Runx2 Regulates Galnt3 and Fgf23 Expressions and Galnt3 Decelerates Osteoid Mineralization by Stabilizing Fgf23.

Runx2 (runt related transcription factor 2) is an essential transcription factor for osteoblast proliferation and differentiation. Uridine diphosphate (UDP)-N-acetylgalactosamine (GalNAc): polypeptide GalNAc-transferase 3 (Galnt3) prevents proteolytic processing of fibroblast growth factor 23 (Fgf23), which is a hormone that regulates the serum level of phosphorus. Runx2 and Galnt3 were expressed in osteoblasts and osteocytes, and Fgf23 expression was restricted to osteocytes in bone. Overexpression and knock-down of Runx2 upregulated and downregulated, respectively, the expressions of Galnt3 and Fgf23, and Runx2 directly regulated the transcriptional activity of Galnt3 in reporter assays. The expressions of Galnt3 and Fgf23 in osteoblast-specific Runx2 knockout (Runx2fl/flCre) mice were about half those in Runx2fl/fl mice. However, the serum levels of phosphorus and intact Fgf23 in Runx2fl/flCre mice were similar to those in Runx2fl/fl mice. The trabecular bone volume was increased during aging in both male and female Galnt3-/- mice, but the osteoid was reduced. The markers for bone formation and resorption in Galnt3-/- mice were similar to the control in both sexes. Galnt3-/- mice exhibited hyperphosphatemia and hypercalcemia, and the intact Fgf23 was about 40% that of wild-type mice. These findings indicated that Runx2 regulates the expressions of Galnt3 and Fgf23 and that Galnt3 decelerates the mineralization of osteoid by stabilizing Fgf23.

Injectable phase-separated tetra-armed poly(ethylene glycol) hydrogel scaffold allows sustained release of growth factors to enhance the repair of critical bone defects.

With the rising prevalence of bone-related injuries, it is crucial to improve treatments for fractures and defects. Tissue engineering offers a promising solution in the form of injectable hydrogel scaffolds that can sustain the release of growth factors like bone morphogenetic protein-2 (BMP-2) for bone repair. Recently, we discovered that tetra-PEG hydrogels (Tetra gels) undergo gel-gel phase separation (GGPS) at low polymer content, resulting in hydrophobicity and tissue affinity. In this work, we examined the potential of a newer class of gel, the oligo-tetra-PEG gel (Oligo gel), as a growth factor-releasing scaffold. We investigated the extent of GGPS occurring in the two gels and assessed their ability to sustain BMP-2 release and osteogenic potential in a mouse calvarial defect model. The Oligo gel underwent a greater degree of GGPS than the Tetra gel, exhibiting higher turbidity, hydrophobicity, and pore formation. The Oligo gel demonstrated sustained protein or growth factor release over a 21-day period from protein release kinetics and osteogenic cell differentiation studies. Finally, BMP-2-loaded Oligo gels achieved complete regeneration of critical-sized calvarial defects within 28 days, significantly outperforming Tetra gels. The easy formulation, injectability, and capacity for sustained release makes the Oligo gel a promising candidate therapeutic biomaterial.

Runt-related Transcription Factors and Gene Regulatory Mechanisms in Skeletal Development and Diseases.

PURPOSE OF REVIEW: Runt-related transcription factors (RUNX) play critical roles in skeletal development, metabolism, and diseases. In mammals, three RUNX members, namely RUNX1, RUNX2, and RUNX3, play distinct and redundant roles, although RUNX2 is a dominant factor in skeletal development and several skeletal diseases. This review is to provide an overview of the current understanding of RUNX-mediated transcriptional regulation in different skeletal cell types. RECENT FINDINGS: Advances in chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) have revealed genome-wide RUNX-mediated gene regulatory mechanisms, including their association with cis-regulatory elements and putative target genes. Further studies with genome-wide analysis and biochemical assays have shed light on RUNX-mediated pioneering action and involvements of RUNX2 in lipid-lipid phase separation. Emerging multi-layered mechanisms of RUNX-mediated gene regulations help us better understanding of skeletal development and diseases, which also provides clues to think how genome-wide studies can help develop therapeutic strategies for skeletal diseases.

Emerging RUNX2-Mediated Gene Regulatory Mechanisms Consisting of Multi-Layered Regulatory Networks in Skeletal Development.

