Involvements of runt-related transcription factor 2 with cell-cycle machinery in osteoblasts and skeletal development: a review of the literature

Introduction Runt-related transcription factor (Runx) 2 acts as a master regulator of osteoblast differentiation and bone development. Runx2-deficient mice die of respiratory failure soon after birth and display a complete lack of bone tissue and differentiated osteoblasts. Heterozygous mice have the characteristics of the human heritable skeletal disorder, known as cleidocranial dysplasia, an autosomal-dominant heritable skeletal disease characterised by openor delayed-closure of calvarial sutures, hypoplastic or aplastic clavicles. Thus far, a number of studies have been performed to elucidate the involvement of Runx2, with factors thought to be important for skeletogenesis, osteoblast proliferation and differentiation. Among these, there have been studies which investigated Runx2 in terms of cell-cycle machinery. In this regard, cell-cycle control factors, such as cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors are particularly important. Herein, we review articles that have investigated the involvement of Runx2 with cell-cycle machinery in osteoblasts, mainly focusing on the interaction of Runx2 with cell-cycle control factors or osteoblast proliferation and its biological behaviour in the cell cycle. Conclusion It is evident that Runx2 generally acts as a negative regulator of osteoblast proliferation and interacts with several cell-cycle control factors in osteoblasts, and that its biological behaviour changes depending on the cell cycle, indicating that in any case, part of Runx2’s function is exerted via the cell-cycle machinery, as well as activation of bone tissue-specific genes, such as osteocalcin. Introduction Runt-related transcription factor (Runx) 2 is a transcription factor that belongs to the runt-domain gene family. Runx2 is a master regulator of osteoblast differentiation and bone development. Mice lacking Runx2 die of respiratory failure, soon after birth and exhibit a complete absence of bone tissue, defective hypertrophic chondrocytes and lack of differentiated osteoblasts. Heterozygous adult mice have characteristics of the human heritable skeletal disorder known as cleidocranial dysplasia (CCD), an autosomal-dominant heritable skeletal disease characterised by openor delayed-closure of calvarial sutures and hypoplastic or aplastic clavicles1,2. However, transgenic mice that overexpress Runx2 in osteoblasts using a type I collagen promoter show osteopenia with multiple fractures3. Thus, it can be considered that strict regulation of Runx2 expression may be required for normal skeletal development (i.e., while Runx2 is essential for osteoblast differentiation and bone development, its expression must be below certain level(s), depending on the developmental stage). To date, numerous studies have been performed to investigate the involvement of Runx2 with factors, which are thought to be important for skeletogenesis, osteoblast proliferation and differentiation. Among these, several studies analysed Runx2 in terms of cell-cycle machinery. From this viewpoint, cell-cycle control factors play extremely important roles. The cell cycle is controlled by many cellcycle control factors namely, cyclins, cyclin-dependent kinases (Cdks) and cyclin-dependent kinase inhibitors (CKIs). Cyclins and Cdks, positively regulate the cell cycle, activating cellcycle factors crucial for the start of the next cell-cycle phase, while CKIs negatively regulate the cell cycle4,5. In mammalian cells, the activities of cyclin D-dependent kinases (Cdk4 and Cdk6) and subsequently those of cyclin E-dependent kinase (Cdk2) are required to go through the Gap 1 (G1) phase and the subsequent synthesis (S)-phase entry4. CKIs are classified into two families, that is, the inhibitor of cyclin-dependent kinase 4 (INK4) family and CIP/KIP family. In general, the INK4 family (p16, p15, p18 and p19) inhibits only Cdk4 and Cdk6, while the CIP/KIP family (p21, p27 and p57) inhibits all the Cdks in vitro5. In this article, we review articles that have investigated the involvement of Runx2 with cell-cycle machinery in osteoblasts, mainly focusing on the interaction of Runx2 with cell-cycle control factors or osteoblast proliferation and its biological behaviour in the cell cycle. Discussion As for the interaction between Runx2 and cell-cycle control factors in osteoblasts, Thomas and co-workers were probably the first to report some interaction between Runx2 and one of the cell-cycle control factors, retinoblastoma (Rb), a potent repressor of * Corresponding author Email: togasawara-tky@umin.ac.jp Department of Oral-Maxillofacial Surgery, The University of Tokyo Hospital, Tokyo 113-8655, Japan Tis su e En gi ne er in g & M ol ec ul ar B io lo gy


Introduction
