Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-κB activation and IFN-β synthesis downstream of Toll-like receptor 3 and 4
Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-κB activation and IFN-β synthesis downstream of Toll-like receptor 3 and 4
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DOI：
10.1002/eji.200425801
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发表时间：
2005-07-01
影响因子：
5.4
通讯作者：
Willems, F
中科院分区：
文献类型：
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作者：
Aksoy, E;Vanden Berghe, W;Willems, F
已知磷酸肌肽3激酶（PI3K）调节toll样受体（TLR）介导的炎症反应，但其对TLR信号传导不同途径的影响尚不清楚。在这里，我们研究了药理抑制PI3K对Toll-IL-1受体结构域适配器诱导的ifn - β (TRIF)依赖信号传导的影响，该信号传导可诱导TLR3和TLR4下游的ifn - β基因表达。首先，发现用wortmannin或LY294002处理单核细胞来源的树突状细胞（DC）可以增强TLR3或TLR4参与时ifn - β的表达。在相同的DC激活模型中，PI3K抑制增加了NF-kappa B的dna结合活性，但不增加干扰素反应因子(IRF)-3，而IRF -3是tlr介导的ifn - β合成所需的关键转录因子。与此同时，wortmaninin处理的DC表现出I κ B激酶(IKK)- α / β磷酸化和I κ B- α降解水平的增强，并伴随nf - κ B核易位的增加。在稳定表达TLR3或TLR4的HEK 293T细胞中进行的实验证实，抑制PI3K活性可以增强nf - κ b依赖性启动子以及ifn - β启动子活性，而不会干扰正调节结构域III-I - i的转录。此外，wortmannin增强了HEK 293T细胞中TRIF过表达诱导的NF-kappa B活性，而催化活性PI3K过表达选择性地减弱了TRIF介导的NF-kappa B转录活性。最后，在共免疫沉淀实验中，我们发现PI3K与TRIF发生物理相互作用。我们得出结论，抑制PI3K活性可增强trf依赖性NF-kappa B活性，从而增加TLR3或TLR4配体诱导的ifn - β合成。
Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF) -dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappa B, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels Of I kappa B kinase (IKK)-alpha/beta phosphorylation and I kappa B-alpha degradation with a concomitant increase in NF-kappa B nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappa B-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappa B activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappa B transcriptional activity. Finally, in coimmunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-kappa B activity, and thereby increases IFN-beta synthesis elicited by TLR3 or TLR4 ligands.