title: DNA methylation contributes to the regulation of sclerostin expression in human osteocytes.
abstract: 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.
authors.affiliation: Department of Internal Medicine, Hospital U.M. Valdecilla-IFIMAV, University of Cantabria, RETICEF, Santander, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Delgado-Calle
authors.forename: Carolina
authors.initials: C
authors.lastname: Sañudo
authors.forename: Alfonso
authors.initials: A
authors.lastname: Bolado
authors.forename: Agustín F
authors.initials: AF
authors.lastname: Fernández
authors.forename: Jana
authors.initials: J
authors.lastname: Arozamena
authors.forename: María A
authors.initials: MA
authors.lastname: Pascual-Carra
authors.forename: José C
authors.initials: JC
authors.lastname: Rodriguez-Rey
authors.forename: Mario F
authors.initials: MF
authors.lastname: Fraga
authors.forename: Lynda
authors.initials: L
authors.lastname: Bonewald
authors.forename: José A
authors.initials: JA
authors.lastname: Riancho
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D001374:Azacitidine / Q000494:pharmacology; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D005819:Genetic Markers / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D010011:Osteocytes / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017384:Sequence Deletion / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D009687:Nuclear Proteins; C422903:SOST protein, human; D001374:Azacitidine