The Effect Of1α,25(OH)2D3on RANKL And OPG MRNA Expression Of Osteoblasts At Various Stages Of Differentiation

Background and objectiveThere are three types of cells in the bone tissue:the osteoblastes,the osteoclasts and the osteocytes.Osteoclasts are derived from hematopoietic stem cells, and RANKL(receptor activator of NF-kB ligand) plays an critical role in osteoclastic bone resorption during bone remodeling. With the synergy effect of M-CSF(macrophage colony-stimulating factor),RANKL binds to its cognate receptor,RANK,which is expressed on the cell surface of osteoclasts and their progenitors, to induce osteoclast differentiation,bone resorption and osteoclast survivability.OPG acts as a decoy receptor, which competitively binds to RANKL and prevents the activation of RANK.Therefore,the ratio of RANKL/OPG could be regarded as the decisive factor of osteoclastic bone resorption.Many in vitro exprements report that the osteoblastic cell line,derived form mesenchymal stem cells,takes a part in regulating the differentiation and function of osteoclasts via expressing RANKL and OPG.la,25(OH)2D3belongs to the steroid hormones.And it is key to maintain normal calcium-phosphorus metabolism in body.1α,25(OH)2D3in physiological dose could induce bone formation.What’s more, the induction is related to the differentiation state of osteoblasts.However,1α,25(OH)2D3of over-dose could induce bone resorption.And the RANKL/RANK/OPG system plays a role in1α,25(OH)2D3inducing bone resorption.Today,the relationship between expression regulation of RANKL/OPG by1α,25(OH)2D3and differentiation stage of osteoblasts is still uncertain.MethodsThe MC3T3-E1murine osteoblast-like cells were cultured with primary media(a-MEM+10%FBS+100U/ml penicillin/streptomycin).After cells proliferation,the cells were seed into6-well plates at an initial density of 5000cell/cm2.Then, changed the primary media with the secondary differentiating culture(addition of50uM ascorbic acid+10-6M dexamethesone+lOmM (3-glycerophosphate to the primary media) at the time of about85%confluence, to induce osteogenic differentiation.The cells were treated with1α,25(OH)2D3(10nM) or absolute ethyl alcohol at each time-point (day0, day7,day14,day21and day28of osteogenic induction) for4h,followed by isolation of total cellular RNA.Then generated cDNA and carried out q-PCR,to explore the relationgship between how to regulate the expression of RANKL or OPG by1α,25(OH)2D3and the differentiation stage of osteoblasts.Meanwhile,alizarin red staining was took to demonstrate progressive mineralization of the differentiating cells in culture.ResultsThe mRNA expression of ALP,Osteocalcin and so on validated that the MC3T3-E1cell line in culture were following a predictable course of differentiation from pre-osteoblasts to mature osteoblast lineage cells.During the process of osteoblastic differentiation,basal RANKL mRNA expression did not alter,but was significantly enhanced by1α,25(OH)2D3.The stimulatory effect of la,25(OH)2D3on RANKL were hightened in more mature cultures,and the RANKL mRNA expression of test group increased by3.4-fold than the basal one at day28(P<0.05). Basal OPG mRNA expression was increased until day14,and then was decreaed.But OPG mRNA expression was always suppressed by1α,25(OH)2D3during culture(P<0.05).The RANKL/OPG ratio, an index of osteoclastogenic stimulus, was therefore increased by1α,25(OH)2D3treatment at all stages of osteoblastic differentiation, but to a higher degree in cultures after the onset of mineralisation.ConclusionsThe RANKL/OPG ratio, an index of osteoclastic bone resorption, was increased by over-dose1,25-(OH)2D3(10"8M) treatment at all stages of osteoblastic differentiation but to a higher degree in cultures after the onset of mineralisation.