Transcriptional control of osteoblast differentiation and function

Chapter 7

Transcriptional control of osteoblast differentiation and function Ge´rard Karsenty Départment of Genetics and Development, Columbia University Medical Center, New York, NY, United States

Chapter outline Runx2, a master control gene of osteoblast differentiation in bony vertebrates Runx2 functions during skeletogenesis beyond osteoblast differentiation Regulation of Runx2 accumulation and function Osterix, a Runx2-dependent osteoblast-specific transcription factor required for bone formation ATF4, a transcriptional regulator of osteoblast functions and a mediator of the neural regulation of bone mass

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Additional transcriptional regulators of osteoblast differentiation and function Transcription factors acting downstream of Wnt signaling in osteoblasts: what do they actually do in differentiated osteoblasts? Regulation of osteoblast differentiation by means other than transcription factors References Further reading

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As is the case for every cell differentiation process, differentiation of a mesenchymal pluripotent cell into any cell type is governed in large part by transcription factors that trigger the entire program of cell differentiation. Our knowledge about the transcriptional control of osteoblast differentiation made its main strides at the end of the 20th century and has been significantly refined since then, with the emergence of novel mechanisms regulating gene expression in addition to transcription factors. Briefly and ideally, a transcription factor that is a differentiation factor for a given cell type should (1) be expressed in progenitors of this cell type, (2) regulate the expression of all cell-specific genes in this cell type, (3) induce expression of the aforementioned genes when ectopically expressed in other cell types (sufficiency criterion), and (4) be necessary for the differentiation of this cell type in vivo, in mice, and at best in humans. As presented in this chapter, the transcription factor currently viewed as the master gene of osteoblast differentiation is one of the very few differentiation factors to fulfill all these criteria.

Runx2, a master control gene of osteoblast differentiation in bony vertebrates The power of a combined effort between molecular biologists and human geneticists in identifying key genes regulating cell differentiation, which has been so beneficial for our understanding of skeletal biology, is best illustrated by the realization at the end of the 1990s that Runt-related transcription factor 2 (Runx2) is a master gene of osteoblast differentiation. Runx2, previously termed Pebp2a1, AmI3, or Cbfa1, was originally cloned in 1993 as one of three mammalian homologs of the Drosophila transcription factor Runt (Kagoshima et al., 1993; Ogawa et al., 1993). Based on the premise that it might be expressed in thymus and T cell lines, but not in B cell lines, Runx2 was thought to be involved in T cell differentiation and was deleted to study T cell differentiation (Ogawa et al., 1993; Satake et al., 1995). However, 4 years after the cloning of Runx2, its crucial role as a transcriptional determinant of osteoblast differentiation was demonstrated by several investigators working independent of one another and using different yet complementary experimental approaches (Ducy et al., 1997; Komori et al., 1997; Lee et al., 1997; Mundlos et al., 1997; Otto et al., 1997).

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One approach, purely molecular, was aimed at the identification of osteoblast-specific transcription factors through the systematic analysis of the promoter of what was then the only osteoblast-specific gene, Osteocalcin. The analysis of a proximal promoter fragment of one of the two mouse Osteocalcin genes led to the identification of the only two known osteoblast-specific cis-acting elements, termed OSE1 and OSE2 (Ducy and Karsenty, 1995). Remarkably, as of this writing, those two cis-acting elements remain the only known strictly osteoblast-specific cis-acting elements. Sequence inspection of OSE2 revealed homology for the DNA-binding site of Runt family transcription factors, and subsequent analysis demonstrated that the factor binding to OSE2 is related immunologically to transcription factors of the Runt family (Geoffroy et al., 1995; Merriman et al., 1995). Eventually, screening of a mouse osteoblast cDNA library revealed that only one of the three mammalian Runx genes, namely Runx2, is expressed predominantly in cells of the osteoblast lineage (Ducy et al., 1997). Indeed, in situ hybridization further revealed that during mouse development, Runx2 expression is first detected in the lateral plate mesoderm at 10.5 days postcoitus (dpc), and later is confined in cells of the mesenchymal condensations. Until 12.5 dpc these cells, which prefigure the future skeleton, represent common precursors of osteoblasts and chondrocytes. At 14.5 dpc osteoblasts first appear and maintain the expression of Runx2, whereas in chondrocytes Runx2 expression decreases significantly and becomes restricted to prehypertrophic and hypertrophic chondrocytes. After birth, Runx2 expression is strictly restricted to osteoblasts and cells of the perichondrium. This spatial and temporal expression pattern suggested Runx2 might play a critical role as a regulator of osteoblast differentiation (Ducy et al., 1997). The demonstration that Runx2 was indeed an osteoblast differentiation factor came from several synergistic lines of molecular and genetic evidence. First, in addition to the Osteocalcin promoter, functional OSE2-like elements were identified in the promoter regions of most other genes that are expressed at relatively high levels in osteoblasts, such as a1(II)-collagen, Osteopontin, and Bone sialoprotein, and eventually many more (Ducy et al., 1997). Second, and more decisively, forced expression of Runx2 in nonosteoblastic cell lines or primary skin fibroblasts induced osteoblast-specific gene expression in these cells, demonstrating that Runx2 acts as a transcriptional activator of osteoblast differentiation in vitro (Ducy et al., 1997). This was subsequently verified in vivo, where it was shown that the constitutive expression of Runx2 at low levels in nonhypertrophic chondrocytes triggered the entire cascade of endochondral bone formation, which normally does not occur in the cartilaginous ribs. Remarkably, however, ectopic expression of Runx in chondrocytes cannot cause transdifferentiation of chondrocytes into osteoblasts (Takeda et al., 2001). This suggests that if transdifferentiation of chondrocytes into osteoblasts can exist it remains a rather rare event. Third, the ultimate demonstration that Runx2 is an indispensable transcriptional activator of osteoblast differentiation came from genetic studies in mice and humans. At the same time, two groups deleted the Runx2 gene from the mouse genome, both expecting an immunological phenotype based on the assumption that Runx2 was involved in T cell differentiation (Komori et al., 1997; Otto et al., 1997). Instead, all Runx2-deficient mice had no skull, because intramembranous bone formation did not occur. In the rest of their skeleton, there are no osteoblasts in Runx2-deficient mice, an observation confirmed by the lack of expression of osteoblast marker genes. The critical importance of Runx2 for osteoblast differentiation was further emphasized by the finding that mice lacking only one allele of Runx2 display hypoplastic clavicles and delayed closure of the fontanelles, i.e., defects of intramembranous ossification (Otto et al., 1997). This phenotype is identical to what is seen in a human disease termed cleidocranial dysplasia (CCD), and subsequent genetic analysis of CCD patients revealed disease-causing heterozygous mutations of the RUNX2 gene, thereby demonstrating the relevance of Runx2 for osteoblast differentiation also in humans (Lee et al., 1997; Mundlos et al., 1997). Taken together, this overwhelming molecular and genetic evidence has led to the generally accepted view that Runx2 is a master control gene of osteoblast differentiation, providing a molecular switch inducing osteoblast-specific gene expression in mesenchymal progenitor cells (Lian and Stein, 2003). Remarkably, many, although not all, of the subsequent advances that have been made in the field are centered around the biology of Runx2. In addition to its prominent role in osteoblast differentiation and skeletogenesis, Runx2 is also involved in the regulation of bone formation beyond development. This has been demonstrated in several ways. First, transgenic mice expressing a dominant-negative variant of Runx2 specifically in fully differentiated osteoblasts are viable, but develop severe osteopenia caused by a decreased rate of bone formation, in the face of normal osteoblast numbers (Ducy et al., 1999). This phenotype is readily explained by the finding that several Runx2 target genes encoding bone extracellular matrix proteins are expressed at much lower levels. Second, mice lacking StatI, a transcription factor attenuating the nuclear translocation of Runx2, as discussed later, display a high-bone-mass phenotype that is explained not only by increased osteoblast differentiation, but also by increased bone matrix deposition (Kim et al., 2003). Third, a similar, but even more severe, phenotype is observed in mice lacking the nuclear adapter protein Shn3. Because Shn3, as discussed later, is involved in the ubiquitination and proteasomal degradation of Runx2, the increased bone formation of the Shn3deficient mice is readily explained by increased Runx2 levels in osteoblasts that in turn lead to enhanced bone matrix deposition (Jones et al., 2006). Given these results, it came as a surprise that another transgenic mouse model,

