Japanese Dental Science Review (2013) 49, 79—87
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Review Article
Cell cycle control factors and skeletal development Toru Ogasawara * Department of Oral and Maxillofacial Surgery, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-8655, Japan Received 5 September 2012; received in revised form 5 February 2013; accepted 9 March 2013 Available online 15 April 2013
KEYWORDS Cell cycle; Transcription factor; Cdk; Cyclin; CKI
Summary In the oral and maxillofacial region, conditions such as delayed bone healing after tooth extraction, bone fracture, trauma-induced bone or cartilage defects, and tumors or birth defects are common, and it is necessary to identify the molecular mechanisms that control skeletogenesis or the differentiation of cells, in order to establish new treatment strategies for these conditions. Multiple studies have been conducted to investigate the involvement of factors that may be crucial for skeletogenesis or the differentiation of cells, including transcription factors, growth factors and cell cycle factors. Several genetically engineered mouse models of cell cycle factors have been generated in research seeking to identify cell cycle factor(s) involved in the differentiation of cells, carcinogenesis, etc. Many groups have also reported the importance of cell cycle factors in the differentiation of osteoblasts, osteoclasts, chondrocytes and other cell types. Herein, we review the phenotypes of the genetically engineered mouse models of cell cycle factors with a particular focus on the size, body weight and skeletal abnormalities of the mice, and we discuss the potential of cell cycle factors as targets of clinical applications. # 2013 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved.
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Introduction. . . . . . . . . . . . . . . Cdks . . . . . . . . . . . . . . . . . . . . 2.1. Cdk2. . . . . . . . . . . . . . . . 2.2. Cdk4. . . . . . . . . . . . . . . . 2.3. Cdk6. . . . . . . . . . . . . . . . Cyclins . . . . . . . . . . . . . . . . . . . 3.1. Cyclin A (A1 and A2). . . . 3.2. Cyclin B (B1 and B2) . . . . 3.3. Cyclin D (D1, D2 and D3) 3.4. Cyclin E (E1 and E2) . . . .
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* Correspondence address: Department of Oral and Maxillofacial Surgery, Graduate School and Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: +81 3 5800 8669; fax: +81 3 5800 6832. E-mail address:
[email protected]. 1882-7616/$ — see front matter # 2013 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdsr.2013.03.001
80 4.
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T. Ogasawara Cip/Kip family . . . . . . . . . . . . . . . . . . . 4.1. p21 (Cip1/Waf1) . . . . . . . . . . . . . 4.2. p27 (Kip1) . . . . . . . . . . . . . . . . . 4.3. p57 (Kip2) . . . . . . . . . . . . . . . . . INK4 family . . . . . . . . . . . . . . . . . . . . . 5.1. p16 (INK4a) . . . . . . . . . . . . . . . . 5.2. p15 (INK4b) . . . . . . . . . . . . . . . . 5.3. p18 (INK4c) . . . . . . . . . . . . . . . . 5.4. p19 (INK4d) . . . . . . . . . . . . . . . . Rb gene family. . . . . . . . . . . . . . . . . . . 6.1. Rb . . . . . . . . . . . . . . . . . . . . . . . 6.2. p107. . . . . . . . . . . . . . . . . . . . . . 6.3. p130. . . . . . . . . . . . . . . . . . . . . . 6.4. Double mutant of Rb and p107 . . 6.5. Double mutant of p107 and p130 Discussion and perspective. . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . .
