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.
Endochondral ossification is regulated by coordinated chondrocyte proliferation and differentiation, and skeletal growth is achieved by chondrocyte proliferation. In the process of endochondral ossification, chondrocytes proliferate and then gradually differentiate, losing the ability to proliferate, and terminally differentiated chondrocytes die by apoptosis, leading to the replacement of the cartilage template with bone. Numerous growth factors and hormones have been implicated in the control of chondrocyte proliferation and differentiation.1 However, the molecular processes coordinating chondrocyte proliferation and differentiation remain largely unknown.
Progression of the cell cycle is controlled by the synthesis of individual cyclins, consequent cyclin-dependent kinase (Cdk) activation, and subsequent inactivation of a series of specific cyclin–Cdk complexes at specific cell stages, coordinating the sequential completion of DNA replication and cell division.2 Cyclin D1, which is a regulator of the G1 phase of the cell cycle, has been shown to be regulated by numerous extracellular growth factors and hormones, including parathyroid hormone-related peptide (PTHrP), transforming growth factor (TGF) β, Indian hedgehog and Wnt5b, in chondrocyte proliferation.3, 4, 5 Further, intracellular signaling molecules, including integrin-linked kinase, small GTPase RhoA and transcription factors c-Fos and activating transcription factor (ATF) 2, also stimulate cyclin D1 expression in cartilage.6, 7, 8, 9 Thus, multiple mitogenic stimuli converge on the induction of cyclin D1. In the growth plate, cyclin D1 is highly expressed in the proliferating layer,5 and Ccnd1-deficient mice show dwarfism with a diminished proliferating layer in the growth plate.3, 10, 11
CCND1 is also a well-established human oncogene, because the involvement of CCND1 amplification and overexpression has been demonstrated in breast cancer, lung cancer, melanoma and oral squamous cell carcinomas.2 Overexpression of Ccnd1 in the mammary gland induces mammary hyperplasia and adenocarcinoma, and overexpression of this gene in stratified squamous epithelium in the tongue and esophagus induces proliferation and dysplasia.12, 13 Overexpression of Ccnd1 in B cells does not lead to the development of lymphoid tumors, but has a strong cooperative effect with the Myc gene in B-cell neoplasia, while overexpression of Ccnd1 in the urothelium is not sufficient to induce hyperplasia.14, 15 Further, overexpression of Ccnd1 shortens the duration of the G1 phase, reduces cell size and reduces serum dependency in rodent fibroblasts.16, 17, 18
Cdk4 and Cdk6 are activated by cyclin Ds (D1, D2 and D3), and the activated Cdk4 or Cdk6 phosphorylates Rb family proteins (retinoblastoma protein: pRb, p130 and p110), resulting in the release of E2f transcription factors. This leads to the transcriptional activation of E2f-responsive genes that are essential for DNA synthesis, including cyclin E and cyclin A, which further promotes pRb phosphorylation by activating Cdk2.2 Rb-related p130 and p107 has an important role in chondrocyte proliferation, because p130−/−p107−/− double knockout mice show enhanced chondrocyte proliferation and retarded chondrocyte maturation.19, 20
We previously investigated the involvement of cell-cycle factors in chondrocyte differentiation and showed that Cdk6 but neither Cdk2 nor Cdk4 is downregulated during differentiation of the chondrogenic cell line ATDC5, and overexpression of Cdk6 blocks chondrocyte differentiation, but that Cdk6 overexpression does not affect proliferation nor cell-cycle progression.21 To investigate whether Cdk6 inhibits chondrocyte differentiation in vivo, we generated chondrocyte-specific Cdk6 transgenic mice using Col2a1 promoter. However, overexpression of Cdk6 in chondrocytes had an effect on neither differentiation nor proliferation. To investigate the possibility that Cdk6 regulates chondrocyte differentiation by activating E2f, we further generated chondrocyte-specific Ccnd1 transgenic mice and Cdk6/Ccnd1 double transgenic mice. Overexpression of Ccnd1 also had no effect on the proliferation and differentiation of chondrocytes. Although overexpression of both Cdk6 and Ccnd1 in chondrocytes increased the number of bromodeoxyuridine (BrdU)-positive cells and induced cell-cycle progression to the S phase, the transgenic mice showed dwarfism due to chondrocyte apoptosis. We show here that overexpression of both Cdk6 and Ccnd1 inhibited chondrocyte maturation and induced chondrocyte apoptosis but not proliferation.
