C/EBPb in Chondrocytes Figure 1. C/EBPb2/2 mice exhibit impaired skeletal growth and endochondral ossification. (A, B) Double stainings with Alizarin red and Alcian blue of the whole skeleton of the wild-type (WT), C/EBPb+/2, and C/EBPb2/2 littermates at E16.5 (A) and at 3 d after birth (B). Scale bar, 2 mm. (C) Double stainings of the upper limbs, hands, and lumbar spines of the two genotypes. (D) Length of humerus, ulna, femur, tibia, vertebra (1st to 5th lumbar spines), and the calvarial length and width of the WT and C/EBPb2/2 littermates. (E) The percent ratio of Alizarin red-positive mineralized area to total length of the long bones of the two genotypes. Data are expressed as means (bars)6SEM (error bars) of 4 bones per genotype. *P,0.01 vs. WT. doi:10.1371/journal.pone.0004543.g001 wild-type cartilage revealed that p57 was localized predominantly in late proliferative and pre-hypertrophic chondrocytes which do not exhibit BrdU uptake (Figure 4C). The p57 expression was confirmed to be decreased in the C/EBPb2/2 cartilage. C/EBPb transactivates p57 through direct binding to a C/ EBP motif To know the mechanism underlying the induction of p57 expression by C/EBPb, we analyzed the promoter activity of p57 using human hepatoma HuH-7 cells and ATDC5 cells transfected with a luciferase reporter gene construct containing the 59-flanking sequences from 21,092 to +226 bp of the p57 promoter (Figure 5A). The transcriptional activity determined by the luciferase-reporter assay was enhanced by co-transfection with C/EBPb in both cells, indicating the transcriptional induction of p57 by C/EBPb. Deletion analysis by a series of 59-deletion constructs identified the responsive element to C/EBPb as being located between 2150 and 2130 bp region. The tandem-repeat constructs of this region were confirmed to respond to the C/ EBPb overexpression depending on the repeat number in both cells (Figure 5B). As this region contained a putative C/EBP- binding motif [15], the site-directed mutagenesis was carried out by creating two mutations in the motif. Both of the mutations caused partial but significant suppression of the promoter activity in both cells, indicating that the C/EBP motif is a responsive PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4543