Effects Of Mechanical Stress On Osteoclast Protein Expression-a Proteomic Study

Osteoclasts formation, differentiation and their function in vitro are affected by mechanical tensile stress, and the response of osteoclasts depends on magnitude and time course of the stress. However, the molecular events of osteoclasts in response to tensile stress are still not well understood so far. Differential proteomics can be applied to evaluate the significant proteins in cellular metabolism, which would aid in better understanding of the molecular mechanisms of osteoclasts in response to tensile stress. Therefore in the present study, we quantitatively and qualitatively analyzed the differentially expressed proteins in human osteoclasts on 5 and 9 days after tensile stress loading in an in vitro osteoclast culture modal by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), computer informatics and computer network communications technology. The aim of our study was to find out the major proteins involved in osteoclast formation and in their functional regulation at different time points after tensile stress loading, to elucidate the molecular mechanisms underlining ostelclast formation under tensile stresss.Objectives: 1. To establish a human bone marrow osteoclasts culture modal suitable for proteomics study; 2. To establish the atlas of protein two-dimensional gel electrophoresis at the early and late phases of human bone marrow osteoclasts induction; 3. To identify the differently expressed proteins at different stages of bone marrow osteoclasts induction under mechanical tensile stress loading; 4. To find out the major proteins involved in the modulation of osteoclasts formation at different stages of mechanical tensile stress loading.Contents: 1. Induced culture of human bone marrow osteoclasts and its identification; 2. Establishment of in vitro cell culture modal under tensile stress loading; 3. Establishment of the atlas of protein two-dimensional gel electrophoresis at different phases of human bone marrow induction with tensile stress loading; 4. Analysis of the protein spots on two-dimensional electrophoresis gels. Comparison of protein expression profiles of the induced osterclast with or without mechanical tensile stress loading; and at different stages of mechanical tensile stress loading 5. PMF analysis of the differentially expressed protein spots on two-dimensional electrophoresis. Obviously differentially expressed protein spots were extracted and applied to PMF analysis; 6. Identification of the major proteins related to osteoclast induction. 7. Discussion of the possible molecular mechanisms involoved in bone marrow cells induction to osteoclasts differentiation and in the modulation of tensile stress on osteoclasts formation and their function.Methods: 1. The human bone marrow osteoclasts were obtained by granulocyte-macrophage colony-stimulating factor (GM-CSF) induction; 2. Tensile stress was loaded on human bone marrow osteoclasts by a multichannel tensile stress loading device developed by the Department of Orthodontics, School of Stomatology and the Schoolof Bio-Medical Engineering of the Fourth Military Medical University; 3. Based on preliminary results, two time points-early phase of cells aggregation and late phase of cell maturation, which corresponded to 5 and 9 days after tensile stress loading, were chosen. Cellular proteins were extracted on these time-points and were subjected to two-dimensional gel electrophoresis; 4. PMF was analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS); 5. the proteins involved in osteoclasts differentiation and tensile stress loading were identified through blasting the MALDI-TOF-MS results with proteome database..Results: 1. 5 days after tensile stress loading, human bone marrow cells started to aggregate. At 9 days of induction, the cells differentiated into typical osteoclasts. However, the cells decreased in number. 2. Two-dimensional gel electrophoresis at different phases of bone marrow osteoclasts induction demonstrated that 13 proteins were differentially expressed at 5 days after stress loading, as compared with unloading group. Among them, 1 protein was found to be a new one, 7 disappeared, 2 were greatly up-regulated and 3 were greatly down-regulated. At 9 days, 15 proteins were differentially expressed compared to unloading group. Among them, 5 were novel, 6 proteins disappeared, 1 was up-regulated and 3 were down-regulated obviously. 3. Among the 15 differentially expressed proteins, the PMFs of 10 proteins were obtained through MALDI-TOF-MS analysis. 4. The PMFs of the 10 proteins were further analyzed by blasting with the protein and pertide databases. Four proteins were identified. They were Glyceraldehyde-3-phosphate dehydrogenase, Actin- cytoplasmic 1,Alpha-enolase and Serine/threonine-protein phosphatase PP1. The other 6 proteins remain unknown.Conclusions: 1. Sufficient osteoclasts can be obtained to do the proteomics study through GM-CSF induced culture of human bone marrow in vitro; 2. It's feasible to investigate the influence of tensile stress loading on osteoclasts through the differential proteomics approach; 3. 28 differentially expressed proteins by tensile stress loading have been detected in this study. 4. Among the 28 differentially expressed proteins, 4 proteins were identified and the PMF of 6 proteins were obtained. Our study provided fundamental information on the differantial expression of proteins of ostelclast under mechanical strain. However, further studies are necessary to elucidate the molecular mechanisms underlining bone remodeling induced by tensile stress loading and to find out the functions and biological signal transduction mechanisms of mechanical stress.