Phospholipase Cγ-2 and Intracellular Calcium Are Required for Lipopolysaccharide-induced Toll-like Receptor 4 (TLR4) Endocytosis and Interferon Regulatory Factor 3 (IRF3) Activation
Phospholipase Cγ-2 and Intracellular Calcium Are Required for Lipopolysaccharide-induced Toll-like Receptor 4 (TLR4) Endocytosis and Interferon Regulatory Factor 3 (IRF3) Activation
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DOI：
10.1074/jbc.c111.328559
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发表时间：
2012-02-03
影响因子：
4.8
通讯作者：
David, Michael
中科院分区：
文献类型：
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作者：
Chiang, Chih-Yuan;Veckman, Ville;David, Michael
Toll样受体4（TLR 4）在TLR中是独特的，其使用多种接头蛋白，导致干扰素调节因子3（IRF 3）和核因子κ B（NF-κ B）途径的激活。以前的工作已经证明，TLR 4启动NF-κ B活化从质膜，但随后的TLR 4易位到内涵体是IRF 3活化所必需的。在这里，我们的特点是几个组件的信号通路，管理TLR 4易位和随后的IRF 3激活。我们发现，磷脂酶C γ 2（PLC γ 2）负责LPS诱导的肌醇1，4，5-三磷酸（IP 3）的生产和随后的钙（Ca 2+）的释放。在RAW 264.7巨噬细胞中，通过抑制剂阻断PLC γ 2功能或通过siRNA敲低PLC γ 2表达导致IRF 3减少，但增强NF-κ B活化。此外，来自PLC γ 2缺陷小鼠的骨髓源性巨噬细胞在LPS刺激后显示IRF 3磷酸化受损和IRF 3调节基因的表达。使用细胞分级分离，我们表明PLC γ 2-IP 3-Ca 2+信号级联是LPS刺激后TLR 4内吞所需的。总之，我们的研究结果描述了PLC γ 2-IP 3-Ca 2+级联在LPS诱导的先天性免疫应答途径中的新作用，其中细胞内Ca 2+的释放介导TLR 4运输和随后的IRF 3激活。
Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kappa B) pathways. Previous work has demonstrated that TLR4 initiates NF-kappa B activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C gamma 2 (PLC gamma 2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP3) production and subsequent calcium (Ca2+) release. Blockage of PLC gamma 2 function by inhibitors or knockdown of PLC gamma 2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-kappa B activation. In addition, bone marrow-derived macrophages from PLC gamma 2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLC gamma 2-IP3-Ca2+ signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLC gamma 2-IP3-Ca2+ cascade in the LPS-induced innate immune response pathway where release of intracellular Ca2+ mediates TLR4 trafficking and subsequent activation of IRF3.