Objective. To determine genome-wide methylation profiles of bone from patients with hip osteoarthritis (OA) and those with osteoporotic (OP) hip fractures.Methods. Trabecular bone pieces were obtained from the central part of the femoral head of 27 patients with hip fractures and 26 patients with hip OA. DNA was isolated, and methylation was explored with Illumina methylation arrays. RNA was extracted, pooled, and deep-sequenced to obtain the whole transcriptome. Differentially methylated regions were identified, and connections between genes with differentially methylated regions were explored by pathway and text-mining analyses.Results. After quality control, methylation of 23,367 CpG sites (13,463 genes) was analyzed. There was a genome-wide inverse relationship between methylation and gene expression in both patient groups. Comparison of OP and OA bones revealed 241 CpG sites, located in 228 genes, with significant differences in methylation (false discovery rate <0.05). Of them, 217 were less methylated in OP than in OA. The absolute methylation differences were >5% in 128 CpG sites and >10% in 45 CpG sites. The differentially methylated genes were enriched for association with bone traits in the genome-wide association study catalog. Pathway analysis and text-mining analysis with Gene Relationships Across Implicated Loci software revealed enrichment in genes participating in glycoprotein metabolism or cell differentiation, and particularly in the homeobox superfamily of transcription factors.Conclusion. Genome-wide methylation profiling of bone samples revealed differentially methylated regions in OP and OA. These regions were enriched in genes associated with cell differentiation and skeletal embryogenesis, such as those in the homeobox superfamily, suggesting the existence of a developmental component in the predisposition to these disorders.Bone increases in size during the growth period by a modeling process driven by the formation of new bone. Thereafter, it is constantly remodeled by the concerted action of bone-resorbing osteoclasts and bone-forming osteoblasts, originating from hematopoietic and mesenchymal precursors, respectively. When remodeling is to start at a certain site, osteoclast precursors are attracted and committed to differentiate into mature osteoclasts. When the resorption phase ends, surrounding osteoblast precursors proliferate and differentiate into mature osteoblasts that synthesize bone matrix that eventually mineralizes and replaces the old bone resorbed by osteoclasts. Thus, bone remodeling requires the cyclic and sequential proliferation and differentiation of osteoclast and osteoblast precursors. Any disturbance of this process will result in abnormal bone mass. This is the case in osteoporosis (OP), which is characterized by a decrease in bone mass, due to high bone resorption and/or low bone formation, and consequently propensity to fracture. In contrast to OP, in osteoarthritis (OA) bone formation is increased in the

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Sclerostin, encoded by the SOST gene, is a potent inhibitor of bone formation, produced by osteocytes, not by osteoblasts, but little is known about the molecular mechanisms controlling its expression. We aimed to test the hypothesis that epigenetic mechanisms, specifically DNA methylation, modulate SOST expression. We found two CpG-rich regions in SOST: region 1, located in the proximal promoter, and region 2, around exon 1. qMSP and pyrosequencing analysis of DNA methylation showed that region 2 was largely methylated in all samples analyzed. In contrast, marked differences were observed in region 1. Whereas the CpG-rich region 1 was hypermethylated in osteoblasts, this region was largely hypomethylated in microdissected human osteocytes. Bone lining cells showed a methylation profile between primary osteoblasts and osteocytes. Whereas SOST expression was detected at very low level or not at all by RT-qPCR in several human osteoblastic and nonosteoblastic cell lines, and human primary osteoblasts under basal conditions, it was dramatically upregulated (up to 1300-fold) by the demethylating agent AzadC. Experiments using reporter vectors demonstrated the functional importance of the region -581/+30 of the SOST gene, which contains the CpG-rich region 1. In vitro methylation of this CpG-island impaired nuclear protein binding and led to a 75 ± 12% inhibition of promoter activity. In addition, BMP-2-induced expression of SOST was markedly enhanced in cells demethylated by AzadC. Overall, these results strongly suggest that DNA methylation is involved in the regulation of SOST expression during osteoblast-osteocyte transition, presumably by preventing the binding of transcription factors to the proximal promoter. To our knowledge, our data provide first ever evidence of the involvement of DNA methylation in the regulation of SOST expression and may help to establish convenient experimental models for further studies of human sclerostin.