The Role Of Sprouty-4 In Osteoblast And Adipocyte Differentiation And The Mechanisms Involved

Objective: Mesenchymal/stromal stem cells(MSCs)have the potential to differentiate into osteoblasts and/or adipocytes.The differentiation process of marrow stromal cells(MSCs)is affected by both internal and external factors.The differentiation of adipogenic and osteogenesis has mutual restraint to achieve a state of dynamic equilibrium.SPRY is a family of soft lipid acylated phosphoproteins that are widely expressed in body tissues and have relatively conserved sequences.It has been found that some SPRY proteins may be involved in bone development.We are going to investigate the role of SPRY4 in the differentiation of osteoblasts and adipocytes and its mechanism.Methods: 1.The expression and distribution of Spry4 in various tissues of wild-type mice were detected by qRT-PCR.The primary bone marrow stromal cells(MSCs)were isolated from C57BL/6J mice and then cultured.The expression of Spry4 during the differentiation of bone and adipocytes were detected by qRT-PCR.2.Spry4 was overexpressed in bone marrow stromal cell line ST2 and MSCs,then the cells were induced to differentiate into osteoblasts and adipocytes.The effect of Spry4 overexpression on differentiation was studied.3.Spry4 expression was silenced in ST2 and MSCs,then the cells were induced to differentiate into osteoblasts and adipocytes,and effect of Spry4 silencing on differentiation were studied.4.To reveal the mechanisms that control the osteogenic differentiation of bone marrow stromal cells by SPRY4,we overexpressed and silenced the expression of Spry4 in ST2 cells.The effect of SPRY4 on Wnt/?-catenin signaling pathway was detected by immunofluorescence and Western blotting.Moreover,the effect of SPRY4 on MAPK signaling pathway was detected by Western blotting.5.To further reveal the mechanisms that control the adipogenic differentiation of bone marrow stromal cells by SPRY4,the effect of SPRY4 on AKT/mTORC1 signaling pathway was detected by Western blotting.We also performed the blocking assays to detecte the effect of SPRY4 on adipocyte differentiation under the background of mTORC1 inhibition in ST2 cells.Results: 1.The expression level of Spry4 was increased during the differentiation of adipocytes in MSCs,and the expression level of Spry4 was the highest at day 2 after induction.The expression level of Spry4 was decreased during the osteogenic differentiation and was the lowest at the day 8 after induction.Spry4 was widely expressed in various tissues,and the expression level in bone tissue was higher than in other tissues.2.Overexpression of Spry4 inhibited the osteogenic differentiation in ST2 cells,as evidenced by attenuated ALP staining,and decreased expression of osteoblast-specific transcription factor Runx2 and the marker genes Alp and OPN.Conversely,overexpression of Spry4 promoted adipogenic differentiation in ST2 cells,as evidenced by enhanced oil red O staining and increased expression of adipocyte-specific transcription factors CEBP? and PPAR?,and marker genes aP2 and adipsin.3.Silencing of Spry4 stimulated the osteogenic differentiation in ST2 cells,as evidenced by enhanced ALP staining,and increased expression of osteoblast-specific transcription factor Runx2 and the marker genes Alp and OPN(Osteopontin).Conversely,silencing of Spry4 inhibited adipogenic differentiation in ST2 cells,as evidenced by attenuated oil red O staining and decreased expression of adipocyte-specific transcription factors CEBP? and PPAR?,and marker genes aP2 and adipsin.Furthermore,silencing of Spry4 in primary MSCs stimulated the osteogenic differentiation and blocked adipogenic differentiation.4.Overexpressing Spry4 in ST2 cells inhibited canonical Wnt pathway,revealed by reduced nuclear transfer of ?-catenin from the cytoplasm to the nucleus.Besides,the protein levels of p-LRP6,total and non-phosphorylated ?-catenin in the total cell lysates as well as ?-catenin and TCF7L2 in the nuclear portion were decreased following Spry4 overexpression.On the contrary,Spry4 silencing in ST2 activated the canonical Wnt pathway,revealed by enhanced nuclear transfer of ?-catenin from the cytoplasm to the nucleus.Besides,the protein levels of p-LRP6,total and non-phosphorylated ?-catenin in the total cell lysates as well as ?-catenin and TCF7L2 in the nuclear portion were increased following Spry4 overexpression.5.Overexpression of Spry4 in ST2 cells inactivated ERK1/2 signaling.In brief,the level of p-ERK1/2 protein is significantly lower in Spry4 overexpressing cells than in control cells.Conversely,silencing of endogenous Spry4 results in the opposite.6.Treatment of ST2 cells with U0126,an inhibitor of ERK1/2,inbited canonical Wnt signaling,revealed by the decreased levels of t-?-catenin,non-p-?-catenin and p-LRP6 protein after treatment.These suggest an interaction between ERK1/2 and canonical Wnt.7.Overexpression of Spry4 in ST2 cells activated the AKT/mTORC1 pathway.Briefly,protein levels of p-AKT,p-mTOR,p-S6K1,p-RPS6,and p-4EBP1 were increased.Conversely,silencing of endogenous Spry4 in ST2 cells inactiavted AKT/mTORC1 pathway,revealed by decreased protein levels in p-AKT,p-mTOR,p-S6K1,p-RPS6 and p-4EBP1.Furthermore,blocking mTORC1 pathway in ST2 cells by rapamycin attenuated the stimulatory effect of SPRY4 on adipocyte differentiation.Conclusion: 1.Spry4 expression decreases during osteoblast differentiation and increases during adipocyte differentiation 2.SPRY4 can inhibit the differentiation of bone marrow stromal cells into osteoblasts and promote their differentiation into adipocytes.3.SPRY4 inhibits osteogenic differentiation of bone marrow stromal cells by inhibiting ERK1/2 and canonical Wnt signaling pathways.4.SPRY4 promotes the differentiation of bone marrow stromal stem cells into adipocytes by activating mTORC1 signaling pathway.Inhibition of mTORC1 can block SPRY4 contribution to adipogenesis.