Effects of neuropeptides and mechanical loading on bone cell resorption in vitro

Abstract

Neuropeptides such as vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are present in nerve fibers of bone tissues and have been suggested to potentially regulate bone remodeling. Oscillatory fluid flow (OFF)-induced shear stress is a potent signal in mechanotransduction that is capable of regulating both anabolic and catabolic bone remodeling. However, the interaction between neuropeptides and mechanical induction in bone remodeling is poorly understood. In this study, we attempted to quantify the effects of combined neuropeptides and mechanical stimuli on mRNA and protein expression related to bone resorption. Neuropeptides (VIP or CGRP) and/or OFF-induced shear stress were applied to MC3T3-E1 pre-osteoblastic cells and changes in receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) mRNA and protein levels were quantified. Neuropeptides and OFF-induced shear stress similarly decreased RANKL and increased OPG levels compared to control. Changes were not further enhanced with combined neuropeptides and OFF-induced shear stress. These results suggest that neuropeptides CGRP and VIP have an important role in suppressing bone resorptive activities through RANKL/OPG pathway, similar to mechanical loading.

Figure 1.

Osteoclast formation and activity. (A) Co-culture of MC3T3-E1 pre-osteoblastic cells and RAW 264.7 machrophage cells resulted in the formation of multinucleated cells with three or more nuclei; and (B) effect of CGRP (calcitonin gene-related peptide) and VIP (vasoactive intestinal peptide) on formation of osteoclasts. Using a microscope with a magnification ×200, Cells with three or more nuclei were considered to be osteoclasts. * p < 0.05 control. The arrow and 1, 2, 3 are nucleus.

Figure 2.

Multinucleated osteoclasts were stained red colors by TRAP (tartrate-resistant acid phosphatase) Assay Kit. (A) Control group (TRAP-positive multinucleated cells with no VIP or CGRP treatment); (B) 10 nM CGRP treatment group; and (C) 1 μM VIP treatment group. To assess the formation and activity of osteoclasts, cells were stained for TRAP activity on Day 9 with a magnification ×200.

Figure 3.

Expression of RANKL (receptor activator of nuclear factor kappa B (NF-κB) ligand) and OPG (osteoprotegerin) mRNA. Change in (A) RANKL mRNA; (B) OPG mRNA; and (C) RANKL/OPG mRNA ratio after neurotransmitter and/or mechanical stimulation. * p < 0.05 control.

Figure 4.

Expression of RANKL and OPG protein. Change in (A) RANKL protein; (B) OPG protein; and (C) RANKL/OPG protein ratio after neurotransmitter and/or mechanical stimulation. * p < 0.05 control.