The Effect And Research Of Smad Pathway Regulate BMP9-induced Osteogenic/Odontogenic Differentiation Of ISCAP

Objective:Although BMP9 has a prominent effect on promoting osteogenic/odontogenic differentiation of stem cells from the apical papilla(iSCAP) in the previous experiment, the mechanism is not clear. Smad signaling pathway is one of the most canonical pathway in BMP signaling pathways. Thus,this research aimed to investigate the effects of Smad signaling pathway on BMP9-induced osteogenic/odontogenic differentiation of iSCAP.Methods:First of all, BMP9 was introduced into the iSCAP by using recombinant adenoviruses method and phosphorylated forms of Smad1/ 5/8 protein was assayed by Western Blot after 36 hours. Subsequently, taking the dominant negative mutant receptor ALK1(dnALK1) recombinant adenoviruses and BMP9 conditioned medium together into the iSCAP. Then, activity of Smad1/5/8 was detected by Western Blot. Chemoluminescence quantitative and staining assay were applied to measure the activity of alkaline phosphatase(ALP), then, Western Blot was conducted to analyze the expression of OCN and OPN and Alizarin red S staining was used to examin calcium deposition. At last, expression of transcription factor RUNX2 and osteogenic/odontogenic target genes(such as OCN、 OPN、DMP1) were measured by RT-PCR.Results:It was found that BMP9 stimulated the activation of Smad1/5/8 in iSCAP,and this phenomenon was decreased by dnALK1 in iSCAP. BMP9-induced osteogenic/odontogenic differentiation(early marker ALP expression of OCN and OPN, late osteogenic/odontogenic marker calcium deposition) were significantly inhibited by dnALK1,which is a competitive inhibitor of BMP9. Furthermore, expression of transcription factor Runx2 and osteogenic/odontogenic target genes were reduced by dnALK1.Conclusion:Taken together, our results revealed that Smad1/5/8 was activated in BMP9-induced osteogenic differentiation of iSCAP. And inhibition of Smad1/5/8 activity resulted in reduction of BMP9-induced osteogenic/odontogenic differentiation of iSCAP, which implies that BMP9 can induce osteogenic/odontogenic differentiation of iSCAP through Smad signaling pathway. AlK1 is an important receptor of BMP9-Smad signaling pathway, dnALK1 can inhibit BMP9 effectively, however, whether can it inhibit the other signal pathways are remained to be studied. In addition, animal experiments should be performed to verify the inhibition of dnALK1 in BMP9-induced osteogenic/odontogenic differentiation of iSCAP.