The Role Of Runx2 In The Process Of Activating Osteoblasts By Excessive Fluoride

Skeletal fluorosis is a disease with multiple pathological featuresincluding osteosclerosis , osteoporosis , osteomalasia , extraperiostealossification and regressive changes of cartilage and articulation.It is reportedthat a variety of cell types functioning in the process of bone turnover areinvolved in the pathogenesis of skeletal fluorosis.The aberrant activation ofosteoblasts in the early stage is one of the critical steps during the pathogenesisof skeletal fluorosis.Fluoride has been recently found to play regulatory roleson promoting osteogenesis.However,the effective dosage range of fluoride onosteoblast activity is still not clear.In addition,the functional role of Runx2,one of the crucial transcription factors in osteoblast,during the pathogenesis ofskeletal fluorosis,as well as the influence of Runx2 on OPG,an regulationfactor of osteoclast activity,and on the process of mineralization have notbeen studied.In this study,we first established an animal model of skeletal fluorosis byfeeding rats with excessive fluoride in drinking water (100mg/L and 50mg/L)with or without calcium-deficient diet.The development of skeletal fluorosisin these animals was evaluated based on the pathological changes of femora,and serum levels of calcium,magnesium,phosphate and ALP were measuredas well.Expressions of Runx2,ALP,Col I and OPG in the femur of fluoroticrat were detected using immunohistochemical method,and the expression ofRunx2 mRNA in the femur was also examined using in situ hybridizationtechnique.Osteoblast cells isolated from calvaria of suckling rats werecultured and treated with fluoride at different concentration,and MTT assaywas performed to evaluate the proliferation/survival rate of osteoblast cellsunder the treatment of fluoride.Slides were made with these culturedosteoblast cells treated with fluoride,and cellular expressions of Runx2,ALP,Col I and OPG protein as well as the expression of Runx2 mRNA weredetermined using immunohistochemical staining and in situ hybridizationtechnique respectively.Cultured osteoblast cells treated with fluoride atdifferent concentrations were harvested at different time points.Cellularexpressions of Runx2,Col I and OPG were detected by RT-PCR,and theexpression of Runx2 protein was detected by Western blotting.In addition,theexpression levels of Runx2,Col I,ALP and OPG protein in cell culturesupernatants were also determined by ELISA method.Ultrastructural changesof cultured osteoblasts under the treatment of fluoride were studied by usingtransmission electron microscope technique and the influence of fluoride onthe formation of mineralization nodules was evaluated under the lightmicroscope after alizarin red stainin.Results were as follows:1.In chronic experimental fluorosis,levels of total serum calciumdecreased , whereas concentration of inorganic phosphate , magnesiumincreased and levels of serum ALP elevated in the groups fed with fluoridizedwater plus low calcium diet.Pathological study showed increase in bothosteoblastic and osteoclastic activities in femora of fluorotic rats,which ismost significant in the group fed with 100mg F-/L water and low calciumdiet.In the group fed with 100mg F-/L water and normal calcium diet,bothmature lamellar bone and woven bone could be observed in the cortex of thediaphysis,implying increased osteoblastic activity.These results indicate thatexcessive fluoride can induce accelerated bone turnover rate and active boneresorption,and that low calcium diet is an important promoting factor ofskeletal fluorosis.2.Expressions of Runx2,OPG,ALP and Col I in osteoblast cells offluorotic rat femora were evaluated by using immunohistochemical stainingmethod . Immunohistochemistry studies showed that Runx2 expressingosteoblast cells in the femur slices increased in all chronic fluorotic groupscomparing to their corresponding normal controls.Though the increase ofRunx2 positive osteoblast cells was more prominent in chronic fluoroticgroups fed with low calcium diet,the differences between low calcium dietgroups and corresponding normal diet groups in chronic fluorotic rats were notstatistically significant.It was also found that OPG positive osteoblastsincreased in all chronic fluorotic groups comparing to normal controls withsignificant difference observed only between 100mg F-/L plus low calciumdiet group and normal control group in short term experiment(p<0.05).Although more ALP positive osteoblasts were seen in all chronicfluorotic groups comparing to normal controls as well,especially in the groupfed with 100mg F-/L water and low calcium diet,the number of ALP positivecells further decreased as the feeding time went on.All groups except for thelow calcium diet group in short term experiment showed significant increasecomparing to the normal control (p<0.05) , and statistically significantdifference was only observed between 100mg F-/L plus low calcium diet groupand normal control group in long term experiment (p<0.05,p<0.01).Theexpression of Runx2 in the femur was also evaluated at mRNA level by usingin situ hybridization method.The results showed more Runx2 mRNA positiveosteoblasts were seen in groups fed with fluoridized water than that in normalcontrol groups,and the expression of Runx2 mRNA further increased as timewent on,but the differences between fluorotic groups and their correspondingcontrols had no statistical significance.3.Osteoblasts were separated from calvaria of suckling rats and culturedin vitro in the presence of fluoride at different concentrations ranging from0.001 to 20.0mg/L.Fluoride at concentrations ranging