DNA methylation contributes to the regulation of sclerostin expression in human osteocytes

Jesus Delgado-Calle, Carolina Sañudo, Alfonso Bolado, Agustín F. Fernández, Jana Arozamena, María A. Pascual-Carra, José C. Rodriguez-Rey, Mario F. Fraga, Lynda Bonewald, José A. Riancho

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)926-937
Number of pages12
JournalJournal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Volume27
Issue number4
DOIs
StatePublished - Apr 2012
Externally publishedYes

Fingerprint

Osteocytes
DNA Methylation
Osteoblasts
Methylation
CpG Islands
Nuclear Proteins
Genetic Promoter Regions
Protein Binding
Osteogenesis
Epigenomics
Genes
Exons
Transcription Factors
Theoretical Models
Bone and Bones
Cell Line

Keywords

  • DNA methylation
  • Osteoblasts
  • osteocytes
  • promoter
  • sclerostin

ASJC Scopus subject areas

  • Orthopedics and Sports Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

DNA methylation contributes to the regulation of sclerostin expression in human osteocytes. / Delgado-Calle, Jesus; Sañudo, Carolina; Bolado, Alfonso; Fernández, Agustín F.; Arozamena, Jana; Pascual-Carra, María A.; Rodriguez-Rey, José C.; Fraga, Mario F.; Bonewald, Lynda; Riancho, José A.

In: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 27, No. 4, 04.2012, p. 926-937.

Research output: Contribution to journalArticle

Delgado-Calle, Jesus ; Sañudo, Carolina ; Bolado, Alfonso ; Fernández, Agustín F. ; Arozamena, Jana ; Pascual-Carra, María A. ; Rodriguez-Rey, José C. ; Fraga, Mario F. ; Bonewald, Lynda ; Riancho, José A. / DNA methylation contributes to the regulation of sclerostin expression in human osteocytes. In: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2012 ; Vol. 27, No. 4. pp. 926-937.
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AU - Delgado-Calle, Jesus

AU - Sañudo, Carolina

AU - Bolado, Alfonso

AU - Fernández, Agustín F.

AU - Arozamena, Jana

AU - Pascual-Carra, María A.

AU - Rodriguez-Rey, José C.

AU - Fraga, Mario F.

AU - Bonewald, Lynda

AU - Riancho, José A.

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AB - 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.

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