摘要
该文研究了Notch信号在骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)诱导小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)成骨分化中的作用及机制。利用过表达Notch配体之一DLL1的腺病毒(adenovirus-delta-like 1,Ad-DLL1)、显性负性突变型Notch1受体的腺病毒(adenovirus-dominant-negative mutant of Notch1,Ad-dn Notch1)或γ-分泌酶抑制剂{N-[N-(3,5-difluorophena-cetyl-L-alanyl)]-S-phenylglycine t-butyl ester,DAPT}处理MEFs,细胞化学染色和/或活性测定检测碱性磷酸酶(alkaline phosphatase,ALP)表达、钙盐沉积;q RT-PCR、Western blot、荧光素酶分别检测BMP2信号I、II型受体和成骨基因表达、Smad1/5/8蛋白磷酸化水平及Smad结合元件(Smad-binding element,SBE)转录活性。结果显示,DLL1促进BMP2介导MEFs早晚期成骨分化,并上调ALK2等受体的m RNA水平、Smad1/5/8的磷酸化水平及SBE转录活性;与之相对应,dn Notch1和DAPT抑制上述指标。Notch经典靶基因发状分裂相关增强子1(hairy/enhancer-of-split related with YRPW motif 1,Hey1)可促进BMP2诱导成骨分化,并逆转DAPT对BMP2诱导成骨分化的抑制作用。该研究结果提示,Notch信号促进BMP2诱导MEFs成骨分化,可能是通过激活BMP2/Smads通路实现的,这一过程中Hey1发挥了重要作用。
This study was aimed to investigate the role of Notch in bone morphogenetic protein 2（BMP2）-induced osteogenic differentiation in mouse embryonic fibroblasts（MEFs）. The over-express DLL1（one of the Notch ligands） adenoviruses（adenovirus-delta-like, Ad-DLL1）, dominant-negative mutant of Notch1（one of the Notch receptors） adenoviruses（adenovirus-dominant-negative mutant of Notch1, Ad-dn Notch1） and specific γ-secretase inhibitor {N-[N-（3,5-difluorophena-cetyl-L-alanyl）]-S-phenylglycine t-butyl ester, DAPT} were used to infect or treat MEFs, respectively. The early osteogenic index alkaline phosphatase（ALP） and late osteogenic index calcium deposits were detected by cytochemical staining and/or activity determination. The expression levels of BMP2 receptors and osteogenesis-related genes, the phosphorylation level of Smad1/5/8, and the transcriptional activity level of Smad-binding element（SBE） were determined by q RT-PCR, Western blot and luciferase reporter assay, respectively. The results showed that, compared with control group, DLL1 could obviously promote BMP2-mediated osteogenic differentiation（P〈0.05）. DLL1 could also increase the m RNA levels of ALK2 and other BMP2 signaling receptors, the phosphorylation level of Smad1/5/8 and the transcriptional activity level of SBE in MEFs（P〈0.05）. Correspondingly, dn Notch1 and DAPT exert negative effects on the indexs mentioned above（P〈0.05）. On the other hand, Hey1, the classical target gene of Notch signal, could promote BMP2-induced osteogenic differentiation. We furthermore found that Hey1 could reverse the inhibitory effect of DAPT on BMP2-induced osteogenic differentiation（P〈0.01）. These results indicated that Notch signaling could remarkably enhances BMP2-induced osteogenesis in MEFs and might exerts its effect though affecting the activation of BMP2/Smads signaling, Hey1 plays an important role in this process.
作者
卫亚琳
牟代勇
廉静
罗进勇
唐敏
Wei Yalin;Mou Daiyong;Lian Jing;Luo Jinyong;Tang Min(Key Laboratory of Clinical Laboratory Diagnostics of Ministry Education, Faculty of Laboratory Medicine Chongqing Medicine University, Chongqing 400016, China)
出处
《中国细胞生物学学报》
CAS
CSCD
2018年第4期478-489,共12页
Chinese Journal of Cell Biology
基金
重庆市基础科学与前沿技术研究项目(批准号:cstc2017jcyj AX0428)资助的课题~~
