C/EBPb in Chondrocytes Figure 3. C/EBPb inhibits proliferation and promotes hypertrophic differentiation in cultured primary chondrocytes. (A) Time course of C/EBPb mRNA level determined by real-time RT-PCR analysis during differentiation of primary chondrocytes and ATDC5 cells cultured for 3 weeks with insulin. (B) Growth curves by the XTT assay of primary chondrocytes derived from ribs of wild-type (WT) and C/EBPb2/2 littermates. (C) Ratio of BrdU-positive cells to total cells after 3 d culture of primary chondrocytes derived from WT and C/EBPb2/2 ribs. (D) ALP and Alizarin red stainings, and relative mRNA levels of COL10, MMP13, and VEGF of the primary chondrocytes from the two genotypes determined by real-time RT-PCR analysis at 2 weeks of culture after confluency. (E) Growth curves of primary WT rib chondrocytes with retroviral transfection of C/EBPb (Rx-C/EBPb) or the control GFP (Rx-GFP). (F) Ratio of BrdU-positive cells to total cells after 4 d culture of primary WT rib chondrocytes with Rx-C/EBPb or Rx-GFP. (G) ALP and Alizarin red stainings, and relative mRNA levels of the chondrocyte hypertrophy markers of the rib chondrocytes with Rx-C/EBPb or Rx-GFP at 2 weeks of culture after confluency. All data are expressed as means (symbols or bars)6SEM (error bars) of 6 wells or dishes per group. *P,0.01 vs. WT or Rx-GFP. doi:10.1371/journal.pone.0004543.g003 compared the cartilage destruction between C/EBPb+/2 and the wild-type littermates that showed similar phenotypes under physiological conditions (Figure 1A) [9]. C/EBPb2/2 mice were not used in this experiment since their skeleton was originally small, the joint shape was abnormal, and the activity was low, so that mechanical stress caused by the joint instability was not assumed to be comparable to that of wild-type mice. The cartilage destruction as well as COL10 expression was suppressed in C/ EBPb+/2 mice, remaining a substantial undegraded matrix even 8 to 12 weeks after the surgery (Figure 6B). Quantification using the OARSI grading system [22] confirmed significant prevention of cartilage destruction by the C/EBPb haploinsufficiency (Figure 6C). Discussion The present study for the first time demonstrated that the transcription factor C/EBPb is essential for physiological skeletal growth and endochondral ossification by analyses of the deficient mice. This function was dependent on the promotion of transition from proliferation to hypertrophic differentiation of chondrocytes through the cell cycle control. Our further screening of cell cycle factors identified the cyclin-dependent kinase inhibitor p57 as the transcriptional target, and detected a responsive element of C/ EBPb in the promoter. We finally showed the functional mediation of p57 in the C/EBPb action, and confirmed the importance of the C/EBPb-p57 signal in the chondrocyte hypertrophy during skeletal growth and osteoarthritis progression. Growth retardation of C/EBPb2/2 mice was seen during embryogenesis only and disappeared as the animals grew up after birth under physiological conditions (Figure 1A & B). This may possibly be due to compensatory mechanisms by other C/EBP family members which are known to control cellular differentia- tion in several lineages [23–26]. Regarding the mesenchymal cell lineage, C/EBPd has been reported to show similar and compensatory actions for adipogenic and osteogenic differentia- tion [11,27–31]. Since the involvement of C/EBPd in chondro- genic differentiation from the mesenchymal precursors remains unknown, we initially examined the expression by immunohisto- chemistry in the limb cartilage (E16.5) (Figure S1A). It was expressed predominantly in late proliferative and pre-hypertrophic chondrocytes, similarly to the C/EBPb expression, and this was not altered in the C/EBPb2/2 cartilage. In addition, retroviral overexpression of C/EBPd enhanced hypertrophic differentiation determined by COL10 and MMP13 mRNA levels in cultured ATDC5 cells (Figure S1B). Furthermore, the p57 promoter activity was enhanced by the C/EBPd overexpression, although the effect was somewhat weaker than that by C/EBPb (Figure S1C). Although we could not detect the distinct regulation of C/ EBPb and C/EBPd expressions in the limb chondrocytes before and after birth, their actions on chondrocyte hypertrophy might be compensatory, especially postnatally. We are now investigating the role of C/EBPd in the skeletal growth using the knockout mice as well as the double knockout mice of C/EBPb and C/EBPd. The runt family transcription factor member Runx2 [1,32,33], parathyroid hormone/parathyroid hormone-related protein (PTH/ PTHrP) [1,34], and cyclic GMP-dependent protein kinase II (cGKII) [35,36] are known as representative regulators of chondrocyte hypertrophy, and interestingly, C/EBPb has been reported to be associated with these representative regulators. C/EBPb acts as a co- activator of Runx2 [6,37]. Generally, the complex of the members of the C/EBP and Runx families is known to interact in the activation of lineage-specific promoters during differentiation of osteoblasts, adipocytes, and granulocytes [6]. Unlike Runx22/2 mice that exhibit a complete lack of bone [32], C/EBPb2/2 mice showed almost normal bone, raising the possibility of functional redundancy with other isoforms such as C/EBPa or C/EBPd in osteoblast differentiation. Contrarily, both Runx22/2 and C/EBPb2/2 mice showed impairment of chondrocyte hypertrophy during cartilage development and growth [32,33], implicating a specific interaction between C/EBPb and Runx2 in cartilage. In the present study, the site-directed mutagenesis in the C/EBP motif of the p57 promoter caused significant but incomplete suppression of the promoter activity induced by the C/EBPb overexpression (Figure 5C). Actually, there is a putative Runx motif which lies close to this C/EBP motif in this region. C/EBPb might therefore stimulate the promoter activity at the Runx motif as a co-activator of Runx2, even after the innate binding was blocked, although our luciferase assay and EMSA so far have failed to find evidence of this. Contrarily to Runx2, PTH/PTHrP keeps chondrocytes prolif- erating and inhibits their hypertrophic differentiation [1,34]. The PTH/PTHrP action via the adenyl cyclase signal in chondrocytes is reported to be dependent on the suppression of p57 expression [38], implicating a possible mediation of C/EBPb in this pathway. However, our present study showed that neither PTH nor the adenyl cyclase activator forskolin affected the C/EBPb protein level in cultured ATDC5 cells or the activity of the p57 promoter (2150 to +226 bp) with or without induction by C/EBPb (Figure S2). Although C/EBPb is therefore unlikely to mediate the p57 suppression by PTH/PTHrP directly, its possible involvement as a co-activator of Runx2 again cannot be denied here, since the PTH/PTHrP action is also at least partly dependent on the Runx2 suppression in chondrocytes [39]. cGKII is a serine/threonine kinase lying downstream of the C- type natriuretic peptide pathway which is essential for skeletal growth [40]. We and others have reported that the deficiency of cGKII in mice and rats caused dwarfism due to impaired hypertrophic differentiation of chondrocytes [35,41], similarly to the present C/EBPb2/2 mice. Interestingly, a previous study showed that cGKII activated C/EBPb through phosphorylation of glycogen synthase kinase-3b (GSK-3b) in osteosarcoma cells [42], and our recent study showed that cGKII induced chondrocyte hypertrophic differentiation through the GSK-3b phosphorylation [36]. These suggest a possible mediation of the present C/EBPb- PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4543