Effect of lacking calcium-sensing receptor on proliferation and differentiation of mouse osteoblasts and its mechanism

Objective To investigate the effect of calcium sensing receptor (CaSR) on proliferation and differentiation of mouse osteoblasts and the regulatory role of Wnt signaling pathway in promotion of proliferation and differentiation of mouse osteoblasts.Methods Four wild-type mice (control group) and 4 CaSR receptor knockout homozygous mice (experimental group) were used for passage of skull osteoblasts.The third generation of osteoblasts was harvested to measure cellular optical density (OD) and alkaline phosphatase (ALP) activity using Cell Counting Kit-8 (CCK-8) on days 2,4,6 and 8,respectively.At the end of day 6,the mRNA levels of core binding factor (Runx2),ALP,osteocalcin (OCN),receptor activator of NF-KB ligand (RANKL),osteoprotegerin (OPG) and β-catenin were determined by RT-qPCR,and the protein expressions of Runx-2,β-catenin,IGF-1 and Wnt-5a were determined by Western blot.Results The OD values (1.55 ±0.05 versus 1.26 ±0.02) and ALP activities (0.023 ±0.002 U/mg · prot versus 0.017 ± 0.001 U/mg · prot) of the osteoblasts reached the peak in the 2 groups on day 6,significantly higher than those at the other time points (P ＜ 0.05).However,the OD value and ALP activity in the experimental group were significantly lower than in the control group at the same time point (P ＜ 0.05).Compared with the control group,the mRNA expressions of IGF-1,OCN,Runx2,ALP,RANKL/OPG and β-catenin and the protein expressions of β-catenin,Wnt-5a,IGF-1 and Runx2 were all significantly reduced in the experimental group (P ＜ 0.05).Conclusions Lack of CaSR may result in obstacles to proliferation and differentiation of osteoblasts.The mechanism may be related to the inhibition of Wnt signaling pathway. Key words: Osteoblasts;  Receptors, G-protein coupled;  Cell proliferation;  Cell differentiation; Signal transduction