Effects of BMP-7 on osteogenic differentiation of prenatal mouse nucleus pulposus cell and Notch signaling pathway

Abstract:

Objective：To investigate the effects of recombinant adenovirus-mediated bone morphogenetic protein-7（Ad-BMP-7） over-expression on osteogenic differentiation of prenatal mouse intervertebral disc nucleus pulposus（NP） cell，and to explore whether BMP-7 regulates classic Notch signal pathway in this process. Methods：Recombinant Ad-BMP-7 and adenovirus-mediated green fluores－cent protein（Ad-GFP） were amplified by HEK293 cell and were used to infect NP cells. The experiment was designed into BMP-7 group（n=3），GFP group（n=3） and blank group（n=3）. Real-time PCR was performed to detect the mRNA expression level of BMP-7，real-time PCR was carried out to detect the mRNA expression level of osteogenesis index osteopontin（OPN），osteoprotegerin（OPG） as well as the classic Notch signal pathway members. Western blot was used to detect the protein expressions level of osteogenesis transcription factor Runx2 and OPN. Alkaline pho-sphatase（ALP） activity was measured by using ALP assay and staining. Results：The high titers Ad-BMP-7 and Ad-GFP were generated by adenovirus amplification in HEK293. The basic mRNA expression of BMP-7 was almost undetectable in NP cell. Compared with those in blank group，Ad-BMP-7 mediated over-expression of BMP-7 significantly increased the mRNA expressions of OPN，OPG and the protein expressions of osteogenesis transcrip－tion factor Runx2，OPN（OPN：F=613.815，P=0.000；BMP-7 group vs. GFP group and blank group，P=0.000；OPG：F=10.046，P=0.012；BMP-7 group vs. GFP group P=0.016；BMP-7 group vs.blank group，P=0.047）. The activity of ALP，a specific indicator for steogenesis in Ad-BMP-7 group was statistically higher than that in blank group（F=513.417，P=0.000；BMP-7 group vs. GFP group and blank group，P=0.000），with enhanced positive purple-blue stained cells. After 3 days of Ad-BMP-7 infection，the mRNA expression of classic Notch signal pathway members Notch-1，Notch-2，Jag-1，Hey-1 significantly increased（Notch-1：F=94.574，P=0.002；BMP-7 group vs. GFP group，P=0.003；BMP-7 group vs. blank group P=0.004）（Notch-2：F=318.962，P=0.001；BMP-7 group vs. GFP group and blank group，P=0.000）（Jag-1：F=166.476，P=0.001，BMP-7 group vs. GFP group and blank group P=0.000）（Hey-1：F=1 082.054，P=0.000；BMP-7 group vs. GFP group and blank group，P=0.000），while reversed to the baseline at day 7. There was no difference in others Notch signal pathway members among treated groups. Conclusion：Adenovirus-mediated BMP-7 over-expression can induce osteogenic dif－ferentiation of NP cell，which partly due to transient up-regulation of some classic Notch signal pathway members.