Whole Aspect of Runx2 Functions in Skeletal Development

Runt-related transcription factor 2 (Runx2) is a fundamental transcription factor for bone development. In endochondral ossification, Runx2 induces chondrocyte maturation, enhances chondrocyte proliferation through Indian hedgehog (Ihh) induction, and induces the expression of vascular endothelial growth factor A (Vegfa), secreted phosphoprotein 1 (Spp1), integrin-binding sialoprotein (Ibsp), and matrix metallopeptidase 13 (Mmp13) in the terminal hypertrophic chondrocytes. Runx2 inhibits the apoptosis of the terminal hypertrophic chondrocytes and induces their transdifferentiation into osteoblasts and osteoblast progenitors. The transdifferentiation is required for trabecular bone formation during embryonic and newborn stages but is dispensable for acquiring normal bone mass in young and adult mice. Runx2 enhances the proliferation of osteoblast progenitors and induces their commitment to osteoblast lineage cells through the direct regulation of the expressions of a hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathway genes and distal-less homeobox 5 (Dlx5), which all regulate Runx2 expression and/or protein activity. Runx2, Sp7, and Wnt signaling further induce osteoblast differentiation. In immature osteoblasts, Runx2 regulates the expression of bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and bone gamma carboxyglutamate protein (Bglap)/Bglap2, and induces osteoblast maturation. Osteocalcin (Bglap/Bglap2) is required for the alignment of apatite crystals parallel to the collagen fibers; however, it does not physiologically work as a hormone that regulates glucose metabolism, testosterone synthesis, or muscle mass. Thus, Runx2 exerts multiple functions essential for skeletal development.


Runx2 and Chondrocyte Differentiation
In the growth plate, chondrocytes proliferate and maturate, making the resting, proliferating, prehypertrophic, hypertrophic, and terminal hypertrophic layers ( Figure 1). Chondrocytes in the resting and proliferating layers are immature, actively proliferate, and highly express Col2a1. Those in the prehypertrophic layer express parathyroid hormone 1 receptor (Pth1r) and Indian hedgehog (Ihh), those in the hypertrophic layer express Col10a1, and those in the terminal hypertrophic layer express vascular endothelial growth factor A (Vegfa), secreted phosphoprotein 1 (Spp1), integrin-binding sialoprotein (Ibsp), and matrix metallopeptidase 13 (Mmp13) [17]. Runx2 is weakly expressed in chondrocytes in the resting and proliferation layers and upregulated in the prehypertrophic chondrocytes, and the expression is maintained until terminal differentiation of hypertrophic chondrocytes [17,18] (Figure 1). Chondrocyte maturation is markedly impaired in Runx2-deficient (Runx2 −/− ) mice, which exhibit an absence of hypertrophic chondrocytes in most of the skeleton, except for the tibia, fibula, radius, and ulna. Overexpression of Runx2 under the control of the Col2a1 promoter, which directs the transgene to chondrocytes, accelerates chondrocyte maturation and endochondral ossification, and chondrocytes in the presumptive permanent cartilage, including articular cartilage, also enter the process of endochondral ossification [19,20]. In contrast, as overexpression of dominant-negative Runx2 under the control of the Col2a1 promoter decelerates chondrocyte maturation, most of the endochondral skeletons are composed of immature chondrocytes, and the immature chondrocytes express tenascin-C (Tnc), which is expressed in the permanent cartilage of wild-type mice [19]. Conditional knockout mice using Col2a1 Cre transgenic mice to delete Runx2 in cartilage (Col2a1 Cre Runx2 fl/fl mice) also exhibit inhibition of chondrocyte maturation [21,22]. express Col10a1, and those in the terminal hypertrophic layer express vascular endothelial growth factor A (Vegfa), secreted phosphoprotein 1 (Spp1), integrin-binding sialoprotein (Ibsp), and matrix metallopeptidase 13 (Mmp13) [17]. Runx2 is weakly expressed in chondrocytes in the resting and proliferation layers and upregulated in the prehypertrophic chondrocytes, and the expression is maintained until terminal differentiation of hypertrophic chondrocytes [17,18] (Figure 1). Chondrocyte maturation is markedly impaired in Runx2-deficient (Runx2 -/-) mice, which exhibit an absence of hypertrophic chondrocytes in most of the skeleton, except for the tibia, fibula, radius, and ulna. Overexpression of Runx2 under the control of the Col2a1 promoter, which directs the transgene to chondrocytes, accelerates chondrocyte maturation and endochondral ossification, and chondrocytes in the presumptive permanent cartilage, including articular cartilage, also enter the process of endochondral ossification [19,20]. In contrast, as overexpression of dominantnegative Runx2 under the control of the Col2a1 promoter decelerates chondrocyte maturation, most of the endochondral skeletons are composed of immature chondrocytes, and the immature chondrocytes express tenascin-C (Tnc), which is expressed in the permanent cartilage of wild-type mice [19]. Conditional knockout mice using Col2a1 Cre transgenic mice to delete Runx2 in cartilage (Col2a1 Cre Runx2 fl/fl mice) also exhibit inhibition of chondrocyte maturation [21,22].   Runx3 is expressed in prehypertrophic and hypertrophic chondrocytes, and chondrocyte maturation is slightly delayed in Runx3 −/− embryos. Furthermore, Runx2 −/− Runx3 −/− mice show a complete absence of chondrocyte maturation, indicating that Runx3 is also involved in chondrocyte maturation, and Runx2 and Runx3 are essential for chondrocyte maturation [5] (Figure 1). Runx2 is involved in chondrocyte proliferation by directly regulating the expression of Ihh, which enhances chondrocyte proliferation [5,23] (Figure 1). In addition, Runx2 interacts with doublesex and mab-3 related transcription factor 2 (Dmrt2) and regulates chondrocyte maturation and Ihh expression [24]. As Ihh induces parathyroid hormone-like hormone (Pthlh), which is expressed in the epiphysis and inhibits chondrocyte maturation by suppressing Runx2 expression through Pth1r, a negative feedback loop of Runx2-Ihh-Pthlh-Pth1r is formed in the regulation of chondrocyte maturation by Runx2 [25][26][27] (Figure 1).