Generation of lacZ, Cdk6 and Ccnd1 transgenic mice and Cdk6/Ccnd1 double transgenic mice
First, a lacZ transgenic line under the control of Col2a1 promoter and enhancer was established to examine the expression pattern of the transgene (Figure 1a). β-Galactosidase activity was specifically detected in chondrocytes as previously described (Figure 1b).22 F0 lacZ transgenic mice also showed similar expression (data not shown). Using the Col2a1 promoter and enhancer cassette, two Cdk6 transgenic lines (K6), a transgenic line with high transgene expression (K6H) and a transgenic line with low transgene expression (K6L), were established (Figure 1a and c). Two Ccnd1 transgenic lines (D1), a transgenic line with high transgene expression (D1H) and a transgenic line with low transgene expression (D1L), were also established (Figure 1a and c). To generate Cdk6/Ccnd1 double transgenic mice, K6H and K6L mice were mated with D1H and D1L mice. All of the single transgenic mice showed normal appearance at E18.5 (Figure 1d, data not shown), and mineralization occurred normally as indicated by the skeletal preparations (Figure 1e). However, double transgenic K6H/D1H mice showed dwarfism, with shortened limbs, domed skull and a protruding tongue with a shortened snout and mandible (Figure 1d). The thoracic cage was small and ribs and vertebrae were thin in K6H/D1H mice at E18.5 (Figures 1e and f). At E15.5, the limbs were also thin, and mineralization was apparently retarded in the endochondral bones of K6H/D1H mice compared with those of wild-type mice (Figures 1g and h). Mineralization was mildly retarded in K6L/D1H mice, while similar levels of mineralization were observed in wild-type, K6H and D1H mice (Figure 1g).
Chondrocyte maturation was retarded in K6H/D1H mice but not in K6H and D1H mice
On histological analysis at E15.5, the length of the layer of terminal hypertrophic chondrocytes, which was mineralized, was similar in the wild-type, K6H and D1H mice, but it was reduced in K6H/D1H mice (Figure 2a). Chondrocyte maturation was examined by in situ hybridization using Col2a1, Col10a1 and Spp1/osteopontin probes. In the wild-type, K6H and D1H mice, the expression of Col2a1, Col10a1 or Spp1 was detected in the epiphysis, metaphysis and diaphysis, respectively (Figure 2b). In K6H/D1H mice, however, Col10a1 was detected in the diaphysis and Spp1 expression was restricted to the mid-diaphysis (Figure 2b). These findings indicate that chondrocyte maturation was retarded in K6H/D1H mice but not in K6H and D1H mice.
Cell cycle was accelerated at the G1 to S phase in chondrocytes of K6H/D1H mice but not in K6H and D1H mice
Chondrocyte proliferation was examined by BrdU labeling. BrdU-positive cells were increased markedly in K6H/D1H mice, mildly in K6L/D1H mice, but not in all of the single transgenic mice and K6H/D1L mice (Figure 3a and b). Next, we examined the cell cycle of chondrocytes in these transgenic mice by fluorescence-activated cell sorting (FACS). The percentages of cells in the G0/G1, S and G2/M phases were similar in the wild-type, K6H and D1H mice, while the percentage of cells in the G0/G1 phase was decreased, and those in the S and G2/M phases were increased in K6H/D1H mice compared with those in the respective phases in wild-type mice (Figure 3c). These findings indicate that overexpression of both Cdk6 and Ccnd1 is required for the acceleration of cell-cycle progression of chondrocytes from the G0/G1 to S phase.
