UNCOR RECT ED PROOF 1 Inhibition of methylation decreases osteoblast differentiation via a 2 non-DNA-dependent methylation mechanism Q1 3 Bart L.T. Vaes a, ⁎, Carolien Lute a , Sebastian P. van der Woning b , Ester Piek b , Jenny Vermeer a , Henk J. Blom c , 4 John C. Mathers d , Michael Müller a , Lisette C.P.G.M. de Groot a , Wilma T. Steegenga a 5 a Division of Human Nutrition, Wageningen University and Research Centre, Wageningen, The Netherlands 6 b Department of Applied Biology, Faculty of Science, Radboud University Nijmegen, Nijmegen, The Netherlands 7 c Department of Clinical Chemistry, VU University Medical Centre, Amsterdam, The Netherlands 8 d Human Nutrition Research Centre, Institute for Ageing and Health, University of Newcastle, Newcastle upon Tyne, UK 9 10 abstract article info 11 Article history: 12 Received 17 April 2009 13 Revised 1 September 2009 14 Accepted 27 September 2009 15 Available online xxxx 16 17 Edited by: J. Aubin 18 19 20 21 Keywords: 22 Osteoblast 23 Methylation 24 Runx2 25 5-Aza-2′-deoxycytidine 26 Periodate oxidized adenosine 27 S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is 28 rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the 29 effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized 30 adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to 31 modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during 32 growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5- 33 Aza-2′-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR 34 induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under 35 osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 36 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers 37 during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only 38 reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter 39 (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken 40 together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired 41 osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a 42 regulator of Runx2-controlled gene expression. 43 © 2009 Published by Elsevier Inc. 44 45 46 47 48 Introduction 49 In adults, newly formed osteoblasts are derived from mesenchy- 50 mal stem cells (MSC) residing in the bone marrow. A large variety of 51 hormones, growth factors, cytokines, and vitamins have been 52 identified that trigger the differentiation of MSC towards osteoblasts. 53 Transcription factors such as Runx2 (Runt-related transcription factor 54 2), Osx (Osterix), and Dlx5 (distal-less homeobox 5), key regulators of 55 the differentiation process, are activated via different signaling 56 pathways. In response, differentiating osteoblasts start to excrete an 57 organic extracellular matrix existing of proteins including collagen 58 type 1, Alp (alkaline phosphatase), and the bone-specific protein Ocn 59 (osteocalcin). Expression of these genes is associated with the late 60 phase of differentiation [1]. 61 Increasing evidence indicates that DNA methylation is an 62 important mechanism underlying the cascade of events turning 63 MSC into osteoblasts. Kang et al. [2] have provided evidence that 64 expression of the master regulators Dlx5 and Osx can be regulated via 65 DNA methylation. They showed that CpG dinucleotides of the Dlx5 66 and Osx promoter were unmethylated in osteogenic cell lines in 67 which these genes are expressed but methylated in non-osteogenic 68 cell lines lacking the expression of the two transcription factors. The 69 significance of promoter methylation in osteoblast-specific gene 70 regulation is further underscored by studies showing that tissue- 71 specific expression of Runx2 [2] and Ocn [2,3] correlates with their 72 promoter methylation status. 73 DNA methylation is dependent on the presence of a methyl donor 74 together with specific enzymes, the so-called DNA methyltrans- 75 ferases. The major methyl donor in all living organisms is S- 76 adenosylmethionine (SAM). SAM donates a methyl group when it is 77 converted into S-adenosylhomocysteine (SAH) (Fig. 1). Subsequently, 78 SAH is hydrolyzed to form homocysteine, which is remethylated to 79 methionine by the enzyme methionine synthase. Finally, methionine 80 can be converted to SAM. The capacity for methyl donation, and hence Bone xxx (2009) xxx–xxx ⁎ Corresponding author. Wageningen University, Nutrition, Metabolism and Geno- mics Group, Division of Human Nutrition, Bomenweg 2, 6703 HD Wageningen, The Netherlands. Fax: +31 317 483342. E-mail address: bart.vaes@wur.nl (B.L.T. Vaes). BON-08629; No. of pages: 10; 4C: 8756-3282/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.bone.2009.09.033 Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone ARTICLE IN PRESS Please cite this article as: Vaes BLT, et al, Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism, Bone (2009), doi:10.1016/j.bone.2009.09.033