Abstract
Development of skeletal cartilage is characterized with coupling growth arrest and cell differentiation. Here, to understand the cyclin-dependent kinase inhibitors involved in the progression of chondrogenic differentiation, we examined changes in the expression levels of cyclin-dependent kinase inhibitor members using mouse ATDC5 prechondrocytes as a widely used in vitro model of cartilage differentiation. Up-regulation of p21 and p27 mRNA was observed following a decrease in growth rate of prechondrocytes, and both transcripts subsequently accumulated during chondrogenic differentiation; p15, p18, and p19 mRNA, in contrast, did not change during differentiation. Only the up-regulation of p21 mRNA during differentiation was prevented by the continuous treatment of early chondrogenic inhibitor, parathyroid hormone, indicating a close correlation between differentiation and p21 induction in ATDC5 cells. Therefore, to examine the role of p21 during chondrogenesis, we established stable cell lines overexpressing full-length p21 antisense RNA in ATDC5. The reduction of endogenous p21 in these cell lines caused inhibition of early chondrogenic differentiation in ATDC5, indicating that p21 gene plays an important role in this process of the cells in vitro. Furthermore, the level of p21 protein and p21.CDK2 complexes transiently increased during differentiation, but not in undifferentiated cells, leading to a decrease in CDK2-associated kinase. However, differentiation-dependent expressed p21 protein was degraded by a proteasome-dependent pathway. Thus, the progression of chondrogenic differentiation requires down-regulation of CDK2-associated kinase with an increase in p21 protein and subsequent degradation of this protein by a proteasomal pathway.
Highlights
Development of skeletal cartilage is characterized with coupling growth arrest and cell differentiation
Effect of Chondrogenic Differentiation Inhibitor on cyclin-dependent kinase inhibitors (CKIs) Gene Expression—To examine whether the up-regulation of CKI genes was associated with the progress of chondrogenic differentiation, we investigated the effect of parathyroid hormone (PTH), which inhibits the progression of early chondrogenesis [24, 33], upon the up-regulation of these genes during differentiation
P21 Antisense RNA Inhibits the Differentiation of Chondrogenic Cell Line ATDC5—To clarify whether the expression of p21 is required for progression of chondrogenic differentiation in ATDC5 cells, we established a cell line expressing antisense RNA of the p21 gene
Summary
Development of skeletal cartilage is characterized with coupling growth arrest and cell differentiation. To understand the cyclin-dependent kinase inhibitors involved in the progression of chondrogenic differentiation, we examined changes in the expression levels of cyclin-dependent kinase inhibitor members using mouse ATDC5 prechondrocytes as a widely used in vitro model of cartilage differentiation. The progression of chondrogenic differentiation requires down-regulation of CDK2-associated kinase with an increase in p21 protein and subsequent degradation of this protein by a proteasomal pathway. The postmitotic chondrocytes terminally differentiate into hypertrophic chondrocytes, which produce type X collagen [19, 20] The progress of these differentiation processes requires crucial regulation by cytokines and hormones [21, 22]. Down-regulation of CDK2-associated kinase during differentiation was accompanied by an increase in p211⁄7CDK2 complexes in accordance with the amount of p21 protein, suggesting that p21 protein contributes to the inhibition of kinase activity during early chondrogenic differentiation. Chondrogenesis may be promoted by p21 protein impairment of the CDK2-associated kinase and by posttranslational modification of itself
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