Effect And Mechanism Of TGF-?? Receptor Inhibitor ITD-1 In Blocking Osteogenic Differentiation Of Bone Marrow Stromal Cells

Objective:Bone marrow stromal cells have the potential to differentiate into a variety of cells and become a good source of cells in the field of bone regeneration.Recent studies have shown that multiple factors play a key role in regulating the osteogenic differentiation of bone marrow stromal cells.TGF-? signaling is involved in regulating bone homeostasis.However,in bones and many other tissues,the role of TGF-? signaling is non-linear.Both the increase and decrease of TGF-? signaling will lead to osteoporosis phenotype,so the mechanism of TGF-? signaling is still to be further explored.T?RII is a key molecule of TGF-? signaling.In this study,the specific inhibitor ITD-1 of T?RII was used to degrade T?RII,to explore the effect of TGF-? signaling on osteogenic differentiation of bone marrow stromal cells and its possible mechanism.Methods:(1)ALP staining experiment was used to test the inhibitory effect of2?M,5?M,10?M ITD-1 on osteogenic differentiation of BMSCs to determine the optimal concentration.Western Blot was used to detect the expression of p-Smad2/3,cck-8 proliferation test was used to detect theproliferation level of cells,and flow cytometry was used to detect the apoptosis level of cells to determine the safety of the optimal concentration.(2)ALP staining,ALP activity detection,alizarin red staining and quantification,qPCR detection of the expression of osteogenic markers to determine the effect of ITD-1 on osteogenic differentiation of BMSCs and ST2 cells induced by Wnt/BMP signaling.(3)RNA-seq was performed to detect the biological process in mRNA of BMSCs treated by ITD-1.(4)Oil red O staining and qPCR detection of adipocyte-related markers was used to clarify the effect of ITD-1 on the differentiation of BMSCs and ST2 cells induced by Wnt/BMP signaling.Results:(1)10?M ITD-1 can almost completely inhibit the ALP activity of BMSCs and the concentration of ITD-1 did not cause obvious cell apoptosis,but reduced the proliferation level of cells.(2)ITD-1 significantly reduced the ALP activity and expression of osteogenic markers in BMSCs and ST2 cells induced by Wnt/BMP signaling.(3)RNA-seq results showed that compared with the DMSO group,the mRNA expression of TGF-? ligands,receptors,and target genes in BMSCs treated with ITD-1 was significantly reduced,the mRNA expression of osteogenic differentiation-related markers was also reduced.Significantly reduced.The results of GO and KEGG enrichment analysis showed that compared with the DMSO group,a large number of genes were enriched in the signal pathways related to adipocytes in the ITD-1 group,suggesting that BMSCs differentiated into adipocytes.(4)ITD-1 can significantly increase the number of adipocytes and theexpression of adipocyte-related markers in BMSCs and ST2 cells induced by Wnt/BMP signaling.Conclusions:(1)TGF-? signal is necessary for osteogenic differentiation of bone marrow stromal cells.Blocking TGF-? signal prevents bone marrow stromal cells from osteogenic differentiation,even under the treatment of Wnt and BMP signaling.(2)Blocking of TGF-? signaling will lead to differentiation of bone marrow stromal cells into adipocytes.TGF-? signal may be a osteogenic switch of bone marrow stromal cells to regulate the differentiation of bone marrow stromal cells.This research may provide a new target for research related to bone repair,as well as clinical prevention and treatment of osteoporosis.