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A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of β-catenin/GSK3 signaling
Klinghoffer R.A.2; Frazier J.2; Annis J.2; Berndt J.D.3; Roberts B.S.2; Arthur W.T.2; Lacson R.1; Zhang X.D.1; Ferrer M.1; Moon R.T.3; Cleary M.A.2
2009-09-03
Source PublicationPLoS ONE
ISSN19326203
Volume4Issue:9
AbstractThe multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/ threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease. © 2009 Klinghoffer et al.
DOI10.1371/journal.pone.0006892
URLView the original
Language英語
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Cited Times [WOS]:14   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionFaculty of Health Sciences
Affiliation1.Merck Research Laboratories
2.Rosetta Inpharmatics LLC
3.University of Washington School of Medicine
Recommended Citation
GB/T 7714
Klinghoffer R.A.,Frazier J.,Annis J.,et al. A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of β-catenin/GSK3 signaling[J]. PLoS ONE,2009,4(9).
APA Klinghoffer R.A..,Frazier J..,Annis J..,Berndt J.D..,Roberts B.S..,...&Cleary M.A..(2009).A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of β-catenin/GSK3 signaling.PLoS ONE,4(9).
MLA Klinghoffer R.A.,et al."A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of β-catenin/GSK3 signaling".PLoS ONE 4.9(2009).
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