Department of Prosthodontics, National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, National Clinical Research Center for Oral Diseases, Beijing Key Laboratory of Digital Stomatology, Peking University School and Hospital of Stomatology, 22 Zhongguancun South Avenue, Haidian District, Beijing, 100081, People's Republic of China.
Stem Cell Res Ther. 2022 Sep 2;13(1):443. doi: 10.1186/s13287-022-03062-0.
Bone is a rigid organ that provides physical protection and support to vital organs of the body. Bone loss disorders are commonly associated with increased bone marrow adipose tissue. Bone marrow mesenchymal stromal/stem cells (BMSCs) are multipotent progenitors that can differentiate into osteoblasts, adipocytes, and chondrocytes. Cell division cycle 20 (CDC20) is a co-activator of anaphase promoting complex/cyclosome (APC/C), and is required for ubiquitin ligase activity. Our previous study showed that CDC20 promoted the osteogenic commitment of BMSCs and Cdc20 conditional knockout mice suggested a decline in bone mass. In this study, we found that knockdown of CDC20 promoted adipogenic differentiation of BMSCs by modulating β-catenin, which suggested a link between adipogenesis and osteogenesis.
Lentivirus containing a CDC20 shRNA was used for CDC20 knockdown in human BMSCs (hBMSCs). Primary mouse BMSCs (mBMSCs) were isolated from Cdc20 and Sp7-Cre;Cdc20 mice. Adipogenesis was examined using quantitative real-time reverse transcription PCR (qRT-PCR) and western blotting analysis of adipogenic regulators, Oil Red O staining, and transplantation into nude mice. CDC20 knockout efficiency was determined through immunochemistry, qRT-PCR, and western blotting of bone marrow. Accumulation of adiposity was measured through histology and staining of bone sections. Exploration of the molecular mechanism was determined through western blotting, Oil Red O staining, and qRT-PCR.
CDC20 expression in hBMSCs was significantly decreased during adipogenic differentiation. CDC20 knockdown enhanced hBMSC adipogenic differentiation in vitro. CDC20-knockdown hBMSCs showed more adipose tissue-like constructs upon hematoxylin and eosin (H&E) and Oil Red O staining. Sp7-Cre;Cdc20 mice presented increased adipocytes in their bone marrow compared with the control mice. mBMSCs from Sp7-Cre;Cdc20 mice showed upregulated adipogenic differentiation. Knockdown of CDC20 led to decreased β-catenin levels, and a β-catenin pathway activator (lithium chloride) abolished the role of CDC20 in BMSC adipogenic differentiation.
Our findings showed that CDC20 knockdown enhanced adipogenesis of hBMSC and mBMSCs adipogenesis in vitro and in vivo. CDC20 regulates both adipogenesis and osteogenesis of BMSCs, and might lead to the development of new therapeutic targets for "fatty bone" and osteoporosis.
骨骼是一种坚硬的器官，为身体的重要器官提供物理保护和支撑。骨丢失疾病通常与骨髓脂肪组织增加有关。骨髓间充质基质/干细胞（BMSCs）是多能祖细胞，可分化为成骨细胞、脂肪细胞和软骨细胞。细胞分裂周期蛋白 20（CDC20）是后期促进复合物/环体（APC/C）的共激活因子，是泛素连接酶活性所必需的。我们之前的研究表明，CDC20 促进了 BMSCs 的成骨分化，Cdc20 条件性敲除小鼠表明骨量减少。在这项研究中，我们发现敲低 CDC20 通过调节β-连环蛋白促进 BMSCs 的脂肪生成分化，这表明脂肪生成和成骨之间存在联系。
使用含有 CDC20 shRNA 的慢病毒对人 BMSCs（hBMSCs）进行 CDC20 敲低。从小鼠骨髓中分离出 Cdc20 和 Sp7-Cre；Cdc20 小鼠的原代小鼠 BMSCs（mBMSCs）。通过定量实时逆转录 PCR（qRT-PCR）和脂肪生成调节剂的 Western 印迹分析、油红 O 染色和裸鼠移植来检测脂肪生成。通过免疫化学、骨髓 qRT-PCR 和 Western 印迹检测 CDC20 敲除效率。通过组织学和骨切片染色测量脂肪堆积量。通过 Western 印迹、油红 O 染色和 qRT-PCR 确定分子机制的探索。
hBMSCs 成脂分化过程中 CDC20 表达明显降低。CDC20 敲低增强了 hBMSC 体外成脂分化。CDC20 敲低的 hBMSCs 在苏木精和伊红（H&E）和油红 O 染色后显示出更多的脂肪组织样结构。与对照小鼠相比，Sp7-Cre；Cdc20 小鼠的骨髓中脂肪细胞增多。Sp7-Cre；Cdc20 小鼠的 mBMSCs 表现出上调的成脂分化。CDC20 敲低导致β-连环蛋白水平降低，β-连环蛋白通路激活剂（氯化锂）消除了 CDC20 在 BMSC 成脂分化中的作用。
我们的研究结果表明，CDC20 敲低增强了 hBMSC 和 mBMSC 的体外和体内成脂分化。CDC20 调节 BMSCs 的成脂生成和成骨生成，可能为“脂肪骨”和骨质疏松症的新治疗靶点的发展提供依据。
