The Mechanism Of Bone Formation And Wnt/β-catenin Pathway Inhibited By AlCl₃

To investigate the molecular mechanism of inhibition of Aluminum(Al) on bone formation. Two hundred 4-week-old healthy male Wistar rats were randomly divided into control group and treatment group. The rats of control group were given distilled water, the rats of treatment group were orally exposed Al3+ 64 mg/kg body weight in drink water for 120 days. The bone mineral density(BMD) of the distal and proximal femoral metaphysis were measured by dual-energy X-ray absorptiometry(DXA); Al contents in femora and serum were measured by graphite furnace atomic absorption spectrophotometry; the m RNA expressions of Dkk1, s FRP1, Wnt3 a, Fzd2, LRP-5, β-catenin, Tcf4, cyclin D1 and c-Myc were detected by real-time fluorescence quantitative PCR(qRT-PCR); the protein levels of Wnt3 a and β-catenin were detected by western blot; the activities of Fzd2 and LRP-5 were detected by ELISA, the transcriptional activity of TCF4 were determined by Electrophoretic Mobility Shift Assay(EMSA) on day 30, 60, 90 or 120, respectively. The results showed as follows:(1)The body weight of rats in treatment group rats were significantly lower than those in control group on day 60, 90 and 120(P<0.01). It indicated that Al Cl3 exposure inhibited normal development and growth of the rats.(2)The BMD of the distal and proximal femoral metaphysis were lower in the treatment group than those in control group on day 120(P<0.05). It indicated that Al Cl3 exposure induced bone lose and inhibited bone formation.(3)The contents of Al in femora and serum were significantly higher in the treatment group than those in control group(P<0.01) during the entire experimental period. It indicated that Al accumulated in bone of rats.(4)The mRNA and protein expressions of Wnt3 a were significantly lower in treatment group than those in control group on day 60, 90 and 120(P<0.05; P<0.01); the mRNA expressions and activities of LRP-5 and Fzd2 were significantly lower in treatment group than those in control group on day 60, 90 and 120(P<0.05; P<0.01). It indicated that Al inactivated of the Wnt3a/β-catenin pathway.(5)The m RNA and protein expressions of β-catenin were significantly lower in treatment group than those in control group on day 60, 90 and 120(P<0.05; P<0.01); The m RNA expression and transcriptional activity of TCF4 were significantly lower in treatment group than those in control group on day 60, 90 and 120(P<0.05; P<0.01). It indicated that Al down-regulated Wnt3a/β-catenin pathway and inhibited the activity of nuclear transcription factors.(6)The mRNA expressions of c-myc and cyclin D1 were significantly lower in treatment group than those in control group on day 60, 90 and 120(P<0.05; P<0.01). It indicated that Al inhibited the targets gene expression of Wnt/β-catenin pathway.(7)The mRNA expressions of Dkk1 were significantly higher in treatment group than those in control group on day 60, 90 and 120(P<0.05; P<0.01); the m RNA expressions of sFRP1 were significantly higher in treatment group than those in control group(P<0.01) on day 90 and 120(P<0.01). It indicated that Al increased the expressions of inhibitors of Wnt3a/β-catenin pathway, which inactivated of the Wnt3a/β-catenin pathway.The above results showed that AlCl3 down-regulated the Wnt/β-catenin pathway and the consequent depression of c-Myc and cyclin D1 mRNA expressions, which inhibited bone formation finally.Taken together, the inhibition of the Wnt/β-catenin pathway and the consequent depression of c-Myc and cyclin D1 m RNA expressions strongly suggest that the Wnt/β-catenin signaling pathway is involved in the inhibition of bone formation caused by AlCl3.