The Influence Of Rho GTPases Signal Pathway During Cadmium Differentiation Of Osteoclasts And Molecular Mechanism

Cadmium(Cd)is a toxic heavy metal and a widely distributed environmental pollutant.The study found that environmental Cd exposure level is closely related to decreased bone density.As the main metabolic process of the body,"bone remodeling" is necessary to maintain the normal development of the bone,and its imbalance may seriously affect the shape and function of the bone.Osteoclasts are the only multinucleated giant cells with bone resorption activity in the body,which play an important role in bone remodeling activities.The changes in their activity or quantity can cause imbalance in bone remodeling and cause osteoporosis.In recent years,studies have found that Rho GTPases play an important role in osteoclast differentiation.However,the effects of cadmium on osteoclast differentiation and the mechanism of Rho GTPases signal path in the process are still unclear.In this paper,the effects of cadmium on osteoclast differentiation and the regulation of Rho GTPases signaling pathway were studied in vivo and in vitro,which provided a scientific basis for further revealing the toxic mechanism of cadmium on bone.1.Effects of Cd exposure on osteoclasts differentiation in mouse bone tissueThirty-six BALB/c female mice of 5 weeks old were randomly divided into 3 groups:0、5、25 mg/L.Drinking water was free for 16 weeks,and water and feed intake were counted weekly.After the end of the experiment,whole blood,serum and bone tissue were collected.Determinate Cd in whole blood,urine and forelimb bone by atomic absorption spectrometry.Determinate serum alkaline phosphatase(ALP)activity,serun calcium and phosphate(Ca5 P)and urine Ca,P by biochemical analyzer.Determinate the contents of elements in the forelimb bone by atomic absorption spectrophotometry.The pathological changes of the hind limbs were observed by paraffin section.The content of TRACP-5b and estradiol(E2)in serum was determined by ELISA.The expression of osteoclast-specific protein,Rho-related protein and mRNA in bone marrow cells were detected by qRT-PCR and Western blot.The distribution and expression of OPG and RANKL in hindlimb bone were observed by immunohistochemistry.The results showed that:① With the increase of cadmium concentration in drinking water,the difference between body weight and food intake was not obvious,while the Cd intake increased significantly,and blood,urinary and bone cadmium increased in a dose-dependent manner,and the difference of 25 mg/L group was extremely significant(P<0.01).②Compared with the control group,the ALP activity and the contents of serum Ca and urinary Ca,P in the cadmium group were not significantly changed(P>0.05),while the serum P content was significantly increased(P<0.01).③ Compared with the control group,the content of Ca,P in the forelimb bone of the Cd-stained group decreased slightly(P>0.05),and Fe,Mn,Zn increased or significantly increased(P<0.05 or P>0.05),and Cu,Se content changes are not obvious(P>0.05).④ With the increase of Cd concentration in drinking water,the trabecular bone of tibia became less and less.⑤ Compared with the control group,TRACP-5b in serum of Cd-treated group increased significantly(P<0.05),and E2 decreased in a dose-dependent manner and the 25 mg/L group showed significant difference(P<0.05).⑥Compared with the control group,the osteoclast-specific mRNA of anti-tartaric acid phosphatase(TRAP),type Ⅱ carbonic anhydrase(CAII),and matrix metalloproteinase-9(MMP-9)in bone marrow cells of 5 mg/L Cd-treated group were significantly increased(P<0.01),and the expression of TRAP and CAII mRNA in the 25 mg/L Cd group was significantly decreased(P<0.01),while the expression of MMP-9 mRNA was significantly increased(P<0.01).The expression of osteoclast-specific protein TRAP,cathepsin K(CTSK),MMP-9 and CAII in Cd-stained bone marrow cells increased or significantly increased(P>0.05,P<0.05 or P<0.01).⑦Compared with the control group,the key regulator of osteoclast differentiation,nuclear factor-kappaB receptor activating factor ligand(RANKL)and osteoprotegerin(OPG)showed significant or extremely significant increase(P<0.01 or P<0.05)in Cd-treated bone marrow cells,while OPG in 5 mg/L Cd group did not change significantly,and RANKL increased more than OPG.⑧ Compared with the control group,the RANKL protein content in the hard tissue of the Cd,treated group increased,and the OPG protein content did not change much.⑨ Compared with the control group,the mRNA expression levels of RhoA,Racl,Cdc42 and RhoU in the Cd-stained group were increased or significantly increased(P>0.05,P<0.05 or P<0.01).⑩Compared with the control group,the expression levels of Rho GTPases key proteins RhoA,Racl,Cdc42 and RhoU in the Cd-treated bone marrow cells increased or significantly increased(P>0.05,P<0.05 or P<0.01).Conclusion:①Cd exposure can destroy the bone micro structure and promote the accumulation of Fe,Mn,Zn elements in bone tissue.