| Osteoblasts, the main function cells of bone formation, play an important role in thesynthesis, secretion and mineralization process of extracellular matrix (ECM), can secreteosteoid to packet itself and transform into bone cells. Osteoblasts also may secrete matrixvesicles (MV). The relation between MV and bone mineralization has been studied formore than40years, but the mechanism of MV promoting hydroxyapatite (HA) formationin ECM and leading to cartilage and bone pathological or physiological mineralization isstill not clear. However there are a large number of studies have shown that MV play animportant role in the process of bone mineralization, and are involved in physiological andpathological mineralization process. MV are small vesicles with a diameter of50-200nm,which contain abundant phospholipid and protein components and play an important rolein the process of bone mineralization. It has been reported that MV proteins deficiencymight participate in the pathological mineralization process of many rare skeletal diseases.Recently, the protein profiling of MV from different origin were analyzed in severalproteomic studies, and more than2,000proteins have been identified in MV includingseveral validated mineralization-related proteins such as alkaline phosphatase (TNAP),annexins. However, the roles of most of the MV proteins in mineralization regulationremain unclear.Saos-2is a human osteoblast-like cell line having significant mineralization abilityunder osteogenic introduction. Meanwhile, another human osteoblast-like cell U2-OS haslow phosphate enzyme activity and could not be mineralized after introduction. Therefore,we speculated that the difference between MV proteins between Saos-2and U2-OS cellscould provide novel clues to clarify the exact roles of MV during mineralization process. In this study, we performed a label-free LC-MSMS proteomic approach to compare the MVprotein profiles between Saos-2and U2-OS cells in order to further screen functional MVproteins for mineralization regulation.Objective: To cultivate osteosarcoma cell line Saos-2and U2-OS which are havesignificantly different mineralization ability. Extracting MV from the cells for comparativeproteomics study and searching differentially expressed MV proteins in bonemineralization for providing new clues to explore the role in mineralization process.Methods: U2-OS and Saos-2cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM; HyClone) and Mccoy′s5A Medium (Gibco) respectively, supplementedwith10%fetal bovine serum and1%penicillin-streptomycin (Beyotime) at37°C under5%(v/v) CO2in a humidified atmosphere. Mineralization was induced on confluent cellsin induction medium supplemented with50μg/mL L-ascorbic acid (Sigma) and10mMβ-glycerophosphate (Sigma). MV were extracted from the cells for comparativeproteomics study and were identified by flow cytometry and electron microscopytechniques. MAS3.0analysis software was used to further analysis the result of proteomicanalysis. The differentially expressed MV proteins were verified by western-blotting.Results: After osteogenic induction, Saos-2cells demonstrated a time-dependentincrease in mineralized nodule formation assessed by Alizarin Red staining, whilemineralization were absent in the matrix of U2-OS cells. Using ExoQuick reagents, MVwere successfully precipitated, which were validated by transmission electron microscopy.The precipitation by ExoQuick reagents were recognized as spherical membrane-boundedvesicle structures with a diameter ranging from50to200nm, and part of vesiclescontained electron dense material. In the proteomic study, we identified a total of175differently expressed proteins (fold>2) in Saos-2MV compared with U2-OS MV,including89up-regulated proteins and86down-regulated proteins. Among theup-regulated MV proteins, alkaline phosphatase (ALP) ranked the most significantlyincreased MV protein in Saos-2cells, which has been proved in many studies.89 up-regulated proteins were further classified on Gene Ontology in terms of biologicalprocesses and molecular function using the MAS3.0software (CapitalBio, Beijing, China).In particular, we observed that12up-regulated MV proteins of Saos-2cells belong tocalcium ion binding and several up-regulated MV proteins of Saos-2cells are involved inthe bone mineralization associated signal transduction pathway.Conclusion: In this study we identified a series of MV proteins specificallyup-regulated in mineralization component osteoblasts, which might provide new clues forthe mechanism study for mineralization control in extracelluar matrix. We also confirmedthat protein Kinase C α (PKCα) and ras-related protein Ral-A (Rala) were novel MVproteins and might be involved in bone mineralization as MV components. |
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