Apoptosis was increased in K6H/D1H mice and K6L/D1H mice but not in all of the single transgenic mice and K6H/D1L mice
Although BrdU-positive cells were apparently increased in K6H/D1H mice, the mice showed dwarfism and had thin cartilages (Figures 1 and 3). Thus, we examined apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Figure 4a). TUNEL-positive cells were increased greatly in K6H/D1H and mildly in K6L/D1H mice, but not in all of the single transgenic mice and K6H/D1L mice (Figure 4b). To further confirm that the chondrocytes in K6H/D1H mice died by apoptosis, we observed them by transmission electron microscopy, which is the most reliable tool for evaluation of the type of cell death. Apoptotic structural alterations, such as cytoplasmic shrinkage, chromatin condensation, nuclear disintegration and apoptotic bodies, were observed in the femurs of K6H/D1H mice (Figure 4c). As it was reported that v-cyclin-Cdk6 inactivates Bcl2 by phosphorylation and induces apoptosis,23 the phosphorylation status of Bcl2 was examined (Figure 4d). Similar levels of phosphorylation of Bcl2 were detected in the wild-type, K6H, D1H and K6H/D1H mice, indicating that overexpression of Cdk6 and Ccnd1 induced apoptosis in a manner independent of Bcl2 phosphorylation. These findings indicate that acceleration of cell-cycle progression in chondrocytes in K6H/D1H mice resulted in an increase in apoptosis, leading to dwarfism with thin cartilaginous skeletons.
High amounts of Cdk6 and cyclin D1 accelerate pRb phosphorylation and upregulate p107 in chondrocytes
To gain insight into the phenotypes of the transgenic mice, the lysates from cartilaginous skeletons of wild-type, K6H, D1H and K6H/D1H mice were used in in vitro studies. High amounts of Cdk6 and cyclin D1 proteins in the cartilaginous skeletons of transgenic mice were detected by western blot analysis (Figure 5a). The cyclin D–Cdk complex is well-known to specifically phosphorylate three pocket proteins, that is, the pRb and two related proteins, p107 and p130. The activity of the E2f family of transcription factors (E2fs) is negatively controlled by physical association of the dephosphorylated pocket proteins. Hence, exogenously expressed Cdk6 and Cyclin D1 under the Col2a1 promoter were expected to dissociate the pocket proteins from E2fs via phosphorylation, and consequently upregulate transcriptional activity of E2fs in chondrocytes.
The levels of E2f-1 and E2f-4, which are expressed in chondrocytes24 and are able to associate with pRb, p107 and p130,25 in the cartilaginous skeletons were not noticeably different among the wild-type, K6H, D1H and K6H/D1H mice (Figure 5b). On the other hand, expression of p107 was elevated in the cartilaginous skeletons of K6H, D1H and K6H/D1H mice compared with that in wild-type mice in much the same way as reported in cells of Rb- or p130-deficient mouse lines,24 although the expression levels of pRb and p130 at both the protein and mRNA levels were not changed (Figure 5b and c). Furthermore, the dephosphorylated form of p107 was predominant especially in K6H/D1H mice (Figure 5b). Importantly, enhanced phosphorylation of pRb was detected in the cartilaginous skeletons of K6H/D1H mice (Figure 5d). These results indicate that pRb phosphorylation and p107 expression were enhanced when high amounts of both Cdk6 and cyclin D1 were supplied, suggesting that pRb function was deficient, but p107 function was elevated in chondrocytes of K6H/D1H mice.
To elucidate the consequence of dysregulation of pocket proteins by overexpressed Cdk6 and Ccnd1 in the cartilaginous skeletons, the expression of established E2f target genes, Ccne1 (cyclin E), dihydrofolate reductase (dhfr), Cdk1 (Cdc2), Cdc6, Cdc25a, Ccna1 (cyclin A), Mybl2 (B-Myb) and thymidine kinase 1,25, 26 was measured. Expression of cyclin E, dhfr, Cdc25a and B-Myb was significantly downregulated, whereas that of Cdc6 was upregulated in the cartilaginous skeletons of K6H/D1H mice compared with wild-type mice (Figure 5e). The dysregulation of E2f target genes was presumably caused by phosphorylation of pRb and upregulation of dephosphorylated p107 in K6H/D1H mice.
