1] Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [2] Department of Molecular Bone Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [3] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki, Japan.
1] Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [2] Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
Oncogene. 2014 Apr 3;33(14):1862-71. doi: 10.1038/onc.2013.130. Epub 2013 Apr 29.
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 (K6(H)/D1(H) 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 K6(H)/D1(H) 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 K6(H)/D1(H) 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.
细胞增殖和分化密切相关。然而，我们之前的研究表明，细胞周期蛋白依赖性激酶（Cdk6）的过表达会阻止软骨细胞分化，而不会影响体外细胞周期的进展。为了研究 Cdk6 是否在体内抑制软骨细胞分化，我们使用 Col2a1 启动子生成了软骨细胞特异性 Cdk6 转基因小鼠。出乎意料的是，Cdk6 转基因小鼠的软骨细胞分化和细胞周期进程与野生型小鼠相似。然后，我们生成了软骨细胞特异性 Ccnd1 转基因小鼠和 Cdk6/Ccnd1 双转基因小鼠，以研究 Cdk6 是否通过 E2f 激活抑制软骨细胞分化的可能性。BrdU 阳性软骨细胞和末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）阳性软骨细胞数量增加，只有 Cdk6/Ccnd1 双转基因小鼠（K6（H）/D1（H）小鼠）显示出软骨细胞成熟受到抑制，这些小鼠表现出矮小。Rb 蛋白高度磷酸化，但 p107 上调，E2f 靶基因的表达失调，表现为 Cdc6 上调，而 cyclin E、二氢叶酸还原酶（dhfr）、Cdc25a 和 B-Myb 下调。同样，在软骨细胞系 ATDC5 中过表达 Cdk6/Ccnd1 高度磷酸化 Rb，上调 p107，诱导细胞凋亡，上调 Cdc6，下调 cyclin E、dhfr 和 B-Myb 和 p107 小干扰 RNA 逆转下调基因的表达。此外，引入激酶失活的 Cdk6 和 cyclin D1 消除了 Cdk6/cyclin D1 在 ATDC5 细胞中的所有作用，表明激酶活性对这些作用的必要性。p53 缺失部分恢复了骨骼的大小，并几乎完全挽救了 K6（H）/D1（H）小鼠中的软骨细胞凋亡，但未能增强这些小鼠中的软骨细胞增殖。这些发现表明，Cdk6/Ccnd1 的过表达通过磷酸化 Rb 抑制软骨细胞成熟并增强 G1/S 细胞周期转换，但软骨细胞未能完成细胞周期，并发生 p53 依赖性凋亡，可能是由于 E2f 靶基因的失调。我们的研究结果还表明，除了 Rb 失活之外，p53 缺失不足以加速软骨细胞增殖，这表明软骨细胞对肉瘤形成的抵抗力。
