title: Overexpression of Cdk6 and Ccnd1 in chondrocytes inhibited chondrocyte maturation and caused p53-dependent apoptosis without enhancing proliferation.
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 (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.
authors.affiliation: 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.
authors.forename: K
authors.initials: K
authors.lastname: Ito
authors.affiliation: 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.
authors.forename: Z
authors.initials: Z
authors.lastname: Maruyama
authors.affiliation: 1] Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [2] Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Sakai
authors.affiliation: Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Izumi
authors.affiliation: Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Moriishi
authors.affiliation: Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: C A
authors.initials: CA
authors.lastname: Yoshida
authors.affiliation: Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Miyazaki
authors.affiliation: Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Komori
authors.affiliation: Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Takada
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kawaguchi
authors.affiliation: Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Komori
mesh_terms: D000818:Animals; D017209:Apoptosis; D001973:Bromodeoxyuridine / Q000737:chemistry; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D051361:Cyclin-Dependent Kinase 6 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism; D012190:Retroviridae; D012509:Sarcoma / Q000473:pathology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: C530104:CCND1 protein, human; C530105:Ccnd1 protein, mouse; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; C495911:CDK6 protein, human; C495912:Cdk6 protein, mouse; D051361:Cyclin-Dependent Kinase 6; D001973:Bromodeoxyuridine