Adiponectin Modulate OPG/RANKL Expression In Osteoblast And TIMP-3 Induce Apoptosis In Osteoblast

Objective: Studies have showed that adiponectin receptor (AdipoR) was detected in osteoblasts, but the related mechanisms remain unclear. The present study was undertaken to investigate the action of adiponectin on the expression of OPG and RANKL in human osteoblasts.Methods: AdipoR in cultured osteoblasts was detected by RT-PCT and western blot. OPG and RANKL expression was determined using Real-time PCR and ELISA. Human osteoblasts were incubated with different doses of adiponectin (0, 3μg/ml, 10μg/ml and 30μg/ml) for 48h to investigate the dose-dependent effects, and with 0 or 30μg/ml of adiponectin for 0h, 12h, 24h, 48h to investigate the time-dependent effects.Results: Our results demonstrated that both AdipoR 1 and 2 mRNA were expressed in normal human osteoblasts. But only AdipoR1 protein was detected in human osteoblasts. Adiponectin could inhibit OPG expression in a dose- and time-dependent manner, and promote RANKL expression in the same way.Conclusion: These results may suggest a new physiological role of adiponectin on bone metabolism by regulating the expression of OPG and RANKL in osteoblasts. Objective: Recently, adiponectin has emerged as a factor in the regulation of bone metabolism, but the mechanism remains unclear. This study was undertaken to investigate the mechanisms of adiponectin on RANKL and OPG expression in osteoblasts.Methods: Real-time PCR was used to detect the expression of RANKL and OPG mRNA in cultured human osteoblasts. RNA interference was used to down-regulate the expression of AdipoR1. JNK, p-JNK, p38, p-p38, ERK1/2 and p-ERK1/2 were analysed by Western blot. The involved signal pathway was studied using mitogen-activated protein kinase (MAPK) inhibitors (SB203580 and SP600125).Results: Suppression of AdipoR1 expression with siRNA abolished the adiponectin-regulated expression of OPG and RANKL mRNA in human osteoblasts. Furthermore, pretreatment of osteoblasts with MAPK inhibitor SB203580 abolished the expression of adiponectin-regulated RANKL and OPG mRNA.Conclusion: The results indicate that adiponectin could induce the expression of RANKL but inhibite the expression of OPG in human osteoblasts through AdipoR1/p3 8 MAPK pathways. Objective: To investigate the action of recombinant tissue inhibitor of metalloproteinase-3 (TIMP-3) protein on the osteoblast apoptosis of cell line MC3T3-El.Methods: Cell survival and apoptosis were measured by MTT and ELISA. The expression of Fas, Fasl, Bcl-2, Bax, caspase-3, caspase-8, cytochrome c, JNK, p-JNK, p38, p-p38, ERK1/2 and p-ERK1/2 were analysed by Western blot.Results: TIMP-3 promoted MC3T3-E1 apoptosis induced by serum deprivation in a dose-and time-dependent manner Western blot analysis showed that TIMP-3 increased Fas and Fasl expression, induced cytochrome c release and raised the activation of caspase-3 and -8 in MC3T3-E1 cells. TIMP-3 activated extracellular signal-regulated kinase ERK1/2 and p38, while ERK and p38 inhibitor PD098059 and SB203580 abolished its apoptotic activities.Conclusion: TIMP-3 can induce MC3T3-E1 cell apoptosis by regulating the expression of Fas and Fasl and by promoting the release of cytochrome c as well as the activation of caspase-3 and -8 via ERK1/2 and p38 signaling pathways. These results may suggest a new physiological role of TIMP-3 on osteoblasts.