Smo-Sufu调控TLR4信号通路及败血症的机制与功能研究

Sepsis, characterized by a system-wide dysregulation of inflammation responses following primary bacterial infection, is one of the leading causes of death in intensive care unit worldwide. TLR4 signaling plays an important role in the regulating the pathogenesis of sepsis, but the upstream regulatory mechanisms of TLR4 signaling are not fully understood. Our previous findings indicate that the canonical Hh signaling, one of the most conserved signaling pathways, has no effects with the development of sepsis, but interestingly a noncanonical Hh signaling mediated by Sufu and Smo plays a critical role in the pathogenesis of sepsis by affecting the LPS-induced TLR4 signaling activation, inflammation and animal survival. It’s worth noting that like the opposite role of Sufu and Smo to Hh signaling pathway, those two proteins also play a contrary role in sepsis development. Sufu and Smo demonstrate their inhibition and promotion role in TLR4 and macrophage activation, and LPS-induced septic death. By biochemistry assays, we found that Sufu can interact with TRAF6，a key effecter of TLR4 signaling, and block its K63-polyUb chain modification, which is essential for NF-kB activation downstream of TLR4 signaling. Interestingly, Smo can inhibit the interaction between Sufu and TRAF6, resulting in rescued NF-kB activation. According to these findings, we hypothesized that Sufu and Smo mediated noncanonical Hh signaling can affect TLR4 signaling activation, NF-kB activation and severity of sepsis by contrarily regulating K63-polyUb chain modification of TRAF6. To test our hypothesis, we will further examine the role of Sufu and Smo in sepsis pathogenesis, TLR4 and NF-kB activation, dissect the underlying mechanism by which Sufu and Smo contrarily regulate TLR4 signaling and result in opposite sepsis outcomes, and evaluate the therapeutic potential by targeting Sufu and/or Smo in sepsis treatment. This study will provide us a solid theoretical base to eventually comprehend the transduction and regulation of Hh signaling, and develop novel and effective therapeutic strategies for Hh signaling related diseases.

败血症是感染引起的系统性炎症反应综合症，严重威胁人类健康。TLR4通路在败血症发生发展中起关键作用。Hedgehog（Hh）通路作为最保守的信号通路之一在败血症中的功能及机制尚不清楚。我们前期研究发现由Hh通路关键因子Sufu和Smo介导的非经典Hh通路能调控败血症发生发展。具体表现为Sufu或Smo能分别抑制或促进败血症时TLR4通路激活，炎症反应及死亡率。生化实验表明Sufu结合TLR4通路关键效应子TRAF6，抑制其K63-泛素链修饰，而Smo则能解除Sufu与TRAF6结合。以上结果提示Sufu和Smo通过TRAF6影响TLR4通路激活并对败血症起调控作用。本项目拟从Smo调控Sufu-TRAF6结合这一表型入手，开展Smo-Sufu调控TLR4通路的分子机制研究，并进一步阐明它们在败血症发生发展中的生物学功能及作用机制，为理解Hh通路信号传导机制及相关疾病诊治提供坚实的理论基础。

败血症是感染引起的系统性炎症反应综合症，严重威胁人类健康。TLR4通路在败血症发生发展中起关键作用。Hedgehog（Hh）通路作为最保守的信号通路之一在败血症中的功能及机制尚不清楚。我们前期研究发现Hh通路关键因子Sufu和Smo能调控败血症发生发展。具体表现为Sufu或Smo能分别抑制或促进败血症时TLR4通路激活，炎症反应及死亡率。生化实验表明Sufu结合TLR4通路关键效应子TRAF6，抑制其K63-泛素链修饰，而Smo则能解除Sufu与TRAF6结合。以上结果提示Sufu和Smo通过TRAF6影响TLR4通路激活并对败血症起调控作用。在前期研究基础上，本项目着重开展了Smo-Sufu调控TLR4通路的分子机制研究，并进一步阐明它们在败血症进展中的生物学功能及作用机制，取得以下重要结果：1）利用经典Hh通路的激活剂SAG或抑制剂Cyclopamine，结合LPS诱导的小鼠败血症死亡模型和TLR4信号通路的激活，我们证明了经典Hh通路的激活或抑制不影响LPS介导的小鼠败血症死亡和TLR4信号通路的激活；2）利用髓系细胞特异性Sufu或Smo敲除小鼠，结合LPS诱导的小鼠败血症死亡模型，我们证明髓系细胞特异性Sufu或Smo缺失能分别恶化或改善LPS诱导的小鼠败血症死亡；3）利用响应TLR4信号通路的NF-kB荧光素酶报告基因系统，我们证明了SUFU能剂量依赖地抑制TLR4信号通路的激活，而SMO能剂量依赖地解除SUFU对TLR4通路的抑制；4）通过基因工程和生化实验，我们证明了SUFU能结合TLR4信号通路的关键效应因子TRAF6，抑制TRAF6激活所必需的K63-泛素链修饰，进而抑制TLR4信号通路的激活。我们进一步证明SMO能抑制TRAF6和SUFU的结合，进而解除SUFU对TRAF6和TLR4信号通路的抑制；5）以TRAF6的K63-泛素链修饰为切入点，我们进一步证明SUFU与TRAF6的结合能促进A20（一种泛素编辑酶）与TRAF6的结合，降低TRAF6的K63-泛素链修饰水平，而SMO过表达则能抑制A20与TRAF6的结合，提高TRAF6的K63-泛素链修饰水平；6）利用持续失活或激活的SUFU及SMO突变体，我们证明SUFU和SMO对TRAF6及TLR4通路的调控不依赖于它们的活性。以上研究结果为研发基于非经典Hh通路的败血症靶向药物提供了理论基础