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overexpressing intact RU/u2 under the control of an osteoblast-specific 0.1 (I)-collagen promoter fragment, did not display the expected high-bone-mass phenotype, but a severe osteopenia accompanied by an increased fracture risk (Liu et al., 2001). Although these mice had increased numbers of osteoblasts, their bone formation rate was strikingly reduced, which likely illustrates the fact that the dosage of Runx2 needs to be tightly regulated to orchestrate proper bone formation in vivo.

Runx2 functions during skeletogenesis beyond osteoblast differentiation Although most of these results demonstrating a key role for Runx2 in osteoblasts were already discussed in the last edition of this book, there is accumulating novel evidence that the role of Runx2 in skeletogenesis is much more complex than previously anticipated. The starting point for these findings was the observation that Runx2-deficient mice also display defects of chondrocyte hypertrophy in some skeletal elements (Inada et al., 1999; Kim et al., 1999). Moreover, because Runx2 is transiently expressed in prehypertrophic chondrocytes of mouse embryos, there was a possibility of a function of Runx2 in chondrocyte differentiation. One way to address this possibility was the generation of a transgenic mouse model expressing Runx2 in nonhypertrophic chondrocytes, using an a1(II)-collagen promoter/enhancer construct (Takeda et al., 2001). In line with the suspected role of Runx2 as a positive regulator of chondrocyte hypertrophy, these transgenic mice displayed accelerated chondrocyte maturation in the growth plates, but also evidence of ectopic cartilage formation in the rib cage or in the trachea, among other locations. Moreover, the presence of this transgene in a Runx2-deficient genetic background prevented the absence of skeletal mineralization that is normally associated with Runx2 deficiency. However, the skeleton of these mice contained only hypertrophic cartilage, and no bone matrix, thereby demonstrating that Runx2 induces chondrocyte hypertrophy, but not a transdifferentiation into osteoblasts (Takeda et al., 2001). Because Runx2 expression in prehypertrophic chondrocytes is transient, the main function of Runx2 here is probably to establish the growth plate. However, through another site of expression, namely in perichondrial cells, Runx2 has an additional function in the regulation of chondrogenesis. In these cells Runx2 positively regulates the expression of Fgfl8, a diffusible molecule that inhibits chondrocyte maturation and osteoblast differentiation (Hinoi et al., 2006; Liu et al., 2002; Ohbayashi et al., 2002). Taken together, these results establish that Runx2 is more than the master gene of osteoblast differentiation; it is, in fact, along with Sox9, the major transcriptional regulator of cell differentiation during skeletogenesis, acting positively and negatively on osteoblast and chondrocyte differentiation.

Regulation of Runx2 accumulation and function In essence, the accumulation and demonstration that Runx2 exhibits, in vivo in mice and humans, all the characteristics of an osteoblast differentiation factor raised the usual questions: (1) What is upstream of Runx2? (2) How is Runx2 function regulated? (3) What is downstream of Runx2? The search for mechanisms regulating Runx2 expression is still ongoing. However, progress in this area of research came when a peculiarity of osteoblast differentiation was confronted in other progress in bone biology. This peculiarity of osteoblast biology is that the synthesis of type I collagen, the overwhelmingly most abundant protein of the bone extracellular matrix, precedes the expression of Runx2 (Wei et al., 2015). The fact that osteoblasts regulate glucose metabolism through the hormone osteocalcin prompted, of course, the study of the regulation of glucose metabolism in osteoblasts (Lee et al., 2007). This 2007 study showed that glucose is the main nutrient of osteoblasts and that, in vivo, glucose is transported in osteoblasts through Glut1, whose expression in cells of the osteoblasts lineage precedes that of Runx2. Glucose uptake favors osteoblast differentiation by preventing the proteasomal degradation of Runx2. Accordingly, Runx2 cannot induce osteoblast differentiation when glucose uptake is compromised, and raising blood glucose levels is sufficient to initiate bone formation in Runx2-deficient embryos. That Runx2 favors Glut1 expression determines the onset of osteoblast differentiation during development (Wei et al., 2015). In addition to this strong metabolic regulation of osteoblast differentiation, other transcription factors have been shown to act upstream of Runx2. There are several lines of evidence showing that, for instance, certain homeodomain-containing transcription factors are involved in the regulation of Runx2 expression. One of these proteins is Msx2, whose role in skeletal development was demonstrated through the identification of gain- and loss-of-function mutations in human patients suffering from Boston-type craniosynostosis or enlarged parietal foramina, respectively (Jabs et al., 1993; Wilke et al., 2000). Likewise, Msx2-deficient mice display defective ossification of the skull and of bones developing by endochondral ossification (Satokata et al., 2000). Moreover, because the expression of Osteocalcin and Runx2 is strongly reduced in Msx2-deficient mice, it appears that Msx2 acts upstream of Runx2 in a transcriptional cascade regulating