from 0.005mg/L to0.5mg/L did not alter mouse osteoblasts proliferation as measured by MTTmethod and exhibited stimulating effect on the proliferation of osteoblast cellsat the concentration over 1.0mg/L (p<0.05) in a time-and dose-dependentpattern with the peak reached at 10mg/L.Higher concentration (20.0mg/L) offluoride exhibited mainly inhibitory effect on osteoblast growth.4.The expression of Runx2,OPG and Col I in osteoblasts cultured invitro were detected by using RT-PCR technique.Fluoride treatment couldsignificantly enhance Runx2 mRNA expression comparing to the controlgroup (p<0.05) at both 48h and 72h with the highest expression at 2.0mgF-/L.Osteoblast OPG mRNA expression was also increased with the treatmentof fluoride at different concentrations comparing to the control (p<0.05) atboth 48h and 72h with the peak at the concentration of 4.0mg F-/L.Inaddition,fluoride treatment could also increase Col I mRNA expression inosteoblasts with significant differences observed at the concentration of 2.0mgF-/L after 48h (p<0.01) and at the concentration of 2.0mg F-/L and 4.0mg F-/Lafter 72h (p<0.01) in comparison with the control.The expression levels ofRunx2,OPG,ALP and Col I proteins in the supernatant of cell culture werealso evaluated by ELISA.The levels of Runx2 protein in cell culturesupernatant slightly increased after treating with fluoride at differentiationconcentrations for 48h without statistical significance in comparison with thecontrol.However,significant increase of Runx2 protein expression wasobserved with the treatment of fluoride at different concentrations for 72h(p<0.01) with the highest expression at 2mg F-/L concentration.OPG proteinexpression in cell culture supernatant was also found increased with thetreatment of fluoride at both 48h and 72h,and the differences were significantat the concentration of 4mg F-/L comparing to the control (p<0.01).ALPexpression was slightly increased after 48h culture in the presence of fluoridewithout statistical significance,and the increase in the expression of ALP after72h treatment of fluoride was statistically significant comparing to the control(p<0.01) with the biggest increase at concentration of 2mg/L.Similarly,increases of Col I expression in cell culture supernatant were also seen after48h treatment of fluoride with significant difference observed at concentrationof 2.0 mg/L (p<0.01) in comparison with the control,and Col I expressionafter treating with fluoride for 72h was significantly increased over the control(p<0.01).To detect the expression of Runx2 protein in osteoblast cells,cellswere harvested after treating with fluoride at different concentrations andwhole cell lysates were made.Western blotting technique was employed todetect the expression of Runx2 protein in cell lysates,and a brown band couldbe seen at the position of ~56.6kDa.Runx2 expression in osteoblasts seemedincreased after treating with fluoride for both 48h and 72h,but there was nosignificant change comparing to the control.5 . We also investigated the influence of fluoride at differentconcentrations on the ultrastructure of osteoblasts by using transmissionelectronic microscope.The results showed that 1mg/L of fluoride couldpromote the function of osteoblast cells,while 20mg/L of fluoride was toxic toosteoblasts with a large amount of collagen fibers appeared around the cellsunder the electronic microscope.Furthermore in mineralization inducingexperiments the congregation of osteoblasts and the sporadic formation ofsmall mineralized nodules was observed under the microscope in the cellstreated with fluoride at 0.01 mg/L.In the osteoblasts treated with 1mg/L offluoride , more medium sized mineralized nodules were formed andinterspersed in the culture.A great deal of mineralized nodules with some atlarge size could be seen in osteoblasts treated with 10mg/L of fluoride.Theseresults coincided with the changes of Runx2 and ALP expression in fluorosis.From this study, we can conclude that:1.In chronic experimental fluorosis in rats,osteogenic activity increasesas is indicated by elevated expression of ALP.Low calcium diet can promoteand aggravate the development of skeletal fluorosis.2.Fluoride can enhance the proliferation and differentiation of in vitrocultured osteoblasts isolated from suckling rats in a dose-and time-dependentmanner at the concentration ranging from 1.0 mg/L to 4.0 mg/L with themaximum effect observed at the concentration of 10.0 mg/L.However,it caninhibit the proliferation and differentiation of osteoblasts when theconcentration of fluoride reaches to 20mg/L.3.Fluoride treatment can enhance the expression of Runx2 at bothmRNA and protein level,which in accord with its proliferation promotingeffect on osteoblasts.The increased expressions of ALP,Col I and OPG inosteoblast cells may be related with the elevated expression of Runx2.4.Fluoride can stimulate the osteogenic activity of osteoblasts as isindicated by increased secretion of collagen type I by osteoblast cells when theconcentration reaches 1 mg/L.Fluoride can also promote the formation of themineralized nodules in cultured osteoblasts induced by ascorbic acid andβ-glyceralphosphate,and such effect is enhanced as the concentration offluoride increased.5 . The influence of fluoride on the metabolism of bone is theconsequence of the interactions between fluoride and an array of other factorsinduced by fluoride,in which Runx2 might play a critical role.These results discover for the first time how fluoride affects Runx2 andOPG expression and mineralization of osteoblasts in chronic fluorosis,whichprovides insight into the mechanisms of pathogenesis of skeletal fluorosis.