BMP-2 Promotes Differentiation of Osteoblasts and Chondroblasts in Runx2-Deficient Cell Lines TINGJIAO LIU, 1,2,3 YUHAO GAO, 4 KEI SAKAMOTO, 1 TOKUTARO MINAMIZATO, 1 KEIZO FURUKAWA, 5 TOMOO TSUKAZAKI, 5 YASUAKI SHIBATA, 6 KAZUHISA BESSHO, 7 TOSHIHISA KOMORI, 8 AND AKIRA YAMAGUCHI 1 * 1 Section of Oral Pathology, Department of Oral Restitution, Graduate School of Tokyo Medical and Dental University, Tokyo, Japan 2 21st Century Center of Excellence (COE) Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, Japan 3 Department of Oral Pathology, Dalian Medical University, Dalian, China 4 Division of Endocrinology, Emory University School of Medicine, Atlanta, Georgia 5 Department of Orthpaedic Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan 6 Department of Oral Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan 7 Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 8 Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD-C6, from Runx2-deficient mouse embryos. RD-C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2) or transduction of BMP-2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD-C6 cells. BMP-2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD-C6 cells with or without rhBMP-2 treatment demonstrated that BMP-2 upregulated 66 genes including 13 transcription-related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD-C6 cells, we transplanted RD-C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP-2. RD-C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP-2 induces Runx2-deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2-independent pathway, but it failed to induce these cells to differentiate into bone-forming osteoblasts and mature chondrocytes. J. Cell. Physiol. 211: 728–735, 2007. ß 2007 Wiley-Liss, Inc. Skeletal tissue is composed of various types of mesenchymal cells such as osteoblasts, chondrocytes, myocytes, and adipocytes. These cells originate from common pluripotent progenitors called mesenchymal stem cell (Caplan, 1991; Pittenger et al., 1999). During the differentiation of these cells, lineage-specific transcription factors critically regulate the pluripotent progenitors in their acquisition of specific phenotypes depending on their maturation (Rodan and Harada, 1997; Yamaguchi et al., 2000). Among these, Runx2 (runt- related gene 2, alternatively called core binding factor alpha 1: Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation (Ducy et al., 1997; Komori et al., 1997; Otto et al., 1997; Stein et al., 2004); this was understood based on the fact that Runx2-deficient mice completely lacked bone formation due to the maturation arrest of osteoblasts (Komori et al., 1997; Otto et al., 1997). These mice also displayed the delayed maturation of chondrocytes (Inada et al., 1999), indicating that Runx2 also regulates the differentiation and maturation of chondrocytes (Enomoto et al., 2000; Ueta et al., 2001; Yoshida et al., 2004). Osterix is another essential transcription factor that regulates osteoblast differentiation and bone formation (Nakashima et al., 2002). Osterix-deficient mice also exhibited lack of bone formation due to the maturation arrest of osteoblast differentiation, but they showed no apparent changes in cartilage (Nakashima et al., 2002). Thus, these two transcription factors govern the critical regulation of osteoblast differentiation and bone formation. Bone morphogenetic proteins (BMPs) were originally identified as proteins that induced ectopic bone formation when implanted into muscular tissue (Urist, 1965), and they are Contract grant sponsor: Scientific Research from the Japan Society for the Promotion of Science; Contract grant number: 14104015. *Correspondence to: Akira Yamaguchi, Section of Oral Pathology, Department of Oral Restitution, Graduate School of Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail: akira.mpa@tmd.ac.jp Received 9 August 2006; Accepted 16 November 2006 DOI: 10.1002/jcp.20988 ORIGINAL ARTICLE 728 Journal of Journal of Cellular Physiology Cellular Physiology ß 2007 WILEY-LISS, INC.