Notch1在BMP9诱导iMEF细胞成骨分化中的作用

Role of Notch1 signaling in the process of BMP9-induced osteogenetic differentiation of immortalized mouse embryonic fibroblasts

目的研究Notch信号中Notch1受体在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)诱导的永生化小鼠胚胎成纤维细胞(immortalized mouse embryonic fibroblasts,i MEF)成骨分化中的作用。方法利用Notch1受体显性负性突变体(dominant negative mutant,dn Notch1)处理i MEF细胞,首先分别采用细胞化学染色、活性测定和RT-PCR了解Notch1对早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP)和晚期成骨指标骨桥蛋白(osteopontin,OPN)、骨钙素(osteocalcin,OCN)的影响;其次用Western blot、荧光素酶和RT-PCR检测Notch1对BMP9经典信号通路中Smad1/5/8磷酸化和Smad结合元件(smad-binding element,SBE)活性影响;最后用结晶紫染色和流式细胞仪分析其对细胞增殖、周期及凋亡的影响。结果与对照组相比,dn Notch1处理i MEF第3、5、7天后,ALP活性均降低(P<0.05),并呈剂量依赖性,11 d时OPN和OCN表达量也下调(P<0.05);BMP9经典通路中Smad1/5/8蛋白量没有明显变化,而p-Smad1/5/8蛋白及SBE活性受到了抑制(P<0.05);结晶紫染色及流式细胞仪分析结果显示dn Notch1能够抑制BMP9诱导的i MEF早期细胞增殖,但对细胞凋亡无影响。结论 dn Notch1竞争抑制Notch1信号,使BMP9诱导i MEF的成骨分化能力显著下降,其可能通过抑制BMP信号通路的活化和i MEF早期增殖两方面来实现。

Objective To determine the role of Notchl in the osteogenic differentiation of immortalized mouse embryonic fibroblasts （iMEF） induced by bone morphogenetic proteins 9 （BMP9）. Methods iMEF cells were treated with dominant negative mutant adenovirus AdRdnNotchl/dnNotchl and BMP9. And the cells treated by the adenovirus vector containing red fluorescence protein （RFP） served as control. The early osteogenic index alkaline phosphatase （ALP） was observed by ALP staining and its activity was determined. While the late osteogenic indexes osteopontin （OPN） and osteoealcin （OCN） were measured with qRT-PCR. The protein expression of p-Smadl/5/8 and Smad binding element （SBE） activity were studied by Western blotting and luciferase detection. The effects of dnNotchl on the cell proliferation, cycle and apoptosis were determined with crystal violet staining and flow cytometry. Results Compared with the control cells, the ALP activity was significantly decreased in 3, 5 and 7 d after the treatment of dnNotchl combined with BMP9 in a dose-dependent manner （P 〈0.05）. On the llth day, the expression of OPN and OCN was obviously decreased （P 〈 0.05 ）, but the level of Smadl/5/8 had no remarkable change though the protein level of p-Smadl/5/8 and the activity of SBE were inhibited significantly （ P 〈 0.05 ）. Crystal violet staining and flow cytometric analysis indicated that dnNotchl suppressed cell proliferation induced by BMP9 at the early stage, but had no effect on cell apoptosis. Conclusion dnNotchl can competitively inhibit the Notchl signal, and thus significantly decrease the osteogenesis induced by BMP9, which might be due to suppressing the activity of BMP signal pathway and the early proliferation of iMEF cells, suggesting that Notch1 may play a promoting role in BMP9-induced osteogenesis.