炎症微环境下TWEAK/Fn14信号调控角质形成细胞双向命运的分子机制与初步应用

TWEAK/Fn14 pathway plays an important role in regional inflammation though its precise mechanism remains unclear. Previously, we demonstrated that TWEAK/Fn14 signaling induces either apoptosis or proliferation of keratinocytes depending on the murine inflammatory models. Such bidirectional modulation of cell fate correlates with inflammatory microenvironment. Therefore, this proposal is aimed to study the effect of cytokines and phenotype-related proteins on the expression of TNF receptors (TNFR) type 1 and 2 in the keratinocytes cultured in inflammatory models that mimic psoriatic, atopic dermatitis or lupus-like microenvironments. The formation and ubiquitination of TRAF2/c-IAP1 complexes will be observed in cells upon TWEAK stimulation. Also, both siRNA and plasmid vector transfection will be adopted for regulating tumor necrosis factor-associated factor 2 (TRAF2) expression while the changes of TNFR subtypes and cell cycle-related Ras/ERK and cyclin/CDK signals are to be determined. Moreover, the two conjugates of Fn14-R2Fab and TWEAK-MV1 that target TNFR2 and TRAF2/c-IAP1 respectively, will be constructed by selecting hydridoma clones and expressing eukaryotic plasmid vector. The influence of these conjugates on keratinocytes in inflammatory models will be analyzed accordingly. Finally, the therapeutic and side effects of two conjugates will be monitored on the K14-VEGF transgenic mice. The results from this study may not only elucidate the roles of TNFR1/2 shift and Fn14-TRAF2-TNFR axis in the bidirectional TWEAK modulation of keratinocyte fate, but also contribute to the development of novel therapeutic approaches for treating skin diseases such as psoriasis.

TWEAK/Fn14信号在组织炎症中发挥关键作用，但确切机制不清楚。我们前期研究提示，TWEAK/Fn14信号激活在不同炎症疾病动物模型中诱导角质形成细胞双向命运（增殖或凋亡）。因此本研究拟建立几种典型炎症微环境体外模型，探讨有关细胞因子对角质形成细胞表达TNFR亚型的影响；观察TWEAK刺激后TRAF2/c-IAP1复合物形成与泛素化水平；利用siRNA转染与质粒载体技术，分析TRAF2变化对TNFR表达及细胞周期相关蛋白Ras/ERK、cyclin/CDK等的作用。同时，还将利用杂交瘤筛选与真核表达技术，构建双效的Fn14-R2Fab及TWEAK-MV1耦合物，观察对体外炎症模型中角质形成细胞命运的纠偏效应，以及对K14-VEGF转基因小鼠的治疗作用。本研究将揭示TNFR1/2漂移与Fn14-TRAF2-TNFR效应轴介导TWEAK调控细胞双向命运的分子机制，并为治疗银屑病找到新靶点。

项目背景：许多慢性皮肤病包括银屑病、红斑狼疮、特应性皮炎等，都有严重炎症反应引起的微结构损伤。我们既往研究提示，肿瘤坏死因子样弱凋亡诱导蛋白（TWEAK）/成纤维细胞生长因子诱导因子14（Fn14）信号在皮肤病的炎症反应中发挥重要作用，但诱导微结构损伤的具体机制不清楚。.主要研究内容：分析银屑病、红斑狼疮、特应性皮炎、鳞状细胞癌患者及动物模型的病变组织，检测TWEAK/Fn14信号激活相关蛋白表达；构建相关疾病的体外炎症微环境模型，分析关键炎症因子对TWEAK/Fn14信号调控细胞周期的影响、cIAP1拮抗剂MV1对TWEAK效应的影响；合成TWEAK胞外区多肽和TWEAK-MV1偶联蛋白，观察其治疗皮肤鳞状细胞癌小鼠模型的动物实验。.重要结果：发现TWEAK/Fn14信号及下游蛋白在上述病变组织中高表达，Fn14基因敲除后多种皮肤病小鼠模型的靶器官炎症反应获得缓解；体外炎症微环境下，Fn14-TRAF2-TNFR2信号轴介导TWEAK调控细胞周期的分子机制；重组TWEAK促进皮肤创伤愈合过程；成功构建TWEAK-MV1偶联蛋白，可抑制小鼠皮肤鳞状细胞癌的生长与显著延长生存率，并缓解银屑病小鼠模型的皮肤损害。.关键数据：迄今发表SCI论文22篇、中文期刊论文6篇（标注资助）；在国际学术会议发言4次，在国内中文会议发言10次；申请发明专利3项；获得中华医学会皮肤性病学分会的优秀SCI论文奖2次、年度学术奖2次，中国皮肤科医师年会优秀论文奖1次；培养博士生7人、硕士生8人（毕业）、陕西省中青年科技创新领军人才1人。.科学意义：本项目阐明不同炎症微环境下TWEAK/Fn14信号调控皮肤固有细胞双向命运方向及微结构损害的分子机制，发现基于Fn14-TRAF2-TNFR2信号轴原理评估红斑狼疮活动度或治疗皮肤创伤愈合、鳞状细胞癌及银屑病等的多种应用策略，为皮肤病早期诊断与靶向治疗奠定理论基础。