targets of the cyclin-CDK complexes that are inhibited by p57 in chondrocytes. More interestingly, the Cip/Kip family proteins have recently been reported to regulate pathways distinct from that of cell cycle control [14]. Since p57 supports skeletal myoblast differentiation by inhibiting phosphorylation of the key transcrip- tion factor MyoD [49], this factor might also induce chondrocyte hypertrophic differentiation by regulating crucial transcription factors like Runx2 or Sox9. This may also explain the direct linkage from the cell cycle arrest to cell differentiation by the C/ EBPb-p57 signaling, unlike the cGKII signal, as mentioned above. We conclude that C/EBPb directly transactivates p57 to promote transition from proliferation to hypertrophic differenti- ation of chondrocytes during endochondral ossification. Besides the anabolic function for physiological skeletal growth, the C/ EBPb haploinsufficiency in adult mice caused resistance to cartilage destruction during osteoarthritis progression in knee joints (Figure 6). Furthermore, C/EBPb has been reported to be induced by proinflammatory cytokines interleukin-1 and tumor necrosis factor-a, and to mediate the decrease of articular cartilage matrix by suppressing the promoter activity of cartilage charac- teristic genes like cartilage-derived retinoic acid-sensitive protein and type II collagen [50–52]. The cytokine-induced C/EBPb also enhances the promoter activity of prostaglandin synthetic enzymes like cyclooxygenase-2 and phospholipase A2 [53,54] and protein- ases like aggrecanase-1 and matrix metalloproteinase-1 [55,56] in chondrocytes. These lines of evidence indicate that the C/EBPb- p57 signal could be a therapeutic target of inflammatory and degenerative joint disorders as well as skeletal growth retardation. Materials and Methods Ethics statement All experiments were performed according to the protocol approved by the Animal Care and Use Committee of the University of Tokyo. Animals C/EBPb deficient mice, kindly provided by Dr. Shizuo Akira (University of Osaka), were maintained in a C57BL/6 back- ground. In each experiment, we compared C/EBPb2/2 or C/ EBPb+/2 mice with the wild-type littermates. Histological analysis The whole skeletons of WT and C/EBPb2/2 littermate embryos (E16.5) were fixed in 99.5% ethanol, transferred to acetone, and stained in a solution containing Alizarin red S and Alcian blue 8GX (Sigma). For histological analysis, tibial limbs were fixed in 4% paraformaldehyde (PFA) buffered with PBS and sectioned in 5-mm slices. Hematoxylin-eosin (HE) stainings were performed according to standard protocols. Alcian blue/von Kossa double stainings were performed with 1% Alcian blue 8GX in 3% acetate and with 5% silver nitrate. For immunohistochemistry, the sections were incubated with antibodies to C/EBPb (C-19), p57 (C- 20), Ihh (C-15), and C/EBPd (M-17) (Santa Cruz Biotechnology Inc.) diluted 1:500 in blocking reagent. The localization of C/EBPb was detected with HRP-conjugated secondary antibody (Promega). For fluorescent visualization, a secondary antibody conjugated with Alexa Fluor 488 (Invitrogen) was used. The p57 detection was performed using a CSA II, Biotin-Free Catalyzed Amplification System (DAKO). In situ hybridization was performed, as we reported previously [21]. Briefly, hybridization with complementary digoxigenin (DIG)-labeled for mouse type X collagen was performed in a humidified chamber for 16 h at 52uC. For the detection of DIG-labeled probes, slides were incubated with HRP- C/EBPb in Chondrocytes conjugated anti-DIG rabbit polyclonal antibody (Dakopatts). The sections were immersed in a diaminobenzidine solution to visualize immunoreactivity. For BrdU labeling, we injected BrdU (Sigma) intraperitoneally to pregnant mice prior to sacrifice, and the sections were stained using a BrdU Immunohistochemistry System (Calbio- chem) and Alexa Fluor 568 (Molecular Probes). Cell cultures Primary chondrocytes were isolated from the ribs of mouse embryos as previously described [57]. The primary chondrocytes, HuH-7 cells and C3H10T1/2 cells were cultured in DMEM with 10% FBS. ATDC5 cells were maintained in DMEM/F12 with 5% FBS. To induce hypertrophic differentiation, the ATDC5 cells were cultured for 3 weeks with insulin. Plasmids and viral vectors C/EBPb and p57 cDNA were cloned into pMx vectors, and retroviral vectors were generated using plat-E cells [58]. The siRNA sequence was designed for the mouse p57 gene (NM_009876.3: nucleotides 925–946 and 307–328) and GFP as previously described [59] and ligated into piGENEmU6 vector (iGENE Therapeutics). The siRNA sequence combined with the promoter was then inserted into a retroviral pMx vector. The adenovirus C/ EBPb and LacZ expression vector were synthesized using an Adeno-X expression system (Clontech). Two weeks after transfec- tion, the cells were harvested and used for subsequent assays. cDNA of cGKII and GSK-3b was ligated into pCMV-HA (Invitrogen). Cell proliferation assay Primary chondrocytes were inoculated at 103 cells per well in a 96-well plate. The proliferation of cells was examined, using an XTT Assay Kit (Roche) at the indicated time point. The absorbance of the product was quantified using a MTP-300 microplate reader (Corona Electric). For BrdU detection analysis, we labeled the chondrocytes with 10 mM BrdU (Sigma) for 18 h and the cells were stained using a BrdU Immunohistochemistry System (Calbiochem). Chondrocyte differentiation assay Primary chondrocytes were cultured for two weeks after confluency, and the total RNA was extracted to assess the COL10, MMP13, and VEGF mRNA levels. For the ALP staining, cells were stained with a solution containing 0.01% Naphthol AS-MX phosphate disodium salt (Sigma), 1% N, N-dimethyl-formamide (Wako), and 0.06% fast blue BB (Sigma). For the Alizarin red S staining, cells were stained with 2% Alizarin red S solution (Sigma). Flow cytometric analysis C3H10T1/2 cells with retroviral transfection with C/EBPb or GFP were incubated for 18 h in the presence of 0.2 mM nocodazole for synchronization at the G2/M phase. Then, cells were suspended in citrate buffer and stained with propidium iodide. DNA content was analyzed with EPICS XL and XL EXPO32 instruments (Beckman). Real-time RT-PCR The total RNA was extracted using an ISOGEN Kit (Wako) and an RNeasy Mini Kit (QIAGEN). One mg of RNA was reverse- transcribed with a Takara RNA PCR Kit (AMV) ver.2.1 (Takara) to generate single-stranded cDNA. PCR was performed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems). All reactions were run in triplicate. Primer sequence information is available upon request. PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4543