High amounts of Cdk6 and cyclin D1 accelerate pRb phosphorylation and upregulate p107 in ATDC5 cells
We previously showed that overexpression of Cdk6 does not affect proliferation nor cell-cycle progression in a mouse chondrogenic cell line, ATDC5.21 ATDC5 cells stably expressing high amounts of exogenous Cdk6/cyclin D1 (K6/D1 cells) were used here to confirm the overdose effect by Cdk6/cyclin D1. Endogenous Cdk6 and cyclin D1 were confirmed to interact in ATDC5 cells (Figure 6a). Exogenous Cdk6/cyclin D1 overexpression accelerated pRb phosphorylation, upregulated p107 and induced apoptosis in ATDC5 cells (Figures 6b and c) as observed in the cartilaginous skeletons of K6H/D1H mice (Figure 4a–c and Figure 5b–d). Further, Cdc6 was upregulated but cyclin E, dhfr and B-Myb were downregulated in K6/D1 cells in an analogous fashion to the observation in K6H/D1H mice, although downregulation of Cdc25a was not observed (Figures 5e and 6d). To investigate whether the upregulation of p107 is a cause of the reduction in the E2f target gene expressions in K6/D1 cells, small interfering RNAs (siRNAs) against p107 were transfected into K6/D1 cells (Figure 6e). All of the E2f target genes downregulated in K6/D1 cells were upregulated (Figure 6f). These findings suggest that high Cdk6/cyclin D1 activity upregulated Cdc6 but downregulated several E2f target genes through the acceleration of pRb phosphorylation and upregulation of p107 expression, respectively. Consequently, the dysregulation of E2f target genes was thought to be the basis for apoptosis in both K6/D1 cells and cartilaginous skeletons in K6H/D1H mice.
To evaluate the effect of Cdk6/cyclin D1 kinase activity in mouse chondrocytes, a kinase-negative form of Cdk627 and cyclin D1 were introduced in ATDC5 cells (KN/D1 cells). The acceleration of pRb phosphorylation, p107 upregulation, dysregulation of E2f target genes and induction of apoptosis, which were observed in K6/D1 cells, were all expunged in KN/D1 cells (Figure 6b–d), indicating that the kinase activity of Cdk6/cyclin D1 is required for the events observed in K6/D1 cells.
Deletion of p53 rescued the dwarfism partially and the apoptosis nearly completely in K6H/D1H mice
The dwarfism phenotype of K6H/D1H mice was mainly attributed to massive apoptosis observed in cartilaginous skeletons. It was assumed that the incomplete cell-cycle progression disturbed by dysregulation of E2f target genes described above was readily sensed by p53, which have a critical role in the checkpoint control of the cell cycle.28 Thus, p53-dependency of the apoptosis was confirmed using the p53−/− genetic background in mice. Growth of the skeleton of p53−/−K6H/D1H mice was partly rescued, although the individual size and thickness of cartilages in p53−/−K6H/D1H mice were still less than those in wild-type mice (Figure 7a). The frequencies of BrdU-positive cells were similar between K6H/D1H and p53−/−K6H/D1H mice (Figure 7b and c). However, the number of TUNEL-positive cells in p53−/−K6H/D1H mice was drastically reduced compared with that in K6H/D1H mice (Figure 7d and e), indicating that the chondrocyte apoptosis in K6H/D1H mice was p53-dependent. However, the chondrocyte densities were similar among the wild-type, K6H/D1H and p53−/−K6H/D1H mice (Figure 7f), indicating that chondrocyte proliferation in p53−/−K6H/D1H mice was still less than that in wild-type mice. These findings suggest that the chondrocytes in p53−/−K6H/D1H mice were accelerated to enter the S phase, but many of them failed to accomplish the cell cycle, although they were protected from p53-dependent apoptosis.
Although Cdk6 but neither Cdk2 nor Cdk4 was downregulated during chondrocyte differentiation of ATDC5 cells and overexpression of Cdk6 impaired the differentiation,21 we failed to show the inhibition of chondrocyte differentiation by Cdk6 in vivo in this study. Cdk6 may have an inhibitory effect on chondrocyte differentiation at the early stage, when mesenchymal cells acquire the chondrogenic phenotype, because overexpression of Cdk6 reduces Sox5 and Sox6 expression in ATDC5 cells.21 Overexpression of p21 and p27 has been shown to promote myogenesis, neurogenesis and hematopoiesis.29, 30, 31, 32 Further, p57KIP2-null mice show dwarfism with increased chondrocyte proliferation rate, increased chondrocyte cell density and delayed ossification, which are also seen in p130−/−p107−/− double knockout mice.19, 20, 33, 34, 35 These previous findings indicate that the cell-cycle inhibition promotes cell differentiation and the cell-cycle promotion inhibits cell differentiation. Our data support the previous findings, further indicating that enhanced cell proliferation is not absolutely required for inhibition of cell differentiation, because chondrocyte differentiation was inhibited in K6H/D1H mice, irrespective of the absence of enhanced chondrocyte proliferation.