Skeletal development is tightly coordinated by chondrocytes and osteoblasts, which are derived from skeletal progenitors, and distinct cell-type gene regulatory programs underlie the specification and differentiation of cells. Runt-related transcription factor 2 (Runx2) is essential to chondrocyte hypertrophy and osteoblast differentiation. Genetic studies have revealed the biological functions of Runx2 and its involvement in skeletal genetic diseases. Meanwhile, molecular biology has provided a framework for our understanding of RUNX2-mediated transactivation at a limited number of cis-regulatory elements. Furthermore, studies using next-generation sequencing (NGS) have provided information on RUNX2-mediated gene regulation at the genome level and novel insights into the multiple layers of gene regulatory mechanisms, including the modes of action of RUNX2, chromatin accessibility, the concept of pioneer factors and phase separation, and three-dimensional chromatin organization. In this review, I summarize the emerging RUNX2-mediated regulatory mechanism from a multi-layer perspective and discuss future perspectives for applications in the treatment of skeletal diseases.

Runx2 and Runx3 differentially regulate articular chondrocytes during surgically induced osteoarthritis development.

The Runt-related transcription factor (Runx) family plays various roles in the homeostasis of cartilage. Here, we examined the role of Runx2 and Runx3 for osteoarthritis development in vivo and in vitro. Runx3-knockout mice exhibited accelerated osteoarthritis following surgical induction, accompanied by decreased expression of lubricin and aggrecan. Meanwhile, Runx2 conditional knockout mice showed biphasic phenotypes: heterozygous knockout inhibited osteoarthritis and decreased matrix metallopeptidase 13 (Mmp13) expression, while homozygous knockout of Runx2 accelerated osteoarthritis and reduced type II collagen (Col2a1) expression. Comprehensive transcriptional analyses revealed lubricin and aggrecan as transcriptional target genes of Runx3, and indicated that Runx2 sustained Col2a1 expression through an intron 6 enhancer when Sox9 was decreased. Intra-articular administration of Runx3 adenovirus ameliorated development of surgically induced osteoarthritis. Runx3 protects adult articular cartilage through extracellular matrix protein production under normal conditions, while Runx2 exerts both catabolic and anabolic effects under the inflammatory condition.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

The transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Involvement of activator protein-1 family members in ß-catenin and p300 association on the genome of PANC-1 cells.

Wnt/ß-catenin is believed to regulate different sets of genes with different coactivators, cAMP response element-binding protein (CREB)-binding protein (CBP) or p300. However, the factors that determine which coactivators act on a particular promoter remain elusive. ICG-001 is a specific inhibitor for ß-catenin/CBP but not for ß-catenin/p300. By taking advantage of the action of ICG-001, we sought to investigate regulatory mechanisms underlying ß-catenin coactivator usage in human pancreatic carcinoma PANC-1 cells through combinatorial analysis of chromatin immunoprecipitation-sequencing and RNA-sequencing. CBP and p300 preferentially bound to regions with the TCF motif alone and with both the TCF and AP-1 motifs, respectively. ICG-001 increased ß-catenin binding to regions with both the TCF and AP-1 motifs, flanking the genes induced by ICG-001, concomitant with the increments of the p300 and AP-1 component c-JUN binding. Taken together, AP-1 possibly coordinates ß-catenin coactivator usage in PANC-1 cells. These results would further our understanding of the canonical Wnt/ß-catenin signaling divergence.

Mesenchymal stromal cells in the bone marrow niche consist of multi-populations with distinct transcriptional and epigenetic properties.

Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.

Induction and expansion of human PRRX1+ limb-bud-like mesenchymal cells from pluripotent stem cells.

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.

The Progress of Stem Cell Technology for Skeletal Regeneration.

Skeletal disorders, such as osteoarthritis and bone fractures, are among the major conditions that can compromise the quality of daily life of elderly individuals. To treat them, regenerative therapies using skeletal cells have been an attractive choice for patients with unmet clinical needs. Currently, there are two major strategies to prepare the cell sources. The first is to use induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), which can recapitulate the skeletal developmental process and differentiate into various skeletal cells. Skeletal tissues are derived from three distinct origins: the neural crest, paraxial mesoderm, and lateral plate mesoderm. Thus, various protocols have been proposed to recapitulate the sequential process of skeletal development. The second strategy is to extract stem cells from skeletal tissues. In addition to mesenchymal stem/stromal cells (MSCs), multiple cell types have been identified as alternative cell sources. These cells have distinct multipotent properties allowing them to differentiate into skeletal cells and various potential applications for skeletal regeneration. In this review, we summarize state-of-the-art research in stem cell differentiation based on the understanding of embryogenic skeletal development and stem cells existing in skeletal tissues. We then discuss the potential applications of these cell types for regenerative medicine.