Runt-related transcription factor (Runx) 2 is a transcription factor that belongs to the runt-domain gene family.Runx2 is a master regulator of osteoblast differentiation and bone development.Mice lacking Runx2 die of respiratory failure, soon after birth and exhibit a complete absence of bone tissue, defective hypertrophic chondrocytes and lack of differentiated osteoblasts.Heterozygous adult mice have characteristics of the human heritable skeletal disorder known as cleidocranial dysplasia (CCD), an autosomal-dominant heritable skeletal disease characterised by open-or delayed-closure of calvarial sutures and hypoplastic or aplastic clavicles 1,2 .However, transgenic mice that overexpress Runx2 in osteoblasts using a type I collagen promoter show osteopenia with multiple fractures 3 .Thus, it can be considered that strict regulation of Runx2 expression may be required for normal skeletal development (i.e., while Runx2 is essential for osteoblast differentiation and bone development, its expression must be below certain level(s), depending on the developmental stage).To date, numerous studies have been performed to investigate the involvement of Runx2 with factors, which are thought to be important for skeletogenesis, osteoblast proliferation and differentiation.Among these, several studies analysed Runx2 in terms of cell-cycle machinery.From this viewpoint, cell-cycle control factors play extremely important roles.The cell cycle is controlled by many cellcycle control factors namely, cyclins, cyclin-dependent kinases (Cdks) and cyclin-dependent kinase inhibitors (CKIs).Cyclins and Cdks, positively regulate the cell cycle, activating cellcycle factors crucial for the start of the next cell-cycle phase, while CKIs negatively regulate the cell cycle 4,5 .In mammalian cells, the activities of cyclin D-dependent kinases (Cdk4 and Cdk6) and subsequently those of cyclin E-dependent kinase (Cdk2) are required to go through the Gap 1 (G 1 ) phase and the subsequent synthesis (S)-phase entry 4 .CKIs are classified into two families, that is, the inhibitor of cyclin-dependent kinase 4 (INK4) family and CIP/KIP family.In general, the INK4 family (p16, p15, p18 and p19) inhibits only Cdk4 and Cdk6, while the CIP/KIP family (p21, p27 and p57) inhibits all the Cdks in vitro 5 .In this article, we review articles that have investigated the involvement of Runx2 with cell-cycle machinery in osteoblasts, mainly focusing on the interaction of Runx2 with cell-cycle control factors or osteoblast proliferation and its biological behaviour in the cell cycle.

Discussion
As for the interaction between Runx2 and cell-cycle control factors in osteoblasts, Thomas and co-workers were probably the first to report some interaction between Runx2 and one of the cell-cycle control factors, retinoblastoma (Rb), a potent repressor of the E2F-DP transcriptional complex, which is important for the S-phase start protein 6 .They discovered the following two points: co-expression of Runx2 and phosphorylate the retinoblastoma protein (pRb) results in association of both proteins, with an osteoblast-specific promoter in vivo, and subsequent transcriptional activation and tumour-derived mutants of pRb fail to activate transcription by Runx2, which implies that Rb acts as a transcriptional co-activator for Runx2 6 .Following Thomas and co-workers, based on the fact that Rb is a key physiological substrate for Cdk6, Ogasawara et al. elucidated some involvement of Cdk6 with Runx2, and showed that Cdk6 blocked Runx2 binding to the osteocalcin promoter, as well as osteoblast differentiation 7 .
Pratap et al. proposed the concept that the function of Runx2 is not limited to activation of bone tissuespecific genes upon differentiation of cells into osteoblast lineages 8 .Using calvarial osteoblasts from wild-type and Runx2-deficient mice, they showed that Runx2 is a cellgrowth inhibitor that contributes to the control of the G 0 /G 1 transition in osteogenic cells and that the carboxy (C)-terminal region of Runx2 is required for that function 8 .The role of Runx2 in modulating cell growth may have something to do with the Runx2dependent modulation of p21 9 and p27 10 .Zaidi and co-workers demonstrated that primary Runx2-null osteoblasts had a high-growth capacity with a decrease of p21 and p19 expression, suggesting that Runx2 is necessary to induce the expression of p21 11 .On the other hand, Westendorf et al. demonstrated that the Runx2 suppresses the p21 promoter and that the C-terminus of Runx2 is required for the repression of the p21 promoter.They also suggested that direct interactions between histone deacetylase (HDAC) 6 and Runx2 might play an important role in Runx2-mediated suppression of the p21 promoter 9 .Thus, it could be considered that mechanisms regulating the modulation of p21 by Runx2 may be stagespecific, bi-functional and complex.As for p27, it was suggested that some interaction between Runx2 and p27 plays a role in controlling basal rates of proliferation in pre-osteoblasts and contributes to the growth arrest associated with osteoblastic differentiation 10 .