Many in vitro and in vivo studies showed that overexpression of Ccnd1 enhances cell proliferation and cyclin D1 have an important oncogenic role in many cancers. Further, overexpression of Cdk6 induces human β-cell proliferation in vitro and enhances human islet engraftment and proliferation in vivo.36 However, overexpression of Ccnd1, Cdk6 or both Ccnd1 and Cdk6 failed to enhance chondrocyte proliferation, indicating that overexpression of cyclin D1 and Cdk6 is not sufficient to enhance chondrocyte proliferation in vivo.
In both transgenic mice and ATDC5 cells, overdose of Cdk6/cyclin D1 kinase activity enhanced the phosphorylation of pRb and increased the amount of p107. In ATDC5 cells, a kinase-negative Cdk6 and cyclin D1 wiped out all effects by Cdk6/cyclin D1, highlighting the requirement of the kinase activity for them. These findings suggest that the accelerated pRb phosphorylation by Cdk6/cyclin D1 upregulated an E2f target gene, Cdc6, meanwhile consequently increased p107 downregulated some of E2f targets, including Ccne1 and Dhfr, which presumably caused dysregulation of cell-cycle progression. Cdc6 is one of the essential and highly regulated components of prereplicative complexes, which are assembled at origins of DNA replication during G1 phase.37, 38 Therefore, upregulation of Cdc6 seemed to be, in part, responsible for the progression to S phase with the marked BrdU incorporation in K6H/D1H mice. On the other hand, Dhfr, for example, has a critical role in regulating the amount of tetrahydrofolate. Tetrahydrofolate and its derivatives are essential for the synthesis of purine and thymidylate, which are indispensable for cell proliferation.39 Thus, the imbalanced regulation of cell-cycle activators observed here, that is, enforced upregulation of Cdc6 but downregulation of E2f target genes, including Ccne1 and Dhfr, was thought to be one of the causes for the promotion of G1/S transition, but failure in the induction of cell proliferation in chondrocytes. Although upregulation of p107 was observed in K6H, D1H and K6H/D1H mice, predominance of the dephosphorylated p107 against phosphorylated p107 in K6H/D1H mice compared with those in D1H and K6H mice may explain why the downregulation of some E2f target genes mainly occurred in K6H/D1H mice. The p107 upregulation might be triggered by the pRb phosphorylation to compensate for the loss of Rb function, because p107 was reported to be induced by E2fs due to a negative feedback regulation during cell-cycle progression.40 These evidences accentuate sensitiveness of Rb under the non-physiological high activity of Cdk/cyclin, and most likely in the process of tumorigenesis in chondrocytes.
