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

Abstract

BackgroundAlthough 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/EBPβ) in chondrocytes during endochondral ossification.Methodology/Principal FindingsMouse embryos with homozygous deficiency in C/EBPβ (C/EBPβ−/−) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPβ−/− chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPβ in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPβ. In fact, a DNA cell cycle histogram revealed that the C/EBPβ 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/EBPβ. p57Kip2 was co-localized with C/EBPβ in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPβ deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPβ 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/EBPβ-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/EBPβ+/− littermates, the C/EBPβ insufficiency caused resistance to joint cartilage destruction.Conclusions/SignificanceC/EBPβ transactivates p57Kip2 to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPβ-p57Kip2 signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis.

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

“…Tissue sections were deparaffinized in xylene and rehydrated through an ethanol series. IHC was performed, as described previously (8,37), using the primary antibodies goat polyclonal anti-GADD45␤ (catalog number sc-8776, Santa Cruz Biotechnology, Inc.; 1:400 dilution, 0.5 g/ml final concentration) and rabbit polyclonal anti-C/EBP␤ (sc-150X, Santa Cruz Biostechnology, Inc.; 1:2000 dilution). Rabbit polyclonal antibody to collagen type X (1:1000 dilution) was kindly supplied by Prof. Ray Boot-Handford (Wellcome Trust Centre for Cell-Matrix Research, Manchester, UK).…”

“…The Gadd45␤ and C/ebp␤ knock-out mice display similar phenotypic changes in the embryonic growth plate, characterized by a delay in terminal chondrocyte differentiation along with decreased expression of genes linked to this process, including Col10a1 and Mmp13 (8,37,69). In addition, the embryonic limbs of C/ebp␤ knock-out mice display impaired osteogenesis (53).…”

p57Kip2 and Cancer: Time for a Critical Appraisal

“…In particular, CKI is able to stabilize MyoD, a transcription factor that is highly specific for muscle involved in myogenic differentiation. p57 Kip2 is also involved in the differentiation of several other cell phenotypes, including podocytes (79), placental cells (65,80,81), keratinocytes (82), pancreatic cells (83), hepatocytes (84,85), T-lymphocytes (86), spermatozoa (87,88), Leydig cells (88), chondrocytes (89)(90)(91), and adrenal cortex cells (92).…”

CCAAT/Enhancer-binding Protein β Regulates the Repression of Type II Collagen Expression during the Differentiation from Proliferative to Hypertrophic Chondrocytes

“…C/EBP␤ induced by pro-inflammatory cytokines such as IL-1␤ and TNF-␣ directly binds to the MMP13 and MMP3 promoter regions and stimulates their expression in chondrocytes, resulting in degradation of cartilage in arthritis (21,22). Furthermore, it was reported that hypertrophic differentiation of chondrocytes was delayed in C/EBP␤ knock-out mice through transactivation of cell cycle factor p57 (23). This was associated with decreased expression of Col10a1 (24) and suggested that C/EBP␤ plays an important role in promoting hypertrophic differentiation of chondrocytes.…”