present study initially investigated skeletal phenotype of C/EBPb- deficient (C/EBPb2/2) mice which have been reported to display mainly hematopoietic and adipogenic defects [9–11]. The mice showed dwarfism with an elongated proliferative zone and delayed chondrocyte hypertrophy in the limb cartilage, implicat- ing the cell cycle control by C/EBPb in chondrocytes. Cell cycle factors appear to play an important role in the control of chondrocyte proliferation and differentiation [12,13]. During the cell cycle activation, complexes of cyclin and cyclin-dependent kinase (CDK) promote G1/S-phase transition from G0/G1 by phosphorylating Rb-related pocket proteins, which activate genes required for the S-phase entry. The cyclin-CDK complexes are inhibited by two major families of CDK inhibitors [14]. The p16 INK4 family specifically binds and inactivates monomeric CDK4 or CDK6, whereas the Cip/Kip family, which includes p21Cip1, p27Kip1, and p57Kip2 (p57), inhibits all G1/S-phase cyclin-CDK complexes. Since the control of these cell cycle factors driving S- phase onset greatly influences the commitment to cell differenti- ation, the present study performed a screen of potential transcriptional targets of C/EBPb using a microarray analysis, and identified p57 as the most probable target during hypertrophic differentiation of chondrocytes. We further investigated the molecular mechanism underlying the regulation of skeletal growth and endochondral ossification through the C/EBPb-p57 signal in chondrocytes. Results C/EBPb2/2 mice exhibit impaired skeletal growth and endochondral ossification To analyze the physiological role of C/EBPb in skeletal growth and endochondral ossification, we investigated the skeletal pheno- types of heterozygous and homozygous C/EBPb-deficient (C/ EBPb+/2 and C/EBPb2/2) mice. Although the C/EBPb+/2 skeleton was normal, C/EBPb2/2 mice exhibited dwarfism as compared to the wild-type littermates from embryonic stages (Figure 1A). After birth, however, the skeletal size of C/EBPb2/2 mice gradually caught up with that of the wild type littermates (Figure 1B), and they became similar after 1 week of age. At the embryos, the limbs and vertebrae which are known to be primarily formed through endochondral ossification were about 20–25% shorter in C/EBPb2/2 mice than the wild-type, although calvarial growth, especially the width, formed through endochondral ossification and intramembranous ossification did not show such a difference (Figure 1D). Skeletal double staining revealed that not only the total bone length, but also the ratio of mineralized area shown by the positive Alizarin red staining to the total length was decreased, confirming that endochondral ossification was impaired by the C/EBPb deficiency (Figure 1C, E). Hypertrophic differentiation of chondrocytes is delayed in the C/EBPb2/2 limb cartilage To know the mechanism underlying the impaired skeletal growth in C/EBPb2/2 mice, we compared the tibial limb cartilage of the wild-type and C/EBPb2/2 littermates at E16.5 (Figure 2A). Among the resting, proliferative, hypertrophic zones, and bone area, the proliferative zone was elongated while the hypertrophic zone was normal in the C/EBPb2/2 limb (Figure 2B). The number of proliferating chondrocytes with BrdU uptake was actually increased in the C/EBPb2/2 cartilage (Figure 2C). C/EBPb was shown by immunohistochemistry to be localized predominantly in late proliferative and pre-hypertrophic chon- drocytes of the wild-type cartilage, but not in the C/EBPb2/2 cartilage (Figure 2D, top). Further histological examination by C/EBPb in Chondrocytes BrdU labeling, in situ hybridization of type X collagen (COL10), immunohistochemistry of indian hedgehog (Ihh), and Alcian blue/ von Kossa double stainings supported the elongation of the proliferative zone and delay of chondrocyte hypertrophy by the C/EBPb deficiency (Figure 2D). C/EBPb inhibits proliferation and promotes hypertrophic differentiation in cultured primary chondrocytes When primary chondrocytes derived from mouse ribs and mouse chondrogenic cell line ATDC5 were cultured in the differentiation medium, the C/EBPb mRNA level was increased with the differentiation (Figure 3A), which was comparable to the in vivo expression pattern of the limb cartilage. We then examined the effects of loss- and gain-of-functions of C/EBPb on proliferation and hypertrophic differentiation of the primary rib chondrocytes. When chondrocytes from wild-type and C/EBPb2/2 littermates were compared, cell number deter- mined by the XTT assay was enhanced in the C/EBPb2/2 chondrocytes at 3 d of culture (Figure 3B). The percentage of BrdU-positive cells was also increased in the C/EBPb2/2 culture at this time point (Figure 3C), indicating that the increased cell number was due to the enhanced proliferation, rather than the effect on cell survival, vitality, or apoptosis. Contrarily, hypertro- phic differentiation determined by alkaline phosphatase (ALP) and Alizarin red stainings, and mRNA levels of COL10, matrix metalloproteinease-13 (MMP13) and vascular endothelial growth factor (VEGF), parameters of chondrocyte hypertrophy, were suppressed by the deficiency (Figure 3D). In contrast, retroviral overexpression of C/EBPb in the wild- type rib chondrocytes suppressed the proliferation and enhanced the hypertrophic differentiation parameters (Figure 3E, F, and G). Collectively, C/EBPb was shown to be essential for cessation of proliferation and promotion of hypertrophic differentiation, suggesting arrest of the cell cycle and exit from it. C/EBPb regulates cell cycle and p57 as the transcriptional target We therefore examined the regulation of cell cycle by C/EBPb. A DNA cell cycle histogram in mouse mesenchymal C3H10T1/2 cells after the cycle synchronization revealed that the C/EBPb overexpression enhanced accumulation of cells in the G0/G1 fraction (Figure 4A). To identify cell cycle factors lying downstream of the C/EBPb signal, we performed a screen of transcriptional targets of C/EBPb using a microarray analysis (Table S1). The C/EBPb overexpression caused downregulation of cyclin B1, B2 and D1, and upregulation of the cyclin-dependent kinase inhibitors p16, p21 and p57, by 50% or more as compared to the empty vector overexpression. Since the above analyses were performed in non-chondrogenic C3H10T1/2 cells, we further examined the expressions of the candidate genes by real-time RT- PCR analysis in the cultures between wild-type and C/EBPb2/2 rib chondrocytes (Figure 4B). Cyclin B1, B2, and p21 were not significantly altered by the C/EBPb deficiency, while p16 showed contradictory upregulation. Cyclin D1 and p57 were confirmed to be upregulated and downregulated, respectively, by the loss-of- function of C/EBPb. However, when the expressions were further compared between primary chondrocytes with retroviral overex- pression of C/EBPb and the control GFP, the cyclin D1 was not downregulated, whereas p57 was upregulated by the C/EBPb overexpression. These indicate that p57 was the only cell cycle factor whose expression was confirmed to be regulated positively and negatively by the gain- and loss-of-functions of C/EBPb, respectively. Double immunofluorescence of p57 and BrdU in the PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4543