From the Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Sendai 980-8578,
From the Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Sendai 980-8578.
J Biol Chem. 2018 Jun 29;293(26):10186-10201. doi: 10.1074/jbc.M117.796631. Epub 2018 May 14.
Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NF-κB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-β pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway.
Toll 样受体 4（TLR4）是革兰氏阴性细菌细胞壁主要成分脂多糖（LPS）的不可或缺的免疫受体。在 LPS 刺激后，TLR4 将信号从细胞表面传递并在内体中内化。然而，TLR4 信号的空间调节尚不完全清楚。在这里，我们研究了 LPS 诱导的 TLR4 内化的机制，并阐明了细胞外 LPS 结合分子 LPS 结合蛋白（LBP）和甘油磷脂酰肌醇锚定蛋白（CD14）的作用。在血清存在的情况下，LPS 刺激表达 CD14 的细胞诱导 TLR4 内化，而抑制性抗 LBP mAb 阻断其内化。将 LBP 添加到无血清培养物中可将 LPS 诱导的 TLR4 内化恢复到与血清相当的水平。CD14 的分泌形式（sCD14）诱导内化，但需要比 LBP 高得多的浓度。抑制性抗 sCD14 mAb 对血清介导的内化无效。缺乏将 LPS 转移到 CD14 的域的 LBP 和具有降低 LPS 结合的 CD14 突变体均减弱了 TLR4 内化。因此，LBP 是 TLR4 内化所必需的重要血清分子，其将 LPS 转移到膜锚定 CD14（mCD14）是前提。LBP 诱导 LPS 刺激的 TBK1、IKKϵ 和 IRF3 的磷酸化，导致 IFN-β 的表达。然而，LPS 刺激的 NF-κB 或坏死性凋亡的晚期激活不受影响。总之，我们的结果表明，LBP 控制 LPS 诱导的 TLR4 内化，该内化诱导 TLR 衔接子 1（TRIF）依赖性 TBK1-IKKϵ-IRF3-IFN-β 途径的激活。综上所述，我们表明 LBP 介导的 LPS 转移到 mCD14 是血清依赖性 TLR4 内化和 TRIF 途径激活所必需的。
