C/EBPb Promotes Transition from Proliferation to Hypertrophic Differentiation of Chondrocytes through Kip2 Transactivation of p57 Makoto Hirata1., Fumitaka Kugimiya1., Atsushi Fukai1, Shinsuke Ohba2, Naohiro Kawamura1, Toru Ogasawara1, Yosuke Kawasaki1, Taku Saito1, Fumiko Yano 2 , Toshiyuki Ikeda1, Kozo Nakamura1, Ung-il Chung 2 , Hiroshi Kawaguchi1* 1 Departments of Sensory & Motor System Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan, 2 Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan Abstract Background: Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein b (C/EBPb) in chondrocytes during endochondral ossification. Methodology/Principal Findings: Mouse embryos with homozygous deficiency in C/EBPb (C/EBPb2/2) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPb2/2 chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPb in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPb. In fact, a DNA cell cycle histogram revealed that the C/EBPb overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57Kip2 as the transcriptional target of C/EBPb. p57Kip2 was co-localized with C/EBPb in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/ EBPb deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/ EBPb in the p57Kip2 promoter between 2150 and 2130 bp region containing a putative C/EBP motif. The knockdown of p57Kip2 by the siRNA inhibited the C/EBPb-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPb+/2 littermates, the C/ EBPb insufficiency caused resistance to joint cartilage destruction. Conclusions/Significance: C/EBPb transactivates p57Kip2 to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPb-p57Kip2 signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis. Citation: Hirata M, Kugimiya F, Fukai A, Ohba S, Kawamura N, et al. (2009) C/EBPb Promotes Transition from Proliferation to Hypertrophic Differentiation of Chondrocytes through Transactivation of p57Kip2. PLoS ONE 4(2): e4543. doi:10.1371/journal.pone.0004543 Editor: Thomas Zwaka, Baylor College of Medicine, United States of America Received September 29, 2008; Accepted January 6, 2009; Published February 20, 2009 Copyright: 2009 Hirata 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 study was supported by a Grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (#19109007). The sponsor had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kawaguchi-ort@h.u-tokyo.ac.jp . These authors contributed equally to this work. Introduction Most skeletal growth is achieved by endochondral ossification. During the process, chondrocytes undergo proliferation, hyper- trophic differentiation, and apoptosis [1], each of which is regulated by distinct signals. Among them, chondrocyte hyper- trophy is a rate-limiting step for the skeletal growth, being responsible for 40–60% of the endochondral ossification [2,3]. The initiation is precisely linked with the cessation of proliferation; however, the molecular mechanism underlying the harmonious transition from the proliferation to hypertrophic differentiation of chondrocytes remains an enigma. CCAAT/enhancer-binding protein b (C/EBPb), also known as nuclear factor-interleukin-6 (NF-IL6), is a member of the C/EBP family of six transcription factors characterized by a carboxyl- terminal leucine zipper dimerization domain and an adjacent highly conserved basic DNA binding domain [4–6]. Contrary to C/EBPa that is purely antiproliferative as a tumor suppressor in several cell types, C/EBPb regulates expression of various genes involved in cell differentiation, proliferation, survival, immune function and female reproduction, as well as tumor invasiveness and progression, through a variety of mechanisms [6].Over the past several years, C/EBPb has been shown to control differentiation of hematopoietic and adipogenic cells [7,8]. The PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4543