In the present study, the massive apoptosis observed in chondrocytes of K6H/D1H mice was dramatically reduced in the background of p53−/−. The function of p53 in regulating cell-cycle arrest and apoptosis is triggered by activated Arf and inhibition of Mdm2.41 In cell-cycle arrest, p21 induced by p53 inhibits cyclin E–Cdk2 activity in G1 and p53 represses cyclin B-Cdc2 expression in G2 phase.42, 43, 44 In apoptosis, Bax, Noxa and Puma regulated by p53 enhance the release of cytochrome c into the cytoplasm from the mitochondria.42 The disruption of p53 almost completely rescued the apoptosis observed in K6H/D1H mice. Therefore, as a major player in the cell-cycle checkpoint, p53 appeared to induce apoptosis in response to the infidelity of cell cycle caused by overexpression of Cdk6 and Ccnd1 in chondrocytes. Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a viral homolog of cyclin D1 called viral cyclin (v-cyclin) or K cyclin, which associates predominantly with Cdk6.45 Although introduction of both Ccnd1 and Cdk6 does not induce apoptosis in vitro, introduction of both v-cyclin and Cdk6 induces apoptosis in a manner independent of p53 and Rb, and apoptosis is observed in the cells with high Cdk6 expression in Kaposi’s sarcoma.46 Further, the inactivating phosphorylation of Bcl2 by v-cyclin-Cdk6 is one of the causes of the apoptosis.23 It has also been reported that v-cyclin expression in primary cells sensitizes the cells to apoptosis p53-dependently; DNA synthesis but not cytokinesis continues in v-cyclin-expressing cells, leading to multinucleation and polyploidy; and the cells survive and expand in the absence of p53, leading to subsequent expansion of tumorigenic clones.47, 48 In cartilage, overexpression of Ccnd1 and Cdk6 was enough to induce chondrocyte apoptosis, which was dependent on p53, but Bcl2 phosphorylation was not enhanced in K6H/D1H mice. Further, we did not observe multinucleation in the chondrocytes of K6H/D1H mice and loss of p53 inhibited apoptosis, but failed to induce cell expansion. Thus, the mechanism of apoptosis induced by cyclin D1-Cdk6 is different from that induced by v-cyclin-Cdk6.
In both sporadic and hereditary genesis of osteosarcomas, inactivation of p53 and RB has been reported to have a key role.49 Mouse models having p53-deficiency reproduced many of the defining features of human osteosarcomas, and osteosarcoma development in these mice was potentiated by loss of Rb functions.50, 51 In our mouse models, again, high doses of Cdk6 and cyclin D1 ultimately resulted in inactivation of the Rb tumor suppressive function. However, p53 deletion in addition to inactivation of Rb was not enough to enhance chondrocyte proliferation, suggesting the presence of multiple inhibitory mechanisms against sarcomagenesis of chondrocytes. This reflects the much lower incidence of chondrosarcoma compared with sarcomas arising in soft tissue.52
Materials and methods
Generation of transgenic mice
The DNA fragment covering the entire coding region of the mouse Cdk6, mouse Ccnd1 or lacZ was cloned into the Not1 site of a Col2a1-based expression vector,22 which contains the promoter and enhancer of mouse Col2a1 gene, in pNASSβ (CLONTECH, Mountain View, CA, USA). The construct inserts were injected into the pronuclei of fertilized eggs from F1 hybrid mice (C57BL/6 X C3H). Transgene integration and expression were identified by Southern blot and Northern blot analyses, respectively, using the whole-length complementary DNA of Cdk6 and Ccnd1. To generate K6/D1 double transgenic mice, K6 transgenic mice were mated with D1 transgenic mice. To generate p53−/−K6H/D1H double transgenic mice, p53+/-K6H transgenic mice were mated with p53+/−D1H transgenic mice. Before the study, all experiments were reviewed and approved by the Animal Care and Use Committee of Nagasaki University Graduate School of Biomedical Sciences.
Skeletal and histological examinations
For staining and visualization of the whole skeleton, cleared skeletons of E15.5 and 18.5 embryos were stained with Alizarin red S and Alcian blue as described previously.53 β-Galactosidase activity was detected as described previously.22, 25 For light microscopy, tissues from E15.5 embryos were fixed in 4% paraformaldehyde/0.1 M phosphate buffer. Sections (7 μm in thickness) were stained with hematoxylin and eosin. Selected samples were stained using von Kossa’s method to identify mineralized tissues.53 In situ hybridization was performed using probes for Col2a1, Col10a1 and Spp1 as described previously.54
BrdU incorporation study and TUNEL
Pregnant female mice were injected intraperitoneally with 50 μg BrdU/gram body weight 1 h before killing. Decalcified sections (3.5 μm in thickness) of the tibiae were prepared and stained with the BrdU staining kit (Zymed, South San Francisco, CA, USA). The number of BrdU-positive cells and the total number of chondrocytes in the layer of proliferating chondrocytes were counted. TUNEL was performed with the ApopTag system (Millipore, Billerica, MA, USA) using decalcified sections (3.5 μm in thickness) of the femurs at E15.5. The number of TUNEL-positive cells and the total number of chondrocytes in the layers of resting chondrocytes were counted.
