title: Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.
abstract: S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.
authors.affiliation: Division of Human Nutrition, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Bart L T
authors.initials: BL
authors.lastname: Vaes
authors.forename: Carolien
authors.initials: C
authors.lastname: Lute
authors.forename: Sebastian P
authors.initials: SP
authors.lastname: van der Woning
authors.forename: Ester
authors.initials: E
authors.lastname: Piek
authors.forename: Jenny
authors.initials: J
authors.lastname: Vermeer
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Blom
authors.forename: John C
authors.initials: JC
authors.lastname: Mathers
authors.forename: Michael
authors.initials: M
authors.lastname: Müller
authors.forename: Lisette C P G M
authors.initials: LC
authors.lastname: de Groot
authors.forename: Wilma T
authors.initials: WT
authors.lastname: Steegenga
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D006801:Humans; D051379:Mice; D009928:Organ Specificity / Q000187:drug effects; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D000469:Alkaline Phosphatase; D000241:Adenosine; D001374:Azacitidine