Role of DNA methylation in the regulation of the RANKL-OPG system in human bone

Abstract

Osteoblasts are specialized cells that form new bone and also indirectly influence bone resorption by producing factors that modulate osteoclast differentiation. Although the methylation of CpG islands plays an important role in the regulation of gene expression, there is still scanty information about its role in human bone. The aim of this study was to investigate the influence of CpG methylation on the transcriptional levels of two osteoblast-derived critical factors in the regulation of osteoclastogenesis: the receptor activator of nuclear factor NF-κB ligand (RANKL) and its soluble decoy receptor osteoprotegerin (OPG). Quantitative methylation specific PCR (qMSP) and pyrosequencing analysis in various cell types showed that the methylation of regulatory regions of these genes, in the vicinity of the transcription start sites, repressed gene transcription, whereas an active transcription was associated with low levels of methylation. In addition, treatment with the DNA demethylating agent 5-azadeoxycitidine promoted a 170-fold induction of RANKL and a 20-fold induction of OPG mRNA expression in HEK-293 cells, which showed hypermethylation of the CpG islands and barely expressed RANKL and OPG transcripts at baseline. Transcriptional levels of both genes were also explored in bone tissue samples from patients with hip fractures and hip osteoarthritis. Although RANKL transcript abundance and the RANKL:OPG transcript ratio were significantly higher in patients with fractures than in those with osteoarthritis (RANKL: 0.76 ± 0.23 vs. 0.24 ± 0.08, p = 0.012; RANKL/OPG: 7.66 ± 2.49 vs. 0.92 ± 0.21, p = 0.002), there was no evidence for differential methylation across patient groups. In conclusion, the association between DNA methylation and the repression of RANKL and OPG expression strongly suggests that methylation-dependent mechanisms influence the transcription of these genes, which play a critical role in osteoclastogenesis. However, other mechanisms appear to be involved in the increased RANKL/OPG ratio of patients with osteoporotic fractures.

Keywords: :

• bone remodeling
• DNA Methylation
• osteoblasts
• osteoporosis

Acknowledgments

This work was supported in part by grants from Instituto de Salud Carlos III-Fondo de Investigaciones Sanitarias (Spanish Ministry of Health) PI09/539, PI06/1267, PS09/02454, the Spanish National Research Council (CSIC 200820I172 to MFF), and the Community of Asturias (FICYT IB09–106). The IUOPA is supported by the Obra Social Cajastur, Spain. Jesús Delgado-Calle is recipient of a grant from IFIMAV. Agustín Fernández-Fernández is supported by the IUOPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.