Role Of PTH And Its Receptor Signaling Pathway In The Mechanism Of Skeletal Fluorosis Based On Osteocytes

Background:Osteocytes are the most abundant cell type in bone,which play an important role in carrying out bone remodeling.Accelerated bone turnover is the key point in the pathogenesis of skeletal fluorosis,fluoride can stimulate osteoblasts and osteoclasts,both of which show varying degrees of activity,but little is known about the effect of fluoride on osteocytes.Parathyroid hormone(PTH)is essential for the maintenance of calcium homeostasis in vivo,regulate bone remodeling,and is involving into the skeletal fluorosis.More recent studies demonstrate that osteocytes are the major effector of PTH.In this study,we investigated the role of PTH in mechanism underlying fluoride regulated osteocytes in vitro and in vivo experiments.The results will make sense for the clarifying the pathogenesis of skeletal fluorosis.Methods:1.In vitro experiment: Primary osteocytes were extracted and cultured in medium rich of collagen for 5 days,and then exposed to varied doses of fluoride(0,1,4,16 mg F-/L)with and without PTH(1-34)(10nmol/L)for 48 h in vitro.Immunohistochemistry of E11/gp38 and OCN were used to further identify the osteocytes.Cell viability was detected by MTT assay.Osteocytes co-cultured with RAW264.7 for 5 days,and comparative analysis of the ability of each group to form osteoclast-like cells was observed under microscope.The real-time PCR and Western bolt were used to test the protein and gene expression of specific factors in osteocytes exposed to different condition,such as sclerostin,RANKL/OPG and Wnt/?-Catenin signal related factors.2.In vivo experiment: 60 Wistar rats were divided into control group,low dose of fluoride group(10mg F-/kg·day)and high dose of fluoride(20mg F-/kg·day)group.After exposure to fluoride for 5 weeks,half of rats in each group were treated with fluoride and PTH(1-34).All the rats were exposed to fluoride with and without PTH for the three weeks.At the end of experimental period,rats were sacrificed by ether anesthesia.Bone tissue such as femur and tibia were collected to measure bone density,and real-time quantitative polymerase chain reaction of related genes,and blood was taken from the eyeball for biochemical analysis(enzyme linked immunosorbent assay).We recorded weigh of rates and their tooth changes weekly during the experiment.The bone density,the level of osteogenic and osteoclast markers and related genes in fluorosis rats were observed.The experiment mainly investigated the replication of skeletal fluorosis model in fluorosis rats,further verifying the results of in vitro experiments and the effect of PTH on accelerated process of bone turnover in skeletal fluorosis.Results:1.In vitro experiment: Osteocytes were isolated from 3-month-old male ICR mice and cultured on collagen coated 24-well plates successfully.The low expression of OCN and high expression of E11/gp38 proteins acted as early markers of osteocytes.The results showed that a low dose of fluoride had a tendency to enhances the cell viability,but high dose of fluoride decreases it.On the other hand,PTH slightly inhibited the cell viability of osteocytes and modulated the effect of fluoride on it.Osteocytes induced osteoclastic differentiation in RAW264.7 cells to validate generation of RANKL by osteocytes in vitro.Fluoride stimulates the osteocyte-derived RANKL,and PTH further enhances the promotion of fluoride.All doses of fluoride with and without PTH treatment markedly inhibited Sost gene expression.The inhibitory action was most obvious with 16 mg/L of fluoride treatment alone and accompanied by PTH.The gene expression of RANKL and RANKL/OPG ratio significantly increased in low and medium dose of fluoride group.Single PTH also much up-regulated RANKL/OPG and heavily enhanced the stimulation effect of fluoride on RANKL/OPG ratio.This data also demonstrated that low and medium dose of fluoride stimulated PTH1 R expression and PTH significantly increased its expression,which was impeded by co-treatment with fluoride and PTH.It indicates fluoride stimulates the expression of wnt/?