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osteoblast differentiation. A similar observation has been described for mice lacking the homeodomain-containing transcription factor Bpx (Tribioli and Lufkin, 1999). These mice die at birth owing to a severe dysplasia of the axial skeleton, whereas the appendicular skeleton is virtually unaffected. Runx2 expression in Bpx-deficient mice is strongly reduced in osteo-chondrogenic precursor cells of the prospective vertebral column, thereby indicating that Bpx is required for Runx2 expression specifically in these skeletal elements. There are also negative regulators of Runx2 expression. One of them is another homeodomain-containing transcription factor, Hoxa2. Hoxa2-deficient mice display ectopic bone formation in the second branchial arch, which is readily explained by an induction of Runx2 expression exactly in this region (Kanzler et al., 1998). Consistent with these observations, transgenic mice expressing Hoxa2 in craniofacial bones under the control of an Msx2 promoter fragment lack several bones in the craniofacial area. Beyond development there is at least one factor required to limit Runx2 expression in osteoblast precursor cells, namely, the high-mobility groupecontaining transcription factor Sox8. Sox8-deficient mice display an osteopenia that is caused by accelerated osteoblast differentiation accompanied by enhanced expression of Runx2 (Schmidt et al., 2005). Likewise, transgenic mice expressing Sox8 under the control of an osteoblast-specific a1(I)collagen promoter fragment virtually lack differentiated osteoblasts because of a decreased expression of Runx2. In addition to the existence of transcriptional regulators of Runx2 expression, there are also factors interacting with the Runx2 protein, thereby activating or repressing its activity. One identified positive regulator of Runx2 action is the nuclear matrix protein Satb2. The importance of this protein in skeletogenesis was first discovered in human patients with cleft palate that carry a heterozygous chromosomal translocation inactivating the SATB2 gene (Fitzpatrick et al., 2003). The generation of a Satb2-deficient mouse model confirmed the importance of this gene in craniofacial development, skeletal patterning, and osteoblast differentiation (Dobreva et al., 2006). The last function was in part attributed to an increased expression of Hoxa2, a negative regulator of bone formation discussed earlier, whose expression is repressed by the binding of Satb2 to an enhancer element of the Hoxa2 gene. In addition to this type of action, there is also a Hoxa2-independent influence of Satb2 on the transcription of Bone sialoprotein and Osteocalcin. Whereas in the case of Bone sialoprotein, Satb2 directly binds to an osteoblast-specific element in the promoter of this gene, the activation of Osteocalcin expression by Satb2 requires a physical interaction with Runx2. This was demonstrated by cotransfection assays using a Luciferase reporter gene under the control of an osteoblast-specific Osteocalcin promoter fragment, but also by coimmunoprecipitation experiments. Moreover; the synergistic action of Satb2 and Runx2 in osteoblasts was genetically confirmed through the generation and analysis of compound heterozygous mice lacking one allele of each gene (Dobreva et al., 2006). Taken together, these results identified Satb2 as an important regulator of osteoblast differentiation in mice and humans. Moreover, the finding that Satb2 also interacts with Activating transcription factor 4 (ATF4), another transcription factor involved in osteoblast differentiation and function that will be discussed later, illustrates that the transcriptional network regulating bone formation is much more complex than previously anticipated. This is further highlighted by the discovery of several other proteins that physically interact with Runx2, thereby attenuating its activity. One of these proteins is Stat1, a transcription factor regulated by extracellular signaling molecules, such as interferons. Stat1-deficient mice, as already mentioned, are viable, but develop a high-bone-mass phenotype explained by enhanced bone formation (Kim et al., 2003). The increase in osteoblast differentiation and function in these mice is molecularly explained by the lack of a Stat1-mediated inhibition of the transcriptional activity of Runx2. Interestingly, the physical interaction of both proteins is independent of Stat1 activation by phosphorylation, and it inhibits the translocation of Runx2 into the nucleus. Thus, overexpression of Stat1 in osteoblasts leads to a cytosolic retention of Runx2, and the nuclear translocation of Runx2 is much more prominent in Stat1-deficient osteoblasts (Kim et al., 2003). Another protein interacting with Runx2, thereby decreasing its availability in the nucleus, is Shn3. Shn3 is a zinc finger adapter protein originally thought to be involved in VDJ recombination of immunoglobulin genes (Wu et al., 1993). Unexpectedly, however, the generation of an Shn3-deficient mouse model revealed that it plays a major function in bone formation. In fact, the Shn3-deficient mice display a severe adult-onset osteosclerotic phenotype owing to a cellautonomous increase in bone matrix deposition (Jones et al., 2006). Interestingly, although several Runx2 target genes are expressed at higher rates in Shn3-deficient osteoblasts, Runx2 expression itself is not affected by the absence of Shn3. Importantly, however, the Runx2 protein level is strikingly increased in Shn3-deficient osteoblasts. This finding is molecularly explained by the function of Shn3 as an adapter molecule linking Runx2 to the E3 ubiquitin ligase WWP1. The Shn3-mediated recruitment of WWP1 in turn leads to an enhanced proteasomal degradation of Runx2, which is best highlighted by the finding that RNA interferenceemediated downregulation of WWP1 in osteoblasts leads to increased Runx2 protein levels and enhanced extracellular matrix mineralization, thus virtually mimicking the defects observed in the absence of Shn3 (Jones et al., 2006). Taken together, these data identify Shn3 as a key negative regulator of Runx2 actions in vivo. Accordingly, and as anticipated, Glut1 expression and glucose uptake are significantly increased in

