title: Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.
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 D3treatment 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.
authors.affiliation: Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL Universidad de Cantabria, 39008, Santander, Cantabria, Spain.|Department of Molecular Biology, University of Cantabria, IDIVAL, Santander, Spain.
authors.forename: Flor M
authors.initials: FM
authors.lastname: Pérez-Campo
authors.affiliation: InSCREENeX GmbH, Brunswick, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: May
authors.affiliation: InSCREENeX GmbH, Brunswick, Germany.
authors.forename: Jeannette
authors.initials: J
authors.lastname: Zauers
authors.affiliation: Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL Universidad de Cantabria, 39008, Santander, Cantabria, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Sañudo
authors.affiliation: Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL Universidad de Cantabria, 39008, Santander, Cantabria, Spain.|Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA.|Roudebush Veterans Administration Medical Center, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Jesús
authors.initials: J
authors.lastname: Delgado-Calle
authors.affiliation: Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL Universidad de Cantabria, 39008, Santander, Cantabria, Spain.
authors.forename: Jana
authors.initials: J
authors.lastname: Arozamena
authors.affiliation: Department of Anatomy and Cell Biology, University of Cantabria, IDIVAL, Santander, Spain.
authors.forename: María T
authors.initials: MT
authors.lastname: Berciano
authors.affiliation: Department of Anatomy and Cell Biology, University of Cantabria, IDIVAL, Santander, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Lafarga
authors.affiliation: Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL Universidad de Cantabria, 39008, Santander, Cantabria, Spain. jose.riancho@unican.es.
authors.forename: José A
authors.initials: JA
authors.lastname: Riancho
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002113:Calcification, Physiologic; D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D019175:DNA Methylation; D000077209:Decitabine; D044127:Epigenesis, Genetic; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D010006:Osteoblasts / Q000166:cytology*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis; D054457:Tissue Scaffolds
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; C422903:SOST protein, human; D015675:Osteocalcin; D000077209:Decitabine; D002117:Calcitriol; D001374:Azacitidine
keywords: DNA methylation; Epigenetics; Immortalized osteoblastic cell lines; SOST; Sclerostin