Runx2 levels are known to fluctuate during the cell cycle.Galindo et al. reported that Runx2 proteins are cell-cycle regulated in normal osteoblastic cells.They showed that the levels of both Runx2 messenger ribonucleic acid (mRNA) and protein were up-regulated in MC3T3-E1 cells upon induction of quiescence by serum starvation 12 .They also showed that Runx2 levels in these cells were elevated in the early G 1 phase and destabilised in the late G 1 phase and that forced expression of Runx2 inhibited cell proliferation, leading to delays of progression into the S phase 12 .By contrast, in the case of chondrocytes, unlike in the case of normal osteoblasts, Runx2 expression is shown to be suppressed during quiescence in human primary chondrocytes or mouse chondrocytic ATDC5 cells, and Runx2 levels are not regulated during the G 1 and S phases in ATDC5 cells 12 , indicating that expression patterns of Runx2 in the cell cycle of osteoblasts and chondrocytes differ.These differences may be associated with the fact that Runx2 and Runx3 are functionally redundant in chondrocyte maturation, as demonstrated by Yoshida et al., who found that chondrocyte maturation is completely absent in Runx2 and Runx3 double-deficient mice, but not in Runx2-deficient mice 13 .
Another finding regarding the modulation of Runx2 levels during the cell cycle is the proposition that Runx2 controls the responsiveness of osteoblasts to mitogenic stimulation by altering the levels of components for various G protein-coupled receptor signalling pathways 14 .
Proper regulation of Runx2 levels have been shown to be disrupted in the context of osteosarcoma.The cellcycle-dependent proteasomal degradation of Runx2 that normally occurs in osteoblastic cells is abrogated in osteosarcoma cells 15 , indicating that the unlimited growth capacity of osteosarcoma cells could be, at least in part, attributed to the lack of proper down-regulation of Runx2 protein, required in some phases of the cell cycle.Besides, because forced expression of Runx2 still suppresses growth in some osteosarcoma cell lines, as well as in normal osteoblasts, it is suggested that in some cases, stimulation of Runx2 beyond its pre-established levels in osteosarcoma cells is capable of triggering an anti-proliferative response 16 .Thus, it was proposed that regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumour growth 16 .
Runx2 is also involved in the proliferation of human mesenchymal stromal cells (hMSC)s, which can differentiate into osteoblasts.It was reported that hMSCs transfected with small interfering RNAs (siRNAs) against Runx2 had higher proliferation rates than control hMSCs transfected with a non-specific siRNA and that this increase in proliferation was accompanied by an up-regulation in cyclin A1, B1 and E1 expressions and a decrease in levels of p21.These findings suggest that Runx2 also acts as a brake on cell proliferation in MSCs as in normal osteoblasts 17 .
Runx2 serves as a pathway intermediate via phosphorylation by cell-cycle control factors in osteoblastic cells.Rajgopal and co-workers reported that Runx2 is hyperphosphorylated by CDK1/cyclin B during mitosis, and that after mitosis, vigorous changes to a hypo-phosphorylated form, which are dependent on protein phosphatases (PP regulation of target genes of Runx2 18 .Furthermore, Shen et al. showed that the cyclin D1-Cdk4 complex inhibits Runx2 via ubiquitin-proteasome pathway.They demonstrated that the cyclin D1-Cdk4 complex phosphorylates Runx2 at serine 472 and induces Runx2 degradation in an ubiquitin-proteasome-dependent manner, leading to the inhibition of osteoblastic differentiation 19 .A report from the same laboratory demonstrated that cyclin D1 and Cdk4 induce Runx2 and Runx3 phosphorylations, ubiquitination and proteasomal degradation in chondrocytes 20 ; thus, it is likely that the mechanism(s) regulating phosphorylation of Runx2 by cell-cycle factors would, to some extent, be common in osteoblasts and chondrocytes.