-Catenin signaling,high dose of fluoride was most obvious.Single PTH had no obvious effect on these factors except wnt10 b,whereas PTH co-treatment significantly enhanced stimulating action of fluoride on them.Western blot results validated that fluoride and single PTH treatment on depressed SOST/Sclerostin expression and increased RANKL expression,and the trend of inhibition was magnified by co-treatment with fluoride and PTH.It was intriguing that co-treatment with a high dose of fluoride and PTH much decreased RANKL but stimulated SOST/Sclerostin.The high dose of fluoride treatment promoted the expression of ?-catenin and smad3 protein.This study demonstrated that osteocytes play a key role in fluoride inducing bone turnover through regulation of RANKL,SOST/ Sclerostin expression and Wnt/?-catenin signaling.The effect of PTH was similar with fluoride.PTH played an important role in the mechanism of fluoride affecting osteocytes via its receptor.2.In vivo experiment: The rats dental injury deteriorated with the extension and increase of fluoride exposure.Administration of PTH(1-34)aggravated the fluoride-induced dental lesion.Accordingly,low dose of fluoride slightly increased the bone density and high dose of fluoride significantly decreased the bone density,co-treatment with PTH and fluoride enhanced bone mineral density.Serum PINP increased significantly in the 20 mg F-/kg·day group and CTx much elevated in all groups showed that fluoride and single PTH promoted bone turnover,high dose of fluoride was more obvious.Meanwhile,fluoride combined with PTH much stimulated serum PINP,indicating that the combination of the fluoride and PTH further enhanced bone turnover especially osteogenesis.It is consistent with the change in rats bone mineral density.Further investigation showed that the effect of fluoride on OCN and Runx2 was not obvious,indicating that fluoride had a weak effect on osteoblast mediated bone formation,however,high dose of fluoride and PTH together inhibited OCN expression but significantly up-regulated the Runx2 suggests that PTH reverses the effect of high fluoride on osteoblast function.the expression of Sost was depressed with fluoride treatment,on the other hand,fluoride increased bone resorption via elevation of RANKL mRNA expression.The similar trend was observed when PTH was administered alone.PTH significantly increased wnt10 expression.Furthermore,PTH combined with low dose of fluoride treatment reinforced the osteogenesis and osteoclastogenesis genes expression,such as Sost,wnt10 and RANKL,which suggested the magnification of PTH action on bone turnover.However,co-treatment with high dose of fluoride and PTH markedly attenuated RANKL expression,but significantly pronounced the Sost along with wnt10.The expression of smad3 gene increased with the increase of fluoride dosage,PTH significantly promoted its expression.Low dose of fluoride and PTH further pronounced the expression of the gene,while high dose of fluoride combined with PTH significantly inhibited the smad3 gene.Different doses of fluoride,PTH,low dose of fluorine combined with PTH all slightly inhibited the expression of TGFR1,but high dose of fluoride and PTH significantly promoted the TGFR1 gene.In addition,PTH significantly enhanced the induction of PTH1 R by fluoride.These results suggest that fluoride cause high bone turnover in rats,which may be partly achieved by down regulating Sost and promoting RANKL expression.PTH alone also enhances bone formation and resorption in varying degree and further promotes the effect of low dose of fluoride,but inverted the action of high dose of fluoride.In general,this data precisely reflects the varied expressions of Sost and RANKL produced by osteocyte under fluoride treatment and PTH mediated this process.In conclusion,this study demonstrated that fluoride inhibited SOST/Sclerostin,and modulated ratio of RANKL/OPG to influence bone formation and bone resorption through mediation of Wnt/?-catenin signaling in osteocytes.Osteocytes probably play a key role in fluoride activated bone turnover,and PTH participates in the process of fluoride modulating SOST/Sclerostin,Wnt/?-catenin signaling and RANKL expression via its receptor.Meantime,PTH administration probably strengthened bone turnover of low dose of fluoride via up-regulation of RANKL and depression of Sost,while influenced the effect of high dose of fluoride through the opposite modulation of these factors.These discoveries have provided new clue to probe into the mechanism underlying development of skeletal fluorosis.