mTORC1信号介导Hippo通路调控FSGS中足细胞凋亡的机制研究

Hippo pathway is an established pathway that regulated cell proliferation and apoptosis. The correlation between Hippo pathway and various nephritis begin to be widely concerned. Recent studies showed that YAP, as a downstream effector of Hippo pathway, could be regulated by mTORC1 and autophagy in tuberous sclerosis complex. However, the relationship of mTORC1 and Hippo pathway in focal segmental glomerulosclerosis(FSGS) still remains unclear. Our preliminary experiments showed that the inhibition of YAP led to podocytes apoptosis in FSGS. Furthermore, inhibition of mTORC1 pathway activated YAP and rescued podocytes from apoptosis. Therefore, in this proposal, we hypothesized that mTORC1 pathway regulated the apoptosis of podocytes in FSGS via Hippo pathway, and intended to reveal the molecular basis of mTORC1 in regulating Hippo pathway either by S6K1, ULK1 or autophagy. To verify these hypotheses, we would treat the primary podocytes with adriamycin as well as induce FSGS model in mT/mG transgenic mice. In these in vitro and in vivo model systems complemented with human renal biopsies, mTORC1 or Hippo pathway would be detected, and furthermore, interfered by Tsc1 gene knockout, lentivirus mediated transfection and pathway targeted small molecular drugs, in order to find out if and how mTORC1 pathway regulated Hippo pathway and finally led to the apoptosis of podocytes in FSGS. The mission of this proposal is to reveal the molecular mechanism underlying the occurrence and development of FSGS from the perspective of important pathways crosstalk, which is fundamental for understanding the pathogenesis and designing new therapeutic strategies in FSGS.

Hippo通路是调控细胞增殖和凋亡的重要通路，其与肾病的相关性开始受关注。研究表明Hippo下游--YAP可受mTORC1信号及自噬的调节，但局灶节段性肾小球硬化(FSGS)中mTORC1是否调控Hippo通路尚不清楚。预实验发现FSGS中，YAP活性抑制，足细胞凋亡增加；下调mTORC1通路可激活YAP并抑制足细胞凋亡。因此，本项目提出mTORC1信号可通过调控Hippo通路，进而影响FSGS中足细胞凋亡的假说，并将深入研究mTORC1下游--S6K1或ULK1及自噬对Hippo通路的调控。为此，我们将利用mT/mG转基因小鼠制备FSGS模型和阿霉素刺激原代足细胞，结合人肾穿标本，采用Tsc1基因敲除、慢病毒转染及小分子药物调节通路活性等手段，探讨mTORC1信号介导Hippo通路调控足细胞凋亡的作用机制。本项目将从重要通路串话角度揭示FSGS发生发展的新机制，为治疗提供新靶点和新策略。

足细胞凋亡是引起局灶节段性肾小球硬化（FSGS）的重要机制之一，探寻针对足细胞凋亡的治疗新靶点和新策略具有重要的科学意义。本课题对Hippo通路及其与mTORC1通路间的串话在足细胞凋亡中的作用机制进行了探讨，在人FSGS肾穿标本和ADR诱导所致体内外FSGS模型中发现：（1）FSGS肾小球足细胞中YAP持续出核，与足细胞凋亡呈正相关，YAP抑制剂Verteporfin可减少核内YAP从而促进足细胞凋亡和FSGS疾病进展，提示YAP入核是FSGS中抗足细胞凋亡的内源性调控机制。（2）接着，我们探索了抑制mTORC1活性对Hippo通路调控足细胞凋亡的影响，结果发现，抑制mTORC1信号通路能有效缓解FSGS疾病进展，同时伴随着Yap激活，核内非磷酸化Yap增多，足细胞凋亡减少。（3）为进一步探讨mTORC1信号与Hippo通路串话的机制，我们针对mTORC1的三个下游及其作用在体内外使用抑制剂或siRNA处理，结果发现，使用3-MA抑制自噬后，非磷酸化YAP减少，足细胞凋亡增加，从而促进了FSGS的疾病进展。因此，本项目的科学意义在于：（1）增加非磷酸化YAP水平可作为足细胞凋亡和FSGS疾病进展的治疗新策略；（2）明确了mTORC1和Hippo通路之间的串话机制，即：mTORC1调控的自噬激活可增加非磷酸化YAP水平，从而减少足细胞凋亡；（3）建立的基于细胞流式技术的原代小鼠足细胞分离法可推动国内足细胞损伤性肾病的研究。