Neuropeptide Y stimulates osteoblastic differentiation of murine MC3T3-E1 cells related to activated Wnt signaling in vitro

Abstract

Objective To investigate the effect of neuropeptide Y (NPY) on the osteoblastic dif-ferentiation of murine MC3T3-E1 cells and its mechanism related to the Wnt signaling pathway. Methods The murine MC3T3-E1 cells were divided into 4 groups according to the stimulators added: phosphate buffered saline (PBS) (control) and different concentrations of NPY (10-8 mol/L, 10-10 mol/L and 10-12 mol/L). The cellular proliferation was detected with MTT assay after 1, 3, 5, 7 and 9 days. The cells were identified with cell immunochemistry and Western Blot to find out the most effective concentration of NPY at different time points under osteoblastic condition. The cells were then divided into 4 groups: PBS, NPY, NPY+NPY receptor antagonist, and NPY+DKK1. Western blot was used to determine the expression of β-catenin and p-GSK-3β in each group. Nuclear signaling activity of β-catenin was observed using immunofluorescence staining. Results NPY significantly improved the proliferation of MC3T3-E1 cells at 7 and 9 days (P< 0.05). NPY (10-8 mol/L and 10-10 mol/L) groups and NPY (10-10 mol/L and 10-12 mol/L) groups signif-icantly improved the ALP activity at 4 and 14 days respectively (P< 0.05). At 4 days, the expression of ALP protein was significantly decreased in the NPY+DKK1 group and the NPY+NPY receptor antagonist group compared with that in the NPY group (P< 0.05). Although the expression levels of β-catenin and p-GSK-3β protein were uninfluenced in either case, NPY significantly stimulated the nuclear signaling activity of β-catenin. Conclusions NPY may significantly increase the expression of ALP protein in MC3T3-E1 cells during osteoblastic differentiation. This effect might be mediated through the canonical Wnt signaling pathway. Key words: Neuropeptide Y; Mice; Cell proliferation; Cell differentiation; Murine MC3T3-E1 cells