Notch pathway inhibits BMP9-induced osteogenic differentiation of mesenchymal stem cells C3H10T1/2 by down-regulating expression of gene JunB

Wang Nan; Tan Tao; Lin Duanyang; Luo Xiaoji

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Notch pathway inhibits BMP9-induced osteogenic differentiation of mesenchymal stem cells C3H10T1/2 by down-regulating expression of gene JunB

更新时间：2023-12-22

- Notch pathway inhibits BMP9-induced osteogenic differentiation of mesenchymal stem cells C3H10T1/2 by down-regulating expression of gene JunB
- Journal of Chongqing Medical University Vol. 43, Issue 10, Pages: 1286(2018)
- 作者机构：
  
  1. 重庆医科大学第一临床学院,重庆,400016
  2. 重庆医科大学附属第一医院骨科,重庆,400016
- 作者简介：
- 基金信息：
- DOI：
  CLC：
- Published：2018，
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Wang Nan, Tan Tao, Lin Duanyang, et al. Notch pathway inhibits BMP9-induced osteogenic differentiation of mesenchymal stem cells C3H10T1/2 by down-regulating expression of gene JunB. [J]. Journal of Chongqing Medical University 43(10):1286(2018)
DOI：

Wang Nan, Tan Tao, Lin Duanyang, et al. Notch pathway inhibits BMP9-induced osteogenic differentiation of mesenchymal stem cells C3H10T1/2 by down-regulating expression of gene JunB. [J]. Journal of Chongqing Medical University 43(10):1286(2018) DOI：

摘要

目的：研究Notch信号通路在骨形态发生蛋白9（bone morphogenetic protein 9，BMP9）诱导小鼠间充质干细胞C3H10T1/2成骨分化过程中的作用及其机制。方法：将细胞分为5组：空白组（Blank，n=3），BMP9处理组（BMP9，n=3），空白质粒对照组（BMP9+Control，n=3），过表达Notch1胞内段质粒（Notch1 intracellular domain，NICD）处理组（BMP9+NICD，n=3），DAPT处理组（BMP+DAPT，n=3）。通过碱性磷酸酶（alkaline phosphatase，ALP）染色和钙盐染色分别验证过表达NICD对成骨分化早晚期情况的影响。进一步采用定量逆转录PCR（quantitative real-time polymerase chain reaction，qRT-RCR）和Western blot检测基因NICD、Hey1及成骨相关基因Smad-1/5/8、p-Smad-1/5/8、OPN、Runx2、OCN、JunB的表达。结果：早期ALP活性及染色结果显示，NICD组较Control组能显著抑制C3H10T1/2细胞成骨分化过程中ALP的形成[5 d：（33 167.66±2 018.45） vs. （146 451.00±14 889.67），P=0.000；7 d：（58 981.33±4 724.70） vs. （89 588.66±5 928.32），P=0.000]，γ-分泌酶抑制剂（N-[N-（3，5-difluorophen-acetyl）-L-alanyl]-S-phenylglycine t-butyl ester，DAPT）完全抑制Notch通路时得到相同的结果[5 d：（44 812.66±4 174.94） vs. （146 451.00±14 889.67），P=0.000；7 d：（64 622.93±4 724.70） vs. （89 588.66±5 928.32），P=0.000]。茜素红S染色结果显示，NCID抑制钙盐结节的形成，而DAPT阻断Notch通路明显促进钙盐结节形成。qRT-PCR结果表明，NCID组中NICD及靶基因Hey1的表达较Control组明显增加[（3.90±0.02） vs. （0.35±0.01），P=0.000；（19.79±0.01） vs. （11.80±0.02），P=0.000]，Western blot得到相同结果[（1.32±0.01） vs. （0.19±0.01），P=0.000]，且NICD明显抑制成骨分化相关基因JunB、Runx2、OCN、OPN等的表达[（0.10±0.01） vs. （0.53±0.01），P=0.000；（0.18±0.01） vs. （0.30±0.02），P=0.000；（0.36±0.01） vs. （0.62±0.02），P=0.000；（0.07±0.01） vs. （0.48±0.01），P=0.000]，而转录因子Smad-1/5/8、p-Smad-1/5/8的表达不受影响[（0.74±0.02） vs. （0.73±0.03），P=0.000；（0.63±0.01） vs. （0.58±0.04），P=0.000]，DAPT阻断Notch通路后上述相关基因的表达明显增加。结论：本实验结果首次证明，NICD激活Notch通路对BMP9诱导的C3H10T1/2细胞成骨分化的抑制作用可能是通过抑制JunB基因发挥作用，而非调控BMP9/Smad（bonemorphogenetic protein 9/drosophila mothers against decapentaplegic）信号通路。

