Regulation Effect And Mechanism Of BCI On Osteoclast Differentiation And Ovariectomy-induced Bone Loss

BackgroundOsteoporosis is one of the most common diseases with a high incidence in middle-aged and elderly populations in China.Osteoporosis is characterized by low bone mass and density,and usually has no obvious clinical manifestations in its early stages.As the disease progresses,it can lead to pain,spinal deformation and fractures,which seriously affect the patient’s quality of life.The pathogenesis of osteoporosis is related to the imbalance of bone remodeling caused by the enhancement of osteoclast bone resorption and the deficiency of osteoblast bone formation.Dual-specificity phosphatases(DUSP6)is a mitogen-activated protein kinase phosphatase responsible for phosphorylation of tyrosine and serine/threonine residues in individual protein substrates and is closely associated with a variety of cell functions,including cell proliferation and differentiation.DUSP6 has been reported to mediate inflammatory T-cell activation and differentiation.BCI,also called(E)-BCI,is a small molecule inhibitor of DUSP6 with a molecular weight of approximately 317,which has been reported to inhibit DUSP6 expression and promote fibroblast growth factor expression in zebrafish embryos.Another study has found that BCI attenuates LPS-induced macrophage inflammation by activating Nrf2 signaling.Current studies associated with BCI are mainly focused on tumor,immune and inflammatory responses.However,the role of BCI in bone metabolism including osteoclast formation and differentiation remains unclear.The purpose of this study was to investigate the effects of DUSP6 inhibitor BCI on osteoclast differentiation and the related regulatory mechanism to provide a new strategy for the treatment of osteoporotic bone loss.MethodsIn the first part,we explored the cytotoxicity of BCI on RAW264.7 cells and bone marrow monocytes/macrophages(BMMs)through the CCK-8 experiment,which was used to determine the optimal concentration for subsequent studies.Besides,we detected the effects of BCI on cell apoptosis and cycle of BMMs.The inhibition effect of BCI on DUSP6 expression in RAW264.7 and BMMs was detected at optimum concentration.In the second part,we comprehensively evaluated the effects of BCI at specific concentrations on RANKL-induced osteoclast formation,fusion and bone resorption activity by TRAP staining,q RT-PCR,Western blot,FAK staining,cell immunofluorescence and bone resorption assay.In the third part,to explore the potential mechanism of BCI inhibiting osteoclast differentiation,we conducted preliminary analysis by transcriptome sequencing,GO and KEGG enrichment analysis,and verified the results by Co-IP and Western blot.In the fourth part,we evaluated the alleviating effect of BCI on bone loss in ovariectomy-induced osteoporotic mice.Results1.Low doses of BCI(≤2μM and ≤4μM)showed no cytotoxicity to RAW264.7 cells and BMMs.Flow cytometry results showed that BCI(≤4 μM)had no significant effect on BMMs cell cycle and apoptosis.BCI inhibited DUSP6 protein expression in RAW264.7 cells and BMMs during RANKL-mediated osteoclast differentiation.These results suggest that BCI can effectively inhibit the expression of DUSP6 protein without affecting cell cycle and apoptosis.2.TRAP staining,q RT-PCR,Western blot and FAK staining showed that BCI at specific concentrations could significantly inhibit RANKL-mediated osteoclast formation and differentiation.FAK and q RT-PCR results showed that BCI at specific concentrations could significantly induce RANKL-mediated osteoclast fusion.Bone resorption experiments showed that BCI could significantly enhance the bone resorption function of RANKL-mediated osteoclasts.3.RNA-Seq analysis was performed to determine the transcriptome of RANKL-induced RAW264.7 cells treated with BCI.In the BCI group,177 genes were down-regulated and 45 genes were up-regulated.The expression of key genes in osteoclast differentiation was significantly decreased in the BCI treatment group,which was consistent with q RT-PCR results.GO and KEGG enrichment analyses predicted the major functions represented by the top 50 most significant DEGs after BCI treatment.The biological processes of these genes are mainly concentrated in receptor-ligand activity,chemokine activity and osteoclast differentiation regulation.Molecular function mainly involves the response to lipopolysaccharide neutrophil migration and neutrophil chemotaxis.KEGG pathway enrichment revealed several major pathways,including cytokine-cytokine receptor interactions,osteoclast differentiation,rheumatoid arthritis,NF-κB and TNF signaling pathways.These results suggest that BCI inhibits osteoclast differentiation and suggests a possible downstream signaling pathway.GSEA results showed that TNF-α signaling via NF-κB was significantly enriched in the RANKL-induced group,but less enriched in the BCI group.In addition,immunofluorescence staining of NF-κB/p65 nuclear translocation showed that NF-κB/p65 nuclear translocation occurred in the RANKL-induced group,but was significantly inhibited in the BCI group.In addition,through the Co-IP experiment,it was found that the protein binding between NF-κB/p65 and NFATc was significantly inhibited by BCI.Western Blot results showed that BCI inhibited RANKL-mediated osteoclast differentiation by decreasing STAT3 and NF-κB signaling.4.Micro-CT was used to evaluate the bone loss in the trabecular region of femur in the osteoporosis model mice.It was observed that bone loss in the trabecular region of femur in the low-concentration and high-concentration BCI groups was alleviated to varying degrees.Quantitative results showed that compared with the OVX group,bone volume/total tissue volume(BV/TV),bone trabecular number(TB.N),bone density(BMD)and bone surface density(BS/TV)of BCI-treated mice were significantly increased.Consistent with micro-CT results,H&E and Masson staining revealed significant bone destruction in the trabecular region of the distal femur in OVX samples.However,this bone destruction was rarely observed in BCI-treated samples.In addition,compared with the Sham group,TRAP staining detected a large number of TRAP+ cells near the bone trabecular region of OVX group samples.Compared with the OVX group,the number of TRAP+ osteoclasts in the trabecular region of the mice treated with high and low concentrations of BCI was significantly reduced.The livers and kidneys of mice were analyzed histologically.HE staining results showed that there were no obvious pathological changes in liver and kidney structure in the BCI group.These results suggest that BCI has a protective effect on OVX-induced bone loss in mice with osteoporosis by inhibiting osteoclast activation in the absence of significant hepatorenal toxicity.ConclusionsOur research is the first to explore the effect of BCI in RANKL-induced osteoclast differentiation and the corresponding mechanism.Our results showed that BCI significantly inhibited RANKL-mediated osteoclast formation,fusion,and bone resorption,and this inhibitory effect might be achieved by blocking STAT3 and NF-κB signaling and inhibiting the binding between NF-κB and NFATc1.In addition,in vivo results showed that BCI effectively ameliorated the bone loss of OVX-induced osteoporotic mice.Our study suggests that BCI therapy is promising as a new treatment strategy for osteoporosis.