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Toll-like receptor 2シグナルにおけるSrcファミリーキナーゼLckの役割

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Title: Toll-like receptor 2シグナルにおけるSrcファミリーキナーゼLckの役割
Authors: 谷詰, 直穂 Browse this author
大谷, 誠 Browse this author
長谷部, 晃 Browse this author →KAKEN DB
佐伯, 歩 Browse this author →KAKEN DB
戸塚, 靖則 Browse this author →KAKEN DB
鄭, 漢忠 Browse this author →KAKEN DB
柴田, 健一郎 Browse this author →KAKEN DB
Keywords: TLR2
Toll-like receptor 2
T cell
Issue Date: Sep-2014
Publisher: 北海道歯学会
Journal Title: 北海道歯学雑誌
Volume: 35
Issue: 1
Start Page: 8
End Page: 15
Abstract: Toll-like receptors (TLRs) are highly expressed on cells of the innate immune system, such as macrophages and dendritic cells, and have been studied most extensively in them. TLRs-mediated signals have been found to enhance antigen presentation by upregulating co-stimulatory molecule expression and promoting the production of pro-inflammatory cytokines. Thus, TLRs are also involved in induction of the acquired immunity in which T cells plays a central role. It has been demonstrated that T cells themselves express TLRs and TLRs also control T cell responses directly by affecting clonal expansion of antigen-specific T cells and enhancing proliferation and survival of T cells and cytokine production by them. However, details on roles of TLRs in T cell responses still remain unknown. Therefore, this study was designed to investigate the roles of TLRs in the T-cell receptor (TCR)-mediated activation of T cells. First of all, the effects of the TLR2 ligand FSL-1 on the TCR-mediated activation of CD4+ T cells derived from C57BL/6 mice induced by anti-CD3ε antibody were investigated. It was found that FSL-1 exhibited synergic effects on the activation. In order to clarify the mechanism on the synergic effects, a crosstalk between the Src family kinase Lck, which plays important roles in the TCR-mediated activation, and TLR2 was investigated. Unexpectedly, overexpression of Lck in HEK293 cells expressing TLR2 (HEK293/TLR2 cells) significantly reduced the TLR2-mediated NF-κB activation induced by stimulation with the TLR2 ligand FSL-1. In addition, deletion mutants of SH3 and SH2 domains of Lck as well as the constitutively active mutant (LckY505F) attenuated the NF-κB activation more strongly than the wild-type Lck (Lckwt), whereas deletion mutants of the kinase domain and the kinase-inactive mutants (LckK273R and LckY394F) significantly reversed the attenuation by the Lckwt. These results suggest that the kinase activity of Lck partially is involved in the attenuation of TLR2 signal by Lck. Furthermore, we found that TLR2 was associated with Lckwt through its kinase domain irrespective of the FSL-1 stimulation. Thus, this study is the first to report that the Src family kinase Lck suppresses the TLR2-mediated signal, although it was not able to explain the mechanism by which TLR2 signal synergistically enhances the TCR-mediated activation of T cells.
TLRは自然免疫系で病原体の侵入を感知するだけでなく,T細胞が中心的な役割を果たす獲得免疫の誘導においても重要な役割を果たしている.これまで,T細胞自身もTLRを発現していることが明らかになっているが,T細胞の機能におけるTLRの役割はいまだ不明な点が多く残されている.本研究では,T細胞受容体を介したT細胞の活性化で重要な役割を果たしているSrcファミリーキナーゼのひとつであるLckに注目し,TLR2とLckのクロストークを検討した.  C57BL/6マウスのCD4陽性T細胞を抗CD3ε抗体およびTLR2のリガンドであるリポペプチドFSL-1で刺激すると,抗CD3ε抗体またはFSL-1で単独刺激したものと比べ,共刺激したものではCD4陽性T細胞の増殖が相乗的に増加した.ヒト胎児腎細胞由来のHEK293細胞にTLR2遺伝子とLck野生型遺伝子をそれぞれ単独,あるいは同時に導入し,NF-κBの活性化を調べた.その結果,予想に反して,TLR2遺伝子を単独導入したものと比較し,TLR2遺伝子とLck野生型遺伝子を同時に導入したものでは,FSL-1刺激によるNF-κBの活性化が有意に抑制されることがわかった.さらに,Lckの各種欠失変異体(キナーゼ不活型点変異体K273R,Y394F,恒常的活性化型点変異体Y505F)を作製し,これらの遺伝子をHEK293細胞に導入し,FSL-1刺激によるNF-κBの活性化を調べたところ,キナーゼ活性がLckによるTLR2シグナルの抑制に部分的に関与するという結果を得た.また,TLR2とLckとの会合が免疫沈降法で確認された.  以上の結果から,TCRを介したT細胞の活性化に関与するLckがTLR2と会合し,下流のシグナルを負に制御することが示唆された.
Type: article
Appears in Collections:北海道歯学雑誌 = Hokkaido Journal of Dental Science > 第35巻 第1号

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