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1. Introduction In the oral and maxillofacial region, conditions such as delayed bone healing after tooth extraction, bone fractures, tumors or birth defects and trauma-induced bone or cartilage defects are common, and it is necessary to elucidate the molecular mechanisms which control skeletogenesis and the differentiation of stem cells, osteoblasts, osteoclasts and chondrocytes to establish new treatment strategies for these conditions. Bone grafts are the current gold-standard strategy to repair irreversible skeletal damage or defects, but the use of bone grafts often entails problems with respect to the availability of bone graft material, difficulties with the donor site, and other factors. Thus, in order to establish new treatment strategies for such conditions, an important goal is to shed light on the molecular mechanisms that control skeletogenesis and the differentiation of cells. Numerous studies have been performed in vitro and in vivo to investigate the involvement of factors that are thought to be crucial for skeletogenesis or the differentiation of cells; such factors include transcription factors, growth factors and cell cycle factors. In particular, cell cycle factors are thought to significantly influence the differentiation of cells, since withdrawal from the cell cycle or a temporal arrest in the G1 phase of the cell cycle is thought to be a requirement for cell differentiation [1—3]. The proliferation of eukaryotic cells depends on their progression through the cell cycle. The cell cycle is controlled by many cell cycle control factors, namely cyclins, cyclin-dependent kinases (Cdks) and cyclin-dependent kinase inhibitors (CKIs). Cyclins and Cdks, which are positive regulators of the cell cycle, activate cell cycle factors that are essential for the start of the next cell cycle phase. In contrast, CKIs function as negative regulator of Cdks by direct binding to cyclins and Cdks [2,4]. In mammalian cells, the activities of the Cyclin Ddependent kinases Cdk4 and/or Cdk6 and those of the Cyclin E-dependent kinase Cdk2 are required to pass through the G1 phase and the subsequent S-phase entry [5].
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Retinoblastoma (Rb) protein is a member of a protein family that also includes p107 and p130. It is a key physiological substrate for Cdk4 and Cdk6, which binds and inactivates the E2F-DP transcription complexes essential for S-phase entry [6,7]. The phosphorylation of pRb by Cdk4/6 and additionally by Cdk2 reverses the growth-suppressive effects of pRb by releasing E2F-DP from inactivation and consequently promoting S-phase entry and progression. Cdk4 and Cdk6 have 71% amino acid identity and are structurally homologous. They share all three D-type cyclins, i.e., CyclinD1, CyclinD2, and CyclinD3, as their catalytic partners to phosphorylate pRb in vitro [6]. As a result, Cdk4 and Cdk6 had been proposed to function redundantly in the G1 phase of the cell cycle. In contrast to the D-type cyclins, Cyclin E is expressed periodically, binding to Cdk2 and inducing Cyclin E-dependent kinase activity to maximal levels at the G1—S transition [8,9]. Once cells enter the S phase, Cyclin E is degraded, and subsequently Cdk2 forms complexes with Cyclin A. CKIs have been classified into two families: the INK4 family and the Cip/ Kip family. Generally, the INK4 family (p16, p15, p18, and p19) inhibits only Cdk4 and Cdk6, whereas the Cip/Kip family (p21, p27, and p57) inhibits all the Cdks in vitro [2]. A schematic presentation of cell cycle regulation in the G1 phase is shown in Fig. 1. Because a temporal cell cycle arrest in the G1 phase or withdrawal from the cell cycle is regarded as a prerequisite for cell differentiation, herein we review the phenotypes of genetically engineered mouse models of the representative G1 cell cycle factors with a particular focus on the size, body weight and skeletal abnormality, and we discuss the potential of cell cycle factors as targets of clinical applications.
2. Cdks 2.1. Cdk2 At birth, homozygous Cdk2-deficient mice did not differ in any obvious way from their control littermates. They
Cell cycle factors in bone
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Figure 1 Cell cycle factors that function in the G1 phase. Cell cycle progression is controlled by the action of cyclins and cyclindependent kinases (Cdks), which phosphorylate and activate cell cycle factors that are crucial for the start of the next cell cycle phase. In contrast, cell cycle arrest is thought to be controlled chiefly by Cdk inhibitors (CKIs), which inhibit Cdks or Cdk-cyclin complexes. In general, the INK4 family (p16, p15, p18, and p19) inhibits only Cdk4 and Cdk6 by interfering with their binding to cyclins (A), whereas the Cip/Kip family (p21, p27, and p57) binds to Cdk-cyclin complexes and inhibits all of the Cdks (B). The phosphorylation of pRb by Cdk4/6 (Cdk2) reverses the growth-suppressive effects of pRb (C). A temporal arrest in the G1 phase or withdrawal from the cell cycle is thought to be a prerequisite for cell differentiation.