Functions of Runx2 in Terminal Hypertrophic Chondrocytes
As chondrocyte hypertrophy is inhibited in Runx2 −/− mice, dominant-negative Runx2 transgenic mice under the control of Col2a1 promoter, and Col2a1 Cre Runx2 fl/fl mice [17][18][19]21,22], the function of Runx2 in the terminal hypertrophic chondrocytes was not clear. After chondrocyte maturation, the vascular invasion occurs at the layer of the terminal hypertrophic chondrocytes, which express Vegfa. As vascular invasion into the cartilage did not occur in Runx2 −/− mice and Vegfa expression was reduced in the terminal hypertrophic chondrocytes, the induction of Vegfa expression by Runx2 in the terminal hypertrophic chondrocytes was considered to be required for the vascular invasion into the cartilage [17,18,28,29]. In Runx2 conditional knockout mice using Col10a1 Cre transgenic mice (Col10a1 Cre Runx2 fl/fl mice) to delete Runx2 in hypertrophic chondrocytes, chondrocyte maturation to terminal hypertrophic chondrocytes and vascular invasion into the cartilage occurred normally, although Vegfa expression in the terminal hypertrophic chondrocytes was reduced [30]. Moreover, Vegfa is strongly expressed in osteoblasts [30]. Ihh, which is expressed in the prehypertrophic chondrocytes, induces Runx2 expression and osteoblast differentiation in the perichondrium, leading to the formation of the bone collar [23,31] (Figure 1). As Ihh was normally expressed in the prehypertrophic chondrocytes of Col10a1 Cre Runx2 fl/fl mice, osteoblast differentiation in the perichondrium and bone collar formation normally occurred, and Vegfa was strongly expressed in the osteoblasts. Thus, Vegfa expression in osteoblasts in the bone collar is sufficient for vascular invasion into the cartilage [30].
Spp1 and Ibsp, which are noncollagenous matrix proteins, are expressed in terminal hypertrophic chondrocytes and osteoblasts, and Mmp13, which is a matrix metalloproteinase that cleaves type II collagen over type I and III, is expressed in terminal hypertrophic chondrocytes [30,32]. Spp1 and Mmp13 have been shown to be regulated by Runx2 in vitro [33][34][35][36][37][38]. Indeed, the expressions of Spp1, Ibsp, and Mmp13 are markedly reduced in Runx2 −/− mice due to the inhibited hypertrophy and absence of osteoblasts [17]. Therefore, the regulation of Spp1, Ibsp, and Mmp13 expression in terminal hypertrophic chondrocytes by Runx2 remains to be clarified. Although terminal differentiation of chondrocytes in Col10a1 Cre Runx2 fl/fl mice occurred normally, the expressions of Spp1, Ibsp, and Mmp13 were reduced in the terminal hypertrophic chondrocytes [30]. Thus, the expressions of Vegfa, Spp1, Ibsp, and Mmp13 in the terminal hypertrophic chondrocytes are regulated by Runx2 in vivo (Figure 1).

