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J Bone Miner Metab. 2017 Mar;35(2):150-160. doi: 10.1007/s00774-016-0753-z. Epub 2016 Apr 2.

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.

Author information

1
Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL Universidad de Cantabria, 39008, Santander, Cantabria, Spain.
2
Department of Molecular Biology, University of Cantabria, IDIVAL, Santander, Spain.
3
InSCREENeX GmbH, Brunswick, Germany.
4
Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA.
5
Roudebush Veterans Administration Medical Center, Indiana University School of Medicine, Indianapolis, IN, USA.
6
Department of Anatomy and Cell Biology, University of Cantabria, IDIVAL, Santander, Spain.
7
Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL Universidad de Cantabria, 39008, Santander, Cantabria, Spain. jose.riancho@unican.es.

Abstract

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

KEYWORDS:

DNA methylation; Epigenetics; Immortalized osteoblastic cell lines; SOST; Sclerostin

PMID:
27038990
DOI:
10.1007/s00774-016-0753-z
[Indexed for MEDLINE]

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