Westendorf1* Abstract Histone Deacetylase 3 Depletion in Osteo/ Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat David F. Razidlo1., Tiffany J. Whitney1., Michelle E. Casper1, Meghan E. McGee-Lawrence1, Bridget A. Stensgard1, Xiaodong Li1, Frank J. Secreto1, Sarah K. Knutson2, Scott W. Hiebert2, Jennifer J. 1 Department of Orthopedic Surgery and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America, 2 Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health. Citation: Razidlo DF, Whitney TJ, Casper ME, McGee-Lawrence ME, Stensgard BA, et al. (2010) Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat. PLoS ONE 5(7): e11492. doi:10.1371/journal.pone.0011492 Editor: Sudha Agarwal, Ohio State University, United States of America Received February 2, 2010; Accepted June 11, 2010; Published July 9, 2010 Copyright: 2010 Razidlo 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: The National Institutes of Health (R01-AR048147 and T32- AR056950) and the Mayo Clinic Foundation supported this project. The funders had no role in study 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: westendorf.jennifer@mayo.edu . These authors contributed equally to this work. Introduction The skeleton is a dynamic organ composed of more than 200 bones that develop via two distinct processes. Intramembranous ossification occurs when mesenchymal cells condense and differentiate into osteoblasts to form the flat bones of the skull and clavicles. In contrast, the long bones develop via a process of endochondral ossification that involves the recruitment of osteoblasts to a cartilaginous template. Mature osteoblasts secrete collagens and other proteins to form an organic matrix (osteoid) that is mineralized when inorganic salts are brought to the site via the circulation. Osteoblasts are derived from several multipotent progenitors, including mesenchymal cells, neural crest cells and pericytes. Osteoblast development requires the expression of several transcription factors (e.g. Runx2 [1,2] and osterix [3]) and the coordinated activation of numerous developmental signaling pathways [4]. Cells committed to the osteoblast lineage mature through successive stages of proliferation (pre-osteoblasts), cell cycle exit and production of matrix proteins (osteoblasts), and finally, terminal differentiation into mechanosensory osteocytes or bone lining cells. If progenitor cells do not receive the proper signals for osteogenesis, as in the case of Runx2-deficiency, they have an increased propensity to differentiate into adipocytes [5,6]. Histone deacetylases (Hdacs) are enzymatic components of large multi-protein complexes that remove acetyl groups from lysine side chains of histones and other proteins. They contribute to epigenetic programming and regulation of gene expression during development and throughout life. The human and mouse genomes encode 18 Hdacs that are classified into four groups on the basis of their structure and function [7]. Hdac3 (OMIM 605166) is part of class I, which also includes Hdac1, 2, and 8. Although class I Hdacs are broadly expressed, these predom- inantly nuclear enzymes have distinct developmental roles [8,9,10,11,12,13]. By comparison, class II Hdacs (Hdacs 4, 5, 6, 7, 9, 10) travel between the nuclear and cytoplasmic compart- ments and have more tissue restricted expression patterns [7,14,15,16,17]. Hdac3 binds several class II Hdacs [18,19] and co-repressor proteins, notably nuclear receptor co-repressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to form large multi-protein complexes [20,21,22]. These multi-protein complexes are recruited to specific DNA sequences and chromatin substrates by transcription factors. Several Hdacs contribute to skeletal development [7,23]. Hdac4 is expressed in prehypertrophic chondrocytes [14]. Germline deletion of Hdac4 caused premature ossification of the developing bones, while Hdac4 overexpression prevented chondrocyte PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11492