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shn3
/
osteoblasts, as is Runx2 expression (Wei et al., 2015). Moreover, given the postnatal onset of the bone phenotype of the Shn3-deficient mice, it has been speculated that compounds blocking the interaction of Runx2, Shn3, and WWP1 may serve as specific therapeutic agents for the treatment of bone loss diseases, such as osteoporosis. This does not exclude the possibility that Shn3 may regulate osteoblast differentiation through additional mechanisms (Shim et al., 2013). While Stat1 and Shn3 exemplify the importance of a negative regulation of Runx2 in postnatal bone remodeling, there is also a need for a negative regulation of Runx2 activity before bone development starts and when Runx2 is already expressed. This is highlighted by the finding that Runx2 expression in the lateral plate mesoderm is already detectable as early as embryonic day (E) 10 of mouse development, whereas the expression of molecular markers of differentiated osteoblasts cannot be detected before E13.5 at the earliest (Ducy, 2000). One molecular explanation for this delay between Runx2 expression and osteoblast differentiation came from the functional analysis of Twist proteins that are transiently coexpressed with Runx2 early during development and inhibit osteoblast differentiation by interacting with Runx2. In essence, the initiation of osteoblast differentiation occurs only when Twist gene expression fades away. In brief, the fact that Runx2 expression precedes osteoblast differentiation by more than 4 days led to the assumption that Runx2 function must be regulated negatively in Runx2-expressing cells. The identification of such a factor relied on human genetic evidence. Haploinsufficiency at the RUNX2 locus causes a lack of bone in the skull, whereas haploinsufficiency at the TWIST1 locus causes essentially too much bone in the skull, a condition called craniosynostosis (EI Ghouzzi et al., 1997; Howard et al., 1997). Because the same phenotypes are observed in the corresponding mouse models lacking one allele of either gene, it was possible to demonstrate a genetic interaction of Runx2 and Twist1 through the generation of compound heterozygous mice. In fact, these mice did not display any detectable defects of skull development and suture fusion (Bialek et al., 2004). In contrast, the defects of clavicle development caused by haploinsufficiency of Runx2 were not rescued by heterozygosity of Twist1, but by the deletion of Twist2. Thus, both Twist proteins have similar functions but in different skeletal elements. This is totally consistent with their expression pattern in mouse embryos (Bialek et al., 2004). Both Twist proteins are presumably basic helixeloopehelix (bHLH) transcription factors, yet this function of Twist is not determined by the bHLH domain, but rather by the C-terminal 20 amino acids, the so-called Twist box, whose sequence is fully conserved in both Twist proteins, in mice and humans. Through the Twist box, Twist proteins interact with the Runx2 DNA-binding domain and prevent its DNA binding. The importance of this sequence motif for Twist function in vivo was confirmed by the existence of a Twist-box mutation within the human TWISTI gene that causes a severe form of craniosynostosis (Gripp et al., 2000). Moreover, an ethylnitrosourea-mutagenesis approach in mice led to the identification of an amino acid substitution within the Twist box of Twist1 that causes premature osteoblast differentiation in vivo (Bialek et al., 2004). Unexpectedly, this mouse model, termed Charlie Chaplin, also displayed decreased chondrocyte maturation, thereby suggesting an additional physiological role of Twist1, independent of its antiosteogenic function (Hinoi et al., 2006). Because Twist1 is not expressed in chondrocytes, but in mesenchymal cells of the perichondrium, it appears that it is required to inhibit the induction of Fgf18 expression in these cells by the action of Runx2, which was described earlier. Indeed, whereas transgenic mice overexpressing Twist1 under the control of an osteoblast-specific 0.1 (I)-collagen promoter fragment displayed enhanced chondrocyte maturation, the decreased chondrocyte maturation in the CC/CC mice was normalized by haploinsufficiency of Runx2 (Hinoi et al., 2006). Taken together, these data demonstrate that Twist1, through inhibition of Runx2 DNA binding, not only limits osteoblast differentiation and bone formation, but also enhances chondrocyte maturation during skeletal development. Other negative regulators of Runx2 function in vivo have been described based on their abilities to influence the CCD phenotype of Runx2þ/
mice. One of them, another zinc fingerecontaining protein termed zinc finger protein 521 (2fp521), antagonizes Runx2 function in a histone deacetylase 3 (HDAC3)-dependent manner.

Osterix, a Runx2-dependent osteoblast-specific transcription factor required for bone formation In addition to Runx2, there is at least one more transcription factor, termed Osterix (Osx), whose activity is absolutely required in mice for osteoblast differentiation. Osx is a zinc fingerecontaining transcription factor that is specifically expressed in osteoblasts of all skeletal elements. Inactivation of Osx in mice results in perinatal lethality owing to a complete absence of bone formation (Nakashima et al., 2002). Unlike the Runx2-deficient mice whose skeleton is completely unmineralized, the Osx-deficient mice lacked a mineralized matrix only in bones formed by intramembranous ossification. In contrast, the bones formed by endochondral ossification contained mineralized matrix, but this resembled

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calcified cartilage, not mineralized bone matrix. This finding suggested that Osx, unlike Runx2, is not required for chondrocyte hypertrophy, thereby demonstrating that it specifically induces osteoblast differentiation and bone formation in vivo. The comparative expression analysis by in situ hybridization further revealed that Osx is not expressed in Runx2deficient embryos, and that Runx2 is normally expressed in Osx-deficient embryos (Nakashima et al., 2002). These results demonstrated that Osx acts downstream of Runx2 in a transcriptional cascade of osteoblast differentiation, and its expression is apparently directly regulated by the binding of Runx2 to a responsive element in the promoter of the Osx gene (Nishio et al., 2006). In contrast to the steadily increasing knowledge about the function of Runx2 and its regulation by other molecules, the molecular mechanisms underlying the action of Osx in osteoblasts are less well understood. Moreover, unlike for RUNX2, no human mutations of the OSX gene have yet been identified that would be associated with decreased bone formation. Nevertheless, one 2005 publication indicates that Osx contributes to the negative effects of nuclear factor of activated T cells (NFAT) inhibitors on bone mass (Koga et al., 2005). NFAT inhibitors, such as FKS06 or cyclosporin A, are commonly used as immune suppressants, e.g., after organ transplantation. However, this treatment is often accompanied by the development of osteopenia in the patient (Rodino and Shane, 1998). Likewise, treatment of mice with FKS06 leads to decreased bone mass owing to impaired bone formation, and the same phenotype was observed in mice lacking the transcription factor Nfatc1. The deduced role of Nfatc1 as a physiological activator of osteoblast differentiation and function can be molecularly explained by an interaction with Osx. In fact, both proteins synergistically activate transcription of a Luciferase reporter gene driven by an osteoblast-specific a1(I)-collagen promoter fragment, which is based on the formation of a DNA-binding complex of Nfatc1 and Osx (Koga et al., 2005). It has been shown that Osx might act as a cofactor for Dlx5 for osteoblast specification (Hojo et al., 2016).