developed normally, and an anatomical and histological examination of the mutant mice showed no obvious malformations. However, they were slightly smaller than wild-type littermates after weaning [10]. It is possible that this smaller size of Cdk2-deficient mice might have something to do with the in vitro finding that the growth-inhibitory effect of fibroblast growth factor (FGF) on chondrocytes appeared to be mediated at least partially through the inactivation of Cyclin E-Cdk2 [11].
2.2. Cdk4 Mice lacking Cdk4 expression (Cdk4neo/neo mice) were reported to be significantly smaller than their wild-type or heterozygous littermates. Their smaller size was noticeable at birth, and the size difference became more apparent throughout postnatal development. The body weights of the adult Cdk4-deficient mice were only 50% of that of their wild-type and heterozygous littermates. An overall reduction in the size of all of the major organs of the Cdk4-deficient mice was also observed [12].
In contrast, mice expressing a mutant Cdk4 protein that cannot be down-regulated by the cell cycle inhibitor p16INK4a (Cdk4R24C/R24C mice) had body weights 5—10% higher than their wild-type and heterozygous littermates, suggesting the occurrence of limited, unscheduled cellular proliferation in at least some tissues [12]. Judging from the phenotypes of Cdk4neo/neo mice and Cdk4R24C/R24C mice, Cdk4 is thought to be a positive regulator of skeletogenesis. However, since Cdk4 bound to Cyclin D1 was shown to be a negative regulator of Runx2, which is essential for osteoblast differentiation [13], the mechanisms controlling osteoblast differentiation and skeletogenesis might be complex. Further studies are necessary to clarify this issue.
2.3. Cdk6 Mice lacking Cdk6 did not display gross anatomical abnormalities or increased mortality for up to two years of their lifespan. However, Cdk6-deficient females were slightly smaller than their wild-type littermates, and they were reported to attain 85% of the body weight of their wild-type
82 littermates at 12 weeks of age [14]. Although the reason why Cdk6-null females were small remains to be determined, their hormonal deficiency was discussed as one of the issues to be studied [14]. Cdk4 and Cdk6 had been considered to function redundantly in the G1 phase of the cell cycle, but it is probable that each protein has one or more distinct role(s) in skeletogenesis that cannot be compensated for by the other, considering the difference between Cdk6-deficient mice and Cdk4-deficient mice. This notion is supported by the fact that several in vitro studies demonstrated that Cdk6 but not Cdk4 was shown to be down-regulated in the differentiation process of osteoblasts, osteoclasts and chondrocytes [15—17]. Moreover, the results of a human genome-wide association analysis suggested that CDK6 may be associated with adult height in humans [18].
3. Cyclins 3.1. Cyclin A (A1 and A2) In mice, Cyclin A1 is expressed exclusively in the germ-cell lineage and was shown to be essential for spermatocyte passage into the first meiotic division in male mice [19]. There was no comment on the size, body weight or skeletal phenotypes of Cyclin-A1 deficient mice [19]. Cyclin A2 proved to be an essential gene because the homozygous Cyclin A2 null mutant is embryonically lethal [20].
3.2. Cyclin B (B1 and B2) Cyclin B1 deletion resulted in embryonic lethality, and Cyclin B1 thus proved to be an essential gene in mice [21]. In contrast to Cyclin B1, homozygous Cyclin B2-deficient mice developed normally, and a thorough anatomical and histological examination of the mutant mice did not reveal any obvious malformations. However, in many cases, Cyclin B2deficient mice weighed less than their heterozygous littermates. It was suggested that these smaller sizes might be due to either lower fertility of the males or females or to embryonic morbidity [21]. In addition, in vitro studies showed that the hyper-phosphorylation of Runx2 during mitosis was associated with Cyclin B in osteoblastic cells [22], and increased cytoplasmic levels of Cyclin B were observed during the differentiation of osteoblasts [23]. The smaller sizes of Cyclin B2-deficient mice could be attributed to a defect in such mechanisms. Although Cyclins B1 and B2 are not G1 cell cycle factors, the mouse models have been reviewed here because Cyclin B2-deficient mice are unusually small.
