Title |
A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling
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Published in |
PLOS ONE, September 2009
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DOI | 10.1371/journal.pone.0006892 |
Pubmed ID | |
Authors |
Richard A. Klinghoffer, Jason Frazier, James Annis, Jason D. Berndt, Brian S. Roberts, William T. Arthur, Raul Lacson, Xiaohua Douglas Zhang, Marc Ferrer, Randall T. Moon, Michele A. Cleary |
Abstract |
The multi-protein beta-catenin destruction complex tightly regulates beta-catenin protein levels by shuttling beta-catenin to the proteasome. Glycogen synthase kinase 3beta (GSK3beta), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark beta-catenin for ubiquitination and subsequent degradation. Because modulation of both beta-catenin and GSK3beta activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the beta-catenin/GSK3beta 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 beta-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3'oxime (BIO), a specific inhibitor of GSK3beta. 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 beta-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear beta-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3beta 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 (TNFalpha, 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 beta-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease. |
Mendeley readers
Geographical breakdown
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France | 1 | 3% |
Unknown | 26 | 90% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 11 | 38% |
Other | 4 | 14% |
Student > Bachelor | 2 | 7% |
Professor | 2 | 7% |
Student > Master | 2 | 7% |
Other | 6 | 21% |
Unknown | 2 | 7% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 6 | 21% |
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Chemistry | 3 | 10% |
Computer Science | 1 | 3% |
Other | 0 | 0% |
Unknown | 2 | 7% |