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Overexpression of Cdk6 and Ccnd1 in chondrocytes inhibited chondrocyte maturation and caused p53-dependent apoptosis without enhancing proliferation

Oncogene volume 33, pages 1862–1871 (2014)Cite this article

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Abstract

Cell proliferation and differentiation are closely coupled. However, we previously showed that overexpression of cyclin-dependent kinase (Cdk6) blocks chondrocyte differentiation without affecting cell-cycle progression in vitro. To investigate whether Cdk6 inhibits chondrocyte differentiation in vivo, we generated chondrocyte-specific Cdk6 transgenic mice using Col2a1 promoter. Unexpectedly, differentiation and cell-cycle progression of chondrocytes in the Cdk6 transgenic mice were similar to those in wild-type mice. Then, we generated chondrocyte-specific Ccnd1 transgenic mice and Cdk6/Ccnd1 double transgenic mice to investigate the possibility that Cdk6 inhibits chondrocyte differentiation through E2f activation. Bromodeoxyuridine (BrdU)-positive chondrocytes and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive chondrocytes were increased in number, and chondrocyte maturation was inhibited only in Cdk6/Ccnd1 transgenic mice (K6H/D1H mice), which showed dwarfism. Retinoblastoma protein (pRb) was highly phosphorylated but p107 was upregulated, and the expression of E2f target genes was dysregulated as shown by upregulation of Cdc6 but downregulation of cyclin E, dihydrofolate reductase (dhfr), Cdc25a and B-Myb in chondrocytes of K6H/D1H mice. Similarly, overexpression of Cdk6/Ccnd1 in a chondrogenic cell line ATDC5 highly phosphorylated pRb, upregulated p107, induced apoptosis, upregulated Cdc6 and downregulated cyclin E, dhfr and B-Myb and p107 small interfering RNA reversed the expression of downregulated genes. Further, introduction of kinase-negative Cdk6 and cyclin D1 abolished all effects by Cdk6/cyclin D1 in ATDC5 cells, indicating the requirement of the kinase activity on these effects. p53 deletion partially restored the size of the skeleton and almost completely rescued chondrocyte apoptosis, but failed to enhance chondrocyte proliferation in K6H/D1H mice. These findings indicated that Cdk6/Ccnd1 overexpression inhibited chondrocyte maturation and enhanced G1/S cell-cycle transition by phosphorylating pRb, but the chondrocytes failed to accomplish the cell cycle, and underwent p53-dependent apoptosis probably due to the dysregulation of E2f target genes. Our findings also indicated that p53 deletion in addition to the inactivation of Rb was not sufficient to accelerate chondrocyte proliferation, suggesting the resistance of chondrocytes to sarcomagenesis.

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Acknowledgements

We thank H Okayama and T Ogasawara for providing DNA plasmids for gene expression, M Hirakawa, M Mori and Y Matsuo for technical assistance, and C Fukuda for secretarial assistance. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the President’s Discretionary Fund of Nagasaki University.

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Authors and Affiliations

  1. Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

    K Ito, Z Maruyama, A Sakai, S Izumi, T Moriishi, C A Yoshida, T Miyazaki, H Komori & T Komori

  2. Department of Molecular Bone Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

    K Ito

  3. Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki, Japan

    K Ito

  4. Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan

    Z Maruyama & H Kawaguchi

  5. Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan

    A Sakai & K Takada

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Ito, K., Maruyama, Z., Sakai, A. et al. Overexpression of Cdk6 and Ccnd1 in chondrocytes inhibited chondrocyte maturation and caused p53-dependent apoptosis without enhancing proliferation. Oncogene 33, 1862–1871 (2014). https://doi.org/10.1038/onc.2013.130

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