Notch signaling-mediated osteocytic overexpression of BMP4 promotes osteogenic differentiation of bone marrow stromal cells

Zhengsong XIE; Yixin ZHAO; Ziling LYU; Hong LIU; Xiaolin TU

doi:10.16016/j.1000-5404.202102063

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Notch signaling-mediated osteocytic overexpression of BMP4 promotes osteogenic differentiation of bone marrow stromal cells

Basic Medicine | 更新时间：2026-01-09

- Notch signaling-mediated osteocytic overexpression of BMP4 promotes osteogenic differentiation of bone marrow stromal cells
- Journal of Third Military Medical University Vol. 43, Issue 16, Pages: 1550-1558(2021)
- 作者机构：
  
  1.400016 重庆，重庆医科大学: 生命科学研究院骨发育与再生实验室
  2.400016 重庆，重庆医科大学: 附属第一医院生殖健康与不孕症中心
  3.400016 重庆，重庆医科大学: 第一临床学院
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81672118);the Special Key Project of Basic Science and Froterier Technology of Chongqing Science and Technology Commission(cstc2015jcyjBX0119)
- DOI：10.16016/j.1000-5404.202102063
  CLC： R322.71;R329.28; R394.3
- Received：18 February 2021，
  
  Revised：2021-03-14，
  
  Published：30 August 2021
- 稿件说明：
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Zhengsong XIE, Yixin ZHAO, Ziling LYU, et al. Notch signaling-mediated osteocytic overexpression of BMP4 promotes osteogenic differentiation of bone marrow stromal cells[J]. Journal of Third Military Medical University, 2021, 43(16): 1550-1558.
DOI：

Zhengsong XIE, Yixin ZHAO, Ziling LYU, et al. Notch signaling-mediated osteocytic overexpression of BMP4 promotes osteogenic differentiation of bone marrow stromal cells[J]. Journal of Third Military Medical University, 2021, 43(16): 1550-1558. DOI： 10.16016/j.1000-5404.202102063.

摘要

目的

探究骨细胞MLO-Y4过表达骨形态发生蛋白4 (bone morphogenetic protein-4

BMP4)通过Notch信号对骨髓基质细胞ST2成骨分化的影响。

方法

使用BMP4重组腺病毒(Ad-BMP4)以及阴性对照(Ad-GFP)分别感染MLO-Y4后与ST2共培养，分为GFP组及BMP4组；Western blot检测MLO-Y4细胞BMP4蛋白表达；采用实时荧光定量PCR(qPCR)检测MLO-Y4的BMP4和Notch配体基因、共培养体系中成骨标志基因、Notch靶基因以及促血管生成因子表达；碱性磷酸酶(alkaline phosphatase

ALP)染色及生化定量检测ALP活性；在共培养体系中加入Notch信号抑制剂DAPT后，再次检测上述指标。

结果

MLO-Y4过表达BMP4，上调Notch配体Jag1、Jag2、Dll4表达；ALP染色以及生化定量结果显示共培养第1天GFP组和BMP4组ALP活性差异无统计学意义，在第3、5天，BMP4组ALP活性更高(

＜0.05)；BMP4组成骨标志基因在第1天仅Runt相关转

录因子2(runt-related transcription factor 2

Runx2)显著升高(

＜0.05)，第3、5天BMP4组Alp、Runx2、骨钙蛋白(osteocalcin，Ocn)、Ⅰ型胶原(collagenⅠ)均显著增高(

＜0.05)；第3天BMP4组Notch信号靶基因Hey1、Hey2，血管内皮生长因子(vascular endothlial growth factor

VEGF)、低氧诱导因子(hypoxia-inducible factor 1α

HIF1α)的表达也显著增高(

＜0.05)；加入DPAT抑制Notch信号后，BMP4组ALP活性、成骨标志基因以及促血管生成因子表达均显著下降(

＜0.05)。

结论

MLO-Y4过表达BMP4可以促进ST2成骨分化，该过程可能与Notch信号增强有关。

Abstract

Objective

To investigate the effect of bone morphogenetic protein 4 (BMP4) overexpression in MLO-Y4 osteocytes on osteogenic differentiation of bone marrow stromal ST2 cells through Notch signaling.

Methods

Ad-GFP or Ad-BMP4 infected MLO-Y4 cells were co-cultured with ST2 cells

and designated as GFP and BMP4 group respectively. Western blotting was used to detect the BMP4 protein level in MLO-Y4 cells. Real-time fluorescence quantitative analysis (qPCR) was applied to determine the mRNA expression of BMP4 and Notch ligands genes as well as osteoblast genes in the MLO-Y4 cells

and Notch target genes and angiogenesis factors in the co-cultured systems. The activity of alkaline phosphatase (ALP) was determined by ALP staining and biochemical quantification. Notch signal inhibitor DAPT was added to the co-culture system to detect the above indexes again.

Results

Overexpression of BMP4 upregulated the expression of Notch ligands Jag1

Jag2 and Dll4 in MLO-Y4 cells. ALP staining and biochemical quantitative results demonstrated that there was no significant difference in ALP activity at day 1 between the GFP and BMP4 groups

and stronger ALP activity was seen in the latter group at day 3 and 5 (

0.05). In the BMP4 group

at day 1 among the osteoblast genes

only runt-related transcription factor 2 (Runx2) was obviously expressed (

0.0

and the levels of Alp

Runx2

osteocalcin (Ocn)

and collagen Ⅰ were all highly expressed in the BMP4 group (

0.05). At day 3

Notch signaling target genes Hey1 and Hey2

angiogenesis factors vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF1α) were also highly expressed in the BMP4 group compared with the control group (

0.05). After addition of DAPT to inhibit Notch signaling

the activity of ALP and expression of osteoblast genes and angiogenesis genes were all decreased (

0.05).

Conclusion

Overexpression of BMP4 in MLO-Y4 cells promotes osteogenic differentiation of ST2 cells

which may be related to the enhancement of Notch signaling.

关键词

Keywords

references

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