Action Of Parathyroid Hormone In Effect Of Fluoride On Osteocyte-Modulated Osteoclastic Differentiation

Background:Fluoride is a kind of trace element that is indispensable for mankind to maintain physical healthy.It may cause various physiological or pathological changes even physical damages to the body when the intake of fluoride is too much,and the damages mainly concentrated in the bone tissue.Endemic fluorosis is one of the endemic diseases that has attract attention from the whole country even the whole world for its universality,stubbornness,serious and irreversibility.And skeletal fluorosis is one of its characteristic damage.It has been found that patients with skeletal fluorosis are often accompanied by characteristic changes in bone mass reduction such as osteomalacia and osteoporosis,and the mechanism of interaction between excess fluoride,osteocyte and osteoclasts is rarely reported.Our studies shown that osteocyte is the main source of the nuclear factor NF-?B receptor activator ligand(RANKL)which is capable of inducing the differentiation of osteoclast.Osteocyte usually buried in the bone matrix and difficult to observe and study both in vitro and in vivo.Which means there are little research about osteocyte in the field of bone metabolism.Parathyroid hormone(PTH)is very important in regulating bone turnover.PTH can bind to the PTH receptor(PTH1R)of osteoblasts and Osteocyte,and induce the differentiation of osteoclast indirectly by regulate the secretion of various cytokines.This research is based on analyzing the activity of primary osteocyte and related factors affected by sodium fluoride,and carried out following work about IDG-SW3 cells.Defining the importance of PTH by studying the mechanism of the differentiation of osteoclast induced by RANKL secreted by osteocyte with of fluoride co-treated with PTH.Which is rarely reported in previous studies on the mechanism of skeletal fluorosis.These results will provide clues and evidences for skeletal fluorosis and related metabolic bone diseases.Methods:1.Experiment of IDG-SW3 induced by co-treatment of PTH and fluoride in vitro:Cells IDG-SW3 can stably expressed sclerostin(SOST)after 28 days of mineralization,which means the cells IDG-SW3 were transformed into osteocytes.Cells IDG-SW3 were mineralized for 28 days then cultured for 7 days with the treatment of a series concentrations of fluoride from 0.001 mg/L to 32 mg/L.After the culture,the cell viability was texted by using MTT.Several groups were chosen to culture for 7 days with the treatment of representative concentrations of fluorine and additional cytokine PTH(1-34)at a concentration of 10 nmol/L.After the culture,the cell viability was analyzed,and the apoptosis rate was measured by using BD's Annexin V-FITC apoptosis detection kit and flow cytometry.Osteoblasts were cultured with treatment of 0.5,4 and 16 mg/L fluoride with or without PTH for 7 days.Western blot was used to detect the expression of protective elements(osteoprotegerin,OPG),SOST and RANKL in IDG-SW3 cells under different experimental conditions.2.Experiment of RAW264.7 induced by fluoride in vitro:In this program,the mouse monocyte cells RAW264.7 was differentiated into osteoclasts by cultured with RANKL,then cultured with the treatment of a series concentrations of fluoride from 0.001 mg/L to 16 mg/L.The viability of cell RAW264.7 was measured by using MTT after the culture.Several groups were chosen to culture for 7 days with the treatment of representative concentrations of fluorine co-treated with 10ng/ml of transforming growth factor beta(TGF-?1),then the apoptosis rate of cells RAW264.7 were measured by using BD's Annexin V-FITC Apoptosis Detection Kit and flow cytometry.3.Experiment of co-cultured osteocytes and osteoclasts induced by co-treatment of PTH and fluoride in vitro:According to the results of previous experiments,4mg/L of fluoride is the most suitable concentration for osteoclast to differentiate,and for cells IDG-SW3 to secrete RANKL to induce RAW264.7 cells differentiate into osteoclasts.In this program,cells IDG-SW3 were mineralized for 28 days,and then co-cultured with cells RAW264.7 for 7 days by using Transwell chamber to induce the differentiation of osteoclast.IDG-SW3 cells were cultured on 24-well plates that pre-plated with type I collagen,and RAW264.7 cells were cultured in the upper layer of Transwell chamber.Both cells were separated into groups which included normal control,0.5,4 and 16mg F~-/L with or without PTH,and were texted by Tartrate-resistant acid phosphatase(TRAP)staining and Diff-Quick staining after co-culture.After the staining,the number of TRAP-positive osteoclast-like cells and cell migration in RAW264.7 cells were measured.Next experiment is in order to make a gene level analysis about the mechanism of osteoblast-induced osteoclast differentiation in different conditions.Cells IDG-SW3 were cultured on 6-well plates that pre-plated with type I collagen,and RAW264.7 cells were cultured in the upper layer of Transwell chamber,co-culture for 7 days.Both kinds of cells were separated into groups which included normal control,0.5,4 and 16 mg F~-/L with or without PTH.At the end of the culture,the expression of related cell signaling pathway and factors of RAW264.7cells and IDG-SW3 cells exposed in different groups were measured at the gene level by using time quantitative PCR.Results:1.Experimental results of IDG-SW3 induced by co-treatment of PTH and fluoride in vitro:The viability analysis showed that fluorine in extremely low concentration inhibits the activity of osteocytes,while fluoride in low and medium concentration can promote their activity,fluoride in high concentration can significantly inhibit the activity of both osteoblasts and osteoclasts.The trend of the activity of osteocytes and osteoclasts exposed to fluoride is like an inverted letter U.It can be observed that PTH significantly inhibits the activity of osteocyte after the treated osteocytes with the treatment of