Runx2 and the Transdifferentiation of Chondrocytes into Osteoblasts
Although terminal hypertrophic chondrocytes were considered to die by apoptosis, cell-lineage tracing experiments using Col10a1 Cre transgenic mice revealed that the terminal hypertrophic chondrocytes in the growth plate transdifferentiate into osteoblasts or osteoblast progenitors [39][40][41][42]. Furthermore, Pthlh-positive chondrocytes in the resting layer of the growth plate and borderline chondrocytes, which are located adjacent to the perichondrium, become stromal cells and osteoblasts through the Col10a1-positive stage [43,44]. In Col10a1 Cre Runx2 fl/fl mice, the transdifferentiation was interrupted, and the frequency of apoptosis of the terminal hypertrophic chondrocytes increased. Therefore, Runx2 is required for the transdifferentiation and survival of the terminal hypertrophic chondrocytes [30] (Figure 1). The trabecular bone formation was delayed in Col10a1 Cre Runx2 fl/fl embryos and newborn mice. Although the frequencies of the transdifferentiated osteoblasts in the trabecular bone and endosteum of cortical bone in femurs or tibiae ranged from 15 to 60% depending on the reports [30,40,41], Col10a1 Cre Runx2 fl/fl mice between 6 and 20 weeks old had similar bone volume and bone formation rate in the trabecular and cortical bone of femurs compared with those in wild-type mice of the same age [30]. Thus, transdifferentiation is required for trabecular bone formation during embryonic and newborn stages but is dispensable for acquiring normal bone mass in young and adult mice; osteoblasts derived from perichondrium/periosteum are likely to be a major source of osteoblasts after birth (Figure 1).

Figure 2.
Regulation of proliferation, differentiation, and bone matrix protein gene expression by Runx2 during osteoblast differentiation: Runx2 induces the commitment of multipotent mesenchymal cells to preosteoblasts and enhances the proliferation of osteoblast progenitors by inducing the expressions of hedgehog, fibroblast grow factor (Fgf), Wnt, and Pthlh signaling pathway genes and distal-less homeobox 5 (Dlx5), which also enhance Runx2 expression and Runx2 protein activity. Runx2 also induces Sp7 expression. Runx2, Sp7 and Wnt signaling induce the differentiation of preosteoblasts into immature osteoblasts. Sp7 and Wnt signaling activate the osteoblast-specific Runx2 enhancer. In immature osteoblasts, Runx2 induces the expressions of bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and bone gamma carboxyglutamate protein (Bglap)/Bglap2, and the osteoblasts maturate and produce abundant bone matrix proteins. Runx2 also induces tumor necrosis factor superfamily member 11 (Tnfsf11) expression and enhances bone resorption. Osteocalcin (Bglap/Bglap2) aligns the apatite crystals parallel to collagen fibers. The collagen fibers run along the longitudinal direction in the dialysis of long bone, which is formed by mature osteoblasts, whereas collagen fibers run irregularly in metaphyseal cortical bone in young mice, which is formed by immature osteoblasts. However, apatite crystals are always aligned parallel to the collagen fibers by osteocalcin [63].

