C/EBPβ Promotes Transition from Proliferation to Hypertrophic Differentiation of Chondrocytes through Transactivation of p57Kip2

Background: Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBP beta) in chondrocytes during endochondral ossification. Methodology/Principal Findings: Mouse embryos with homozygous deficiency in C/EBP beta (C/EBP beta-/-) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBP beta-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBP beta in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBP beta. In fact, a DNA cell cycle histogram revealed that the C/EBP beta overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57(Kip2) as the transcriptional target of C/EBP beta. p57(Kip2) was co-localized with C/EBP beta in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBP beta deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBP beta in the p57(Kip2) promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57(Kip2) by the siRNA inhibited the C/EBP beta-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBP beta+/- littermates, the C/EBP beta insufficiency caused resistance to joint cartilage destruction. Conclusions/Significance: C/EBPb transactivates p57(Kip2) to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBP beta-p57(Kip2) signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis.