Id4, a New Candidate Gene for Senile Osteoporosis, Acts as a Molecular Switch Promoting Osteoblast Differentiation Yoshimi Tokuzawa 1. , Ken Yagi 1. , Yzumi Yamashita 1 , Yutaka Nakachi 1 , Itoshi Nikaido 1 , Hidemasa Bono 1 , Yuichi Ninomiya 1 , Yukiko Kanesaki-Yatsuka 1 , Masumi Akita 2 , Hiromi Motegi 3 , Shigeharu Wakana 3 , Tetsuo Noda 3,4 , Fred Sablitzky 5 , Shigeki Arai 6 , Riki Kurokawa 6 , Toru Fukuda 7 , Takenobu Katagiri 7 , Christian Scho ¨ nbach 8,9 , Tatsuo Suda 1 , Yosuke Mizuno 1 , Yasushi Okazaki 1 * 1 Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan, 2 Division of Morphological Science, Biomedical Research Center, Saitama Medical University, Iruma-gun, Saitama, Japan, 3 RIKEN BioResource Center, Tsukuba, Ibaraki, Japan, 4 The Cancer Institute of the Japanese Foundation for Cancer Research, Koto-ward, Tokyo, Japan, 5 Developmental Genetics and Gene Control, Institute of Genetics, University of Nottingham, Queen’s Medical Center, Nottingham, United Kingdom, 6 Division of Gene Structure and Function, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan, 7 Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan, 8 Division of Genomics and Genetics, Nanyang Technological University School of Biological Sciences, Singapore, Singapore, 9 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan Abstract Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparc2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparc2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis. Citation: Tokuzawa Y, Yagi K, Yamashita Y, Nakachi Y, Nikaido I, et al. (2010) Id4, a New Candidate Gene for Senile Osteoporosis, Acts as a Molecular Switch Promoting Osteoblast Differentiation. PLoS Genet 6(7): e1001019. doi:10.1371/journal.pgen.1001019 Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America Received January 27, 2010; Accepted June 4, 2010; Published July 8, 2010 Copyright: ß 2010 Tokuzawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants-in-aid of the Genome Network Project and Support Project of Strategic Research Center in Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to Saitama Medical University Research Center for Genomic Medicine. The funders had no role in study design, data collection and analysis, design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: okazaki@saitama-med.ac.jp . These authors contributed equally to this work. Introduction Senile osteoporosis or age-related osteopenia is accompanied by increased bone marrow tissue adiposity [1]. Bone marrow adipocytes and osteoblasts are thought to originate from common mesenchymal stem cells (MSCs). Therefore, it has been suggested that the excessive accumulation of marrow adipocytes following bone loss is caused by unbalanced differentiation of MSCs into marrow adipocytes and osteoblasts [2]. Support for this hypothesis comes from studies of peroxisome proliferators-activated receptor-c (Pparc), a master regulator of adipocyte differentiation, deficient embryonic stem cells that showed an increase in osteoblast differentiation [3]. In contrast, calvarial adipocyte differentiation is augmented when runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation has been knocked out [4]. Transcription factors Runx2 and Sp7 transcription factor 7 (Sp7) regulate MSC commitment to osteoblast differentiation along with bone morphogenetic protein (BMP) signaling pathway [5]. Conversely, Pparc and CCAAT/enhancer binding protein (C/EBP) transcription factor family members drive MSCs differentiation toward adipocytes [6]. Other proteins that regulate the balance between adipocyte and osteoblast differentiation are tafazzin, Wnt5a, Wnt10b, Msx2, C/ EBPb and basic helix-loop-helix (bHLH) family member e40 (Bhlhe40) [6–8]. Aforementioned transcription factors suppress adipocyte differentiation and promote osteoblast differentiation. Regardless of PLoS Genetics | www.plosgenetics.org 1 July 2010 | Volume 6 | Issue 7 | e1001019