Runx2 and Proliferation of Osteoblast Progenitors
Mesenchymal cells derived from the presumptive calvaria of Runx2 -/embryos proliferate faster than osteoblastic cells derived from wild-type calvaria in vitro [64]. Therefore, Runx2 was considered to inhibit the proliferation of osteoblast progenitors. However, the number of mesenchymal cells in the presumptive calvaria, mandible, and limbs in Runx2 -/embryos is extremely low. Although there are no osteoblasts in the whole skeleton of Sp7 -/embryos and Runx2 -/embryos, Sp7 -/embryos have sufficient preosteoblasts in the calvaria, mandible, and limbs, in which Runx2 is expressed [65]. Furthermore, the amount of the preosteoblasts in Sp7 -/-Runx2 +/embryos is half that of Sp7 -/embryos, indicating that the amount of the preosteoblasts in Sp7 -/embryos are dependent on the gene dosage of Runx2. Runx2 induces the proliferation of osteoblast progenitors and enhances the proliferation induced by Fgf2. In addition, Runx2 directly regulates the expression of Fgfr2 and Fgfr3 [65]. Thus, Runx2 is required for the proliferation of osteoblast progenitors and induces it, at least in part, through the induction of Fgfr2 and Fgfr3 expression ( Figure 2).

Runx2 Functions in Differentiated Osteoblasts
Runx2 induces the expression of bone matrix protein genes, including Col1a1, Spp1, Ibsp, and Bglap/Bglap2, in vitro [66]. However, the phenotypes of Runx2 conditional knockout mice using Cre transgenic mice under the control of the 2.3 kb Col1a1 promoter,  Figure 2. Regulation of proliferation, differentiation, and bone matrix protein gene expression by Runx2 during osteoblast differentiation: Runx2 induces the commitment of multipotent mesenchymal cells to preosteoblasts and enhances the proliferation of osteoblast progenitors by inducing the expressions of hedgehog, fibroblast grow factor (Fgf), Wnt, and Pthlh signaling pathway genes and distal-less homeobox 5 (Dlx5), which also enhance Runx2 expression and Runx2 protein activity. Runx2 also induces Sp7 expression. Runx2, Sp7 and Wnt signaling induce the differentiation of preosteoblasts into immature osteoblasts. Sp7 and Wnt signaling activate the osteoblast-specific Runx2 enhancer. In immature osteoblasts, Runx2 induces the expressions of bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and bone gamma carboxyglutamate protein (Bglap)/Bglap2, and the osteoblasts maturate and produce abundant bone matrix proteins. Runx2 also induces tumor necrosis factor superfamily member 11 (Tnfsf11) expression and enhances bone resorption. Osteocalcin (Bglap/Bglap2) aligns the apatite crystals parallel to collagen fibers. The collagen fibers run along the longitudinal direction in the dialysis of long bone, which is formed by mature osteoblasts, whereas collagen fibers run irregularly in metaphyseal cortical bone in young mice, which is formed by immature osteoblasts. However, apatite crystals are always aligned parallel to the collagen fibers by osteocalcin [63].

Runx2 and Proliferation of Osteoblast Progenitors
Mesenchymal cells derived from the presumptive calvaria of Runx2 −/− embryos proliferate faster than osteoblastic cells derived from wild-type calvaria in vitro [64]. Therefore, Runx2 was considered to inhibit the proliferation of osteoblast progenitors. However, the number of mesenchymal cells in the presumptive calvaria, mandible, and limbs in Runx2 −/− embryos is extremely low. Although there are no osteoblasts in the whole skeleton of Sp7 −/− embryos and Runx2 −/− embryos, Sp7 −/− embryos have sufficient preosteoblasts in the calvaria, mandible, and limbs, in which Runx2 is expressed [65]. Furthermore, the amount of the preosteoblasts in Sp7 −/− Runx2 +/− embryos is half that of Sp7 −/− embryos, indicating that the amount of the preosteoblasts in Sp7 −/− embryos are dependent on the gene dosage of Runx2. Runx2 induces the proliferation of osteoblast progenitors and enhances the proliferation induced by Fgf2. In addition, Runx2 directly regulates the expression of Fgfr2 and Fgfr3 [65]. Thus, Runx2 is required for the proliferation of osteoblast progenitors and induces it, at least in part, through the induction of Fgfr2 and Fgfr3 expression (Figure 2).