3.3. Cyclin D (D1, D2 and D3) It was reported that all Cyclin D1-deficient mice were abnormally small compared to their heterozygous and wild-type littermates [24]. During subsequent growth to adulthood, Cyclin D1-deficient mice remained proportionately smaller (between 10% and 40%) than their heterozygous littermates, and they exhibited skeletal abnormalities. Approximately 50% of the Cyclin D1-deficient mice showed a malformation of the jaw (lateral distortion of the mandibles), and this led to unchecked growth of the incisor teeth because of their
T. Ogasawara misalignment. This malformation of the jaw was not seen in any wild-type or heterozygous littermates [24]. In contrast to Cyclin D1-deficient mice, Cyclin D2- or Cyclin D3-deficient mice exhibited no whole-body phenotype. Cyclin D2 / mice were reported to be indistinguishable phenotypically from their wild-type littermates [25]. Similarly, Cyclin D3-deficient mice were reported to appear normal during the 1.5 year observation period [26]. Taken together, the findings indicate that among the D-type cyclins, Cyclin D1 is apparently a major regulator of skeletogenesis, and its role in skeletogenesis cannot be compensated for by Cyclin D2 or Cyclin D3.
3.4. Cyclin E (E1 and E2) Cyclin E1 / mice were reported to be indistinguishable from their wild-type littermates [27]. In-depth histopathological analyses showed normal morphogenesis in all tissues examined. Likewise, mice lacking Cyclin E2 were initially indistinguishable from control littermates. As for Cyclin E1 / ; E2 / mice (double-mutant mice), no double-mutant mice were born alive. Approximately 50% of the double-mutant mice were alive at embryonic day 10.75, and these mutant embryos appeared growth-retarded. Geng et al. found that the tetraploid complementation rescue method fully rescued the embryonal lethality, and they were able to recover viable Cyclin E1 / ; E2 / embryos at all points of development and at postnatal day 1 [27]. Although Geng et al. reported that the rescued E1 / E2 / mice at postnatal day 1 exhibited a normal appearance, the mice appeared to have shorter limbs than their wild-type littermates, judging from the wholebody photographs presented in their paper.
4. Cip/Kip family 4.1. p21 (Cip1/Waf1) It was reported that p21 / mice developed normally and their size was normal; histological sections from several organs including vertebral bones, muscle, testis, and brain were examined and were found to be normal [28]. However, several studies have shown the importance of p21 in osteoblast, osteoclast and chondrocyte differentiation [11,29— 32]. It is thus possible that the role of p21 in skeletogenesis can be compensated for by other CKIs, such as p27 or p57, which are classified in the Cip/Kip family.
4.2. p27 (Kip1) The generation and characterization of p27-deficient mice (p27 / mice) was first reported by three independent groups at the same time. Nakayama et al. [33] reported that p27 / mice were not distinguishable at birth from their wild-type and heterozygous littermates. However, by 4—6 weeks of age, it became evident that many (but not all) p27 / mice weighed more than the littermate control mice. This weight difference became more obvious with age. Although the body size of the p27 / mice was increased, the outward appearances of these mice were normal [33]. Fero et al. [34] observed that p27 / mice were significantly heavier than their control littermates, and that p27 heterozygotes were
Cell cycle factors in bone intermediate in size. The weight difference was not evident at birth, but it became considerable between 2 and 3 weeks of age and was maximal by 10 weeks of age, and it was maintained throughout adulthood. Except for their increased size, p27-deficient mice were morphologically normal. Those authors considered that an enlargement of all internal organs was one of the reasons for the increased weight of the p27 / mice [34]. Kiyokawa et al. [35] also reported that the p27 / mice weighed 20—40% more than their littermate controls after weaning. To determine whether there was a correlation between weight and growth, they examined skeletal growth and organ weight. By radiographical analysis, they observed differences in the length of the skull and longitudinal bones, including the femur, tibia, and humerus, that corresponded to the increase in the size of the mice [35]. In accordance with the phenotypes of p27 / mice, there have been reports indicating that osteogenic differentiation in vitro is associated with p27 [36,37].
