Epigenetic modifications and canonical wingless/int-1 class (WNT) signaling enable trans-differentiation of nonosteogenic cells into osteoblasts

Mesenchymal cells alter and retain their phenotype during skeletal development through activation or suppression of signaling pathways. For example, we have shown that Wnt3a only stimulates osteoblast differentiation in cells with intrinsic osteogenic potential (e.g. MC3T3-E1 pre-osteoblasts) and not in fat cell precursors or fibroblasts (3T3-L1 pre-adipocytes or NIH3T3 fibroblasts, respectively). Wnt3a promotes osteogenesis in part by stimulating autocrine production of the osteoinductive ligand Bmp2. Here, we show that the promoter regions of the genes for Bmp2 and the osteoblast marker Alp are epigenetically locked to prevent their expression in nonosteogenic cells. Both genes have conserved CpG islands that exhibit increased CpG methylation, as well as decreased acetylation and increased methylation of histone H3 lysine 9 (H3-K9) specifically in nonosteogenic cells. Treatment of pre-adipocytes or fibroblasts with the CpG-demethylating agent 5'-aza-2'-deoxycytidine or the histone deacetylase inhibitor tri-chostatin-A renders Bmp2 and Alp responsive to Wnt3a. Hence, drug-induced epigenetic activation of Bmp2 gene expression contributes toWnt3a-mediateddirect trans-differentiation of pre-adipocytes or fibroblasts into osteoblasts.Wepropose that direct conversion of nonosteogenic cells into osteoblastic cell types without inducing pluripotency may improve prospects for novel epigenetic therapies to treat skeletal afflictions.