Roles Of TGF-?1 Signaling Pathway In The Mechanism Of Fluoride Regulating Bone Turnover In The Development Of Skeletal Fluorosis

Background: Previous study shows that TGF-? superfamily has influence on osteoblast and osteoclast viability.As one of this family,TGF-?1 mainly exists in bone tissue,and plays an important role in bone turnover.Activin receptor-like kinase(ALK)5 as one of T?R1 subtypes,can activate Smad2,Smad3 protein and regulate bone turnover.The present study was designed to observe the relationship between TGF-?1 signaling pathway and fluoride-induced bone formation,and aimed to detect the mechanism of TGF-?1 and its receptor signaling pathway in the bone turnover underlying the process of skeletal fluorosis.Based on this study,we hope to provide a theoretical basis forskeletal fluorosis treatment and precaution.Method and results: In this study,the in vivo and in vitro experiment were used to explore the role of TGF-?1 and its signaling pathway in regulating bone turnover induced by fluoride.Wistar rats were treated with fluoride for two months to establish skeletal fluorosis model.One month later,rats were treated with or without SB431542 for next one month,which as an inhibitor of TGF-?1 subtypes recepitor ALK5.Dental fluorosis was appeared after rats were treated with fluoride for one month,and teeth were easily broken after the increasing of fluoride concentration.Rats were anesthetized with ether,and blood was taken from the venous plexus of the eye,then we separated femur,tibia ofrats.We determined the characteristics of skeletal fluorosis through bone tissue pathology observation.After that,X-ray bone density equipment,conventional pathology,immunohistochemistry,real-time PCR and Elisa analysis were used in the present study.Results showed in high fluoride(20 mg F-/kg)group,bone density decreased compared with control.Moreover,cortical bone was disordered and osteocalcin protein expression showed decreased.It was also showed high fluoride inhibited Runx2 gene expression and stimulated RANKL expression.Fluoride also stimulated PINP and CTX expression in serum.Besides,Smad3 expression showed increased,but P38 MAPK expression decreased by fluoride treatment.These results indicated activated bone turnover mainly characterized as bone resorption in rats after treated by fluoride for two months.This process accompanied with the Smad3 expression increased and P38 MAPK expression decreased which as downstream factors of TGF-?1.To detect the relationship between TGF-?1 and fluoride-induced in bone turnover,SB431542 was used in this study.Results showed with SB431542 treated in rats,bone density increased compared with control.Fluoride combined with SB431542 treated in rats enhanced bone density compared with fluoride treated alone,which was indicated that SB431542 may inhibited bone turnover and lead to bone density increased.This trend was demonstrated in osteoblast and osteoclast gene expression.Compared with fluoride treatment alone in rats,fluoride combined with SB431542 inhibited the expression of Runx2 and RANKL.The expression of PINP and CTX was also decreased by SB431542 treated in rats.Results showed ALK5 expression decreased by SB431542 treated in rats,and the expression of Smad3 also reduced compared with fluoride treated in rats.SB431542 treated in rat inhibited P38 MAPK expression,high fluoride combined with SB431542 also decreased its expression.These in vivo results indicated that TGF-?1 may regulate fluoride-induced bone turnover,which mainly characterized as bone resorption,via Smad3 and P38 MAPK pathway.RAW264.7 cells were treated with fluoride and SB431542 in vitro study.MTT and bone lacuna analysis were used to observe cell viability and differentiation.Results showed low fluoride stimulated cell viability compared with control.In low fluoride(1mg F-/L,4mg F-/L)groups,bone lacuna areas increased compared with control,which indicated that low fluoride stimulated cells osteoclastic differentiation.With SB431542 treatment in cells,cells viability obviously declined compared with fluoride treated alone.In low fluoride combined with SB431542 groups,bone lacuna areas decreased compared with fluoride treatment alone,which indicated that SB431542 inhibited cells osteoclastic differentiation.In the present study,we focus on detecting the role of TGF-?1 in fluoride-induced osteoblastic differentiation,thus the MC3T3-E1 