CaN通过抑制转录辅因子YAP放大NFAT转录活性的机制研究

Transcription factor NFAT (Nuclear factor of activated T-cells, NFAT) plays an important role in podocyte injury. Transcription cofactor YAP rugulates many transcription factors. We recently observed that YAP silencing could activate NFAT and the YAP was combined with NFAT in podocyte nucleus, which demonstrated that YAP was a nuclear repressor of NFAT. A calcineurin (CaN) mediated nuclear translocation is a basical mechanism for NFAT activation. Our recent preliminary study study showed that CaN activation could inhibite YAP. Therefore, we proposed that, besides the pathway of nuclear translocation, CaN promotes NFAT activation via inhibition of YAP. To identify the detailed mechanism of the inhibition of NFAT by YAP, we plan to use tagged protein pull down to explore the interaction domains of both NFAT and YAP, and the effect of YAP on NFAT-regulated target gene by ChIP. Futhermore, to identify the effects on NFAT and biologic changes, we plan to silence or mutate YAP in vitro, and construct LPS-induced mouse model in vivo using podocyte-conditional mutation/knockout mice. Our research will first demonstrate a new amplification pathway of NFAT activation, which can provide new theory for podocyte protection and potential targets for clinical therapy.

NFAT是足细胞损伤的重要转录因子。YAP是调控多种转录因子的转录辅因子。我们前期研究发现：沉默YAP能增加NFAT转录活性，且YAP和NFAT在细胞核内相互结合，提示YAP是NFAT的阻遏因子。 此前认为，CaN促使NFAT进入细胞核是其活化的基本模式。我们最近发现：CaN活化时还能抑制YAP。因此，我们提出：除了介导NFAT进入细胞核，CaN还能通过抑制YAP来放大对NFAT的激活效应。 为阐明YAP阻遏NFAT的机制，本课题拟采用标签蛋白pull down探索NFAT和YAP相互作用的结构域；用ChIP研究YAP对NFAT目的基因的影响；用基因转染、突变技术干预足细胞YAP，用足细胞特异性YAP基因突变和敲除小鼠构建脂多糖肾病模型，观察干预YAP对NFAT的影响及相应的生物学效应。本研究将首次阐明一种增强CaN/NFAT信号的新途径，为保护足细胞提供新的理论和治疗靶点。

慢性肾脏病是重要的全球性公共卫生问题。足细胞是肾小球滤过膜屏障的重要组成细胞，足细胞损伤是慢性肾脏病进展的一个重要原因。但在肾脏疾病中导致足细胞损伤的分子机制仍不完全清楚。活化T细胞核因子（nuclear factor of activated T cells, NFAT2）是足细胞损伤的重要转录因子。钙调磷酸酶（Calcineurin, CaN）促使NFAT2进入细胞核是其活化的基本模式。YAP (Yes-associated protein)是调控多种转录因子的重要转录辅因子, 具有抗调亡作用。本项目前期实验发现，CaN活化时还能抑制YAP入核发挥作用。因此，我们提出：除了介导NFAT进入细胞核，CaN还能通过抑制YAP来放大对NFAT的激活效应。.研究发现：（1）通过Co-IP实验发现在足细胞核中YAP和NFAT相互结合，而二者在细胞浆中并不结合。（2）通过荧光素酶报告实验及Q-PCR实验发现足细胞中沉默YAP增加NFAT转录活性及其目的基因表达，YAP是阻遏NFAT的转录辅因子。（3）通过体内外实验发现CaN/CDC42/Nwasp/stress fibers/YAP信号通路在损伤环境中参与调控足细胞凋亡，CaN能抑制YAP进入细胞核中发挥作用。（4）体外实验中，沉默足细胞YAP可致NFAT核蛋白表达增加、CaN活性升高、细胞活动力增加、细胞骨架损伤、足细胞凋亡增多。（5）在体实验中建立足细胞特异性YAP敲除小鼠，给予腹腔注射CaN抑制剂FK506。在足细胞特异性YAP敲除小鼠中，FK506显著降低蛋白尿、血肌酐，抑制肾脏系膜基质增生、肾小球基底膜增厚及减轻足突融合。本研究首次阐明一种增强CaN/NFAT信号的新途径，为保护足细胞提供新的理论和治疗靶点。CaN/YAP信号通路介导足细胞骨架破坏、细胞凋亡，蛋白尿发生及肾功能进展，可能是治疗蛋白尿相关肾病的有效靶点。