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

Abstract:

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.