Inhibition of Osteogenic Differentiation of Human Adipose-Derived Stromal Cells by Retinoblastoma Binding Protein 2 Repression of RUNX2-Activated Transcription†‡§

Histone methylation is regarded as an important type of histone modification defining the epigenetic program during the lineage differentiation of stem cells. A better understanding of this epigenetic mechanism that governs osteogenic differentiation of human adipose-derived stromal cells (hASCs) can improve bone tissue engineering and provide new insights into the modulation of hASC-based cell therapy. Retinoblastoma binding protein 2 (RBP2) is a histone demethylase that specifically catalyzes demethylation of dimethyl or trimethyl histone H3 lysine 4 (H3K4me2 or H3K4me3), which is normally associated with transcriptionally active genes. In this study, the roles of RBP2 in osteogenic differentiation of hASCs were investigated. We found that RBP2 knockdown by lentiviruses expressing small interfering RNA promoted osteogenic differentiation of hASCs in vitro and in vivo. In addition, we demonstrated that knockdown of RBP2 resulted in marked increases of mRNA expression of osteogenesis-associated genes such as alkaline phosphatase (ALP), osteocalcin (OC), and osterix (OSX). RBP2 was shown to occupy the promoters of OSX and OC to maintain the level of the H3K4me3 mark by chromatin immunoprecipitation assays. Furthermore, coimmunoprecipitation and luciferase reporter experiments suggested that RBP2 was physically and functionally associated with RUNX2, an essential transcription factor that governed osteoblastic differentiation. Significantly, RUNX2 knockdown impaired the repressive activity of RBP2 in osteogenic differentiation of hASCs. Altogether, our study is the first to demonstrate the functional and biological roles of H3K4 demethylase RBP2 in osteogenic differentiation of hASCs and to link RBP2 to the transcriptional regulation of RUNX2. STEM CELLS 2011;29:1112–1125