Runx2 Functions in Differentiated Osteoblasts
Runx2 induces the expression of bone matrix protein genes, including Col1a1, Spp1, Ibsp, and Bglap/Bglap2, in vitro [66]. However, the phenotypes of Runx2 conditional knockout mice using Cre transgenic mice under the control of the 2.3 kb Col1a1 promoter, which directs the Cre expression in differentiated osteoblasts, were controversial in two groups, although they used the same Col1a1 Cre transgenic mouse line [21,67]. The deletion of the runt domain, which is essential for DNA binding, in osteoblasts resulted in no phenotypes [21], whereas the deletion of exon 8, which creates a cryptic Runx2 protein that maintains DNA binding capacity but has a lower capacity for transcriptional activation, in osteoblasts results in reduced bone mass [67]. The cryptic Runx2 protein may have interrupted the binding of Runx3, which regulates osteoblast proliferation [6].
Runx2 conditional knockout mice have been generated by deleting the runt domain using newly generated 2.3 kb Col1a1 Cre transgenic mice [68]. The body weight was lower, clavicles were shorter, the unmineralized area in calvaria was wider, and incisors were shorter in the Runx2 conditional knockout mice compared with those in the control mice at 6 weeks of age. The trabecular bone volume was lower and cortical bone was thinner in femurs; the bone formation was lower in the trabecular and cortical bone; the bone volume of vertebrae was lower, and the serum markers for bone formation and resorption were lower in the Runx2 conditional knockout mice than those in the control mice. These observations were seen in both males and females. Furthermore, osteoblast proliferation was reduced in the Runx2 conditional knockout mice. The expressions of bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap/Bglap2, were reduced in the Runx2 conditional knockout mice and the osteoblasts had a lower amount of cytoplasm compared with those in control mice, indicating that Runx2 is required for the major bone matrix protein gene expression ( Figure 2). Moreover, the expressions of Runx2 target genes, including Ihh, Ptch1, Fgfr1, Fgfr2, Fgfr3, Tcf7, Wnt10b, Pth1r, Sp7, and Dlx5, were also reduced. Thus, Runx2 regulates Ihh, Fgf, Wnt, and Pthlh signaling pathways and the expression of transcription factors, Sp7 and Dlx5, after differentiation into osteoblasts and during osteoblast differentiation. As hedgehog and Wnt signaling and Sp7 are also involved in bone formation by mature osteoblasts [69][70][71][72], Runx2 regulates bone formation not only through the direct regulation of bone matrix protein gene expression but also through the regulation of these target gene expressions. The proteins transcribed by these target genes also regulate Runx2 gene transcription and Runx2 protein activity and stability [36,[56][57][58][59][60][61]. In addition, Runx2 enhances bone resorption through the regulation of Tnfsf11 [68,73] ( Figure 2).
Osteocalcin is the most abundant non-collagenous bone matrix protein and is transcribed from Bglap and Bglap2, whose expressions are directly regulated by Runx2 [63]. Bglap/Bglap2 −/− mice are normal in bone mass, bone formation, and bone resorption [74,75]. Although collagen fibers run along the longitudinal direction of the long bone in wild-type and Bglap/Bglap2 −/− mice, the orientation of apatite crystals, which is parallel to collagen fibers in wild-type mice, is markedly disrupted in Bglap/Bglap2 −/− mice, and the bone strength is reduced [74]. Therefore, osteocalcin is required for the alignment of apatite crystals parallel to collagen fibers ( Figure 2). Although osteocalcin has been shown to regulate glucose metabolism, testosterone synthesis, and muscle mass, these are normal in Bglap/Bglap2 −/− mice [74,75]. Thus, osteocalcin is important for bone quality but does not work as a hormone [63].

Conclusions
Runx2 has multiple functions in skeletal development, including chondrocyte maturation, osteoblast differentiation, the proliferation of chondrocytes and osteoblast progenitors, transdifferentiation of chondrocytes, and matrix protein gene expression in chondrocytes and osteoblasts. Runx2 is a good target for the treatment of osteoporosis by increasing bone formation. As Runx2 has multiple functions in osteoblasts and chondrocytes, however, it is important to specifically upregulate Runx2 expression or activity in osteoblast lineage cells. The upregulation of Runx2 in the articular cartilage will lead to osteoarthritis due to the maturation of chondrocytes and degradation of the cartilage matrix [19,76]. In contrast, the suppression of Runx2 in the articular cartilage will be beneficial for osteoarthritis [77][78][79]. Thus, the lineage-specific regulation of Runx2 is required for clinical application.

Conflicts of Interest:
The author declares no conflict of interest.