C/EBPβ promotes transition from proliferation to hypertrophic differentiation of chondrocytes through transactivation of p57Kip2

Abstract

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 β (C/EBPß) in chondrocytes during endochondral ossification. Methodology/Principal Findings: Mouse embryos with homozygous deficiency in C/EBP-bs (C/EBPß-/-) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPß-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPß in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPß. In fact, a DNA cell cycle histogram revealed that the C/EBPß 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 p57Kip2 as the transcriptional target of C/EBPß. p57Kip2 was co-localized with C/EBPß in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPß deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPß in the p57Kip2 promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57Kip2 by the siRNA inhibited the C/EBPß-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/ EBPß+/- littermates, the C/EBPß insufficiency caused resistance to joint cartilage destruction. Conclusions/Significance: C/EBPß transactivates p57Kip2 to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/ EBPb-p57Kip2 signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis. Copyright: © 2009 Hirata et al.