ATF4, a transcriptional regulator of osteoblast functions and a mediator of the neural regulation of bone mass The role of ATF4 in skeletal biology also arose from a combination of molecular biology and human and mouse genetic data. RSK2, which encodes a kinase, is the gene mutated in CoffineLowry syndrome, an X-linked mental retardation condition associated with skeletal abnormalities (Trivier et al., 1996). Like Rsk2-deficient mice, Atf4
/
mice display a lower bone mass owing to impaired bone formation (Yang et al., 2004). In vitro kinase assays demonstrated that ATF4 is strongly phosphorylated by Rsk2, and that this phosphorylation is undetectable in osteoblasts derived from Rsk2-deficient mice. The subsequent analysis of an Atf4-deficient mouse model revealed that this transcription factor plays a crucial role in bone formation. In fact, Atf4-deficient mice display a delayed skeletal development and thereafter develop a severe lowbone-mass phenotype caused by a decrease in bone formation (Yang et al., 2004). Molecularly, ATF4 was identified as the factor binding to the osteoblast-specific element OSE1 in the Osteocalcin promoter, thereby directly activating the transcription of this gene. Moreover, ATF4 is required for proper synthesis of type I collagen, although this function is not mediated by a transcriptional regulation of type I collagen expression. In fact, because type I collagen synthesis in the absence of nonessential amino acids is specifically reduced in primary osteoblast cultures lacking ATF4, it appears that ATF4 is required for efficient amino acid import in osteoblasts, as has been described for other cell types (Harding et al., 2003). Because a reduced type I collagen synthesis was subsequently also observed in mice lacking Rsk2, these data provided evidence that the diminished ATF4 phosphorylation in the absence of Rsk2 may contribute to the skeletal defects associated with CoffineLowry syndrome (Yang et al., 2004). In addition to its role in bone formation, ATF4, through its expression in osteoblasts, regulates bone resorption (Elefteriou et al., 2005). This function is molecularly explained by the fact that ATF4 binds to the promoter of the Rankl (receptor activator of NF-kB ligand) gene to promote osteoclast differentiation (Teitelbaum and Ross, 2003). As a result, ATF4-deficient mice have a decreased number of osteoclasts because of their reduced Rankl expression. Most importantly, this function of ATF4 is involved in the control of bone resorption by the sympathetic nervous system. In fact, treatment of normal osteoblasts with isoproterenol, a surrogate of sympathetic signaling, enhanced osteoclastogenesis of cocultured bone marrow macrophages through an induction of osteoblastic Rankl expression (Elefteriou et al., 2005). As expected, this effect was blunted when the osteoblasts were derived from mice lacking the b2-adrenergic receptor Adrb2. However, the effect of isoproterenol was also blunted by an inhibitor of protein kinase A, or by using osteoblasts derived from ATF4deficient mice (Elefteriou et al., 2005). Taken together, these results demonstrated that ATF4 is an important mediator of extracellular signals, such as b-adrenergic stimulation, in osteoblasts. Thus, it is not surprising that the function of ATF4 is mostly regulated posttranslationally. For example, as already mentioned, ATF4 also interacts with other proteins, such as the nuclear matrix protein Satb2 (Dobreva et al., 2006). Also as

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described earlier, the proximal Osteocalcin promoter contains two osteoblast-specific elements, termed OSE1 and OSE2, that serve as binding sites for ATF4 and Runx2, respectively (Ducy and Karsenty, 1995; Ducy et al., 1997; Schinke and Karsenty, 1999; Yang et al., 2004). Because of the proximity of both elements, there is indeed a physical interaction of the two proteins, which is stabilized by Satb2, which acts as a scaffold enhancing the synergistic activity of Runx2 and ATF4, which is required for optimum Osteocalcin expression (Xiao et al., 2005; Dobreva et al., 2006). Other aspects of ATF4 biology are also regulated posttranslationally. In fact, even the osteoblast specificity of ATF4 function is not determined by osteoblast-specific ATF4 expression, but by a selective accumulation of the ATF4 protein in osteoblasts, which itself is explained by the lack of proteasomal degradation (Yang and Karsenty, 2004). This is best demonstrated by the finding that the treatment of nonosteoblastic cell types with the proteasome inhibitor MG 115 leads to accumulation of the ATF4 protein, thereby resulting in ectopic Osteocalcin expression. Taken together, these data provided the first evidence for the achievement of a cell-specific function of a transcriptional activator by a posttranslational mechanism. They are therefore of general importance for our understanding of the transcriptional networks controlling cellular differentiation and function. Remarkably, ATF4 biology further illustrates how the molecular understanding of a disease-causing gene can translate into therapeutic interventions. Indeed, an increased Rsk2-dependent phosphorylation of ATF4 may also be involved in the development of the skeletal abnormalities in human patients suffering from neurofibromatosis (Ruggieri et al., 1999; Stevenson et al., 1999). This disease, which is primarily known for tumor development within the nervous system, is caused by inactivating mutations of the NF1 gene, which encodes a Ras-GTPase-activating protein (Klose et al., 1998). The generation of a mouse model lacking Nf1 specifically in osteoblasts (Nf1
/
ob ) led to the demonstration that this gene plays a major physiological role in bone remodeling. In fact, the Nf1
/
ob mice displayed a high-bone-mass phenotype that is caused by increased bone turnover and is accompanied by enrichment of unmineralized osteoid (Elefteriou et al., 2006). The analysis of this phenotype revealed an increased production of type I collagen in the absence of Nf1, which is molecularly explained by an Rsk2-dependent activation of ATF4. Likewise, transgenic mice overexpressing ATF4 in osteoblasts display a phenotype similar to that of the Nf1
/
ob mice, and the increased type I collagen production and osteoid thickness in the latter are significantly reduced by haploinsufficiency of ATF4 (Elefteriou et al., 2006). These molecular findings may also have therapeutic implications. Given the previously discussed function of ATF4 in amino acid import, it appeared reasonable to analyze whether the skeletal defects of the Nf1
/
ob mice can be affected by dietary manipulation. Indeed, the increased bone formation and osteoid thickness of Nf1
/
ob mice can be normalized by a low-protein diet, and the same was the case in the transgenic mice overexpressing ATF4 in osteoblasts (Elefteriou et al., 2006). Likewise, the defects of osteoblast differentiation and bone formation observed in both the ATF4- and the Rsk2deficient mice were corrected by feeding a high-protein diet. Taken together, these data not only emphasize the importance of ATF4 in osteoblast biology, but also demonstrate how knowledge about its specific functions in osteoblasts can be useful for the treatment of skeletal diseases.

