蛋白质去泛素化酶USP7调节脂肪基质干细胞成骨向分化的研究

Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. The main reason lies in the fact that hASCs can be obtained easily from adipose tissues, which are carrying a more abundant and accessible pool of mesenchymal stem cells, with less invasive and less expensive procedures. How to effectively promote osteogenic differentiation of hASCs has become a core issue in bone tissue engineering fields. Recently, it has become increasingly clear that chromatin regulators play an important role in cell fate determination and lineage commitment. Thereby, a better understanding of how epigenetic regulation contributes to osteogenic differentiation of hASCs will provide critical insights into the modulation of hASCs based cell therapy...In our previous work, we demonstrated that histone demethylase RBP2 and LSD1 play a key role in osteogenic differentiation of hASCs. To further understand the epigenetic mechanisms governing osteogenic differentiation of hASCs, we established a luciferase reporter system to screen and identify key regulators involved in this process. In our preliminary work, we found that USP7 (Ubiquitin specific protease 7) knockdown was associated with a decreased activity of the luciferase reporter gene. Meanwile, USP7 deficiency resulted in an impairment of hASCs osteogenic differentiation, manifested by in vitro ALP staining and Alizarin red S (ARS) staining assays. Next, immunopurification of USP7 containing protein complex followed by mass spectrometry analysis revealed that JmjC domain containing histone demethylase PHF8 (Plant homeodomain finger-containing proteins 8) is co-purified with USP7. Co-Immunoprecipitation experiment demonstrated that USP7 is physcially associated with PHF8. In addition, we found that USP7 depletion in hASCs was associated with decreased expression of PHF8 at the protein level, the effect of which could be reverted by proteasome inhibitor MG132, suggesting that PHF8 is a candidate substrate of USP7. Furthermore, we demonstrated that, similar to USP7, PHF8 is required for luciferase reporter gene activation and osteogenic differentiation of hASCs in vitro. Importantly, we found that USP7 depletion resulted in luciferase reporter gene downregulation could be restored by PHF8 overexpression. These reuslts suggest that USP7 and PHF8 are funtionally connected and USP7-promoted PHF8 stabilization plays an important role in osteogenic differentiation of hASCs. In the future, we will further identify whether USP7 is a bona fide deubiquitinase for PHF8 and explore the epigenetic mechanism of how USP7-promoted PHF8 stabilization influences on osteogenic differentiation of hASCs. Our study provides a mechanistic link of USP7 to osteogenic differentiation of hASCs, and supports the pursuit of USP7 and PHF8 as potential targets for modulation of hASCs based cell therapy and bone tissue engineering with epigenetic intervention.

人脂肪基质干细胞 (hASCs) 是骨组织工程理想的细胞来源。申请人以往研究证实，组蛋白甲基化修饰在hASCs成骨分化中起着重要作用。为更深入的了解hASCs成骨分化的表观遗传机理，我们构建了可评价其成骨分化的荧光素酶报告系统。初步筛选发现蛋白质去泛素化酶USP7可以促进hASCs的成骨分化；蛋白质组学鉴定和后续实验表明USP7与组蛋白去甲基化酶PHF8相互作用并稳定PHF8表达；功能学实验显示PHF8过表达可以有效地逆转USP7缺失引起的成骨能力下降。以上结果提示USP7和PHF8功能上密切相关，且在hASCs的成骨分化中起重要作用。在今后的工作中我们将进一步探索USP7/PHF8在hASCs成骨分化中的作用，并明确USP7/PHF8调控hASCs成骨分化的表观遗传机制。该工作有利于深刻认识hASCs成骨分化的表观遗传调控机理，为基于hASCs的骨组织工程学提供了潜在的表观遗传干预靶点。

骨缺损是临床常见问题，如何更好地修复缺损的骨组织，成为修复重建领域关注的热点。人脂肪基质干细胞 (hASCs) 是骨组织工程理想的细胞来源。申请人以往研究证实，组蛋白甲基化修饰在hASCs成骨分化中起着重要作用。为更深入的了解hASCs成骨分化的表观遗传机理，我们构建了可评价其成骨分化的荧光素酶报告系统。初步筛选发现蛋白质去泛素化酶USP7可以促进hASCs的成骨分化；我们将USP7敲低及过表达，通过体外（碱性磷酸酶，茜素红染色及定量实验，成骨相关基因表达的检测等）和体内（裸鼠异位成骨实验）实验明确USP7在hASCs成骨分化中的作用。为了验证结果的普适性，我们在hBMMSCs细胞系也证明USP7能够促进hBMMSCs的成骨向分化。为了探讨该作用的发挥是否依赖于USP7的去泛素化酶活性，我们引入了去泛素化酶抑制剂。HBX 41,108是去泛素化酶抑制剂，可抑制USP7的酶活性。我们通过抑制USP7去泛素化酶活性，观察hASCs在成骨向分化的变化。结果表明USP7抑制剂HBX 41,108能够抑制hASCs的成骨向分化。该结果一方面证明了USP7对hASCs成骨向分化的调控依赖于酶活性，另一方面也为应用转化提供了思路。即可通过小分子药物来治疗骨异常增生等疾病。. 本项目在我们以往研究的基础上，探讨蛋白质去泛素化酶USP7如何调控hASCs的成骨向分化及其应用拓展。该研究将是对以往组蛋白甲基化修饰影响间充质干细胞定向分化的拓展和深入。阐明去泛素化酶USP7如何调控hASCs的成骨向分化及调控hASCs成骨向分化的表观遗传机制，对于建立hASCs成骨向分化的表观遗传学调控网络和深入认识hASCs成骨向分化的表观遗传学机理（即以染色质为基础的基因表达调控）具有重要意义，并且可以为表观干预（小分子药物）hASCs的成骨向分化能力提供潜在的靶点。