cells and bone marrow mesenchymal stem cells(BMSCs)were used in the study.MC3T3-E1 cells were treated with fluoride and SB431542 for 4 and 7 days,then cells viability was detected by MTT analysis.Results showed low fluoride stimulated cells viability but high fluoride inhibited it.With SB431542 co-treatment,cells viability declining compared with fluoride treatment alone.We attempted to confirm the crosstalk among the identified mi RNAs by using Affymetrix mi RNA 4.0 software in this study.Result showed some reported mi RNA were also increased significantly in MC3T3-E1 cells by fluoride treated.KEGG analyzed showed fluoride stimuted TGF-?1 and its downstream Smad3,MAKP signaling pathways.Besides,some other signaling pathways were also regulated MC3T3-E1 cells differentiation such as Hedgehog,Notch and Wnt.It showed fluoride activated many osteoblastic signaling network to regulate bone formation and also indicated TGF-?1 plays a key role in skeletal fluorosis.BMSCs were isolated from the femurs of young Kunming mice(3 weeks old,30g)in a sterile environment.After 3 generations,cells were cultured with ?-sodium glycerolphosphate,vitamin C and dexamethasone to induce cells osteoblastic differentiation.Cells were treated with fluoride and SB431542 to observe osteoblastic differentiation.MTT analysis was used to detect cells viability.Alkaline phosphatase and alizarin red staining were used to observe cells differentiation.Besides,immunocytochemical analysis,RT-PCR and western blot analysis were used to detect the related factors expression of osteoblast and TGF-?1.Results showed that low fluoride stimulated cells viability while high fluoride inhibited it.Compared with control,low fluoride enhanced ALP activity,but high fluoride inhibited it.In low fluoride groups,calcium deposition showed clearly and the area of mineralized nodules is larger.In high fluoride group,mineralized nodules showed obviously decreased compared with control.Low fluoride increased Runx2 gene and protein expression,but high fluoride inhibited its expression.Besides,the expression of TGF-?1 relative factors was also influenced by fluoride.Results showed that 1mg F-/L fluoride stimulated ALK5 expression while 16 mg F-/L fluoride inhibited it.Fluoride inhibited Smad3 gene expression and stimulated MAPK expression.With SB431542 co-treatment,cells viability and ALP activity was receded compared with fluoride treatment alone.The mineralized nodules disappeared,OCN and Runx2 protein expression also reduced compared with fluoride treatment alone.Besides,the Runx2 gene expression reduced compared with fluoride treatment alone.With fluoride and SB431542 co-treatment in cells,ALK5 expression was reduced compared with fluoride treated alone.In 4mg F-/L and 16 mg F-/L fluoride combined with SB431542 groups,MAPK expression reduced compared with fluoride treatment alone.Besides,Smad3 gene expression and Smad2/3 protein expression were reduced in low fluoride and SB431542 co-treatment groups,while they were increased in high fluoride and SB431542 co-treatment group.The expression of P-Smad2/3 protein was decreased by flouride combined with SB431542 treatment.These results indicated that TGF-?1 play a key role in fluoride-induced osteoblasts viability and differentiation via the MAPK pathway,and Smad3 pathway may also has a negative work.Conclusion: The in vivo study indicated that excessive fluoride leads to skeletal fluorosis,which mainly characterized as active bone turnover.The in vitro study showed that low fluoride stimulated osteoblast and osteoclast viability and differentiation,but high fluoride inhibited cells viability and differentaition.In this study,rats and cells were treated with fluoride and SB431542.Results indicated TGF-?1 played a key role in fluoride-induced bone turnover,osteoclast and osteoblast differentiation.This study indicated that as an TGF-?1 receptor inhibitor,SB431542 significantly influenced bone resorption,the ability of osteoblast and osteoclast differentiation,finally interfered the process of bone turnover.Besides,gene analysis indicated TGF-?1 regulated bone resorption process by stimulated RANKL expression and negative regulated osteoblastic differentiation by inhibited Runx2 expression via Smad3 pathway.Moreover,it was also indicated TGF-?1 has positive regulation in fluoride-induced bone formation and osteoblastic differentiation via MAPK pathway.