Application of hemodynamic forces on endothelial cells affects the formation of reactive oxygen species (ROS). Major cellular sources of ROS are NADPH oxidase (NOX) complexes. We could show that NOX4 is the major NOX isoform in endothelial cells. NOX4 expression is downregulated by long-term exposure to hemodynamic forces like laminar shear stress or cyclic strain. The transcription factor NRF2 is the main mediator of cellular adaptation to redox stress. Recent data from our laboratory support an induction of NOX4 after downregulation of NRF2, but the underlying molecular mechanism is not well understood.
In this study, we analyzed the cross-talk between NOX4 and NRF2 in human endothelial cells. Transduction of HUVEC with lentiviral particles containing scrambled shRNA, shNRF2, or shNOX4 did not result in alterations of cell viability or cell proliferation. However, regulation of NOX4 by transcription factor NRF2 was dependent on the cellular proliferation and/or status of confluence. Transduction with shNRF2 induced NOX4 in non-proliferating HUVEC and human microvascular endothelial cells (HMEC-1). Next, we cloned 1490 bp of the 5'-regulatory sequence and additional terminal deletions of the human NOX4 gene. We could show a high basal activity of the cloned NOX4 promoter constructs by dual-luciferase reporter assay 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. Application of shear stress resulted in significant downregulation of NOX4 and twofold upregulation of NRF2 and NRF2 target genes NQO-1 and HO-1. Downregulation of NRF2 by shNRF2 inhibited shear stress-dependent induction of NRF2, NQO1 and HO-1. In contrast, NOX4 downregulation in response to laminar shear stress was not affected by NRF2 downregulation. Finally, transduction of HUVEC with shNOX4 inhibited the flow-dependent upregulation of NRF2, suggesting a novel feedback mechanism between NOX4 and NRF2.
In conclusion, our data suggest a novel feedback mechanism involving the NADPH oxidase isoform NOX4 and the transcription factor NRF2 in human endothelial cells. Furthermore, NOX4 might control basal ROS production and antioxidative capacity in endothelial cells.