ROR2 Promoter Methylation Change in Osteoblastic Differentiation of Mesenchymal Stem Cells

OBJECTIVE Osteoblasts arise from multipotent mesenchymal stem cells (MSCs) present in the bone marrow stroma and undergo further differentiation to osteocytes or bone cells. Many factors such as proteins present in the Wnt signaling pathway affect osteoblast differentiation. ROR2 is an orphan tyrosine kinase receptor that acts as a co-receptor in the non-canonical Wnt signaling pathway. However, ROR2 has been shown to be regulated by both canonical and non-canonical Wnt signaling pathways. ROR2 expression increases during differentiation of MSCs to osteoblasts and then decreases as cells differentiate to osteocytes. On the other hand, research has shown that ROR2 changes MSC fate towards osteoblasts by inducing osteogenic transcription factor OSTERIX. Here we speculated whether ROR2 gene expression regulation during osteoblastogenesis is epigenetically determined. MATERIALS AND METHODS MSCs from bone marrow were isolated, expanded and characterized in vitro according to standard procedures. ROR2 promoter methylation status was determined using methylation specific PCR in a multipotent state and during differentiation to osteoblasts. RESULTS We determined that the demethylation process in ROR2 promoter occurs during the differentiation process. The process of demethylation begins at day 8 and continues until 21 days of differentiation. CONCLUSION This result is in concordance with previous works on the role of ROR2 on osteoblast differentiation, which have shown an upregulation of ROR2 expression during this process.