Additional transcriptional regulators of osteoblast differentiation and function Activator protein 1 (AP-1) is a heterodimeric transcription factor composed of members of the Jun and Fos families of basic leucine zipper proteins (Karin et al., 1997). These include the Jun proteins c-Jun, JunB, and JunD, as well as the Fos proteins c-Fos, Fra1, Fra2, and Fosb, respectively. Although AP-1 transcription factors have been demonstrated to fulfill various functions in different cell types, it is striking that some of the family members play specific roles in bone remodeling, as demonstrated by several loss- or gain-of-function studies in mice (Wagner and Eferl, 2005). For instance, the deletion of c-Fos from the mouse genome results in severe osteopetrosis owing to an arrest of osteoclast differentiation, whereas the transgenic overexpression of c-Fos results in osteosarcoma development (Grigoriadis et al., 1993, 1994). Moreover, transgenic mice overexpressing either Fml or i1fosB, a splice variant of FosB, display a severe osteosclerotic phenotype caused by increased osteoblast differentiation and function (Jochum et al., 2000; Sabatakos et al., 2000). Likewise, mice lacking Fra1 in extraplacental tissues display an osteopenia associated with reduced bone formation, indicating a physiological role of Fra1 in osteoblasts (Eferl et al., 2004). When the same approach was used to inactivate JunB in extraplacental tissues, thereby circumventing the embryonic lethality caused by a complete genomic deletion of JunB, the resulting mice developed a state of low bone turnover, owing to cell-autonomous defects in osteoblasts, but also in osteoclast differentiation (Kenner et al., 2004). Taken together, these data provide evidence for a crucial role of AP-1 transcription factors in the regulation of bone formation, although their connection to the other transcriptional regulators described earlier still needs to be further investigated. For instance, it is known from other cell types that Jun proteins can also interact with ATF family members, thus raising the possibility that heterodimerization with ATF4 may be one mechanism by which these proteins can regulate

170 PART | I Basic principles

osteoblast-specific gene expression (Chinenov and Kerppola, 2001). Interestingly, it has been demonstrated that the osteosarcoma development of c-Fos transgenic mice is dramatically decreased in an Rsk2-deficient genetic background (David et al., 2005). This observation is molecularly explained by the lack of c-Fos phosphorylation by Rsk2, thereby leading to increased proteasomal degradation. Thus, Rsk2 apparently not only is involved in the physiological regulation of bone formation via phosphorylation of ATF4, but may also have an influence on the development of osteosarcomas via phosphorylation of c-Fos. Another potential mechanism by which AP-1 family members might be involved in the regulation of bone formation came- from the analysis of mouse models with impaired circadian regulation. These mice, which lack components of the molecular clock, namely the Per or Cry genes, display a high-bone-mass phenotype caused by increased bone formation (Fu et al., 2005). Moreover, they respond to intracerebroventricular infusion of leptin with a further increase in bone mass, suggesting that the components of the molecular clock are involved in the regulation of bone formation via the sympathetic nervous system. Interestingly, virtually all genes encoding members of the AP-1 transcription factor family were expressed at higher levels in osteoblasts derived from mice lacking either the Per genes or the b2-adrenergic receptor Adrb2 (Fu et al., 2005). This increase was especially pronounced in the case of the c-Fos gene, whose expression can also be induced by the addition of isoproterenol in wild-type osteoblasts. In turn, c-Fos leads to a direct activation of c-Myc transcription, thereby indirectly increasing the intracellular levels of cyclin D1 and promoting osteoblast proliferation. Taken together, these data demonstrated that the expression of AP-1 components is activated via sympathetic signaling, and that this induction is counteracted by the activity of clock gene products. Finally, another transcription factor that, like AP-1, is not cell specific but plays a great role in osteoblast biology, is the cAMP-responsive element binding protein (CREB), a transcription factor mediating changes in gene expression caused by signaling through various G-protein-coupled receptors. The demonstration that gut-derived serotonin is an inhibitor of bone formation in mice, rats, and humans, the regulation of the synthesis of which is under the control of Lrp5 signaling in the gut, raised the question of how serotonin signals in osteoblasts (Yadav et al., 2008; Frost et al., 2011). Expression analysis and cell-specific gene deletion experiments in the mouse showed that Htr1B is the receptor of serotonin, mediating its function in osteoblasts, namely, an inhibition of proliferation, and that the transcription factor mediating this effect is CREB (Yadav et al., 2008).

Transcription factors acting downstream of Wnt signaling in osteoblasts: what do they actually do in differentiated osteoblasts? The discovery of the LRP5 gene as a major determinant of bone mass in humans because of its homology to a Wnt coreceptor in Drosophila has generated an enormous interest and hope in what the canonical Wnt signaling could do in cells of the osteoblast lineage and especially in differentiated osteoblasts. An obligatory implication of this belief, an implication that cannot be ignored or dismissed, is that ablating canonical Wnt signaling in differentiated osteoblasts should lead to an osteopetrotic phenotype. Unfortunately, when taken at face value as they should, the experiments performed to test this hypothesis not only did not provide any evidence that it is the case, but in fact clearly showed that it is not the case. Indeed, the osteoblast-specific inactivation of b-catenin, the molecular node of the canonical Wnt signaling pathway, resulted in the expected low-bone-mass phenotype; however, this was caused by an isolated and massive increase in bone resorption (Glass et al., 2005). The molecular explanation for the regulation of osteoclast differentiation by b-catenin expression in osteoblasts came from the expression analysis of Osteoprotegerin (Opg), a well-known inhibitor of bone resorption, blocking the activity of Rankl (Simonet et al., 1997; Yasuda et al., 1998). Surprisingly, although Opg expression was markedly increased in osteoblasts from mice expressing the stabilized form of b-catenin, its expression was decreased in mice harboring an osteoblast-specific deletion of b-catenin (Glass et al., 2005). That similar results were observed when b-catenin was deleted from osteocytes established that it is a general rule of bone biology. Of note, these results need to be considered together with the experimental evidence gathered by two different laboratories, using for that purpose mutant mouse strains also generated in two different laboratories, that mice lacking Lrp5 signaling in osteoblasts only do not have any bone phenotype to speak of (Yadav et al., 2008; Kode et al., 2014). One of the transcription factors activated by b-catenin in osteoblasts has already been identified as Tcfl, whose importance for the regulation of gene expression in osteoblasts is highlighted by several lines of evidence. First, in situ hybridization revealed that Tcfl is expressed in osteoblasts during bone development and after birth. Second, Tcfl-deficient mice display a low-bone-mass phenotype caused by an increase in bone resorption. Third, the compound heterozygosity of Tcfl and b-catenin in osteoblasts also results in low bone mass, which is not observed when one allele of each gene is

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inactivated alone. Fourth, Opg expression is decreased in osteoblasts of Tcfl-deficient mice, and the molecular analysis of the Opg promoter revealed the existence of a Tcf1-binding site, whose functional activity was subsequently proven by chromatin immunoprecipitation and DNA cotransfection assays (Glass et al., 2005). The implication of these results cannot be underestimated, without taking great and yet predictable risks, when proposing to harness the canonical Wnt signaling pathway for therapeutic purposes in the context of osteoporosis.