4.3. p57 (Kip2) To date, at least three individual groups have generated p57deficient mice (p57 / mice), and all groups pointed out defects in the skeletogenesis of these mice [38—40]. Yan et al. [38] reported that most p57 / mice died after birth and displayed various anatomical defects. They also observed that the heterozygous mice that inherited a maternal targeted allele exhibited similar deficiencies and neonatal death. The presence of developmental defects in the heterozygous animals was thought to be a consequence of paternal imprinting in this locus. Developmental defects of p57 mutant mice included cleft palate. The cleft palate seen in p57 / and imprinted p57+/ mice had defects in both the hard and soft palates. Most p57 mutant mice had short limbs, a defect attributable to abnormal endochondral ossification caused by delayed cell cycle exit during chondrocyte differentiation. Since most of the cranial bones (which develop through intramembranous ossification) in the p57 mutant mice appeared to be normal, Yan et al. considered that this defect was specific to bones formed through endochondral ossification. In agreement with this concept, the interparietal bone that forms at the base of the skull through endochondral ossification was also significantly underdeveloped in the p57 / mice [38]. Around the same time, Zhang and co-workers also reported that mice lacking p57 had altered cell proliferation and differentiation, leading to cleft palate and endochondral bone ossification defects with incomplete differentiation of hypertrophic chondrocytes [39]. Takahashi and co-workers reported the same phenotypes of p57-deficient mice (cleft palate and defective bone formation, etc.) [40]. Though most of the p57-deficient mice died within 24 h after birth, about 10% of them survived beyond the weaning period. Those authors investigated the phenotypes of the surviving p57deficient mice and discovered that they all showed severe growth retardation [40]. Concerning molecular mechanisms, Hirata and co-workers demonstrated that C/EBPb directly transactivates p57 to promote the transition of chondrocytes from proliferation to hypertrophic differentiation during endochondral ossification [41], and other studies have also explored the molecular
83 mechanisms of p57 in skeletogenesis [42,43]. In addition to these in vivo studies, there is evidence of p57 being involved in the proliferation and differentiation of osteoblasts or chondrocytes [44—46].
5. INK4 family 5.1. p16 (INK4a) Serrano et al. reported the phenotype of mice carrying a targeted deletion of the INK4a locus which eliminated both p16INK4a and p19ARF [47]. The mice were viable and did not display gross congenital defects, but they developed spontaneous tumors at an early age and were highly responsive to oncogenic treatments. Sharpless et al. later generated and characterized p16 Ink4a-specific knockout mice that maintained normal p19ARF function; these mice exhibited normal development except for thymic hyperplasia [48]. It is thus possible that the role of p16INK4a in skeletogenesis or body size can be compensated for by other CKIs.
5.2. p15 (INK4b) Mice deficient in p15INK4b (p15INK4b / mice) were born at the expected Mendelian ratios, were fertile and did not exhibit gross morphological or behavioral abnormalities [49]. As in the case of p16INK4a, it is possible that the role of p15INK4b in skeletogenesis or body size can be compensated for by other CKIs.
