Hdac3 Regulates Bone Density Figure 7. Cortical bone is thinner in Hdac3 CKO mice. A. MicroCT reconstructions of tibia and fibulas from WT and Hdac3 CKO mice. Arrows point to fractures. B–F. Calcein incorporation (B), bone formation rates (C), mineral apposition rates (D), mineralized surfaces (E) were decreased, while mineralization lag time (F) was increased in Hdac3 CKO mice (n = 5 WT and CKOOsx, n = 2 CHet, n = 3 CKO). G–J. Cortical measurements from microCT reconstructions demonstrate that periosteal area (Ps.Ar, G), endocortical area (Ec.Ar, H), Cortical area (Ct.Ar), which estimates axial strength (I), and section (Sec) modulus, an estimate of bending strength (J), were reduced in Hdac3 CKO mice (n = 3 per group). *p,0.05, **p,0.01, #p,0.0001. doi:10.1371/journal.pone.0011492.g007 consistent with our recent studies examining how the Hdac inhibitor vorinostat (SAHA) affects skeletal health in a preclinical cancer model. Although vorinostat inhibited tumor burden in murine long bones, bone mass of contralateral limbs was significantly reduced [55]. Vorinostat represses multiple Hdacs and the data reported here suggest that Hdac3 suppression is sufficient to decrease bone volume. Our results indicate that sustained Hdac3 suppression is likely to produce osteopenia and potentially fractures. Hdac inhibitors are in many clinical cancer trials and vorinostat is already approved in the United States for treating cutaneous T cell lymphomas [56]. Hdac inhibitors are also being considered as possible therapies for several non- cancerous conditions, including degenerative and inflammatory disorders [57]. Our data strongly suggest that bone density should be monitored in any individuals receiving Hdac inhibitors as therapy. Moreover, future generations of Hdac inhibitors should be designed to avoid targeting Hdac3 because of multiple organ (heart, liver and bone) failures in Hdac3-deficient mice [12,31]. Materials and Methods Mice/Genotyping Mice were genotyped for Hdac3 alleles as reported [11]. Osx-Cre transgenic animals were previously described [30]. Animals were housed in an accredited facility under a 12-hour light/dark cycle and provided water and food (PicoLabH Rodent Diet 20, LabDiet) ad libitum. Diets were supplemented three times per week with Nutri-CalH (Ve´toquinol Canada Inc, Lavaltrie, QC) and Napa Nector (SE Lab Group Inc, Napa, CA). For dynamic histomor- phometry experiments, 5.5 and 12 week-old mice were injected subcutaneously with tetracycline (25 mg/kg) 7 and 14 days, respectively, before euthanasia. They were also injected subcuta- neously twice with calcein (10 mg/kg) at four days and one day before ending the experiment. All animal research was conducted according to guidelines provided by the National Institute of Health and the Institute of Laboratory Animal Resources, National Research Council. The Mayo Clinic Institutional Animal Care and Use Committee approved all animal studies. Whole Mount Embryo Staining Newborn pups were euthanized and placed in chloroform. Skeletons were dissected and fixed overnight in ethanol. Cartilage elements were stained with a 30% Alcian blue dye (dissolved in 80 ml 95% ethanol and 20 ml glacial acetic acid). Skeletons were washed twice with 95% ethanol and then placed in 2% KOH until the remaining soft tissues were dissolved. Bones were stained with 75 mg/ml Alizarin red S (Sigma) in 1% KOH overnight and then PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11492