A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling.
A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling.
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DOI：
10.1371/journal.pone.0006892
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发表时间：
2009-09-03
期刊：
影响因子：
3.7
通讯作者：
Cleary MA
中科院分区：
文献类型：
--
作者：
Klinghoffer RA;Frazier J;Annis J;Berndt JD;Roberts BS;Arthur WT;Lacson R;Zhang XD;Ferrer M;Moon RT;Cleary MA
多蛋白质β-连环蛋白破坏复合物通过将β-连环蛋白穿梭于蛋白酶体来紧密调节β-连环蛋白蛋白水平。糖原合成酶激酶3β（Glycogen synthase kinase 3 β，GSK 3 β）是破坏复合物中的一个关键丝氨酸/苏氨酸激酶，负责几个磷酸化事件，标志着β-catenin的泛素化和随后的降解。由于β-catenin和GSK 3 β活性的调节可能对治疗疾病具有重要意义，因此需要完全了解调节β-catenin/GSK 3 β相互作用的机制。我们筛选了表达靶向5，201个人类可药用基因的小发夹RNA（shRNAs）的阵列慢病毒文库，以沉默激活β-连环蛋白通路报告基因（BAR）与6-溴靛玉红-3 ′肟（BIO）协同作用的事件，6-溴靛玉红-3 ′肟是GSK 3 β的特异性抑制剂。最高筛选命中包括靶向二氢叶酸还原酶（DHFR）的shRNA，其是抗炎化合物甲氨蝶呤的靶标。将细胞暴露于BIO加甲氨蝶呤导致BAR活性的有效协同活化、GSK 3特异性位点处β-连环蛋白磷酸化的减少以及核β-连环蛋白的积累。此外，观察到的协同作用与抑制GSK 3 β磷酸化相关，并在抑制磷脂酰肌醇3-激酶（PI 3 K）后被中和。将这些观察结果与炎症联系起来，我们还观察到脂多糖（LPS）诱导的促炎细胞因子（TNFα、IL-6和IL-12）产生的协同抑制，以及暴露于GSK 3抑制剂和甲氨蝶呤的外周血单核细胞中抗炎细胞因子IL-10产生的增加。我们的数据确立了DHFR作为β-连环蛋白和GSK 3信号传导的新型调节剂，并对临床使用甲氨蝶呤和GSK 3抑制剂治疗炎症性疾病提出了几点启示。
The 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.
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影响因子：
64.5
作者：
GRODEN, J;THLIVERIS, A;WHITE, R
通讯作者：
WHITE, R
DOI：
10.1002/art.23716
发表时间：
2008-06-15
期刊：
ARTHRITIS & RHEUMATISM-ARTHRITIS CARE & RESEARCH
影响因子：
--
作者：
Buchbinder, Rachelle;Barber, Melissa;Jolley, Damien
通讯作者：
Jolley, Damien
影响因子：
11.4
作者：
Aberle, H;Bauer, A;Kemler, R
通讯作者：
Kemler, R
影响因子：
9.7
作者：
Jaszewski, R;Millar, B;Majumdar, APN
通讯作者：
Majumdar, APN
影响因子：
30.8
作者：
Rubinson, DA;Dillon, CP;Van Parijs, L
通讯作者：
Van Parijs, L