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

Background: CCAAT/enhancer-binding protein (C/EBP) promotes hypertrophic differentiation of chondrocytes. Results: C/EBP directly represses the expression of type II collagen by interacting with its intronic enhancer. Conclusion: C/EBP biphasically functions to repress genes characteristic of proliferative chondrocytes while stimulating genes expressed by hypertrophic chondrocytes. Significance: C/EBP is a key regulator to trigger phenotypic conversion from proliferative to hypertrophic chondrocytes. CCAAT/enhancer-binding protein (C/EBP) is a transcription factor that promotes hypertrophic differentiation by stimulating type X collagen and matrix metalloproteinase 13 during chondrocyte differentiation. However, the effect of C/EBP on proliferative chondrocytes is unclear. Here, we investigated whether C/EBP represses type II collagen (COL2A1) expression and is involved in the regulation of sex-determining region Y-type high mobility group box 9 (SOX9), a crucial factor for transactivation of Col2a1. Endogenous expression of C/EBP in the embryonic growth plate and differentiated ATDC5 cells were opposite to those of COL2A1 and SOX9. Overexpression of C/EBP by adenovirus vector in ATDC5 cells caused marked repression of Col2a1. The expression of Sox9 mRNA and nuclear protein was also repressed, resulting in decreased binding of SOX9 to the Col2a1 enhancer as shown by a ChIP assay. Knockdown of C/EBP by lentivirus expressing shRNA caused significant stimulation of these genes in ATDC5 cells. Reporter assays demonstrated that C/EBP repressed transcriptional activity of Col2a1. Deletion and mutation analysis showed that the C/EBP core responsive element was located between +2144 and +2152 bp within the Col2a1 enhancer. EMSA and ChIP assays also revealed that C/EBP directly bound to this region. Ex vivo organ cultures of mouse limbs transfected with C/EBP showed that the expression of COL2A1 and SOX9 was reduced upon ectopic C/EBP expression. Together, these results indicated that C/EBP represses the transcriptional activity of Col2a1 both directly and indirectly through modulation of Sox9 expression. This consequently promotes the phenotypic conversion from proliferative to hypertrophic chondrocytes during chondrocyte differentiation.