南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (4): 697-705.doi: 10.12122/j.issn.1673-4254.2024.04.11

• • 上一篇    下一篇

锌指蛋白-36缺陷抑制小鼠的成骨细胞分化：基于激活ERK/MAPK通路

戎圣炜，李宏芳，魏怡然，冯子航，甘 露，邓仲豪，赵 亮   

  1. 南方医科大学南方医院关节与骨病外科，广东 广州 510515；北京怡健殿诊所，北京 100033；北京大学国际医院健康管理中心，北京 102206
  • 发布日期:2024-04-29

Zinc finger protein-36 deficiency inhibits osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells and preosteoblasts by activating the ERK/MAPK pathway

RONG Shengwei, LI Hongfang, WEI Yiran, FENG Zihang, GAN Lu, DENG Zhonghao, ZHAO Liang   

  1. Department of Orthopedics, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; Beijing Yijiandian Clinic, Beijing 100033, China; Health Management Center, Peking University International Hospital, Beijing 102206, China
  • Published:2024-04-29

RichHTML

19

PDF

248

摘要： 目的 探究锌指蛋白-36（ZFP36）对成骨细胞分化的调控作用及机制。方法 通过提取小鼠原代骨髓间充质干细胞，结合小鼠成骨细胞前体细胞系MC3T3-E1，在体外成骨分化诱导状态下观察Zfp36（编码ZFP36）的表达变化。通过干扰RNA技术构建Zfp36缺陷的细胞，观察成骨分化作用的改变。通过第二代转录组测序技术探究Zfp36缺陷细胞成骨分化过程中的转录组水平改变。通过ERK/MAPK信号抑制分子U0126验证Zfp36缺陷对成骨分化作用的调控机制。结果 小鼠原代骨髓间充质细胞以及MC3T3-E2细胞中Zfp36表达在成骨分化0~14 d过程中呈逐渐升高趋势，在第7天到达峰值时较第0天升高3.85倍（P<0.0001）。抑制上述细胞Zfp36的表达后，成骨分化过程中碱性磷酸酶染色及茜素红染色弱于对照组，成骨分化标志基因Alpl（P<0.01）、Sp7（P<0.001）、Bglap（P<0.01）、Ibsp（P<0.0001）表达显著减弱。转录本测序结果提示Zfp36缺陷细胞的下调基因富集到骨矿化相关基因集中，且与ERK信号相关。蛋白表达检测显示Zfp36缺陷细胞的磷酸化ERK蛋白比例较对照组升高2.1倍(P=0.0274)。通过分子化合物U0126抑制Zfp36缺陷细胞中被激活的ERK/MAPK信号，可观察到表型挽救现象，并且呈剂量依赖。Zfp36缺陷细胞在10 μmol/L 度U0126作用下碱性磷酸酶染色增强，成骨细胞分化标志基因Runx2（P<0.05）及Bglap（P<0.05）表达显著增高。结论 ZFP36参与了小鼠成骨细胞的分化调控过程，Zfp36缺陷会引起ERK/MAPK信号通路的激活，进而抑制成骨细胞向骨细胞的分化。

关键词: 锌指蛋白-36；成骨细胞分化；ERK/MAPK信号通路；骨髓间充质干细胞

Abstract: Objective To explore the role of zinc finger protein 36 (ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. Methods ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. Results During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7 (P<0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P<0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36- deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P<0.05). Conclusions ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.

Key words: zinc finger protein 36; osteoblast differentiation; ERK/MAPK signaling pathway; bone-marrow-derived mesenchymal stem cells