The Effect Of17β-estradiol And Wnt/β-catenin Signaling Pathways On Expression Of Matrix Gla Protein In Human Osteosarcoma Cells

Objective:To observe the effect of17β-E2and Wnt/β-catenin signaling pathways on expression of matrix Gla protein (MGP) in human osteosarcoma cells (MG63) in vitro, to investigate the potential role and the possible molecule mechanism of MGP in postmenopausal osteoporosis.Methods:Human osteosarcoma MG63cells were recoveried, and cultured at370C in a humidifier5%CO2incubator. The MEM medium which was phenol red free contained10%fetal bovine serum(FBS).When the cells confluenced, placed the cells into25cm2culture bottle with5x105cells each bottle. The medium changed every2days. When90%confluence,the MG63cells placed into1%BSA serum-free MEM medium for2days, and then carried out the following drug intervention respectively:(1)17β-E210-10,10-8,10-6mol/1, ICI10-7mol/1, ICI10-7mol/1+17β-E2108-mol/1intervented for48hours;(2)17β-E210-8mol/1, DKK-1200ng/ml,17β-E210-8mol/1+DKK1200ng/ml intervented for48hours;(3)17β-E210-8mol/1, warfarin10-5mol/ml,17p-E210-8mol/1+warfarin10-5mol/ml intervented for48hours, all refered1%BSA serum-free MEM culture as the control group, then total RNA and protein was extracted and examined by Real-Time quantitative RT-PCR and Western blot. Measured the expression of MGP、β-catenin、Runx2、LRP5mRNA and protein.Results:(1)17β-E2can up-regulate the expression of MGP, at the concentration of10-10mol/l、10-8mol/l、10-6mol/l,the MGP mRNA expression were4.63-fold,7.16-fold,2.95-fold (P<0.05),the MGP protein expression were1.17-fold,1.58-fold,1.28-fold, which was compared with the control group. At the concentration of10-8mol/l,the effects of the17β-E2was more obvious.The difference was statistically significant (β<0.05).After administrated with ICI182780which is the inhibitor of estrogen receptor at the concentration n of10-7mol/1, the expression of MGP showed no significant difference compared with the control group (P>0.05), while combined with17β-E2, the modulation effect of MGP by17β-E2was blocked, the difference was statistically significant (P<0.05).(2),17β-E2increased the expression of Wnt-associated protein such as β-catenin、LRP5、Runx2, DKK-1which is the Wnt/β-catenin signaling pathway inhibitor down-regulated the expression of β-catenin、LRP5、Runx2。DKK-1associated with17β-E2can inhibit the upregulation effect of MGP by17β-E2, the difference was statistically significant (P<0.05).(3)The MGP inhibitors warfarin suppressed the expression of MGP, while warfarin associated with17β-E2can inhibited the upregulation effect of β-catenin LRP5、Runx2by17β-E2,the difference was statistically significant (P<0.05).Conclusion:17β-E2significantly upregulated the expression of MGP, the classic Wnt/β-catenin signaling pathway may be one of the key signaling pathways involved in the expression of MGP. MGP may promote the expression of Wnt β-catenin signaling pathway associated protein such as β-catenin、LRP5、Runx2to promote the bone formation. MGP may play an important role in the prevention and therapy of postmenopausal osteoporosis, and Wnt/β-catenin signaling pathway involved in the process.