Role Of TGF-? Signaling Pathway In Fluoride Modulating Osteoclastic Differentiation Of RAW264.7 Cells

Background:Transforming growth factor-beta(TGF-?)is a kind of multifunctional cytokine in the transforming growth superfamily,which is produced in all stages of cell differentiation and is particularly abundant in bone tissue.TGF-? can be produced by many cells in the bone and secreted in the bone matrix,which play an important role in osteoclastogenesis differentiation and bone resorption.In the onset of skeletal fluorosis,osteoblasts and osteoclasts show varying degrees of activity,and osteoclasts is responsible for the process of bone reabsorption.In this study,we investigated the role of TGF-? in mechanism underlying fluoride regulated osteoclasts differentiation in vitro and in vivo experiments,which provided the theoretical basis for the study of skeletal fluorosis and metabolic bone diseases.Methods:1.In vivo experiment: 60 Wistar rats were divided into control group,low dose of fluoride group and high dose of fluoride.After exposure to fluoride for 1 month,half of rats in each group were treated with fluoride and SB431542.All the rats were exposed to fluoride with or without Sb431542 for the next month.At the end of experimental period,rats were sacrificed by ether anesthesiabone tissue,and samples were collected for analysis.One side of femur was fixed for HE staining and immunohistochemistry to observe the pathological changes of bone tissue in rats.One side of tibia was used to measure the bone density.Bone metabolic markers were detected by enzyme linked immunosorbent assay.We mainly observed the skeletal pathology of femur tissue,protein expression of OPG and level of PINP and CTX in serum of rats treated by fluoride with or without SB431542.2.In vitro experiment:RANKL was presented in the medium to induce RAW264.7 cells into osteoclastic cells,which were divided into control group.The RANKL-induced RAW264.7 cells acted as in vitro model to be exposed to varying dose of fluoride with or without SB431542.The concentration of fluoride was designed as 0,1,4,16 mg F-/L,the concentration of inhibitor SB431542 was 10 ?mol/L.The RAW264.7 cells in the presence of RANKL were divided into eight groups according to different treatment.The cells were cultured with differential treatment for 4 days and 7 days and test cell viability by MTT method.The RANKL-induced RAW264.7 cells were seeded on the bone slice,and cultured for 7 days.The bone resorption pits stained with toluidine blue were observed under micrpscope and their areas were measure to analyze the the ability of bone resorption.The realtime PCR and Western bolt were used to test the protein and gene expression of specific factors in RANKL-induced RAW264.7 cells exposed to different condition.RAW264.7 cells were induced by RANKL.The experiment was divided into control group,fluoride group and fluoride combined with SB431542 group.After 7 days of culture,the bone pieces were stained with toluidine blue,and the morphology of the bone lacunae was observed under light microscope.3.Mi RNA assay: The RANKL-induced RAW264.7 cells were exposed to 8 mg/L of fluoride for 7 days.A ffymetrix mi RNA 4.0 array was used to analyzed the mi RNA expression profile,and compared the significant difference of mi RNA expression between fluoride-treated group and control(fold change ?2.0 or ?-2.0).Data was extracted and normalized using Expression console and Transcriptome Analysis console.Results:1.In vivo experiment: The dental and skeletal fluorosis injury deteriorated with the extension and increase of fluoride exposure.Administration of SB431542 aggravated the fluoride-induced dental and skeletal lesion,but the character of bone turnover was inhibite by SB431542.Accordingly,Fluoride obviously prompted the level of PINP and CTX in serem,and single SB431542 Administration reduced theiconcentration in serum.Fluoride treatment decreased the bone density of tibia of rats,the lower bone density was observed in high dose of fluoride group comparerd to the low dose group,which was consistent with above skeletal pathology.The protein expression of OPG in B cells decreased in fluoride-treated groups compared to the control,but up-regulated expression was observed in SB431542 groups.Fluoride treatment caused active bone turnover accompanied with bone formation and bone resorption.SB431542 acted as inhibitor of intracellular sub-type TGF-?1 receptor and inhibitor bone turnover,which implied the TGF-?1 might play a role in fluoride-induced bone turnover.2.In vitro experiment: Results showed that low dose of fluoride markedly stimulated cell viability and early stage of high dose of fluoride also do it in the RANKL-induded RAW264.7 cells.Low dose of fluoride prompted osteoclastic differentiation and bone resorption ability,and high fluoride inhibited them.The gene expression of TRAP and RANK increased in RANKL-induded RAW264.7 cells exposed to low dose of fluoride.All of these results demonstrated fluoride could stimulate osteoclastic differention and function and activated intracellular signaling pathway of TGF-?1 through TGF-?1 receptor and phosphorylation of Smad3.The stimulation of fluoride on RANKL-induded RAW264.7 cells was inhibited by SB431542 administration accompanied with decrase of TGF-?1 receptor and phosphorylation of Smad3 expressions.The in vitro data suggested that TGF-?1 signaling pathway played a key role in fluoride mediation osteoclastion differentiation and function.3.mi RNA array analysis: Results indicated that fluoride increased mi RNAs expression which were involved in activating signaling pathays associated osteoclastic differention.The KEGG enrichment analysis found that fluride can stimulated many signaling pathways which mediated osteoclastic differention,such as IGF and TGF-?1 signaling.Conclusion:Our results demonstrated that low dose of fluoride stimulated cell viability,differention,mature and function,but high dose inhibited them and impeded osteoclastic differentiation.TGF-?1 signaling pathway played a key role in fluoride mediation osteoclastion differentiation and function by activating intracellular T?R1 and phospholated Smad3.Similarly,fluoride treatment can stimulated multiple signal pathways in osteoclastic cells at the same time,which can be cross-talk and mediate fluoride-sitmulated osteoclastic character.These data provide new clue to probe into fluoride regulating bone resorption.