Regulation of osteoblast differentiation by means other than transcription factors Chromatin structure, which influences by posttranslational modifications of histone proteins around which the DNA is wrapped, is a major determinant of gene expression (Allis et al., 2007). Histone acetylation promotes gene transcription by relaxing the chromatin structure, whereas deacetylation of histones by HDACs induces chromatin condensation and transcriptional repression (Berger, 2002; Verdin et al., 2003; Allis et al., 2007). Class II HDACs contain a poorly active catalytic domain and a long N-terminal extension to which transcription factors can bind. The existence of this domain has suggested that class II HDACs can link extracellular cues to the genome of a given cell (Verdin et al., 2003; Haberland et al., 2009). Several class I HDACs (Hdac1 and Hdac3) are expressed in osteoblasts, and conditional deletion of Hdac3 within osteo-chondroprogenitor cells decreases osteoblast number, and, as mentioned earlier, one mechanism whereby Zpf521 inhibits Runx2 functions during osteoblast differentiation is by interacting with HDAC3. On the other hand, one class II HDAC, HDAC4, acts as a central integrator of two extracellular cues acting on osteoblasts. One is parathyroid hormone, which targets HDAC4 for degradation and thereby releases MeF2c that can now transactivate Rankl, and thus favors osteoclast differentiation. The other is sympathetic tone, which instead favors HDAC4 accumulation in the nucleus, its association with ATF4, and again Rankl expression and osteoclast differentiation (Obri et al., 2014). Another mode of regulation of osteoblast differentiation that has received much attention since the last edition of this book is the one fulfilled by small noncoding RNAs. MicroRNAs (miRNAs) are small noncoding RNAs that downregulate expression of their target genes by either mRNA degradation or translational inhibition (Valencia-Sanchez et al., 2006; Bartel, 2009; Djuranovic et al., 2011; Huntzinger and Izaurralde, 2011). Although most miRNAs are broadly expressed, some have a more restricted pattern of expression and influence cell differentiation (Poy et al., 2004). The importance of this mode of gene regulation during skeletogenesis is inferred from the observation that inactivation of

Perichondrium

Growth Plate Cartilage

Perichondrium

Proliferating Chondrocyte

Runx2

Pre-hypertrophic Chondrocyte

Runx2

Runx2 Fgf18

Fgf18 Hypertrophic Chondrocyte (Type X collagen)

Bone Runx2 Mesenchymal Progenitor

FIGURE 7.1

Runx2 Differentiated Osteoblast

Bone matrix Production (Type I collagen, Osteocalcin, Osteopontin, Bone sialoprotein, and others)

Schematic representation of the functions of Runx2 during chondrogenesis and osteoblast differentiation.

172 PART | I Basic principles

Msx2, Bpx

Hoxa2 Proliferation

Twist AP1 Mesenchymal Progenitor

Runx2

Runx2

Osteoblast Precursor

Chondrocyte

Runx2, Satb2

Fra1

Immature Osteoblast

β-catenin

FIGURE 7.2

Osx

Mature Osteoblast

Tcf1 Opg Rankl

ATF4

Collagen Production

Osteoclast

Schematic representation of the transcriptional control of osteoblast differentiation and functions.

DICER, a protein necessary for processing of miRNAs, affects osteoblast differentiation (Gaur et al., 2010). Accordingly, it has been suggested that miRNAs may be involved in osteoblast proliferation and/or differentiation (Hassan et al., 2010). However, these observations were derived from gain-of-function experiments performed in cell culture. Few miRNAs had been shown, through cell-specific loss-of-function experiments performed in the mouse, to regulate osteoblast proliferation and/or differentiation in vivo. A first example of these was miRNA-2561, whose silencing in the mouse reduces Runx2 accumulation without, however, causing a characteristic CCD phenotype (Li et al., 2009). Two other miRNAs, miR-34b and miR-34c, were shown through cell-specific gene deletion to inhibit osteoblast proliferation by suppressing cyclin D1, CDK4, and CDK6 accumulation (Wei et al., 2012). They also hampered osteoblast differentiation by inhibiting the function of SATB2, a protein interacting with Runx2 and ATF4 (Wei et al., 2012). Another miRNA, miR-124, inhibits bone formation by targeting another transcription factor implicated in osteoblast differentiation, ATF4, whereas miR-188 was shown to favor bone formation in an HDAC9-dependent manner (Li et al., 2015) (Fig. 7.1 and 7.2).

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Stevenson, D.A., Birch, P.H., Friedman, J.M., Viskochil, D.H., Balestrazzi, P., Boni, S., Buske, A., Korf, B.R., Niimura, M., Pivnick, E.K., Schorry, E.K., Short, M.P., Tenconi, R., Tonsgard, J.H., Carey, J.C., 1999. Descriptive analysis of tibial pseudarthrosis in patients with neurofibromatosis 1. Am. J. Med. Genet. 84, 413e419. Takeda, S., Bonnamy, J.P., Owen, M.J., Ducy, P., Karsenty, G., 2001. Continuous expression of Cbfal in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfal-deficient mice. Genes Dev. 15, 467e481. Teitelbaum, S.L., Ross, E.P., 2003. Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 4, 638e649. Tribioli, C., Lufkin, T., 1999. The murine Bapxl homeobox gene plays a critical role in embryonic development of the axial skeleton and spleen. Development 126, 5699e5711. Trivier, E., De Cesare, D., Jacquot, S., Pannetier, S., Zackai, E., Young, L., Mandel, J.L., Sassone-Corsi, P., Hanauer, A., 1996. Mutations in the kinase Rsk-2 associated with Coffin-Lowry syndrome. Nature 384, 567e570. Valencia-Sanchez, M.A., Liu, J., Hannon, G.J., Parker, R., 2006. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 20 (5), 515e524. Verdin, E., Ott, M., 2013. Acetylphosphate: a novel link between lysine acetylation and intermediary metabolism in bacteria. Mol Cell 51 (2), 132e134. Wagner, E.F., Eferi, R., 2005. Fos! AP-I proteins in bone and the immune system. Immunol. Rev. 208, 126e140. Wei, J., Shi, Y., Zheng, L., Zhou, B., Inose, H., Wang, J., Guo, X.E., Grosschedl, R., Karsenty, G., May 14, 2012. miR-34s inhibit osteoblast proliferation and differentiation in the mouse by targeting SATB2. J. Cell Biol. 197 (4), 509e521. Wei, J., Shimazu, J., Makinistoglu, M., Maurizi, A., Kajimura, D., Zong, H., Takarada, T., Iezaki, T., Pessin, J.E., Hinoi, E., Karsenty, G., 2015. Glucose uptake and Runx2 synergize to orchestrate osteoblast differentiation and bone formation. Cell 161, 1576e1591. Wilke, A.O.M., Tang, Z., Eianko, N., Walsh, S., Twigg, S.R.E., Hurst, J.A., Wall, S.A., Chrzanowska, K.H., Maxson, R.E.J., 2000. Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification. Nat. Genet. 24, 387e390. Wu, L.C., Mak, C.H., Dear, N., Boehm, T., Foroni, L., Rabbitts, T.H., 1993. Molecular cloning of a zinc finger protein which binds to the heptamer of the signal sequence for V(D)J recombination. Nucleic Acids Res. 21, 5067e5073. Xiao, G., Jiang, D., Ge, C., Zhao, Z., Lai, Y., Boules, H., Phimphilai, M., Yang, X., Karsenty, G., Franceschi, R.T., 2005. Cooperative interactions between activating transcription factor 4 and Runx2! Cbfal stimulate osteoblast-specific osteocalcin gene expression. J. Biol. Chem. 280, 30689e30696. Yadav, V.K., Ryu, J.H., Suda, N., Tanaka, K., Gingrich, J., Schutz, G., Glorieux, F.H., Insogna, K., Mann, J.J., Hen, R., Ducy, P., Karsenty, G., 2008. Lrp5 control bone mass by inhibiting serotonin synthesis in the duodenum. Cell 135, 825e837. Yang, X., Karsenty, G., 2004. ATF4, the osteoblast accumulation of wwich is determined post-translationally, can induce osteoblastspecific gene expression in non-osteoblastic cells. J. Biol. Chem. 279, 47109e47114. Yang, X., Matsuda, K., Bialek, P., Jacquot, S., Masuoka, H.C., Schinke, T., Li, L., Brancorsini, S., Sassone-Corsi, P., Townes, T.M., Hanauer, A., Karsenty, G., 2004. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry syndrome. Cell 117, 387e398. Yasuda, H., Shima, N., Nakagawa, N., Mochizuki, S.I., Yano, K., Fujise, N., Sato, Y., Goto, M., Yamaguchi, K., Kuriyama, M., Kanno, T., Murakami, A., Tsuda, E., Morinaga, T., Higashio, K., 1998. Identity of osteociastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 139, 1329e1337.

