RNAi Screen for NRF2 Inducers Identifies Targets That Rescue Primary Lung Epithelial Cells from Cigarette Smoke Induced Radical Stress.
Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism.
MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Center, University of Dundee, Dow Street, Dundee, DD1 5EH, United Kingdom.
Frances-Rose
FR
Schumacher
Cenix BioScience GmbH, 01307, Dresden, Germany.
Steffen
S
Schubert
Cenix BioScience GmbH, 01307, Dresden, Germany.
Michael
M
Hannus
Cenix BioScience GmbH, 01307, Dresden, Germany.
Birte
B
Sönnichsen
Boehringer Ingelheim Pharma GmbH & Co. KG, Research Germany, 8400, Biberach a. d. Riss, Germany.
Carina
C
Ittrich
Boehringer Ingelheim Pharma GmbH & Co. KG, Research Germany, 8400, Biberach a. d. Riss, Germany.
Stefan
S
Kreideweiss
MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Center, University of Dundee, Dow Street, Dundee, DD1 5EH, United Kingdom.
Thimo
http://orcid.org/0000-0003-0700-6993
T
Kurz
Boehringer Ingelheim Pharma GmbH & Co. KG, Research Germany, 8400, Biberach a. d. Riss, Germany.
Jörg F
JF
Rippmann
D001980:Bronchi / Q000166:cytology* / Q000378:metabolism; D016923:Cell Death; D002470:Cell Survival; D002478:Cells, Cultured; D055808:Drug Discovery; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000235:genetics; D058990:Molecular Targeted Therapy; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000235:genetics*; D018384:Oxidative Stress; D029424:Pulmonary Disease, Chronic Obstructive / Q000188:drug therapy / Q000235:genetics; D034622:RNA Interference; D020545:Respiratory Mucosa / Q000166:cytology* / Q000378:metabolism; D012907:Smoking / Q000009:adverse effects*; D015854:Up-Regulation
C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human