fluoride ion(F~-)co-treated of PTH.The results of apoptosis experiments showed that fluoride in high concentration significantly increased the apoptosis of osteocytes,and the apoptosis rate of group with of fluoride co-treated with PTH was significantly higher than the fluoride-treated group.These results agreed with the cell activity results of cells IDG-SW3The expression of SOST and RANKL in osteocytes shows that,cells IDG-SW3 started to secret SOST after 21days of mineralization,while RANKL protein was secreted at whole process of mineralization.That proved that cells IDG-SW3 differentiated into osteocytes after 21days of mineralization,and RANKL was expressed from the anaphase of osteoblasts to osteocytes.These results agreed with the characteristic of cells IDG-SW3.THE result of western blot showed that single treatment of fluoride or PTH inhibited the expression of SOST protein in osteocytes,and treatment of fluorine in low concentration co-treated with PTH also inhibited the expression of SOST.Single fluoride treatment had no significant effect on the expression of RANKL protein in osteocytes,while fluorine co-treated with PTH significantly increased the expression of RANKL.In contrast,fluoride inhibited the expression of OPG,co-treated with PTH aggravated the decrease in the expression of OPG,indicating,which shows out the specific effect of PTH on the expression of OPG under the action of fluorine.2.Experimental results of RAW264.7 induced by fluoride in vitro:According to the cell activity result of cells RAW264.7,with the inducement of RANKL,cells RAW264.7 differentiated into osteoclasts,The cell activity of 0.01mgF~-/L group(fluoride in extremely low concentration)was significantly lower than that of the normal control group,while the 4 mgF~-/L group(fluoride in medium concentration)reached the peak,and the cell activity of 16 mgF~-/L group(fluoride in high concentration)is the smallest among all,And it proves that the trend of osteoclast activity stimulated by fluoride is an inverted letter U.The results of apoptosis experiments showed that fluoride in low and medium concentration had no significant effect on the apoptosis of osteoclast,while fluoride in high concentration promoted the apoptosis of osteoclasts,and TGF-?1 aggravated this promotion of high concentration fluoride.3.Experimental results of co-cultured osteocytes and osteoclasts induced by co-treatment of PTH and fluoride in vitro:Co-culture is a better way to simulate the in vivo environment and to observe the associations and signaling pathways between cells.With the treatment of fluoride in varies concentration co-treated with PTH,cells IDG-SW3 secreted RANKL to induce RAW264.7 to differentiate into osteoclasts in co-culture.The result of TRAP staining showed that fluoride in high and medium concentration promoted the differentiation of osteoclast,and treatment of single PTH or PTH co-treated with fluoride can also promote the differentiation of osteoclast.The result of cell migration showed that the migration rate of osteoclasts was highest in the 4 mg F~-/L group.The result of time quantitative PCR showed that fluorine significantly inhibited the expression of the osteogenic inhibitor DKK1,while fluoride co-treated with PTH significantly increased the expression of SOST and DKK1;The expression of OPG in osteocytes was significantly decreased with fluoride co-treated with PTH while the expression of RANKL was significantly increased;the ratio of RANKL:OPG was significantly increased with the PTH treatment.The gene level analysis of RAW264.7 showed that fluoride co-treated with PTH promoted the gene expression of TRACP in cells RAW264.7,and the expression of cathepsin K increased under the action of single PTH.The key transcription factor of osteoclast differentiation.The gene expression of RANK,JNK and NFATc1 is similar to that of the TRAP,demonstrated that treatment of fluorine promotes the differentiation of osteoblast-induced osteoclast.At last,the expression of Wnt10b in RAW26.7 cells was significantly increased with the treatment of fluoride and PTH,the treatment of PTH significantly reduced the gene expression of Notch1 and?-catenin.Conclusion:The cell viability trend of osteocytes and osteoclasts exposed to fluoride is an inverted U shape.A trace amount of fluorine treatment inhibited cell viability of osteoblasts and osteoclasts,while low amount of fluoride promoted their activity,and high concentration of fluoride significantly inhibited cell viability of osteoblasts and osteoclasts.By comparison with osteoclast,osteocytes owned a better toleration towards high dose of fluoride.Treatment of single PTH not only inhibited the activity of osteocytes,but also increases the apoptosis of osteocytes when co-treating with fluoride.Fluoride treatment inhibits the expression of SOST and OPG proteins in osteocytes;the co-treatment of PTH and fluoride can enhance the inhibition to the expression of SOST and OPG proteins,and increased the expression of RANKL protein.Fluoride in an appropriate concentration can induce the differentiation and migration of co-cultured osteoclasts by inhibiting the expression of OPG in osteocytes and increase the ratio of RANKL and OPG.The present study found that fluoride promoted the differentiation of osteoclast through the RANK-JNK-NFATc1 pathway.Treatment of PTH and fluoride increased the expression of Wnt10b in osteoclast,which stimulated osteoblast differentiation and bone formation.PTH treatment can also promote the differentiation of osteoclasts by indirectly inhibiting the expression of Notch1,which upregulated RANK expression.In the other hand,PTH treatment can also promote the differentiation of osteoclasts by inhibiting the expression of?-Catenin which regulated the expression of OPG in osteocyte.Actually,our conclusions were based on results at gene level,all of which demanded further study,and used gene interfering technique to clarify target that osteocyte modulated osteoclast under fluoride exposure.