Synergistic Effect And Mechanism Of Triiodothyronine On Osteogenic Differentiation Of Mesenchymal Stem Cells Induced By BMP9

[OBJECTIVE] Thyroid hormone(TH)plays a key role in the bone growth and metabolism balance by regulating bone formation and bone resorption.The regulation of thyroid hormone on the osteogenic differentiation of mesenchymal stem cells is an important link in the process of regulating bone formation by thyroid hormone,the mechanism of which is not very clear.Bone morphogenetic protein 9(BMP9)plays an important role in promoting osteogenic differentiation.Moreover,many regulatory factors can interact with BMP9-induced signal pathways to synergistically regulate osteogenesis.The aim of this study was to investigate the effects and mechanism of triiodothyronine(T3)on osteogenic differentiation of mesenchymal stem cells(MSCs)through bone morphogenetic protein 9(BMP9).[METHODS] 1、The effects of T3 on BMP9 induced osteogenic differentiation of MSCs.ALP activity and staining were measured using a modified Great Esc APe SEAP Chemiluminescence assay and a BCIP/NBT Alkaline Phosphatase staining assay kit respectively.Immunohistochemical staining and western blot were used to detect the expression of osteocalcin and osteopontin.Alizarin red staining was used to measure mineral nodule formation.2、We next examined whether T3 and BMP9 synergistically promote bone formation in by using the foetal limb culture approach and stem cell implantation experiment.The foetal limbs of mouse embryos(E18-21)were cultured in vitro,and the changes of endochondral osteogenesis were observed by calcein.Our previous studies demonstrated that MSC implantation is a reliable and effective approach to investigating ectopic bone formation.Subcutaneous injection of C3H10T1/2 cells infected by adenovirus was performed.The effect of T3 on ectopic bone formation induced by BMP9 was verified by Micro-CT analysis and Masson’s staining of subcutaneous nodules.3、Crosstalk between T3 and BMP9 signalling pathway in the regulation of MSC osteogenic differentiation.We analysed the effect of T3 on BMP9-induced Smad1/5/8 phosphorylation and the nuclear and cytoplasmic expression of Smad1/5/8 by Western blotting and immunofluorescence assay.The effect of AMPK pathway on T3 and BMP9 induced MSC osteogenic differentiation.C3H10T1/2 cells were treated with T3 and BMP9.Seventy-two hours after treatment,the expression and phosphorylation of AMPK and p38 protein were analysed by Western blotting with the indicated antibodies.C3H10T1/2 cells were pre-treated with Compound.C or si RNA.Seventy-two hours post treatment,Western blotting analysis was performed with the indicated antibodies to detect the effects of above factors on the expression and phosphorylation of AMPK and p38 protein as well as alkaline phosphatase.[RESULTS] 1、The synergistic treatment of T3 could significantly increase BMP9 induced expression of alkaline phosphatase,osteocalcin and osteopontin,and promote the formation of mineralized nodules.2、T3 and BMP9 synergistic have stronger positive effects on promoting hypertrophic cartilage area in fetal rat tibia tissue than BMP9 alone.No significant effect was found in group Control group and T3 group.In stem cell transplantation experiment,the density of bone nodules formed by the synergistic action of BMP9 and T3 co-treatment was significantly higher than that in BMP9 group.We didn’t detect any ectopic bone formation in Control group and T3 group.3、The results of the immunofluorescence staining and Western blot suggest that T3 promotes smad1/5/8 phosphorylation and nuclear translocation induced by BMP9,and activates the BMP/Smad pathway.4、Further study showed that T3 and BMP9 combined promote activation of AMPK/p38 signaling pathway to induce osteogenesis.And the activation of p38 phosphorylation and the activity of alkaline phosphatase induced by T3 and BMP9 were inhibited by AMPK inhibitor and si AMPK.SB203580 is an inhibitor of p38 and can inhibit alkaline phosphatase activity induced by combined action of T3 and BMP9.[CONCLUSION] 1、T3 promotes osteogenic differentiation of mesenchymal stem cells induced by BMP9 in vitro and in vivo.2、T3 promotes BMP9-induced classical BMP/Smad signaling pathway,enhancing phosphorylation and nuclear translocation of smad1/5/8.3、BMP9 synergizes the effect of T3 by promoting the activation of AMPK/p38 pathway.