Further reading Boyden, L.M., Mao, J., Belsky, J., Mitzner, L., Farhi, A., Mitnick, M.A., Wu, D., Insogna, K., Lifton, R.P., 2002. High bone density due to a mutation in LDL-receptor-related protein 5. N. Engl. J. Med. 346, 1513e1521. Day, T.E., Guo, X., Garrett-Beal, L., Yang, Y., 2005. Wntlbetacatenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739e750. Gong, Y., Slee, R.B., Fukai, N., Rawadi, G., Roman-Roman, S., Reginato, A.M., Wang, H., Cundy, T., Glorieux, E.H., Lev, D., Zacharin, M., Oexle, K., Marcelino, J., Suwairi, W., Heeger, S., Sabatakos, G., Apte, S., Adkins, W.N., Allgrove, J., Arslan-Kirchner, M., Batch, J.A., Beighton, P., Black, G.c., Boles, R.G., Boon, L.M., BOlTone, c., Brunner, H.G., Carle, G.E., Dallapiccola, B., De Paepe, A., Floege, B., Halfhide, M.L., Hall, B., Hennekam, R.C., Hirose, T., Jans, A., Juppner, H., Kim, C.A., Keppler-Noreuil, K., Kohlschuetter, A., LaCombe, D., Lambert, M., Lemyre, E., Letteboer, T., Peltonen, L., Ramesar, R.S., Romanengo, M., Somer, H., Steichen-Gersdorf, E., Steinmann, B., Sullivan, B., Superti-Furga, A., Swoboda, W., van den Boogaard, M.J., Van Hul, w., Vikkula, M., Votruba, M., Zabel, B., Garcia, T., Baron, R., Olsen, B.R., Warman, M.L., 2001. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107, 513e523. Hecht, J., Seitz, V., Urban, M., Wagner, E., Robinson, P.N., Stiege, A., Dieterich, c., Kornak, U., Wilkening, U., Brieske, N., Zwingman, c., Kidess, A., Stricker, S., Mundlos, S., 2007. Detection of novel skeletogenesis target,genes by comprehensive analysis of a Runx 2(-/-) mouse model. Gene Expr. Patterns 7, 102e112. Hill, T.P., Spater, D., Taketo, M.M., Birchmeier, W., Hartmann, C., 2005. Canonical Wntlbeta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev. Cell 727e738. Huelsken, J., Birchmeier, W., 2001. New aspects of Wnt signaling pathways in higher vertebrates. Curr. Opin. Genet. Dev. 11, 547e553. Kato, M., Patel, M.S., Levasseur, R., Lobov, I., Chang, B.H., Glass II, D.A., Hartmann, C., Li, L., Hwang, T.H., Brayton, C.F., Lang, R.A., Karsenty, G., Chan, L., 2002. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J. Cell Biol. 157, 303e314.

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Little, R.D., Carulli, J.P., Del Mastro, R.G., Dupuis, J., Osborne, M., Folz, C., Manning, S.P., Swain, P.M., Zhao, S.C., Eustace, B., Lappe, M.M., Spitzer, L., Zweiei, S., Braunschweiger, K., Benchekroun, Y., Hu, X., Adair, R., Chee, L., FitzGerald, M.G., Tulig, C., Caruso, A., Tzellas, N., Bawa, A., Franklin, B., McGuire, S., Nogues, X., Gong, G., Allen, K.M., Anisowicz, A., Morales, A.J., Lomedico, P.T., Recker, S.M., Van Eerdewegh, P., Recker, R.R., Johnson, M.L., 2002. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant highbone-mass trait. Am. J. Hum. Genet. 70, 11e19. Mao, J., Wang, J., Liu, B., Pan, W., Farr III, G.H., Flynn, C., Yuan, H., Takada, S., Kimelman, D., Li, L., Wu, D., 2001. Low-density lipoprotein receptorrelated protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol. Cell 7, 801e809. Vaes, B.L., DlIey, P., Sijbers, A.M., Hendriks, J.M., van Someren, E.P., de Jong, N.G., van den Heuvel, E.R., Olijve, W., van Zoelen, E.J., Dechering, K.J., 2006. Microarray analysis on Runx2-deficient mouse embryos reveals novel Runx2 functions and target genes during intrame! llbranous and endochondral bone formation. Bone 39, 724e738. Zheng, Q., Zhou, G., Morello, R., Chen, Y., Garcia-Rojas, X., Lee, B., 2003. Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo. J. Cell Biol. 162, 833e842.