Polycystin 2-Scribble-YAP通路在常染色体显性多囊肾病囊肿形成中的作用及机制

Autosomal dominant polycystic kidney disease (ADPKD) is the most common fatal hereditary kidney disease caused by mutations in PKD1 and/or PKD2 genes. However, the pathogenesis of the disease is still unclear and there is no effective treatment for it. In our previous studies, we found that knockdown of pkd2 in zebrafish embryos (pkd2 morphants) would decreased the expression of PC2, Scribble and phosphorylated YAP; overexpression of Scribble and phosphorylated YAP could reduce cyst formation in pkd2 morphants; Scribble could regulate YAP phosphorylation to influence the cyst formation. Accordingly, we propose a new mechanism hypothesis that the PC2-Scribble-YAP pathway plays an important role of in regulating cyst formation in ADPKD. In this study, we used the techniques of confocal microscopy and co-immunoprecipitation to identify the interaction between Scribble and PC2, investigated the role of Scribble in regulating the phosphorylation of YAP by using microinjection and zebrafish mutational analyses, and explored the role of YAP phosphorylation in the treatment of ADPKD. Thus, we will elucidate the effect and a novel mechanism of the PC2-Scribble-YAP pathway in the cyst formation of ADPKD, which may provide a new potential therapeutic target for ADPKD.

常染色体显性多囊肾病是最常见的致死性遗传性肾病，由PKD1和/或PKD2基因突变所致，发病机制未完全阐明，缺乏有效治疗手段。我们前期研究发现，敲低斑马鱼pkd2基因表达可降低PC2、Scribble及磷酸化YAP表达；过表达Scribble及磷酸化YAP可降低pkd2缺失斑马鱼胚胎的囊肿比例；Scribble可调控YAP的磷酸化水平，影响囊肿形成。据此，我们提出PC2-Scribble-YAP通路在多囊肾病囊肿形成中发挥作用这一新的机制假说。我们利用激光共聚焦和免疫共沉淀等技术研究Scribble与PC2的相互作用；运用显微注射和斑马鱼突变体构建等技术探究Scribble对YAP磷酸化的调控；采用药物干预实验研究提高磷酸化YAP水平干预多囊肾病的作用。最终阐明PC2-Scribble-YAP通路在多囊肾病囊肿发生发展中的重要作用及机制，为寻找治疗多囊肾病的全新靶标提供理论和实验依据。

常染色体显性多囊肾病是最常见的致死性遗传性肾病，发病机制未完全阐明，缺乏有效治疗手段。我们通过本项研究发现：①Scribble在多囊肾细胞及斑马鱼模型（pkd2 morphants）中表达下调；Scribble蛋白定位于斑马鱼前肾管上皮细胞的基底侧膜上，且Scribble可与PC2在细胞膜上共定位；过表达Scribble和Scribble-PDZ可抑制pkd2 morphants前肾囊肿形成，而过表达Scribble-LRR不具有类似效应。证实了Scribble与PC2的相互作用，其影响多囊肾相关表型是通过PDZ结构域实现的。②在多囊肾细胞及斑马鱼模型中，YAP蛋白由细胞质转移到细胞核内，而细胞质中YAP蛋白减少可导致斑马鱼产生多囊肾相关表型；在多囊肾细胞及斑马鱼模型中过表达Scribble可影响YAP蛋白的亚细胞定位变化，影响肾囊肿相关表型。③构建斑马鱼YAP突变体，下调细胞质YAP水平可产生肾脏囊肿相关表型。④利用生物信息学手段，发现NS398可能延缓ADPKD疾病进展，并在多囊肾细胞、斑马鱼及小鼠模型中证实了其延缓ADPKD疾病进展的作用。⑤利用生物信息学手段分析YAP蛋白的其它可能调控基因时，发现小窝蛋白CAV1在ADPKD中可能发挥重要调控作用，CAV1在ADPKD中表达明显升高，且与疾病进展密切相关。综上，我们证实了Polycystin 2-Scribble-YAP通路在常染色体显性多囊肾病囊肿形成中的作用及机制；筛选并证实小分子化合物NS398可延缓ADPKD疾病进展；初步发现了CAV1在ADPKD疾病进展中的重要作用，为后续研究打下基础。

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