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PLoS One. 2009 Sep 3;4(9):e6892. doi: 10.1371/journal.pone.0006892.

A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling.

Author information

1
Rosetta Inpharmatics, LLC, Seattle, Washington, United States of America. richard_klinghoffer@merck.com

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.

PMID:
19727391
PMCID:
PMC2731218
DOI:
10.1371/journal.pone.0006892
[Indexed for MEDLINE]
Free PMC Article
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