Abstract

Objective：To investigate the role and mechanism of Notch signaling pathway in bone morphogenetic protein 9（BMP9） in－duced osteogenic differentiation of mesenchymal stem cells line C3H10T1/2. Methods：The cells were divided into five groups：the blank（n=3），BMP9（n=3），BMP9+Control（n=3），BMP9+NICD（n=3），BMP9+DAPT（n=3）. The effect of Notch pathway on early and late osteogenic differentiation was detected by ALP and calcium staining. Furthermore，the expressions of NICD，Hey1 and the osteogen－esis-related genes Smad-1/5/8，p-Smad-1/5/8，OPN，Runx2，OCN，JunB were detected by qRT-RCR and Western blot after transfecting C3H10T1/2 cells with NICD plasmid. Results：The staining and activity of ALP showed that the NICD-activated Notch pathway could significantly inhibit the formation of ALP on 5 d and 7 d during the C3H10T1/2 cell differentiation[5 d：（33 167.66±2 018.45） vs. （146 451.00±14 889.67），P=0.000；7 d：（58 981.33±4 724.70） vs. （89 588.66±5 928.32），P=0.000]，and when DAPT inhibited Notch pathway，it came to the same result [5 d：（44 812.66±4 174.94） vs. （146 451.00±14 889.67），P=0.000；7 d：（64 622.93±4 724.70） vs. （89 588.66±5 928.32），P=0.000]. Alizarin S staining showed that NICD inhibited the formation of alcium salt nodules，while DAPT blocking Notch pathway significantly promoted the formation of calcium salt nodules during the osteogenic differentiation of C3H10T1/2 cells. qRT-RCR results showed that the expres－sion of NICD and target gene Hey1 in NCID group was significantly higher than that in the control group[（3.90±0.02） vs. （0.35±0.01），P=0.000；（19.79±0.01） vs. （11.80±0.02），P=0.000]，and Western blot got the same result. Furthermore，the expressions of JunB，Runx2，OCN and OPN were markedly inhibited by NICD[（0.10±0.01） vs. （0.53±0.01），P=0.000；（0.18±0.01） vs. （0.30±0.02），P=0.000；（0.36±0.01） vs. （0.62±0.02），P=0.000；（0.07±0.01） vs. （0.48±0.01），P=0.000]，but the expressions of Smad-1/5/8 and p-Smad-1/5/8 were not affected[（0.74±0.02） vs. （0.73±0.03），P=0.000；（0.63±0.01） vs. （0.58±0.04），P=0.000]；however，the expres－sions of the above-mentioned related genes significantly increased after DAPT blocking Notch pathway. Conclusion：The results first demonstrate that the inhibitory effect of NICD on the BMP9-induced osteogenic differentiation of C3H10T1/2 cells may be mediated by the inhibition of JunB gene，rather than the regulation of BMP9/Smad signaling pathway.

关键词

骨形态发生蛋白9Notch信号通路间充质干细胞成骨分化

Keywords

bone morphogenetic protein 9Notch signaling pathwaymesenchymal stem cellsosteogenic differentiation

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