The Role And Mechanism Of Rho Effector Protein Rtkn In Regulating Osteoblast And Adipocyte Differentiation

Objective The balance between adipogenesis and osteogenesis is critical for bone homeostasis,otherwise pathological changes occurrs.The Rho effector protein Rtkn(Rtkn)has been demonstrated to play significant roles in the proliferation and migration of tumors,as well as in the development and differentiation of neurons.Our preliminary data showed that Rtkn mRNA was abundant in bone,and changes in the expression level during the adipogenic/osteogenic differentiation of BMSCs.The current study mainly focuses on the regulation and mechanism of Rtkn in the process of BMSCs differentiation in vitro and in vivo.Methods:1.Use quantitative real-time polymerase chain reaction(qRT-PCR)to detect the Rtkn mRNA in tissues of mice;culture BMSCs and its cell line ST2 for adipogenesis/osteogenic induction,the mRNA expression of Rtkn was detected.Overexpression or knockdown of Rtkn in cell line ST2,C3H10T1/2,MC3T3-E1,and BMSCs,observe mRNA and protein levels of key factors after adipogenesis or osteogenesis by qRT-PCR and Western blotting(WB).The oil red O staining and alkaline phosphatase(ALP)staining were taken to research the differentiation status.2.Detect the changes of key factors in the Wnt/β-catenin signaling pathway after overexpression of Rtkn by WB;verify its effect on β-catenin nuclear transport by immunofluorescence;through bioinformatics databases and literature reports which we screen out the human T cell leukemia virus type Ⅰ binding protein 3(Taxlbp3)which was verified to interaction with Rtkn and β-catenin by co-immunoprecipitation(Co-IP).The effect of Taxlbp3 on Wnt/β-catenin signaling pathway was confirmed by WB and immunofluorescence.The function of Taxlbp3 in the regulation of differentiation of BMSCs was confirmed by gain/deletion experiments.3.Injected Rtkn siRNA to tibia cavity,after a month,make paraffin sections then do HE staining to observe adipocyte,use immunohistochemical staining to detect osteoblast.Three months after injection,analyzes tibia bone mass and related parameters by μCT.Construct tissue-specific knock-in Tax1bp3 mice,culture their primary calvarial cells and conduct adipogenesis/osteogenesis,then probe the effect in vivo.Results 1.Rtkn was abundant in bone,the expression level changed during differentiation of stromal ST2 cells and BMSCs.2.Enforced expression of Rtkn in ST2,C3H10T1/2 and MC3T3E1 cells promoted osteogenesis at the expense of adipogenesis.Inactivation of Rtkn blocked osteogenic Differentiation and promoted adipogenesis in cell lines and BMSCs.3.Overexpression of Rtkn will increase p-LRP6(Ser1490),non-p-β-catenin and TCF7L2,thereby activates the Wnt/β-catenin pathway.At the same time,promote β-catenin transfer to the nucleus.4.The coIP experiment verified that Rtkn,Taxlbp3,t-β-catenin and non-p-β-catenin can bind to each other.5.Enforced expression of Taxlbp3 downregulated p-LRP6(Ser1490),non-p-β-catenin and p-Gsk3β(Ser9),thereby inhibiting the activity of the Wnt/β-catenin signaling pathway.The knockdown of Taxlbp3 can promote the transfer of β-catenin to the nucleus.6.Enforced expression of Taxlbp3 in ST2,C3H10T1/2 and MC3T3E1 cells promoted adipogenesis at the expense of osteogenesis.7.After one month injection of Rtkn siRNA into the tibia cavity of mice,the number and area of adipocytes increased significantly and the number of osteoblasts reduced.Three months after the injection of Rtkn siRNA,the μCT showed that bone mass of tibia was reduced,and the related bone static parameters also demonstrated that.8.Culture calvarial primary cells from Coll-cre;lsl-Taxlbp3 mice will favored adipocyte differentiation and impaired osteoblast formation after inducing.Conclusion 1.Rtkn was abundant in bone and the expression level changed during differentiation of stromal ST2 cells and BMSCs;2.Rtkn promoted osteogenesis at the expense of adipogenesis.3.Rtkn activates the Wnt/β-catenin signaling pathway,and its interaction with Taxlbp3 and β-catenin may be the molecular mechanism.Taxlbp3 promotes adipogenesis instead of osteogenesis.4.Rtkn silencing favored adipocyte differentiation and crippled osteoblast formation in vivo.5.The specific increase in the expression of Taxlbp3 favored adipocyte differentiation and impaired osteoblast formation in vivo.