C/EBPb in Chondrocytes Figure 2. Hypertrophic differentiation of chondrocytes is delayed in the C/EBPb2/2 limb cartilage. (A) HE staining of whole tibias of wild-type (WT) and C/EBPb2/2 littermate embryos (E16.5). Orange, red, blue, and green bars indicate layers of resting zone, proliferative zone, hypertrophic zone, and bone area, respectively. Scale bars, 200 mm. (B) Length of proliferative and hypertrophic zones of the two genotypes. (C) Number of BrdU-positive cells in the proximal tibia of the two genotypes. Data are expressed as means (bars)6SEM (error bars) of 5 mice per genotype. *P,0.05 vs. WT. (D) Immunostaining with an antibody to C/EBPb (a-C/EBPb), BrdU labeling, in situ hybridization of type X collagen (COL10), immunostaining with an antibody to Ihh (a-Ihh), and Alcian blue/von Kossa double stainings of the tibial cartilage in two genotypes. Color bars indicate layers as above. Scale bars, 100 mm. doi:10.1371/journal.pone.0004543.g002 element (Figure 5C). EMSA revealed the specific binding of the nuclear extract from C/EBPb-overexpressed ATDC5 cells with the oligonucleotide probe containing the identified responsive element above (Figure 5D). The mutagenesis in the C/EBP motif of the probe resulted in a failure to form the complex. Cold competition with excess amounts of an unlabeled wild-type probe, but not the mutated probe, suppressed the complex formation, confirming the specific binding to the C/EBPb motif. Specificity of C/EBPb binding was further verified by the antibody supershift. These lines of results demonstrate that C/EBPb transactivates the p57 promoter, at least in part, through direct binding to a C/EBP motif between the 2150 and 2130 bp region. The C/EBPb-p57 signal induces chondrocyte hypertrophic differentiation To know the functional interaction between C/EBPb and p57 during chondrocyte hypertrophic differentiation, we established two small interfering RNA (siRNA) constructs of p57 for the gene silencing. We initially confirmed significant decreases of p57 protein and mRNA levels by stable transfection of the two siRNAs (Figure 5E). The C/EBPb-induced hypertrophic differentiation of cultured rib chondrocytes determined by ALP staining and COL10 expression was suppressed by the p57 knockdown through the siRNA (Figure 5F), indicating the mediation of p57 in the C/ EBPb induction of hypertrophic differentiation. We confirmed that retroviral overexpression of p57 enhanced the hypertrophy markers in cultured ATDC5 cells (Figure 5G). C/EBPb is involved in cartilage destruction during osteoarthritis progression In addition to the physiological role in skeletal growth in embryos, we finally examined the contribution of C/EBPb in chondrocytes under pathological conditions. We and others have reported that endochondral ossification including chondrocyte hypertrophy is a crucial step for cartilage destruction during osteoarthritis progression [16–20]. We therefore created an experimental osteoarthritis model that induces instability to the knee joints in 8-week-old wild-type mice [17,21], and found that C/EBPb was localized at the frontline of cartilage degradation in the central and peripheral areas of the joint cartilage during osteoarthritis progression (Figure 6A). To know the functional involvement of C/EBPb under the pathological conditions, we PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4543