去甲斑蝥素通过抑制PP2A介导β-catenin去磷酸化修饰的抗肾间质纤维化分子机制研究

Wnt/β-catenin is one of the important signaling pathway of TGF-β induced renal interstitial fibrosis. The phosphorylation of β-catenin is regarded as the key part to block Canonical Wnt signaling transduction. It has been reported that the protein phosphatase 2A(PP2A) can dephosphorylate β-catenin and regulate Wnt signal transduction, but the detailed role and possible molecular mechanisms are not very clear so far. NCTD is known as a compound inhibitor of protein phosphatase including PP2A. We have found it can attenuate renal tubulointerstitial fibrosis by in vivo and in vitro experiments. The antifibrotic effect of NCTD may related to its role of PP2A inhibition blocking TGF-β/Smad3 signal pathways. In addition, our preliminary study also found NCTD could inhibit the expression of β-catenin and nuclear translocation in tubular epithelial cell stimulated by TGF-β1. Therefore, we suppose that NCTD may play the antifibrotic role through inhibiting PP2A to positively regulate Wnt /β-catenin signaling pathway. This study intends to explore: (1)The effect and underlying mechanisms of PP2A positively regulating Wnt signal pathway by mediating β-catenin dephosphorylation in renal tubular cell; (2) The relationship between the antifibrotic effect of NCTD and its role of PP2A inhibition regulating Wnt signaling transduction. This study will reveal the molecular mechanism of renal fibrosis from the aspect of β-catenin dephosphorylation for the first time, which will provide novel insights into the development of new therapeutic strategy.

Wnt /β-catenin是TGF-β介导肾间质纤维化的重要通路之一，β-catenin磷酸化是阻断Wnt信号转导的关键环节。蛋白磷酸酶(PP2A)能去磷酸化β-catenin调控Wnt信号，但对肾间质纤维化的具体作用和机制尚不清楚。去甲斑蝥素(NCTD)是PP2A抑制剂，我们前期研究证实NCTD具有抗肾间质纤维化作用，其机制与其抑制PP2A阻断TGF-β/ Smad3信号通路有关, 预实验还发现NCTD能抑制肾脏β-catenin表达和核转录。因此，我们推测NCTD可能抑制PP2A对Wnt /β-catenin信号的正向调控。本课题拟探讨1) PP2A介导β-catenin去磷酸化，正向调控Wnt信号的作用和机制；2）NCTD抗肾间质纤维化是否与其抑制PP2A，削弱PP2A对Wnt信号正向调控有关。首次从β-catenin去磷酸化修饰角度揭示肾间质纤维化的分子机制，为其防治开辟新思路。

Wnt/β-catenin是TGF-β1介导肾间质纤维化的重要通路之一，β-catenin磷酸化是阻断Wnt信号转导的关键环节。蛋白磷酸酶（PP2A）能去磷酸化β-catenin正向调控Wnt信号，但对肾间质纤维化的具体作用和机制尚不清楚。去甲斑蝥素（NCTD）是PP2A抑制剂，我们前期研究发现NCTD具有抗肾间质纤维化作用，它能抑制肾脏β-catenin表达和核转录。因此我们推测NCTD可能抑制PP2A对Wnt/β-catenin信号的正向调控。本课题拟探讨 1）PP2A介导β-catenin去磷酸化，正向调控Wnt信号的作用和机制；2）NCTD抗肾间质纤维化是否与其抑制PP2A，削弱PP2A对Wnt信号正向调控有关。本研究通过体内外实验研究发现： NCTD可以下调UUO大鼠肾脏及TGF-β1刺激的人肾小管上皮细胞（HK-2）Wnt4、β-catenin蛋白表达，但PP2A可以促进TGF-β1刺激的HK-2细胞内的β-catenin磷酸化，下调β-catenin表达，抑制Wnt/β-catenin信号通路。因此，NCTD抗肾间质纤维化的作用并非通过负向调控PP2A介导的β-catenin磷酸化实现。由于TGF-β1/Smad3是介导肾间质纤维化的另一条主要的信号转导通路，我们前期研究证实NCTD抗肾间质纤维化的作用与其抑制TGF-β1/Smad3信号通路有关，但具体分子机制尚不清楚。本研究进一步发现PP2A能调控Smad3 C端磷酸化和核转位，NCTD能抑制PP2A介导的Smad3 C端磷酸化和核转位，从而负向调控TGF-β1/Smad3信号通路发挥抗肾间质纤维化的作用。越来越多的研究表明，长链编码RNA（lncRNA）参与了许多疾病的发生发展，本研究中我们还利用高通量测序在UUO小鼠上进一步筛选了NCTD抗肾间质纤维化相关lncRNA及mRNA，为后续进一步研究NCTD抗肾间质纤维化的新机制及靶点奠定了基础。以上研究揭示了NCTD抗肾间质纤维化的新机制，并为NCTD抗肾间质纤维化的防治提供了新思路和策略。