摘要
目的探讨Notch信号是否通过MAPK通路影响BMP9诱导C2C12细胞成骨分化。方法利用BMP9腺病毒处理C2C12细胞5d后,用PCR和Western blot检测MAPK通路中P38、Erk1/2和JNK总蛋白以及磷酸化水平。用Notch信号特异性抑制剂DAPT阻断Notch信号后,细胞化学染色分析ALP和钙盐沉积,以及早晚期成骨指标Runx2和OCN在RNA以及蛋白水平上的变化。同时检测BMPs靶基因Id1、Id2和Id3 RNA水平的表达和MAPK信号中P38、Erk1/2和JNK总蛋白以及磷酸化水平变化。结果 BMP9能上调Hey1和Notch信号的配、受体表达;BMP9不影响P38、Erk1/2和JNK总蛋白的表达,但能上调其磷酸化水平;DAPT能抑制BMP9诱导C2C12细胞的ALP活性以及钙盐沉积;影响Runx2和OCN成骨标志物的表达;阻断Notch信号后Id1、Id2和Id3表达受到抑制,P38、Erk1/2磷酸化水平下调,而JNK磷酸化水平无明显变化。结论 Notch信号通过MAPK途径参与了BMP9诱导的C2C12细胞的成骨分化。
Objective To analysis the role of Notch signaling pathway in bone morphogentic protein 9 （BMP9） induced osteogenic diffierentiaion of C2C12 cells through MAPK signaling. Methods C2C12 cells were infected by recombinant adenovirus expressing BMP9,then the total protein level and phosphorylated form of p38,Erk1/2,JNK MAPKs were determined by RT-PCR and Western blot. After treatment C2C12 cells with DAPT,the early osterogenic marker,ALP activity were detected by quantitative and staining assay, later ostergenic marker calcium deposition was determined by Alizarin Red S staining. The expression of ostergenic marker genes Runx2 and OCN, BMPs target genes Id1,Id2,Id3 were analysed by RT-PCR and Western Blot.The total protein level and phosphorylated form of p38, Erk1/2 and JNK MAPKs were determined by Western blot. Results BMP9 increased Hey1 and Notch ligands and receptors mRNA level and the protein expression of Hey1 and phosphorylated form of p38,Erk1/2 and JNK MAPKs, not the total protein level. The Notch specific inhibitor DAPT dose-dependently decreased ALP activity and calcium deposition. Furthermore, DAPT inhibited the RNA expression of BMPs target genes Id1, Id2, Id3. Moreover, The BMP9-induced phosphorylated form of p38, Erk1/2 were reduced by treatment with DAPT. Conclusion Notch signaling may involve in BMP9 induced osteoblast commitment of C2C12 mesenchymal stem cells through MAPK signaling pathway.
出处
《基础医学与临床》
CSCD
北大核心
2013年第12期1564-1571,共8页
Basic and Clinical Medicine
基金
重庆市自然科学基金(cstc2012jjA10004)
