Identification of Gli1-interacting proteins during simvastatin-stimulated osteogenic differentiation of bone marrow mesenchymal stem cells
Identification of Gli1-interacting proteins during simvastatin-stimulated osteogenic differentiation of bone marrow mesenchymal stem cells
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辛伐他汀刺激骨髓间充质干细胞成骨分化过程中 Gli1 相互作用蛋白的鉴定
DOI：
10.1002/jcb.29221
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发表时间：
2019-11-01
影响因子：
4
通讯作者：
Tian, Faming
中科院分区：
文献类型：
--
作者：
Chi, Bojing;Fan, Xinhao;Tian, Faming
辛伐他汀可促进骨髓间充质干细胞(BMSCs)的成骨分化。我们的研究旨在阐明这一过程的潜在机制，特别是刺猬信号在这一过程中的作用。用10(-7)m ol/L辛伐他汀或不加10(-7)m ol/m o l辛伐他汀培养骨髓间充质干细胞，观察其成骨分化能力。用Western印迹和实时定量聚合酶链式反应(PCR)检测成骨标记物I型胶原(COL1)和骨钙素(OCN)以及Hedgehog信号分子的关键分子。免疫共沉淀和质谱分析用于筛选Gli1相互作用蛋白。用环多巴胺(CPN)作为Hedgehog信号的抑制剂。结果表明，辛伐他汀可增加碱性磷酸酶(ALP)活性、细胞外基质矿化、ALP、CO1和OCN的mRNA表达以及Gli1的表达和核转位。在接触CPN的组中观察到了相反的效果，但辛伐他汀治疗部分挽救了这一效果。基因本体论和京都百科全书的基因和基因组丰富分析表明，Gli1相互作用的蛋白主要与丝裂原活化蛋白激酶(MAPK)(P=7.04E(-04))、河马、胰岛素和胰高血糖素信号有关。此外，通过蛋白质-蛋白质相互作用网络分析鉴定的HUB基因包括Gli1相互作用蛋白，如Ppp2r1a、rac1、ETF1和XPO1/CRM1。综上所述，目前的研究表明，辛伐他汀刺激BMSCs成骨分化的机制涉及激活Hedgehog信号，如与Gli1的相互作用，尤其是MAPK信号通路。
Simvastatin has been shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Our study aimed to illuminate the underlying mechanism, with a specific focus on the role of Hedgehog signaling in this process. BMSCs cultured with or without 10(-7) mol/L simvastatin were subjected to evaluation of osteogenic differentiation capacity. Osteogenic markers such as type 1 collagen (COL1) and osteocalcin (OCN), as well as key molecules of Hedgehog signaling molecules, were examined by Western blot and real-time polymerase chain reaction (PCR). Co-immunoprecipitation and mass spectrometry assays were applied to screen for Gli1-interacting proteins. Cyclopamine (Cpn) was used as a Hedgehog signaling inhibitor. Our results indicated that simvastatin increased alkaline phosphatase (ALP) activity; mineralization of extracellular matrix; mRNA expression of ALP, COL1, and OCN; and expression and nuclear translocation of Gli1. Contrasting effects were observed in Cpn-exposed groups, but were partially rescued by the simvastatin treatment. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that Gli1-interacting proteins were primarily associated with mitogen-activated protein kinase (MAPK) (P = 7.04E(-04)), hippo, insulin, and glucagon signaling. Further, hub genes identified by protein-protein interaction network analysis included Gli1-interacting proteins such as Ppp2r1a, Rac1, Etf1, and XPO1/CRM1. In summary, the current study showed that the mechanism by which simvastatin stimulates osteogenic differentiation of BMSCs involves activation of Hedgehog signaling, as indicated by interactions with Gli1 and, most notably, the MAPK signaling pathway.