C/EBPb in Chondrocytes Figure 6. C/EBPb is involved in cartilage destruction during osteoarthritis progression. (A) Time course of joint cartilage destruction and C/EBPb expression in the medial portion of cartilage after creating an experimental osteoarthritis model that induces instability to the knee joints of 8-week-old wild-type mice. Safranin-O staining and immunohistochemical staining with an antibody to C/EBPb (a-C/EBPb) or the non-immune control IgG were performed at the indicated weeks after surgery. A sham operation was performed using the same approach, and assessed after 12 weeks. Boxed areas with solid and dotted lines in the top row indicate the regions of the other rows. Scale bars, 100 mm (top) and 400 mm (others). (B) HE and Safranin-O stainings, and immunohistochemical stainings with a-C/EBPb and a-COL10 or the non-immune IgG in the tibial cartilage of wild-type (WT) and C/EBPb+/2 littermates 8 weeks and 12 weeks after the surgery. Boxed areas in the top row indicate the regions of the other rows. Scale bars, 200 mm (top), 400 mm (others). (C) Cartilage destruction according to the OARSI grading system. Data are expressed as means (bars)6SEM (error bars) for 5 mice per genotype at 8 and 12 weeks. *P,0.05 vs. WT doi:10.1371/journal.pone.0004543.g006 ylation targets of cGKII in other types of cells [43]. In addition to the abovementioned GSK-3b, C/EBPb is also targeted by multiple protein kinases including protein kinase A, calmodulin- dependent protein kinase, Erk-1/2, ribosomal protein S6 kinase, and CDK2 [44]. The skeletal abnormalities of C/EBPb2/2 mice were much milder than those of the p572/2 mice which were perinatally lethal due to various defects analogous to Beckwith-Weidemann syndrome in children, including cleft palate and body wall dysplasia besides severe dwarfism [38,45–47]. This may be because p57 is more crucial for chondrocyte hypertrophic differentiation than C/EBPb whose function could be substituted by several upstream signals of p57. In fact, the C/EBPb deficiency did not abrogate, but partially suppressed the p57 expression in chondrocytes (Figure 4B, C), while the knockdown of p57 strongly suppressed the C/EBPb-induced hypertrophic differentiation of chondrocytes (Figure 5F). Among other cell cycle factors, mice lacking the Rb-related pocket proteins p107 and p130 show skeletal phenotype very similar to that of the p572/2 mice [48], indicating that these proteins are likely to be major downstream PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4543