Nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for proteasome inhibitor-mediated cardioprotection.
OBJECTIVE
We have shown previously that non-toxic inhibition of the ubiquitin-proteasome system upregulates antioxidative defence mechanisms and protects endothelial cells from oxidative stress. Here, we have addressed the question whether the induction of antioxidative enzymes contributes to cardioprotection by non-toxic proteasome inhibition.


RESULTS
Treatment with 0.5 micromol/L MG132 for 48 h proved to be non-toxic and protected neonatal rat cardiac myocytes against H(2)O(2)-mediated oxidative stress in lactate dehydrogenase assays. This correlated with reduced levels of intracellular reactive oxygen species as determined by loading myocytes with dichlorofluorescein. Immunoblots showed significant upregulation of superoxide dismutase 1 (SOD1), haem oxygenase 1, and catalase upon proteasome inhibition. Luciferase assays using a reporter driven by the SOD1 promoter revealed proteasome inhibitor-mediated induction of luciferase activity. Deletion and mutation analyses identified an antioxidant response element (ARE) in the SOD1 promoter to be not only essential but also sufficient for transcriptional upregulation by proteasome inhibition. An essential role for the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)-which was stabilized by proteasome inhibition-in ARE-mediated transcriptional activation was revealed in cardiac myocytes from Nrf2 wild-type and knockout mice: proteasome inhibition upregulated antioxidative enzymes and conferred protection against H(2)O(2)-mediated oxidative stress in Nrf2 wild-type cells. In contrast, the induction of antioxidative enzymes and cytoprotection were completely abolished in cardiac myocytes from Nrf2 knockout mice.


CONCLUSIONS
Non-toxic proteasome inhibition upregulates antioxidative enzymes via an Nrf2-dependent transcriptional activation of AREs and confers cardioprotection.
Dreger
Henryk
H
Medizinische Klinik für Kardiologie und Angiologie, Campus Mitte, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Westphal
Kera
K
Weller
Andrea
A
Baumann
Gert
G
Stangl
Verena
V
Meiners
Silke
S
Stangl
Karl
K
D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000378:metabolism*; D001665:Binding Sites; D002317:Cardiovascular Agents / Q000494:pharmacology*; D002318:Cardiovascular Diseases / Q000201:enzymology / Q000517:prevention & control; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D051267:NF-E2-Related Factor 2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D020218:Response Elements; D013482:Superoxide Dismutase / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D000975:Antioxidants; D002317:Cardiovascular Agents; D007976:Leupeptins; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; C495637:Nfe2l2 protein, rat; D016877:Oxidants; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D051547:Heme Oxygenase-1; C000606291:Sod1 protein, mouse; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde