Department of Endocrinology, Endocrinology Research Center, Xiangya Hospital of Central South University, Changsha, China.
National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan, P. R. China.
Cell Prolif. 2022 Feb;55(2):e13178. doi: 10.1111/cpr.13178. Epub 2022 Jan 11.
DNA N6-methyladenine (N6-mA) demethylase Alkbh1 participates in regulating osteogenic differentiation of mesenchymal stem cell (MSCs) and vascular calcification. However, the role of Alkbh1 in bone metabolism remains unclear.
Bone marrow mesenchymal stem cells (BMSCs)-specific Alkbh1 knockout mice were used to investigate the role of Alkbh1 in bone metabolism. Western blot, qRT-PCR, and immunofluorescent staining were used to evaluate the expression of Alkbh1 or optineurin (optn). Micro-CT, histomorphometric analysis, and calcein double-labeling assay were used to evaluate bone phenotypes. Cell staining and qRT-PCR were used to evaluate the osteogenic or adipogenic differentiation of BMSCs. Dot blotting was used to detect the level of N6-mA in genomic DNA. Chromatin immunoprecipitation (Chip) assays were used to identify critical targets of Alkbh1. Alkbh1 adeno-associated virus was used to overexpress Alkbh1 in aged mice.
Alkbh1 expression in BMSCs declined during aging. Knockout of Alkbh1 promoted adipogenic differentiation of BMSCs while inhibited osteogenic differentiation. BMSC-specific Alkbh1 knockout mice exhibited reduced bone mass and increased marrow adiposity. Mechanistically, we identified optn as the downstream target through which Alkbh1-mediated DNA m6A modification regulated BMSCs fate. Overexpression of Alkbh1 attenuated bone loss and marrow fat accumulation in aged mice.
Our findings demonstrated that Alkbh1 regulated BMSCs fate and bone-fat balance during skeletal aging and provided a potential target for the treatment of osteoporosis.
DNA N6-甲基腺嘌呤（N6-mA）去甲基酶 Alkbh1 参与调节间充质干细胞（MSCs）的成骨分化和血管钙化。然而，Alkbh1 在骨代谢中的作用尚不清楚。
使用骨髓间充质干细胞（BMSCs）特异性 Alkbh1 敲除小鼠来研究 Alkbh1 在骨代谢中的作用。Western blot、qRT-PCR 和免疫荧光染色用于评估 Alkbh1 或 optineurin（optn）的表达。微 CT、组织形态计量分析和 calcein 双标记测定用于评估骨表型。细胞染色和 qRT-PCR 用于评估 BMSCs 的成骨或成脂分化。点印迹用于检测基因组 DNA 中的 N6-mA 水平。染色质免疫沉淀（Chip）实验用于鉴定 Alkbh1 的关键靶标。Alkbh1 腺相关病毒用于在老年小鼠中过表达 Alkbh1。
BMSCs 中 Alkbh1 的表达随年龄增长而下降。Alkbh1 敲除促进了 BMSCs 的成脂分化，同时抑制了成骨分化。BMSC 特异性 Alkbh1 敲除小鼠表现出骨量减少和骨髓脂肪增多。机制上，我们通过 Chip 实验鉴定了 optn 是 Alkbh1 介导的 DNA m6A 修饰调控 BMSCs 命运的下游靶标。过表达 Alkbh1 可减轻老年小鼠的骨丢失和骨髓脂肪堆积。
我们的研究结果表明，Alkbh1 调节了骨骼衰老过程中 BMSCs 的命运和骨-脂平衡，并为骨质疏松症的治疗提供了一个潜在靶点。
