This study investigated the role of transcription factor AP-2alpha in chondrocyte differentiation in vitro. AP-2alpha mRNA declined during differentiation, and overexpression of AP-2alpha inhibited differentiation. The results demonstrated that AP-2alpha plays a negative role in chondrocyte differentiation.
Introduction: Transcription factor AP-2alpha has been detected in growth plate and articular chondrocytes and has been shown to regulate cartilage matrix gene expression in vitro. However, the precise functional role of AP-2alpha in chondrocyte differentiation is not known. In this study, we assessed the expression and the function of AP-2alpha in chondrocyte differentiation of ATDC5 cells.
Materials and methods: Chondrocyte differentiation of ATDC5 cells was induced with insulin or transforming growth factor beta (TGF-beta). Proteoglycan production was assessed by alcian blue staining, and expression levels of chondrocyte marker genes and AP-2 gene family were determined by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). Overexpression of AP-2alpha in ATDC5 cells was accomplished by retroviral infection. Infected cells were selected for G418 resistance and pooled for further analysis.
Results and conclusions: Quantitative real time RT-PCR analysis showed that among the four members of the AP-2 gene family, AP-2alpha mRNA was the most abundant. AP-2alpha mRNA levels progressively declined during the differentiation induced by either insulin or TGF-beta treatment. Retroviral expression of AP-2alpha in ATDC5 cells prevented the formation of cartilage nodules, suppressed the proteoglycan production, and inhibited the expression of type II collagen, aggrecan, and type X collagen. Expression profile analysis of key transcription factors involved in chondrogenesis showed that overexpression of AP-2alpha maintained the expression of Sox9 but suppressed the expression of SoxS and Sox6. Taken together, we provide, for the first time, molecular and cellular evidence suggesting that AP-2alpha is a negative regulator of chondrocyte differentiation.