Abstract 15323: Hypoxia Upregulates NADPH Oxidase 4 by a HIF-Independent Mechanism in Endothelial Cells
Endothelial dysfunction and reactive oxygen species (ROS) play an important role in hypoxia-induced vascular disorders. One of the most important sources of ROS in the vasculature is NADPH oxidase 4 (NOX4). We could previously show that NOX4 is the main NOX isoform in endothelial cells. However, the mechanism of NOX4 regulation under hypoxia in endothelial cells remains to be elucidated. First, we found a significant upregulation of NOX4 mRNA level after 24 h of hypoxia (1 % O2) in human umbilical vein endothelial cells (HUVEC). H2O2 production of HUVEC was 2-fold elevated after hypoxia. Lentiviral downregulation via shNOX4 restored the peroxide levels to normoxic values, suggesting a crucial role of NOX4 in this context. Next, we studied the role of hypoxia-inducible factor (HIF) 1α. Hypoxia increased HIF-1α expression on protein level. We cloned NOX4 promoter deletion constructs containing the first 1251 bp upstream from the transcription start site and the first 239 bp of exon 1. The -119/+239 NOX4 promoter construct had an increased basal activity compared to the promoterless vector using dual-luciferase assay. Full-length and partially deleted NOX4 promoter constructs showed a comparable activity under hypoxic and normoxic conditions in highly transfectable human microvascular cells (HMEC-1), suggesting that the recently discovered hypoxia responsive element (HRE) in the NOX4 promoter may be non-functional in endothelial cells. Upregulation of the VEGF promoter activity and of a hypoxia responsive elements (HRE3x) containing promoter have been used as positive controls. We further overexpressed HIF-1α in HEK 293T cells and stabilized HIF-1α under normoxic conditions by treatment with DMOG (24 h, 10 mM) in HUVEC. In addition, we blocked the HIF-pathway by adenoviral overexpression of a HIF-2α dominant-negative mutant in HUVEC. None of these experiments resulted in a significant change of NOX4 on the mRNA or promoter level. In contrast, stimulation of endothelial cells with Actinomycin D indicates increased stability of the NOX4 mRNA under hypoxic conditions. In conclusion, our data strongly suggest a HIF-independent increase in mRNA stability as major mechanism of NOX4 upregulation under hypoxic conditions in human endothelial cells.
- © 2012 by American Heart Association, Inc.