Low-intensity Pulsed Ultrasound Inhibited Osteoclast Differentiation Through Up-regulating FTO Expression To Regulate N6-methyladenosine Modifications

Objective: Bone tissue undergoes constantly remodeling over time in a dynamic balance between bone resorption and bone formation.The break of such balance would lead to a number of associated hyperostosis or osteolytic diseases.Bone resorptive diseases share a common molecular basis in both orthopedic and dental context,including joint replacement,bone resorption caused by implants,and bone resorption caused by periodontitis,where the activity or number of osteoclasts exceeds more than that of osteoblasts.Osteoclasts are derived from bone marrow cells,where bone-marrow-derived progenitor cells fuse with each other to form giant multinucleated cells.The differentiation of osteoclasts mainly depends on the binding of receptor activator for nuclear factor-B(RANK)and receptor activator for nuclear factor-B ligand(RANKL)secreted by osteoblasts with the contribution of macrophage colony stimulating factor(M-CSF).Thereafter,tumor necrosis factor-associated factor 6(TRAF6)is recruited to initiate intracellular signaling cascades,followed by activation of NF-kB signaling pathway to induce the activation of nuclear factor of activated T cells 1(NFATc1).NFATc1 initiates an autoregulatory loop that cooperates with other transcription factors to activate osteoclast specific gene expression including cathepsin K(CTSK)and matrix metalloproteinase 9(MMP9).Low-intensity pulsed ultrasound(LIPUS)is a non-invasive,pulsed mechanical wave,which could impose therapeutic and protective effects on fractures,bone defects and osteoarthritis where the underlying molecular mechanisms,whether by promoting osteoblast differentiation or by anti-inflammation remain unelucidated systimatically.LIPUS has been reported participating in regulating gene expression and protein translation,in terms of epigenetic modifications to activate the relevant signaling pathways,thereby affecting cell proliferation and differentiation,so as to exert its tissue regeneration,anti-inflammatory,and immune regulation performances.Since the physiological reaction of bone repair is a complex process,which involves cells derived from hematopoietic system,the strategy to mobilize osteogenic activity and the ability of overall bone regeneration in the bone injury microenvironment give rise to the significance of further exploration of LIPUS for its corresponding performance in molecular levels.As emerging studies have revealed the important biological role of epigenetic regulation in maintaining bone homeostasis,post-transcriptional regulation and modification have gradually provided research interests for researchers.M6A(N6-methyladenosine,m6A)modification is the most common type in eukaryotic m RNA modifications,where the N6 site of RNA was methylated with an adenosine residue.M6 A is mainly completed by methyltransferase complex,including methyltransferase like 3(mettl3),mettl14 and WTAP(Human Wilms Tumor 1 Associated Protein,WTAP);methylated eraser FTO(FAT and obesity related gene)and ALKBH5;and m6 A readers,members of the YTH domain family.As a dynamic RNA modification process,m6 A is thought to be involved in multiple aspects of m RNA metabolism including alternative splicing,RNA stability,RNA nucleation,and translation.In the bone microenvironment,m6 A modification also affects the process of bone formation.In the process of osteoclast differentiation,the level of m6 A modification is upregulated.To conclude,this study intends to explore from the perspective of RNA modification,in terms of m6 A modifications,where LIPUS presents its role on osteoclast differentiation to explore the influences of LIPUS and the relative molecular mechanism of inhibiting osteoclast differentiation,aiming to explain the role of LIPUS for bone microenvironment in view of epigenetics,giving rise to the theoretical evidence for its role to prevent bone resorption,which would provide alternatives for resorptive bone diseases.Methods: 1.To observe the osteoclast differentiation ability of RAW264.7 induced by50ng/m L RANKL with or without LIPUS loading.The number and size of osteoclasts were detected by tartrate-resistan acid phosphatase(TRAP)staining and F-actin fluorescence staining.The RT-q PCR and Western blot were applied to determine the m RNA and protein level of osteoclast markers including CTSK,TRAF6,MMP9,NFATc1,NF-kB1;The expression of lysosomal associated membrane protein 1(LAMP-1)was observed by immunofluorescence staining.2.The effects of LIPUS on the level of m6 A modification during osteoclast differentiation was detected by enzyme-linked immunosorbent assay.Bioinformatic method was used to predict m6A-related enzymes that could bind to TRAF6,a key gene of osteoclast differentiation,and verified by RT-q PCR.m RNA stability of Traf6 and Ctsk in osteoclasts after transfected with FTO was determined by RT-q PCR.3.To observe the effect of LIPUS on osteoclast differentiation when interfering with m6 A modification enzyme where chemical and biological methods to inhibit FTO expression were employed.RT-q PCR,Western blot and immunofluorescence were used to observe the m RNA and protein levels of osteoclast differentiation markers.Results: 1.Compared with the control group,the number and size of osteoclast differentiation were decreased in LIPUS treatment group,where the m RNA and protein expressions of TRAF6,CTSK,MMP9,NFATc1 and NF-kB1 representing osteoclast differentiation were significantly decreased(P <0.05).The number of lysosomes in LIPUS group is also reduced,compared to control group.2.LIPUS can reduce the modification level of m6 A in the process of osteoclast differentiation and up-regulate the expression of FTO(P <0.05).After interference with FTO expression,the degradation rate of TRAF6 and CTSK m RNA was delayed.3.After biological transfection of si-FTO,the expressions of osteoclast differentiation markers Ctsk?Traf6?Mmp9?Nfact1and Nfkb1 in the si-NC transfection group and without LIPUS were significantly up-regulated compared with the corresponding si-NC group(P<0.05).Meanwhile,LIPUS inhibited the expression of osteoclast differentiation genes Traf6,Ctsk,Mmp9,Nfatc1 and Nfkb1 were significantly down-regulated.In si-FTO transfection group,the inhibitory effect of LIPUS on osteoclast differentiation markers disappeared,and CTSK and NFATC1 were significantly increased compared with si-FTO transfection group without LIPUS(P<0.05).The detection results of m6 A modification level in each group showed that after si-FTO transfection in LIPUS group,the modification level of m6 A was higher than that in si-NC group.However,in the LIPUS group,the m6 A modification of si-FTO and si-NC transfected was down-regulated compared with that of the RANKL group,but the difference was not statistically significant.After chemically inhibited FTO activity,the expressions of osteoclast differentiation markers CTSK,TRAF6,MMP9,NFATC1 and NFkB1 in FTO inhibited group were increased compared with those in non-inhibited FTO group(P<0.05).In the inhibitor group,the protein expression levels of osteoclast markers MMP9,CTSK,TRAF6 and NFATC1 were slightly decreased after LIPUS treatment,except for NFATc1(P>0.05).Immunofluorescence results showed that the inhibition effect of LIPUS on CTSK and MMP9 expression disappeared after transfection of si-FTO or inhibition of FTO activity.Conclusion: 1.LIPUS can inhibit the differentiation and formation of osteoclasts,and can downregulate expression of TRAF6,a marker of osteoclast differentiation.2.LIPUS can promote the expression of demethylase and reduce m6 A modification in the process of osteoclast differentiation.3.FTO can maintain the degradation rate of TRAF6 and CTSK.4.The effect of LIPUS on osteoclast differentiation depends on the presence of FTO.