and proliferation. However, regulation of NOX4 by transcription factor NRF2 was depending on the cellular proliferation and/or status of confluence. Transduction with shNRF2 induced NOX4 in proliferating HUVEC and human microvascular endothelial cells (HMEC-1). To verify these data on transcriptional level, we cloned 1490 bp 5'-regulatory sequence of human NOX4 gene and 5'-terminally deleted fragments. Using dual-luciferase reporter assay, all NOX4 promoter constructs showed high basal activity in endothelial cells. Transduction with shNRF2 increased NOX4 promoter activity significantly. Long-term exposure of HUVEC to arterial laminar shear stress (30 dyne/cm2) induced elongation of endothelial cells in the direction of the flow. Lentiviral downregulation of NOX4 using shNOX4 inhibited this elongation of cell shape in response to flow. In contrast, transduction with shNRF2 increased cell elongation. In addition, application of shear stress resulted in significant downregulation of NOX4 and 2-fold upregulation of NRF2 and NRF2 target genes NQO-1 and HO-1. Downregulation of NRF2 using shNRF2 caused inhibition of shear stress-dependent induction of NRF2, NQO1 and HO-1. NOX4 downregulation in response to laminar shear stress was unaffected by shNRF2. However, transduction of HUVEC with shNOX4 inhibited flow-dependent upregulation of NRF2. In conclusion, ROS production by NOX4 might control the antioxidative capacity and cellular adaptation to hemodynamic forces. Our data suggest a novel feedback mechanism involving the major NADPH oxidase isoform NOX4 and the transcription factor NRF2 in human endothelial cells.
- © 2011 by American Heart Association, Inc.