② Exposure to Cd increases the number of osteoclasts in the body,possibly affecting osteoclast activity via the estradiol or indirectly promoting osteoclast differentiation in bone tissue via the OPG/RANKL/RANK pathway.③ Cd up-regulates the expression of key Rho proteins in bone marrow cells.2.The regulation mechanism of Rho GTPases signal in the process of Cd affecting osteoclast differentiationRAW264.7 cells were treated with very low dose Cd(0,5,10,20 nM)or low dose Cd(0,2,5,10 μM)on the basis of M-CSF and RANKL-induced differentiation for 4 days.CCK-8 method was used to detect cell viability during osteoclast differentiation.TRAP staining was used to observe osteoclastogenesis.qRT-PCR and Western blot were used to detect the expression of specific proteins and mRNAs related to osteoclast differentiation.On this basis,different concentrations of Cd(0,10 nM,2 μM,5 μM)were used to treat 24 h on the second and third day respectively.Immunofluorescence(IF)was used to observe the changes of osteoclast precursor cells and osteoclasts’ cytoskeleton.Transwell assay was used to detect the migration ability of precursor cells.qRT-PCR was used to detect the expression of Rho GTPases mRNA in precursor cells and osteoclasts,respectively.The expression of key proteins downstream of Rho GTPases was detected by Western blot separately.The results showed that:Compared with the control group,① nanomolar(very low dose)level Cd can up-regulate cell viability(P>0.05),and micromolar(low dose)level Cd can significantly down-regulate cell activity in a dose-dependent manner(P<0.01).② the number and area of osteoclasts produced by some groups were significantly greater than those of the control group after nanomolar level of Cd treated(P<0.05 or P<0.01),and the number and area of osteoclasts were smaller than the control group after micromolar Cd treated(P<0.05 or P<0.01).③ 5 nM and 10 nM Cd group can up-regulate or significantly up-regulate the expression of osteoclast-specific proteins TRAP,CTSK,CAII and MMP9 mRNA(P>0.05,P<0.05 or P<0.01),and 20 nM Cd group can significantly down-regulate TRAP,MMP-9 mRNA and the expression of CTSK and CAII mRNA were significantly up-regulated(P<0.05 or P<0.01).Nanomolar level of Cd up-regulated or significantly up-regulated the expression of TRAP,CTSK,integrin 3(Beta3)protein(P>0.05,P<0.05 or P<0.01),and micromolar level of Cd can significantly down-regulate the expression of TRAP,CTSK,CAII,MMP-9 mRNA(P<0.05 or P<0.01)and down-regulate or significantly down-regulate the expression of TRAP,CTSK,Beta3 protein(P>0.05,P<0.05 or P<0.01).④the precursor cells of the 10 nM Cd group became longer,and the pseudopods of the micromolar Cd group became shorter.⑤ 10 nM Cd promoted the migration of precursor cells(P<0.01),micromolar Cd significantly inhibited cell migration(P<0.05 or P<0.01).⑥10 nM,2μM Cd group up-regulated the expression of Cdc42 and RhoQ mRNA in the precursor cells,and 5 μM Cd significantly down-regulated the expression of Rac1,Rac2 and RhoU mRNA in the precursor cells(P<0.05 or P<0.01),and the expression of mRN As of other Rho family genes were not obvious.⑦ 10 nM Cd up-regulated or significantly up-regulated the expression of ROCK1 and ROCK2 proteins in the downstream of Rho GTPases(P>0.05 or P<0.05).The micromolar Cd group significantly down-regulated the expression of ROCK1 and ROCK2 proteins(P<0.05 or P<0.01),and the expression of Cofilin protein in Cd-exposed group was not significantly changed(P>0.05),and 5μM Cd significantly down-regulated the expression of p-Cofilin protein(P<0.01),and 10 nM,2μM,5 μM Cd group was significantly down-regulated the expression of ARP2/3 protein(P<0.01).⑦10 nM Cd promoted the formation of the osteoclast sealing zone,and the 2μM,5 μM Cd group inhibited the fusion of the precursor cells.⑨ 10 nM,2 μM Cd group up-regulated the expression of Cdc42 mRNA in bone cells(-P>0.05),and 5μM Cd significantly down-regulated the expression of RhoB,Racl,Rac3 and RhoU mRNA in osteoclasts(P<0.05 or P<0.01),and the expression of other Rho family mRNAs was not obvious.⑩ the expression of ROCK2 and ARP2/3 protein in osteoclasts of 10 nM and 2 μM Cd groups increased,but the difference was not significant(P>0.05).10 nM Cd significantly down-regulated the expression of p-Cofilin protein in osteoclasts.(P<0.01),and 5 μM Cd significantly down-regulated the expression of ROCK1,p-Cofilin and ARP2/3 protein in osteoclasts(P<0.01).Conclusion:① Long-term exposure of Cd at nanomolar level can promote osteoclast differentiation,and micromolar Cd exposure for a long time can inhibit osteoclast differentiation in a dose-dependent manner.② Rho GTPases signaling pathway plays an important role in the process of Cd affecting osteoclast differentiation.Regulation of the formation of filamentous pseudopods and cell migration and formation of blocking bands in precursor cells may be the main mechanism of action,ultimately affecting osteoclasts formation and bone resorption function.