5.3. p18 (INK4c) For p18INK4c, wild-type, heterozygous, and null mice appeared indistinguishable at birth. However, within 2—3 weeks, the p18INK4c / mice became distinctly larger than their wild-type littermates. By the end of 1 month, the p18INK4c / mice were 35—45% larger than their p18INK4c+/+ littermates. The body weights of the p18INK4c / mice were increased by 20%, 40%, and 30% at 1, 2, and 3 months, respectively. There was no obvious difference in the levels of p18INK4c protein between p18INK4c+/+ and p18INK4c+/ tissues, and there was no significant difference in the body and organ size between the wild-type and heterozygous mice. These results suggested that there is no gene dose-dependence for the p18INK4c protein expression [50]. Latres and co-workers [49] also reported that p18INK4c / mice were larger than their wild-type littermates. However, the mice generated in their laboratory showed only 20% weight increases at most compared to the wild-type mice. They attributed the quantitative differences seen in the two groups to the different genetic backgrounds of the mice [49]. It is true that p18INK4c was shown to be larger, but the mechanisms involved are not yet fully understood, and extensive studies are needed to elucidate the roles of p18INK4c in skeletogenesis and body size.
5.4. p19 (INK4d) In a study by Zindy et al. [51], p19INK4d-deficient mice were born at a normal Mendelian ratio, developed into adulthood,
84 had a normal life span. Except for abnormalities in testicular size and male germ-cell maturation, no other obvious developmental anomalies were observed in the p19INK4d-deficient animals. They did not spontaneously develop tumors [51]. Here again, as in the case of p16INK4a and p15INK4b, it is possible that the role of p19INK4d in skeletogenesis or body size can be compensated for by other CKIs. In conclusion, regarding the INK4 family, these findings raise the hypothesis that p18INK4c has the most important role in skeletogenesis and/or body size.
6. Rb gene family 6.1. Rb Because Rb / embryos which were generated by a conventional knockout strategy died by the 16th embryonic day, Rb was shown to be essential for normal mouse development [52]. In contrast, the Rb+/ mice were developmentally normal except for a pituitary tumor predisposition with nearly complete penetrance [52]. In an in vitro study, it was demonstrated that Rb acts as a transcriptional coactivator of Runx2, which is a master regulator of osteogenic differentiation [53]. Further studies may elucidate the role of Rb in skeletogenesis. Toward this end, conditional knockout strategies using osteoblast- or chondrocyte-specific promoters will be valuable.
6.2. p107 pl07 / and pl07+/ mice did not display any increased morbidity or mortality up to 24 months of age, and comparative gross and histological surveys of the internal organs of 4— 12-month-old pl07 / mice did not reveal any developmental or pathological abnormalities [54]. However, upon careful examination of pl07 / embryos, Cobrinik and co-workers discovered a subtle thickening of the radius, ulna, and humerus [55].
6.3. p130 It was reported that the size and appearance of homozygous p130 mutant mice were normal at birth and that these mice displayed no detected histological abnormalities at birth and at 2 months of age, and they reproduced normally [55]. Unlike the p107 / embryos, the forelimb development of pl30 / embryos appeared normal [55].
6.4. Double mutant of Rb and p107 Though Rb+/ ; pl07 / mice were not distinguishable from their littermates at birth, they exhibited severe growth retardation for several weeks, averaging 50% of the weight of their littermates [54]. The average body weight of 1-weekold Rb +/ ; pl07 / mice was about one-half of that of Rb+/ ; pl07+/ mice, which was equivalent to the other mutant genotypes and wild-type. This tendency persisted at 2 weeks of age. Approximately 25% of the Rb+/ ; pl07 / mice survived further than 3 weeks of age. Most of these surviving animals gained weight slowly to reach 70—90% of normal
T. Ogasawara weight after 3 months. Surviving mice subsequently died from pituitary tumors associated with their Rb+/ status after 12 months. Therefore, it was considered that there was no apparent additional tumor phenotype associated with this mutant combination, at least up to 1 year of age [54].
