过表达NICD通过下调基因JunB的表达抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化

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

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-difluorophenacetyl)-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。

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.