| Objective:Glucocorticoids(GC) play an important role in modifying the metabolic activity of osteoblasts.However,their mechanisms remain unknown.Osteoblast-derived tissue inhibitor of metalloproteinase-1(TIMP-1) was recently reported to be involved in cell apoptosis of osteoblasts.Therefore,we tested the effects of the GC(dexamethasone) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells.Method:murine osteoblastic MC3T3-E1 cells were cultrured in DMEM medium.And then were treated with 10-6～10-9mol/L Dex dexamethasone,10-6mol/L dexamethasone+10-5mol/L RU486, 200～1000ng/ml TIMP-1+10-6mol/L Dex for indicated time.Real-time quantitative reverse-transcription polymerase chain reaction(RTQ-PCR) was used for determining TIMP-1 mRNA expression.Western blot and sandwich-enzyme-immunoassay(ELISA) analysis were performed for determining TIMP-1 protein levels in both cells and culture media, respectively.Cell apoptosis was measured by ELISA.Caspase-3 activation was also measured by the use of its substrates.Result:1.RTQ-PCR showed that dexamethasone inhibited TIMP-1 mRNA expression in MC3T3-E 1 cells in a does-dependent manner.2.Western blot showed dexamethasone inhibited TIMP-1 production in a does and time-dependent manner.3.TIMP-1 ELISA showed dexamethasone inhibited the secrection of TIMP-1,in a does and time-dependent manner.4.All these effects were blocked by a glucocorticoid receptor(GR) antagonist,RU486.5.Dexamethasone induced apopsis of MC3T3-E1 cells in a does-dependent manner.6.Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by dexamethasone in MC3T3-E1 cells. Conclusion:GC as Dex suppresses TIMP-1 production in osteoblasts and this effect is associated with its induction of osteoblast apoptosis.A decrease in TIMP-1 production caused by GC contributes to the mechanisms of GC-induced bone loss.
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