Overexpression of Cdk6 and Ccnd1 in chondrocytes inhibited chondrocyte maturation and caused p53-dependent apoptosis without enhancing proliferation
Overexpression of Cdk6 and Ccnd1 in chondrocytes inhibited chondrocyte maturation and caused p53-dependent apoptosis without enhancing proliferation
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DOI：
10.1038/onc.2013.130
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发表时间：
2014-04-03
期刊：
影响因子：
8
通讯作者：
Komori, T.
中科院分区：
文献类型：
--
作者：
Ito, K.;Maruyama, Z.;Komori, T.
细胞增殖和分化是密切相关的。然而，我们以前表明，细胞周期蛋白依赖性激酶（Cdk 6）的过度表达在体外阻断软骨细胞分化，而不影响细胞周期进程。为了研究Cdk 6是否抑制体内软骨细胞分化，我们使用Col 2a 1启动子产生软骨细胞特异性Cdk 6转基因小鼠。出乎意料的是，Cdk 6转基因小鼠中软骨细胞的分化和细胞周期进展与野生型小鼠中的相似。然后，我们建立了软骨细胞特异性Ccnd 1转基因小鼠和Cdk 6/Ccnd 1双转基因小鼠，以研究Cdk 6通过E2 f激活抑制软骨细胞分化的可能性。溴脱氧尿苷（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）阳性软骨细胞的数量增加，软骨细胞成熟仅在Cdk 6/Ccnd 1转基因小鼠（K6 H/D1 H小鼠）中受到抑制，表现出侏儒症。K6 H/D1 H小鼠软骨细胞中视网膜母细胞瘤蛋白（pRb）高度磷酸化，p107上调，E2 f靶基因表达失调，表现为Cdc 6上调，cyclin E、二氢叶酸还原酶（dhfr）、Cdc 25 a和B-Myb下调。类似地，在软骨形成细胞系ATDC 5中过表达Cdk 6/Ccnd 1，高度磷酸化pRb，上调p107，诱导凋亡，上调Cdc 6和下调细胞周期蛋白E，dhfr和B-Myb和p107小干扰RNA逆转下调基因的表达。此外，引入激酶阴性Cdk 6和细胞周期蛋白D1消除了ATDC 5细胞中Cdk 6/细胞周期蛋白D1的所有作用，表明这些作用需要激酶活性。在K6（H）/D1（H）小鼠中，p53缺失部分恢复了骨骼的大小，几乎完全挽救了软骨细胞的凋亡，但未能增强软骨细胞的增殖。这些结果表明，Cdk 6/Ccnd 1过表达通过磷酸化pRb抑制软骨细胞的成熟并促进G1/S细胞周期转换，但软骨细胞不能完成细胞周期，并可能由于E2 f靶基因的失调而发生p53依赖性凋亡。我们的研究结果还表明，除了Rb的失活，p53的缺失是不足以加速软骨细胞的增殖，这表明软骨细胞肉瘤的抗性。
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 wildtype 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 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.