Differential Proteomics Research Of 1?,25-?OH?₂D₃ On Osteoclast Differentiation

Vitamin D is a fat-soluble steroid derivative,which plays an important role in the maintenance of calcium and phosphorus metabolism and the normal physiological function of bone in human,1?,25-dihydroxyvitamin D3[la,25-(OH)2D3]is the main active form of vitamin D.Studies show that la,25-(OH)2D3 regulates the differentiation and function of osteoclast(OC),but the specific mechanism is not clear.In this study,RAW264.7 cells were used as osteoclast precursor cell(OPC)and were induced differentiation into OC by 25 ng/mL macrophage colony stimulatory factor(M-CSF)and 30 ng/mL receptor activator of nuclear factor-?B ligand(RANKL).The distribution of vitamin D receptor(VDR)in the cells was detected by immunofluorescence and Western blot,real-time cell analysis(RTCA)was used to detect the proliferation of RAW264.7 cells,quantitative real-time PCR(qRT-PCR)was used to detect the expression of OC marker genes,two-dimensional electrophoresis(2-DE)was used to detect the differentially expressed proteins during OC differentiation treated with 1?,25-(OH)2D3,and the differential expressed proteins were identified by mass spectrometry.The main results were as follows:1.RAW264.7 cells and OC could express VDR,which existed in the membrane,cytoplasm and nucleus of them,but there was no significant difference in cell proliferation when treated with 0,10-9,10-8,10-7 mol/L 1?,25-(OH)2D3.10-8 mol/L 1?,25-(OH)2D3 treatment significantly promoted OC differentiation.The expression of TRAP,CK and MMP-9 genes were significantly up-regulated while CA? gene was significantly inhibited at day 5.2.Differentially expressed protein profilings of 10-8 mol/L 1?,25-(OH)2D3 treatment groups and blank control groups at day 1,day 3,day 5 of OC differentiation were constructed,23 differentially expressed protein spots were detected,which were identified as 22 proteins by mass spectrometry.19 proteins were analyzed by Gene Ontology(GO)and STRING.The results showed that based on molecular functions,differential proteins could be classified into 12 GO terms,such as protein binding;based on biological process,these proteins could be classified into 14 GO terms,such as negative regulation of apoptotic process;based on cellular component,these proteins could be classified into 14 GO terms,and distribute inside or outside the cells.STRING analysis showed most of the proteins existed or may have interaction among them.According to the results,1?,25-(OH)2D3 may promote OC differentiation by promoting energy metabolism,cell adhesion and inhibiting cell apoptosis.3.The gene expression level of pyruvate kinase,nucleophosmin 1,protein disulfide isomerase A3,macrophage migration inhibitory factor,Galectin-3,annexin A2,SH3 domain-binding glutamic acid-rich-like protein 3,elongation factor-1 alpha 1 were analyzed by qRT-PCR.The results showed that except the expression of the elongation factor 1-?1 gene which had no significant change,the expression level of pyruvate kinase gene at day 3 and protein disulfide isomerase A3 gene at day 5 were consistent with protein abundance change in 2-DE,the gene expression level of other proteins was not consistent with the change of protein abundance.The effects of la,25-(OH)2D3 on these proteins during OC differentiation are worthy of further study.