Aims: Increased levels of reactive oxygen species cause oxidative stress and severely damage lipids, proteins, and DNA. We have previously shown that partial proteasome inhibition induces an antioxidative gene pattern in endothelial cells. Here, we elucidate the mechanisms of proteasome inhibitor-mediated upregulation of antioxidative enzymes and cytoprotection.
Methods and results: Non-toxic proteasome inhibition upregulated mRNA and protein expression of superoxide dismutase 1 (SOD1) and haem oxygenase 1 (HO1) in several human endothelial and vascular smooth muscle cell types. Transcriptional activation of these enzymes was shown by inhibition of RNA polymerase II and nuclear run-on assays. Transfection of endothelial cells with luciferase reporter constructs revealed that upregulation can be largely confined to an antioxidant response element (ARE), which proved to be sufficient for transcriptional activation of SOD1 and HO1. Co-transfection studies and bandshift analyses confirmed binding of the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)-which was stabilized by proteasome inhibition as shown by immunoblots-to the ARE site of HO1. Experiments with aortic endothelial and smooth muscle cells from Nrf2 wild-type and knockout mice revealed an essential role of Nrf2: in wild-type cells, proteasome inhibitor-mediated induction of SOD1 and HO1 was accompanied by protection of vascular cells against oxidative stress as determined by lactate dehydrogenase release assays. In contrast, proteasome inhibitor-mediated induction of antioxidative enzymes and cytoprotection were completely lost in cells from Nrf2 knockout mice.
Conclusion: Nrf2-dependent transcriptional activation of antioxidative enzymes is crucial for proteasome inhibitor-mediated protection of vascular cells against oxidative stress.