6.5. Double mutant of p107 and p130 In work by Cobrinik et al. [55], pl07+/ ; pl30+/ compound heterozygotes appeared normal. However, double homozygous pl07 / ; pl30 / mice died soon after birth. Although the neonates were born alive, they had evident breathing abnormalities and poor oxygenation that were apparent until they died at various times up to 6 h after birth. On embryonic day 18.5, the pl07 / ; pl30 / embryos were up to 30% smaller than their littermates and they had distinctive external features, including dramatically shortened limbs, moderately protruding abdomen, a shortened snout. In addition, there were obvious aberrations in bone structure and in the timing of bone deposition in the pl07 / ; pl30 / embryos. At 16 days post-coitum (d.p.c.), the pl07 / ; pl30 / embryos exhibited reduced rib cage size and significantly reduced bone deposition in each of the long bones of the limbs. In contrast to the abnormal development of the ribs and long bones, which form through the process of endochondral ossification, most of the cranial bones (which form through intramembranous ossification) of the pl07 / ; pl30 / embryos developed normally in general. Concerning the cranium, only the interparietal bone, which forms at the posterior of the skull through endochondral ossification, was markedly underdeveloped in 19 d.p.c. pl07 / ; pl30 / embryos [55]. There was a more subtle delay detected in the formation of the supraoccipital bone, which also forms by endochondral ossification. Taken together, the findings indicate that p107 and p130 are needed for endochondral but not intramembranous bone development. Regarding the pl07 / ; pl30+/ mice, although the mice were within the normal weight range at birth, they attained only 65% of the normal weight between 2 and 3 weeks of age, and they died at increased frequency during the first and second weeks. As described in Section 6.2, since pl07 / ; pl30+/+ mice displayed none of these phenotypes, p130 is thought to have limited ability to compensate for p107 loss in pl07 / ; pl30+/ mice. In contrast, p107 was able to compensate more fully for the loss of p130, because the pl07+/ ; pl30 / mice showed only a modest and temporary growth delay from which they recovered at 3 weeks of age. The finding that developmentally significant growth control by p107/pl30 is principally restricted to chondrocytes suggests that these cells may be governed by growth-regulatory programs [55].
7. Discussion and perspective The possible involvement of cell cycle factors in skeletogenesis and the phenotypes of genetically engineered mouse models of the G1 cell cycle factors were reviewed, with a particular focus on the size, weight and skeletal abnormalities of the mice (for this reason, it should be noted that several important phenotypes of each mouse model, such as
Cell cycle factors in bone Table 1
85
Sizes of the genetically engineered mouse models.
Genotype
Size
Cdk2 / Cdk4 / Cdk4 R24C/R24C Cdk6 /
Slightly small Significantly small Large Slightly small (female)
Cyclin A1 / Cyclin A2 / (embryonically lethal)
Not described Not applicable
Cyclin B1 / (embryonically lethal) Cyclin B2 /
Not applicable
Cyclin D1 Cyclin D2 Cyclin D3
/
Significantly small Normal Normal
/ /
Cyclin E1 / Cyclin E2 / Cyclin E / ; E2 / (embryonically lethal) p21 (Cip1/Waf1) p27 (Kip1) / p57 (Kip2) / p16 p15 p18 p19
(INK4a) (INK4b) (INK4c) (INK4d)
Small
/
/ / / /
Rb / (embryonically lethal) Rb+/ p107 / p130 / Rb+/ ; pl07 / pl07+/ ; pl30+/ pl07 / ; pl30 / (embryo) pl07 / ; pl30+/ pl07+/ ; pl30 /
Normal Normal Small? Normal Significantly large Small Normal Normal Large Normal Not applicable Normal Normal Normal Small Normal Small Small Normal (with modest and temporary delay)
Table 2 Skeletal abnormalities of genetically engineered mouse models. Genotype Cdk2 Cdk4 / Cdk4 R24C/R24C Cdk6 /
None None None None
Cyclin A1 / Cyclin A2 / (embryonically lethal)
Not described Not applicable
Cyclin B1 / (embryonically lethal) Cyclin B2 /
Not applicable
Cyclin D1 Cyclin D2 Cyclin D3
/
/
p21 (Cip1/Waf1) p27 (Kip1) / p57 (Kip2) / p16 p15 p18 p19
(INK4a) (INK4b) (INK4c) (INK4d)
/
/
/ /
/
pl07+/ ; pl30+/ pl07 / ; pl30 / /
; pl30+/
pl07+/ ; pl30
None None Short limbs? None None Short limbs and cleft palate None None None None
/
Rb / (embryonically lethal) Rb+/ p107 / p130 / Rb+/ ; pl07
