C/EBPb in Chondrocytes Figure 5. C/EBPb transactivates p57 through binding to a C/EBP motif and the C/EBPb-p57 signal induces chondrocyte hypertrophic differentiation. (A) Deletion analysis using luciferase-reporter constructs containing the 59-flanking sequences from 21,092 to +226 bp of the p57 promoter and a series of deletion fragments in HuH-7 and ATDC5 cells transfected with C/EBPb or the control GFP. (B) Dose-response analysis of the tandem repeats of the identified responsive element (2150/2130) by the luciferase-reporter assay in the HuH-7 and ATDC5 cells. (C) Site-directed mutagenesis analysis by two mutations (mt1 and mt2) in the C/EBPb motif (2147/2141) as compared to the wild-type (WT) construct by the luciferase-reporter assay in HuH-7 and ATDC5 cells. *P,0.01 vs. WT-C/EBPb. (D) EMSA for specific binding of the nuclear extract from C/EBPb- transfected ATDC5 cells with the oligonucleotide probe (2160/2120) containing the WT or mt1 C/EBPb motif. The arrowhead indicates the complex. Cold competition with 50-fold excess of unlabeled WT or mt1 probe, and supershift by an antibody to C/EBPb of the complex are also presented. (E) The protein and mRNA levels of p57 determined by immunoblotting and real-time RT-PCR, respectively, by the p57 siRNA. Stable lines of C3H10T1/2 cells retrovirally transfected with two kinds of siRNA of p57 (Rx-si-p57-1 and Rx-si-p57-2) or the control GFP siRNA (Rx-si-GFP) were established. *P,0.01 vs. Rx-si-GFP. (F) Effects of the p57 siRNA above on the C/EBPb-induced hypertrophic differentiation of chondrocytes. Primary rib chondrocytes were transfected with Rx-si-p57-1, Rx-si-p57-2, or Rx-si-GFP, and further adenovirally co-transfected with C/EBPb or the control LacZ (Ax-C/EBPb or Ax-LacZ). Hypertrophic differentiation was determined by ALP staining and relative COL10 mRNA level by real-time RT-PCR analysis at 2 weeks of culture after confluency. *P,0.01 vs. Ax-C/EBPb with Rx-si-GFP. (G) Hypertrophic differentiation of ATDC5 cells stably transfected with the retrovirus expressing p57 (Rx-p57) or the empty vector (Rx-EV) cultured for 3 weeks with insulin and further for 2 d with inorganic phosphate. *P,0.01 vs. Rx-EV. All graphs are expressed as means (bars)6SEM (error bars) for 6 wells/group. doi:10.1371/journal.pone.0004543.g005 cartilage only exhibited an elongated proliferative zone and delayed chondrocyte hypertrophy (Figure 2D). Hence, the cell cycle arrest in chondrocytes caused by C/EBPb appears to immediately link to the start of the differentiation. The discrepancy may be due to the diversity of signaling pathway(s) lying downstream of cGKII and upstream of C/EBPb. We previously reported that cGKII phosphorylated Sox9, an inhibitor of chondrocyte hypertrophy, and suppressed its nuclear entry [35]. Besides Sox9 and GSK-3b, vasodilator-stimulated phosphoprotein and cysteine- and glycine-rich protein 2 are putative phosphor- PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4543