The Effect Of Mineralizing Solution, Dexamethasone And 1α, 25 Dihydroxyvitamin D3 On RANKL Protein Expression In Murine Bone Marrow Stromal Cells

AbstractBackgroundOsteoclast differentiated from bone marrow hemopoietic stem cell, a multinuclear giant cell, is the major cell causing bone resorption, which plays an important role in maintaining bone form and bone rebuilding. Bone tissue microenvironment support Osteoclast information indispensablely. It is known that osteoblasts or stromal cells are essential in active Osteoclast information induced by bone resorption factors such as PTH and 1,25(OH)2D3. It is thought that the action of Bone resorption stimulating factors is to induce osteoblasts or stromal cells to paracrine and accelerate osteoclast precursors differentiation and confluencingto osteoclasts , other than acting on osteoclast precursors directly. Therefore, the action of osteoblasts or stromal cells on osteoclast precursors is necessary to osteoclast differentiation and information.The receptor activator of nuclear factor kappaB ligand [RANKL, also known as osteoclast differentiation factor, osteoprotegerin ligand, and tumor necrosis factor-related activation-induced cytokine] is acrucial factor to regulate bone metabolism, which expressed in osteoblasts, stromal cells, thymus gland, lymph node and spleen. It is showed that RANKL promotes osteoclast differentiation, maturation and activation, by which binding to RANK that is one of tumor necrosis factor superfamily members in presence of M-CSF.Rat bone marrow stromal cells were induced to osteoblasts by mineralizing solution (αMEM medium supplemented with 20% FBS, 10?(-8) M Dexamethasone,10 mM Na-β -Glycerophosphate, 50 ug/ml L-ascorbic acid). RANKL mRNA level did not unchange in progress of osteoblast precursors differentiating into osteoblasts. It is recognized that dexamethasone and 1,25(OH)2D3 can promote osteoclast information and activation. But The effect of mineralizing solution, dexamethasone and 1,25(OH)2D3 on RANKL protein expression in murine bone marrow stromal cells are unknown yet.ObjectiveThe objective of this study is to investigate the effect of mineralizing solution, dexamethasone and 1,25(OH)2D3 on RANKL protein expression in murine bone marrow stromal cells.Materials and methodsThe third generation of rat bone marrow stromal cells cultivated with mineralizing solution for 48 h were treated with immunofluorescence staining and the change of RANKL protein expression was observed by laser confocal microscopy. In addition, after MC3T3-G2/PA6 stromal cell line was treated with 10?(-8) dexamethasone and/or 10?(-8) 1,25(OH)2D3 for 72 h, cell protein was extracted and was used to protein quantity, SDS-PAGE and Western Blot analysis in order to examine the RANKL protein expression.ResultsIt was showed that yellow-green fluorescence labelled at RANKL protein in rat bone marrow stromal cells could be detected in mineralizing group and the control. And the fluorescence intensity in mineralizing group is higher than that of the control, which is markedly different according to simple sample T test. RANKL protein was observed in all four groups. Compared to the control, three experimental groups displayed the increased RANKL protein expression significantly. But there was no marked difference among three experimental groups.ConclusionsIt was concluded that mineralizing solution increased RANKL protein expression in rat bone marrow stromal cells. Data were showed that both dexamethasone and 1,25(OH)2D3 enhanced the expression of RANKL protein in MC3T3-G2/PA6 stromal cells, but their effect were not synergetico It also suggested that osteoclast information and bone loss resulted from dexamethasone or 1,25(OH)2D3 were correlated to increased expression of RANKL protein.