The Effect Of Ca²⁺/CaM/CaMKs Signaling In Cadmium Induced Osteoclast Differentiation

With the acceleration of industrialization,cadmium level in the environment enters the organism's body via the food chain and polluted atmosphere and causes chronic cadmium poisoning.Bones are important targets for cadmium.Previous studies showed that long-term low-dose cadmium exposure decreases bone mineral density and bone mineralization.Cadmium could increase RANKL expression in OB and inhibit the expression of OPG.In RAW264.7 cell line,cadmium induced osteoclast(OC)differentiation.The results from in vivo experiments have proved to be consistent with those carried out in vitro.So far,the molecular mechanism underlying cadmium regulation of osteoclasts is unclear.Calcium ions are biologically important ions that regulate the proliferation,differentiation,and apoptosis of both normal and cancer cells.This study utilized C57BL/6 5-6 weeks old mice as subjects,and evaluated the effect of cadmium concentrations on the differentiation of BMM(Bone Marrow-Derived Macrophages)into OC.It also investigated the role of Ca2+/CaM/CaMKs pathway in cadmium induced osteoclast differentiation.The experimental content is as follows:1.Effect of cadmium on osteoclastsPrimary hepatocytes were isolated from 5-6 weeks old male C57BL/6 mice,and treated with 0,20,50,100 nM cadmium in conjunction with 30 ng/mL M-CSF and 60 ng/mL RANKL in a-MEM medium.The medium was cultured for 5 days and then stained for TRAP to identify OC.Western blot was used to detect the protein expression levels of NFATcl,TRAP,CAII,and CK in OC.RT-qPCR was used to detect bone resorption-related gene expression in OC.The results showed that as the concentration of cadmium increased,the number of differentiated OC increased.50 and 100 nM of cadmium had a significant effect on OC.In these concentrations,NFATc1,TRAP,CAII,and CK had significant increase,but there was no significant effect on the gene expression level of NFATc1.This experiment shows that low concentrations of cadmium(50,100 nM)induced OC differentiation.2.The role of Ca2+ in the regulation of cadmium induced osteoclast differentiationIn order to reveal the role of Ca2+ in the process of cadmium-mediated osteoclast differentiation,co-treatment of BMM with BAPTA-AM(calcium chelator),2-APB(IP3 receptor inhibitor)and TG(STIM1 calcium channel inhibitor)with cadmium.BMM were cultured in OC,and the expression levels of bone resorption activity-related proteins:NFATc1,TRAP,CAII,and CK were measured using Western blot.In addition,[Ca2+]i was detected by confocal microscopy,TRAP staining to identify OC.The results showed that as the concentration of cadmium increased(0,20,50,and 100 nM),Ca2+ concentration increased.However,BAPTA-AM significantly decreased the effect of cadmium treatment on OC.Co-treatment with cadmium and BAPTA-AM significantly inhibited the expression of bone resorption-associated proteins in OC.Co-treatment with 2-APB,TG inhibitors and cadmium significantly inhibited the expression of bone resorption marker proteins.In summary,cadmium activates the endoplasmic reticulum to release calcium ions into the cytoplasm,which increases the intracellular calcium concentration.This in turn activates the CaM/CaMKs pathway.NFATc1 is a downstream protein of CaM/CaMKs pathway,and it is also the key to osteoclast differentiation.Cadmium activates CaM/CaMKs/NFATc1 pathway and regulates osteoclast differentiation by increasing intracellular calcium concentration.3.The effect of Ca2+/CaM/CaMKs signaling pathway on cadmium-mediated osteoclastsIn order to study the role of Ca2+/CaM/CaMKs signaling pathway in cadmium-mediated osteoclast differentiation,KN-93(CaMKII inhibitor),W-7(CaM inhibitor),and STO-609(CAMKK inhibitor)were treated.P-CaM,P-CaMKII,P-CaMKIV expression was determined.The results showed that KN-93,W-7,STO-609 in co-treatment with cadmium statistically decreased the number of OC on TRAP staining and inhibited the expression levels of P-CaM,P-CaMKII and P-CaMKIV compared with cadmium group.NFATc1 is downstream to Cat+/CaM/CaMKs,the transcription factor of OC.The results show that cadmium activate CaM/CaMKs pathway,and CaM directly activate CaMKV/CaMKII.Cadmium promotes osteoclast differentiation by activating CaM/CaMKs while activating NFATc1.