Retroviral infectants and stable transfectants of ATDC5 cells and siRNAs
ATDC5 cells were maintained in Dulbecco's Modified Eagle medium/Ham’s F-12 supplemented with 5% fetal bovine serum and 10 μg/ml transferrin. The cells stably expressing human Cdk6/cyclin D1 or a kinase-negative form of human Cdk627/cyclin D1 were generated by retroviral infection using pDON-5 Neo vector (Takara, Otsu, Japan). Infectants were selected using 0.8 mg/ml G418 (Invitrogen, Carlsbad, CA, USA) to isolate clones (K6/D1-1/-2 and KN/D1-1/-2). Transfectants of ATDC5 cells with expressing human Cdk6 and rat cyclin D1 (K6/D1-1′ and -2′ clones) were selected using 0.8 mg/ml G418 (Invitrogen) and subsequently 0.8 mg/ml hygromycin B (WAKO, Osaka, Japan) as described previously.27
To knockdown p107 expression, K6/D1-1′ and -2′ clones were transfected with siRNAs against murine p107 (ON-TARGETplus SMARTpool no. L-042276, Dharmacon, Lafayette, CO, USA) and non-targeting siRNA (ON-TARGETplus non-targeting siRNA no. 1, Dharmacon) as a control using Lipofectamine LTX and PLUS Reagents (Invitrogen).
Flow cytometric analysis
Mouse chondrocytes were isolated from vertebrae, ribs and limbs at E15.5 according to the method described previously.55 Isolated chondrocytes were fixed with 10% neutral formalin on ice for 30 min, treated with RNase (100 μg/ml) and propidium iodide (40 μg/ml) at room temperature for 30 min. Guava Cell Cycle Reagent (Millipore) was used for the determination of sub-G1 phase of ATDC5 cells. The cells were analyzed by FACS Caliber (Beckton Dickinson, Franklin Lakes, NJ, USA). The percentage of cells in each cell cycle was determined using cell-cycle analysis software (ModFitLT, Verity Software House Inc., Topsham, ME, USA).
Western blot analysis and immunoprecipitation
Lysates prepared from mouse vertebrae, ribs and limbs at E15.5 and ATDC5 cells containing 30 μg of proteins were analyzed by western blot using anti-Cdk6 (MBL, Nagoya, Japan; #K0066-3), anti-cyclin D1 (EPITOMICS, Burlingame, CA, USA; #2261), anti-Rb (Santa Cruz, Santa Cruz, CA, USA; sc-50), anti-p107 (Santa Cruz; sc-318), anti-p130 (Santa Cruz; sc-317), anti-E2f1 (Santa Cruz; sc-139), anti-E2f4 (Santa Cruz; sc-866), anti-phosphorylated Rb (Ser780) (MBL; #555) and anti-β-actin (Sigma, St Louis, MO, USA; A5441) antibodies.
Proteins from whole-cell extracts of ATDC5 cells were immunoprecipitated with anti-Cdk6 antibody (MBL; #K0066-3) or normal mouse immunoglobulin G, followed by western blot analysis using anti-Cdk6 or anti-cyclin D1 antibodies.
Quantitative reverse transcription –PCR
Quantitative reverse transcription–PCR was performed using an RNeasy Kit (QIAGEN, Hilden, Germany), ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan; FSQ-101), THUNDERBIRD Probe qPCR Mix (Toyobo; QPS-101), LightCycler 480 (Roche, Indianapolis, IN, USA), and TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA; Mm00485586_m1 for Rb, Mm01250721_m1 for p107, Mm00487954_m1 for p130, Mm00432367_m1 for Ccne1, Mm00772472_m1 for Cdk1, Mm00515662_m1 for Dhfr, Mm03048221_m1 for Cdc6, Mm00483162_m1 for Cdc25a, Mm01289638_m1 for Ccna1, Mm00485340_m1 for Mybl2, Mm01246403_g1 for thymidine kinase 1 and Mm00607939_s1 for β-actin for normalization).
Statistical evaluation was performed by Mann–Whitney U-test. Data are presented as mean±s.e.m. P<0.05 was considered statistically significant.
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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.
The authors declare no conflict of interest.
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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) doi:10.1038/onc.2013.130
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