A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling.
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.
Klinghoffer
Richard A
RA
Rosetta Inpharmatics, LLC, Seattle, Washington, United States of America. richard_klinghoffer@merck.com
Frazier
Jason
J
Annis
James
J
Berndt
Jason D
JD
Roberts
Brian S
BS
Arthur
William T
WT
Lacson
Raul
R
Zhang
Xiaohua Douglas
XD
Ferrer
Marc
M
Moon
Randall T
RT
Cleary
Michele A
MA
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002460:Cell Line; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology; D008954:Models, Biological; D010091:Oximes / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism*
C483321:6-bromoindirubin-3'-oxime; D000893:Anti-Inflammatory Agents; D007211:Indoles; D015850:Interleukin-6; D010091:Oximes; D014409:Tumor Necrosis Factor-alpha; D051176:beta Catenin; D018664:Interleukin-12; D013762:Tetrahydrofolate Dehydrogenase; D038362:Glycogen Synthase Kinase 3; D008727:Methotrexate