BMP9 Induces Osteogenic/odontogenic Differentiation of ISCAP through the Smad Pathway

doi:10.13523/j.cb.20160803

China Biotechnology

2016, Vol. 36

Issue (8): 16-22 DOI: 10.13523/j.cb.20160803

BMP9 Induces Osteogenic/odontogenic Differentiation of ISCAP through the Smad Pathway

CHEN Bing, KONG Ling-jiao, LEI Jin-xia, SHEN Lu, ZHANG Cai, WANG Jin-hua

Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Stomatological Hospital of Chongqing Medical University, Chongqing 401147, China

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Abstract

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,the aims are 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), Alizarin red S staining was used to examine 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, these results reveal that Smad1/5/8 was activated in BMP9-induced osteogenic differentiation of iSCAP.And inhibition of Smad1/5/8 activity results 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.

Key words： Immortalized dental apical papilla stem cells Dominant negative mutant receptor ALK1 Osteogenic/odontogenic differentiation Bone morphogenetic proteins9

Received: 26 April 2016 Published: 25 August 2016

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CHEN Bing, KONG Ling-jiao, LEI Jin-xia, SHEN Lu, ZHANG Cai, WANG Jin-hua. BMP9 Induces Osteogenic/odontogenic Differentiation of ISCAP through the Smad Pathway. China Biotechnology, 2016, 36(8): 16-22.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20160803 OR https://manu60.magtech.com.cn/biotech/Y2016/V36/I8/16

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