None Malformation of the jaw None None
/
Cyclin E1 / Cyclin E2 / Cyclin E / ; E2 / (embryonically lethal)
pl07
carcinogenesis and abnormalities in tissues other than bone were not discussed herein). As described above and summarized in Tables 1 and 2, several mouse models display phenotypes in their size and weight. However, body size or weight does not always reflect bone quantity or quality. Since significant skeletal abnormalities are observed in Cyclin D1 / , p57 / , Rb +/ ; pl07 / , pl07 / ; pl30+/ and pl07 / ; pl30 / animals, and it can be safely concluded that some of the G1 cell cycle factors regulate skeletogenesis in vivo, mainly via endochondral ossification. It is noteworthy that most of the skeletal abnormalities of mice seem to be closely associated with the proliferation of chondrocytes, as seen in the Cyclin D1 / , p57 / , Rb +/ ; pl07 / , pl07 / ; pl30+/ and pl07 / ; pl30 / animals. These findings suggested that these cell cycle factors function as critical regulators of the growth plate chondrocytes, probably by controlling the transition from proliferation to hypertrophic differentiation, leading to normal skeletal
Skeletal abnormality
/
/
Not applicable None Subtle thickening of the radius, ulna, and humerus in embryos None Subtle thickening of the radius, ulna, and humerus in embryos? None Abnormal development of the ribs and long bones in embryos Abnormal development of the ribs and long bones in embryos None
development. To this point, although several studies have investigated the underlying molecular mechanisms [41—43], the details are still largely unknown and further studies are desired. Regarding the in vitro studies, many G1 cell cycle factors have been reported to control the differentiation of osteoblasts, osteoclasts, chondrocytes and other types of cells. For example, many cell-based studies have demonstrated a certain correlation between the induction of p21 and/or p27 and differentiation [29—32,37,56]. Likewise, the potential roles of Cdks and cyclins in the differentiation of osteoblasts, osteoclasts or chondrocytes have been studied [15— 17,57,58]. Among these reports, I would like to point out that we discovered that Cdk6 is a critical regulator of the
86 differentiation of osteoblasts, osteoclasts and chondrocytes [15—17]. However, it is true that there have been reports indicating that the role of Cdk6 in osteoclast differentiation is multifaceted and diverse [57,58], and thus extensive studies are necessary to accurately clarify the role of Cdk6 in osteoclast differentiation. Nevertheless, because individual groups also reported the significance of Cdk6 in the differentiation of multiple types of cells [59—62], it is apparent that Cdk6 is one of the key regulators in the differentiation of multiple types of cells. This importance of Cdk6 in differentiation has been emphasized in several review articles [63—65]. Lastly, I would like to discuss the possibility of cell cycle factors as targets in bone or cartilage regenerative medicine. As noted above, several cell cycle factors have been reported to control differentiation both in vivo and in vitro. In addition, since cell cycle factors are thought to function downstream of various transcription factors, growth factors or cytokines, etc., if they were used in clinical applications, their effects might be specific and safe. It is thus natural to ask whether cell cycle factors might have potential as targets of regenerative medicine. To address this question, we are studying the effects of gain- and loss-of-function experiments with several G1 cell cycle factors by overexpression of their dominant negative form and using gene silencing through RNA interference or supplementation of inhibitors, etc. At the present time, it seems that some of the G1 cell cycle factors could indeed be a candidate target in bone or cartilage regenerative medicine (Ogasawara et al., unpubl. data). Our findings will be reported, and we expect that they will contribute to the greater understanding of regenerative medicine.
Conflict of interest statement There is no conflict of interest.
Acknowledgement This work was partly supported by KAKENHI (Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, No. 22659365 to T.O.).
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