Hippo信号通路调控Leydig干细胞增殖和分化的机制

The proliferation/differentiation of the stem Leydig cell (SLC) is regulated by the niche signaling pathways. However, which pathways are and how these pathways act are not entirely clear. Hippo signaling has been shown to modulate proliferation/differentiation of cells in several tissues. Hippo signaling consists of regulators acting in a kinase cascade to negatively regulate the activity of the two homologous transcriptional co-activators; YAP and TAZ. Our preliminary data shows that the activation of Hippo signaling inhibits the proliferation while promotes the differentiation of SLC. We hypothesize that YAP/TAZ inhibits the proliferation of SLC while Hippo signaling activation, through the inhibition of YAP/TAZ, promotes the differentiation of SLC by regulating the expression of genes involved in androgen biosynthesis. In the present proposal, we will evaluate the specific roles of YAP and TAZ in MA-10 cells transfected with siRNA directed against Yap, Taz and Yap/Taz by performing dual-crosslink chromatin immunoprecipitation, real-time qPCR and dual-luciferase reporter assay. We will also use the mice of Leydig cell conditional Yap ablation to examine whether SLC proliferation and/or differentiation will be affected. The expression of steroidogenic and proliferation related genes, Intratesticular and serum androgen levels will be measured. The work outlined in this proposal will provide new insights into the roles and mechanisms of action of Hippo signaling and its effectors YAP/TAZ in SLC. This research will provide new clues regarding Leydig cell development and steroid biosynthesis as well as the etiology of fertility disorders.

Leydig干细胞（SLC）增殖和分化受到微环境信号通路的调控，但其调控通路及机制未知。Hippo信号可调节组织细胞的增殖或分化，其负向调控转录共激活因子YAP和TAZ。预实验显示Hippo信号通路抑制SLC增殖而刺激分化。我们假设：Hippo信号通路激活可通过磷酸化YAP/TAZ而抑制YAP/TAZ，从而抑制SLC增殖，同时通过调控雄激素合成相关基因的表达而刺激SLC分化。拟用Yap、Taz和Yap/Taz siRNA转染MA-10细胞，用qPCR、双荧光基因报告和染色质免疫共沉淀技术评估Hippo信号通路在MA-10的作用及机制。通过检测Leydig条件敲除YAP小鼠的雄激素水平及雄激素合成和增殖相关基因表达以探究Hippo信号通路调控SLC增殖和分化机制。本项目将为Hippo信号通路及其效应蛋白YAP/TAZ在SLC中的作用及机制提供新的见解，为生育障碍病因学提供新线索。

Leydig干细胞（SLC）增殖和分化受到微环境信号通路的调控，但其调控通路及机制未知。Hippo信号可调节组织细胞的增殖或分化，其负向调控转录共激活因子YAP和TAZ。本项目目的是探究YAP/TAZ信号通路对SLC增殖、分化及类固醇合成的影响及机制。首先我们使用YAP/TAZ抑制剂维替泊芬（VP）处理MLTC-1细胞株24小时，发现VP能够显著促进MLTC-1细胞孕酮（P4）的分泌，且VP上调Star和Nr4a1基因水平，siRNA沉默Yap1后出现相同结果，荧光素酶基因报告显示Yap1通过Star和Nr4a1靶向促进MLTC-1细胞类固醇激素合成；同时，体外分离成年小鼠曲细精管，用EDS处理24小时去除原有的成年LC，分别在增殖期及分化期用VP处理， EdU结果显示VP抑制了SLC增殖，分化期VP抑制睾酮产生，说明VP也抑制SLC分化；利用Yap1flox/Tazflox和Cyp17a1cre小鼠条件敲条Leydig细胞的Yap1和Taz证实Yap1/Taz缺失抑制睾酮合成及睾酮合成关键基因和蛋白；此外，采用曲细精管培养系统和体内EDS再生模型，研究胰高血糖素样肽-1（GLP-1）对SLC增殖和分化的影响；最后，探究了环境污染物（四甲基双酚A、双酚AF、全氟十一酸、全氟十三酸和二甲苯）对Leydig细胞发育的分子调节机制。总之，本项目揭示了YAP/TAZ和GLP-1在Leydig细胞发育中的作用及机制提供新的见解，为生育障碍病因学提供新线索。为治疗男性性功能减退，提供科学依据，具有重大的医疗意义和社会经济意义。