Incidentally, the extracellular signal-regulated kinase (ERK)mitogen activated protein kinase (MAPK) pathway can stimulate cell proliferation 21 and Xiao et al. reported that phosphorylation of Runx2 is, at least in part, mediated by MAPK signalling pathways, including ERK 22 .They conducted an in vitro study demonstrating that Runx2 is controlled by MAPKs and suggested that this pathway has a vital role in the control of osteoblast-specific gene expression 22 .
Given Xiao et al. discovery, and with the purpose of elucidating the role of MAPK skeletogenesis in vivo, Ge et al. generated transgenic mice, which express constitutively active MAPK/ERK1 (MEK-SP) or dominant-negative (MEK-DN) forms of the MAPK intermediate MEK1, by using a 647-bp mouse osteocalcin gene 2 (mOG2) promoter 23 .Crossing TgMEK-SP or MEK-DN mice, with Runx2 +/− mice, they proved that the CCD phenotype in Runx2 +/− mice was partially rescued by MEK-SP.Conversely, the severity of the CCD phenotype in Runx2 +/− mice was increased by MEK-DN 23 .These data indicate that Runx2 is an in vivo, as well as in vitro, target of the ERK-MAPK pathway.Nevertheless, growth curves of osteoblasts from wild-type, TgMEK-SP and TgMEK-DN animals were identical, while differentiation of osteoblasts from TgMEK-SP was promoted and that from TgMEK-DN was reduced relative to the wild type 23 .Taken together, it seems that the in vivo Runx2-modulating role of the ERK-MAPK pathway demonstrated by Ge and co-workers may be mainly mediated not by the modulation of the cell cycle, but by other mechanisms, such as the direct promotion of differentiation, etc.This notion is consistent with the facts that they used an osteocalcin gene promoter and that osteocalcin is expressed in differentiated osteoblasts, which almost lose their proliferation ability.Therefore, in order to investigate the effects (mainly in osteoblast proliferation or the cell cycle) of the ERK-MAPK pathway on Runx2 in vivo, transgenic mouse model(s) using other osteoblastspecific promoters, which are activated earlier than the osteocalcin promoter, would be valuable.
Glycogen synthase kinase (GSK)-3β was recently proven to be very important for CCD phenotype rescue.Kugimiya and co-workers studied genetically engineered Gsk-3β +/− , Runx2 +/− mice, which demonstrated remarkable rescue of both the fontanelle and clavicle abnormalities of Runx2 +/− mice 24 .Because GSK-3β plays a key role in determining the cyclin D1 expression level by regulating mRNA transcription and protein degradation 25 , there is a possibility that cyclin D1, which is a positive regulator of the cell cycle, plays some role in this phenomenon.
As it has been shown that the range of bone phenotypes in CCD patients is attributable to quantitative reduction in the functional activity of Runx2 and that Runx2 dosage insufficiency causes promotion of osteoblast proliferation 26 , it might be possible that modulation of the cell cycle or cell growth through Runx2 function would at least partially contribute to normal skeletal development.
The findings of these three reports together imply that the modulation of Runx2 via a cell-cycle control factor may lead to novel therapeutics to treat bone catabolic disorder, or CCD.

Conclusion
Because this review was aimed to describe the possible involvement of Runx2 with cell-cycle machinery, we mainly reviewed findings on the interaction of Runx2 with cellcycle control factors, proliferation of osteoblasts and its behaviour during the cell cycle of osteoblasts; for this reason, important points about osteoblast differentiation, apoptosis, etc., did not receive comprehensive treatment.As already described in this review, it is evident that Runx2 generally acts as a negative regulator of osteoblast proliferation and interacts with several cell-cycle control factors in osteoblasts, and that its biological behaviour changes depending on the cell cycle, indicating that, in any case, part of Runx2's function is exerted via the cell-cycle machinery, as well as activation of bone tissuespecific genes such as osteocalcin.However, many questions remain to be solved in this field, and further studies will be required.The results of these future studies will certainly contribute to advancements in bone biology, the clinical treatment of bone catabolic disorders, and bone regenerative medicine as a whole.

Abbreviations list
)1/ PP2A, help maintain the post-mitotic Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) FOR CITATION PURPOSES: Ogasawara T. Involvements of runt-related transcription factor 2 with cell-cycle machinery in osteoblasts and skeletal development: a review of the literature.Hard Tissue 2013 May 01;2(3):29.
Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) FOR CITATION PURPOSES: Ogasawara T. Involvements of runt-related transcription factor 2 with cell-cycle machinery in osteoblasts and skeletal development: a review of the literature.Hard Tissue 2013 May 01;2(3):29.