The Role And The Mechanism Of R-Spondin 1 In Mechanical Loading Regualted Bone Metabolism

Objective: Appropriate mechanical stimuli are essential to bone metabolism and functional homeostasis via a series of molecular cascades,which have been not yet fully elucidated.Recent studies showed that the role of R-spondins(Rspos),which were known as key agonists of Wnt/β-catenin signaling,in bone metabolism has been attracting more and more attention of scientists.However,the role of Rspos in mechanical loading regulated bone metabolism has never been investigated.The present research was aimed to clarify the role and the potential mechanism of Rspo1 in mechanical loading influenced bone metabolism.Methods:(1)The BMSCs were loaded by cyclic mechanical stretch(CMS)using the FX-5000 T system and then the expression and secretion levels of Rspo1 in the cells were assessed by qRT-PCR,Western-blot and ELISA analysis.The unloading induced osteoporosis mouse model was constructed by tail suspension(TS)and then primary BMSCs was isolated from the bone marrow.After adherent culture and purification in vitro,the expression and secretion level of Rspo1 in the unloading BMSCs was also evaluated by qRT-PCR,Western-blot and ELISA analysis.(2)The BMSCs were infected by adenovirus to overexpress Rspo1 or by lentivirus to knockdown Lgr4.During osteogenic induction,the Rspo1 overexpressed cells were treated with or without Dkk-1,the Lgr4 silenced cells were treated with or without recombinant Rspo1,and the CMS loaded cells were treated with or without Rspo1 neutralizing antibody(Rspo1 Ab).After induction,the osteoblastic differentiation ability was assessed by analysis of the osteogenic marker genes(OCN,ALP,Col-1a1)expression,the ALP activity,the OCN secretion level,the ALP staining and alizarin red staining,and the activity of the Wnt/β-catenin signaling was evaluated by analysis of the Wnt target genes(Axin2 and Tcf1)mRNA expression,the intracellular protein expression of β-catenin and the TOPFlash luciferase activity.(3)The unloading mice were weekly subjected to intra-femoral injection of adenovirus carrying overexpressed Rspo1.After the TS experiment,the unloading femurs were subjected to Micro-CT scanning analysis to assess the bone mass and microstructure,and the osteoblastic parameters and new bone formation were evaluated by the bone histomorphometry analysis.Results:(1)The expression and supernatant level of Rspo1 in BMSCs was consistently and significantly upregulated by CMS and downregulated by mechanical unloading.(2)The overexpression of Rspo1 and the recombinant Rspo1 all resulted in significantly enhanced osteoblastic differentiation ability and Wnt/β-catenin signaling activity in BMSCs,but when the Wnt/β-catenin signaling in BMSCs was inhibited by Dkk-1,the promoting effect of Rspo1 on osteogenic differentiation was completely abolished,and when the Lgr4 was knocked down in BMSCs,the promoting effect of Rspo1 on osteoblastic differentiation ability and Wnt/β-catenin signaling activity were consistently disappeared.(3)The osteoblastic differentiation ability and Wnt/β-catenin signaling activity in BMSCs were markedly enhanced by CMS,and then were partly but significantly counteracted by the presence of Rspo1 Ab.(4)In the unloading femurs,Rspo1 overexpression lead to the enhanced osteoblastic activity and new bone formation,increased bone mass and improved bone microstructure.Conclusions:(1)The mechanical stimuli could regulate the expression and secretion of Rspo1 in BMSCs,and the Rspo1 was identified to be a new mechano-sensitive protein.(2)Rspo1 could promote the osteoblastic differentiation of BMSC and attenuate the unloading induced bone loss via activating the Wnt/β-catenin signaling pathway in which Lgr4 was essential.(3)Rspo1/Lgr4 was suggested to be a novel signal responsible for bone mechano-transduction and a novel potential target for development of preventive drugs for disuse osteoporosis.