Sakuma Chisato, Sato Mitsuru, Takenouchi Takato, Kitani Hiroshi
Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
Mol Immunol. 2015 Feb;63(2):328-36. doi: 10.1016/j.molimm.2014.08.004. Epub 2014 Sep 10.
Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1β and phosphorylation of inhibitor of κB α/β (IKKα/β) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages.
威斯科特-奥尔德里奇综合征蛋白（WASP）是免疫细胞中的一种衔接分子。最近，我们发现WASP通过布鲁顿酪氨酸激酶（Btk）与WASP N端结构域的结合参与巨噬细胞中的脂多糖-TLR4信号传导。Btk已被证明在几种TLR的信号传导中起重要作用，并调节巨噬细胞中的炎症反应。在本研究中，我们通过使用来自表达抗WASP N端结构域单链可变片段（scFv）或VL单结构域细胞内抗体的转基因（Tg）小鼠的骨髓源性巨噬细胞系，评估了Btk与WASP之间的相互作用在TLR2信号传导中的重要性。在这种Tg骨髓源性巨噬细胞中，WASP N端结构域中结合位点的掩盖强烈抑制了WASP与Btk之间的特异性相互作用。在用TLR2配体刺激后，TNF-α、IL-6和IL-1β的基因表达以及κBα/β抑制剂（IKKα/β）和核因子（NF）-κB的磷酸化受到损害。此外，如脂多糖刺激后WASP酪氨酸磷酸化受损所示，在Tg骨髓源性巨噬细胞中，TLR2配体刺激后WASP的酪氨酸磷酸化受到严重抑制。这些结果强烈表明，WASP N端结构域与Btk之间的关联在巨噬细胞的TLR2信号通路中起重要作用。
