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- Title
A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β- Catenin/GSK3 Signaling.
- Authors
Klinghoffer, Richard A.; Frazier, Jason; Annis, James; Berndt, Jason D.; Roberts, Brian S.; Arthur, William T.; Lacson, Raul; Zhang, Xiaohua Douglas; Ferrer, Marc; Moon, Randall T.; Cleary, Michele A.
- Abstract
The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3b (GSK3b), 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 GSK3b activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3b 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 bcatenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3b. 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 GSK3b 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.
- Subjects
TETRAHYDROFOLATE dehydrogenase; GLYCOGEN synthase kinase-3; PHOSPHORYLATION; RNA; CELLS; METHOTREXATE
- Publication
PLoS ONE, 2009, Vol 4, Issue 9, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0006892