Effects of alpha-calcitonin gene-related peptide on osteoprotegerin and receptor activator of nuclear factor-κB ligand expression in MG-63 osteoblast-like cells exposed to polyethylene particles

Abstract

Background: Recent studies demonstrated an impact of the nervous system on particle-induced osteolysis, the major cause of aseptic loosening of joint replacements.

Methods: In this study of MG-63 osteoblast-like cells we analyzed the influence of ultra-high molecular weight polyethylene (UHMWPE) particles and the neurotransmitter alpha-calcitonin gene-related peptide (CGRP) on the osteoprotegerin/receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factorκB (OPG/RANKL/RANK) system. MG-63 cells were stimulated by different UHMWPE particle concentrations (1:100, 1:500) and different doses of alpha-CGRP (10-7 M, 10-9 M, 10-11 M). RANKL and OPG mRNA expression and protein levels were measured by RT-PCR and Western blot.

Results: Increasing particle concentrations caused an up-regulation of RANKL after 72 hours. Alpha-CGRP showed a dose-independent depressive effect on particle-induced expression of RANKL mRNA in both cell-particle ratios. RANKL gene transcripts were significantly (P < 0.05) decreased by alpha-CGRP treatment after 48 and 72 hours. OPG mRNA was significantly down-regulated in a cell-particle ratio of 1:500 after 72 hours. Alpha-CGRP concentrations of 10-7 M lead to an up-regulation of OPG protein.

Conclusion: In conclusion, a possible osteoprotective influence of the neurotransmitter alpha-CGRP on particle stimulated osteoblast-like cells could be shown. Alpha-CGRP might be important for bone metabolism under conditions of particle-induced osteolysis.

Figure 1

RANKL and OPG mRNA levels in MG-63 cells after particle and alpha-CGRP treatment. Time course of (A) RANKL and (D) OPG mRNA expression of MG-63 cells after stimulation with different UHMWPE particle concentrations. Time course of UHMWPE particle-induced RANKL mRNA expression after treatment with different alpha-CGRP doses in (B) a cell-particle ratio of 1:100 and (C) a cell-particle ratio of 1:500. Time course of UHMWPE particle-induced OPG mRNA expression after treatment with different alpha-CGRP doses in (E) a cell-particle ratio of 1:100 and (F) a cell-particle ration of 1:500. Significant differences are marked. ((a) P < 0.05).

Figure 2

RANKL protein levels in MG-63 cells after particle and alpha-CGRP treatment. Time courses of RANKL protein levels in MG-63 osteoblast-like cells stimulated by UHMWPE particles and alpha-CGRP using Western blot analysis. (A) Representative Western blot for RANKL in untreated group and the alpha-CGRP-incubated groups. Densitometric quantification of RANKL in (B) a cell-particle ratio of 1:100 and (C) a cell-particle ratio of 1:500 with and without alpha-CGRP-incubation. RANKL protein levels are expressed relatively to GAPDH. Data are reported as mean ± standard deviation (n = 5). ((a) P < 0.05).

Figure 3

OPG protein levels in MG-63 cells after particle and alpha-CGRP treatment. Time courses of OPG protein levels in MG-63 osteoblast-like cells stimulated by UHMWPE particles and alpha-CGRP using Western blot analysis. (A) Representative Western blot for OPG in untreated group and the alpha-CGRP-incubated groups. Densitometric quantification of OPG in (B) a cell-particle ratio of 1:100 and (C) a cell-particle ratio of 1:500 with and without alpha-CGRP-incubation. OPG protein levels are expressed relatively to GAPDH. Data are reported as mean ± standard deviation (n = 5). ((a) P < 0.05).