Hdac3 Regulates Bone Density Figure 5. Osteoblast number is decreased in Hdac3-CKO mice. Distal femurs from 5.5 week-old male mice were sectioned and stained with Goldner’s Trichrome (A and B) or TRAP (C). Osteoblast surface as a proportion of bone surface (Ob.S/BS; A), osteoblast number per tissue area (N.Ob/ T.Ar; B) and osteoclast surface per bone surface (Oc.S/BS; C) were measured with Osteometrics software. n = 627, *p,0.05. doi:10.1371/journal.pone.0011492.g005 activity might be necessary to complete chondrocyte differentia- tion, promote apoptosis, and/or for the onset of ossification. Interestingly, only animals in the CKO groups (not the Het or CHet groups) exhibited significant changes in trabecular and cortical bone structure (Figures 4–7), which likely explains their smaller stature. Preliminary studies suggest that endochondral ossification of the bone collar was delayed in neonatal Hdac3 CKO mice (data not shown). More focused studies are in progress to determine how Hdac3 participates in the temporal events of endochondral bone formation. Two p53 inducible genes, p21CIP1 and Phlda3 [39,40], were upregulated in Hdac3 CKO mice. This raises the possibility that DNA damage might be contributing to defective osteoblast maturation in the CKO animals (Figure 9B). The cyclin- dependent kinase inhibitor, p21 (CIP1/WAF1), was the most upregulated gene in Hdac3 CKO mice and Hdac3 repressed the basal activity of the p21Cip1 promoter fragment. We previously showed that Runx2 represses p21Cip1 via Hdac-dependent mechanisms in osteoblasts [25]. p21Cip1 was one of the first genes described to be upregulated by Hdac inhibitors [35,36,37] and is elevated in Hdac1-deficient embryonic stem cells, Hdac2 KO mice [9,41] and in liver-specific Hdac3 CKO mice [31]. Overexpression of p21 in osteoblasts blocked proliferation and differentiation [42]. Thus, elevated levels of p21 may interfere with osteoblast maturation and activity in our Hdac3 CKO mice. Several other genes and signaling pathways that were affected by Hdac3 depletion have known roles in osteoblast maturation. Notably, antagonists of matrix mineralization, matrix gla protein (Mgp) and ectonucleotide pyrophosphatase (Enpp)1[43,44]were elevated. We did not observe changes in osteocalcin levels; however, osteoblast lineage cells are indeed inhibited as a result of Hdac3 depletion as evidenced by reduced bone formation rates and the downregulation of genes (e.g. MEPE) expressed by mature osteoblasts. Inhibitors (Axin2, Ror2, Sfrp4) of the canonical Wnt signaling pathways were also increased (Figure 9B). Suppression of Wnt10b signaling may decrease the osteogenic potential and increase adipogenesis of multipotent mesenchymal cells [45]. Additional experiments beyond the scope of this study are needed to determine how canonical Wnt signaling is affected by Hdac3- depletion. Conditional Hdac3 deletion disrupts metabolic pathways and activates Ppar nuclear receptors. For example, elimination of Hdac3 in postnatal liver cells increased the expression of Ppar- gamma 2 target genes and caused lipid accumulation, hepatocyte hypertrophy and hepatomegaly [31]. Conditional deletion of Hdac3 in cardiomyocytes elevated expression of Ppar-alpha target genes and increased fatty-acid-induced myocardial lipid accumu- lation and cardiac hypertrophy [12]. Another mouse model, which globally disrupted the interaction between Hdac3 and Ncor1 (an activating component of Hdac3 protein complexes), revealed that this protein pair contributes to circadian and metabolic physiology [46]. The data reported within show that Hdac3-depletion in osterix-positive progenitor cells impairs the activities of osteoblasts derived from these multipotent cells and promotes adipocyte differentiation. Osterix was previously described as a product of preosteoblasts and immature chondrocytes [3,32]. Our data suggest that some of these cells retain the potential to differentiate into the adipocyte lineage in vivo. The absence of Hdac3 may promote the activity of Ppar-gamma 2, which is an essential transcription factor for adipogenesis and binds to Hdac3- containing co-repressor complexes [47]. Several Ppar-gamma 2- regulated genes (e,g., Angiopoietin-like 4, Mmp2 [48,49]) were increased in CKO mice (Table 1). Elevated serum levels of adiponectin, another Ppar-gamma 2-activated gene, in Hdac3 CKO mice are also consistent with increased Ppar-gamma2 activity (data not shown). Together, the existing mouse models agree that Hdac3 is a crucial regulator of lipid accumulation in multiple tissues. Multiple Hdacs, including Hdac3, regulate osteoblast maturation in vitro [24,26,27,29,50]. RNAi-mediated Hdac3 suppression in an immortalized and lineage-committed calvarial cell line, MC3T3- E1, promoted calcium deposition in the extracellular matrix, and increased the expression of osteoblast maturation genes [24]. Broad- acting Hdac inhibitors also promoted osteogenic maturation of several cell lines, primary calvarial cells and bone marrow-derived mesenchymal progenitor cells [28,50,51,52,53]. Thus, the results of this study are inconsistent with several in vitro studies. An explanation for this discrepancy is that Cre-mediated recombina- tion of Hdac3 in progenitor cells is a permanent event. By comparison, small molecule Hdac inhibitors have short half-lives in serum and might only temporally change chromatin structure and gene expression in relatively short-term cell culture assays. Moreover, non-proliferating cells and cells with intact cell cycle checkpoints are resistant to the toxic effects of Hdac inhibitors [11,54]. However, this population of cells may still be susceptible to the sustained effects of Hdac3 deletion in vivo. Thus, the fact that the decreased osteoblast viability we observed with the in vivo knockout of Hdac3 was not seen after siRNA suppression of Hdac3 in vitro may be due to differences in the composition and/or maturity of the targeted cell populations as well as to the methods used to reduce Hdac3 expression or function. In conclusion, Hdac3 is essential for bone formation. Condi- tional deletion of Hdac3 with osterix-driven Cre recombinase drastically reduces trabecular and cortical bone formation rates, bone volume and trabecular number while increasing bone marrow adipogenesis. Preliminary experiments show that condi- tional Hdac3 deletion with other osteoblast-specific Cre drivers (i.e. Col1a1 and osteocalcin) also causes bone loss. These results are PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11492