Hedgehog Activation Regulates Human Osteoblastogenesis.

Two genetic diseases, Gorlin syndrome and McCune-Albright syndrome (MAS), show completely opposite symptoms in terms of bone mineral density and hedgehog (Hh) activity. In this study, we utilized human induced pluripotent stem cell (iPSC)-based models of the two diseases to understand the roles of Hh signaling in osteogenesis. Gorlin syndrome-derived iPSCs showed increased osteoblastogenesis and mineralization with Hh signaling activation and upregulation of a set of transcription factors in an osteogenic culture, compared with the isogenic control. MAS-specific iPSCs showed poor mineralization with low Hh signaling activity in the osteogenic culture; impaired osteoblastogenesis was restored to the normal level by treatment with an Hh signaling-activating small molecule. These data suggest that Hh signaling is a key controller for differentiation of osteoblasts from precursors. This study may pave a path to new drug therapies for genetic abnormalities in calcification caused by dysregulation of Hh signaling.

Antxr1, Which is a Target of Runx2, Regulates Chondrocyte Proliferation and Apoptosis.

Antxr1/Tem8 is highly expressed in tumor endothelial cells and is a receptor for anthrax toxin. Mutation of Antxr1 causes GAPO syndrome, which is characterized by growth retardation, alopecia, pseudo-anodontia, and optic atrophy. However, the mechanism underlying the growth retardation remains to be clarified. Runx2 is essential for osteoblast differentiation and chondrocyte maturation and regulates chondrocyte proliferation through Ihh induction. In the search of Runx2 target genes in chondrocytes, we found that Antxr1 expression is upregulated by Runx2. Antxr1 was highly expressed in cartilaginous tissues and was directly regulated by Runx2. In skeletal development, the process of endochondral ossification proceeded similarly in wild-type and Antxr1-/- mice. However, the limbs of Antxr1-/- mice were shorter than those of wild-type mice from embryonic day 16.5 due to the reduced chondrocyte proliferation. Chondrocyte-specific Antxr1 transgenic mice exhibited shortened limbs, although the process of endochondral ossification proceeded as in wild-type mice. BrdU-uptake and apoptosis were both increased in chondrocytes, and the apoptosis-high regions were mineralized. These findings indicated that Antxr1, of which the expression is regulated by Runx2, plays an important role in chondrocyte proliferation and that overexpression of Antxr1 causes chondrocyte apoptosis accompanied by matrix mineralization.

Generation of orthotopically functional salivary gland from embryonic stem cells.

Organoids generated from pluripotent stem cells are used in the development of organ replacement regenerative therapy by recapitulating the process of organogenesis. These processes are strictly regulated by morphogen signalling and transcriptional networks. However, the precise transcription factors involved in the organogenesis of exocrine glands, including salivary glands, remain unknown. Here, we identify a specific combination of two transcription factors (Sox9 and Foxc1) responsible for the differentiation of mouse embryonic stem cell-derived oral ectoderm into the salivary gland rudiment in an organoid culture system. Following orthotopic transplantation into mice whose salivary glands had been removed, the induced salivary gland rudiment not only showed a similar morphology and gene expression profile to those of the embryonic salivary gland rudiment of normal mice but also exhibited characteristics of mature salivary glands, including saliva secretion. This study suggests that exocrine glands can be induced from pluripotent stem cells for organ replacement regenerative therapy.

Bone regeneration by human dental pulp stem cells using a helioxanthin derivative and cell-sheet technology.

BACKGROUND: Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells (BMSCs). Meanwhile, the treatment of severe bone defects, such as fractures, cancers, and congenital abnormalities, remains a great challenge, and novel bone regenerative techniques are highly anticipated. Several studies have previously shown that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of preosteoblastic and mesenchymal cells. However, the osteogenic differentiation activities of TH have only been confirmed in some mouse cell lines. Therefore, in this study, toward the clinical use of TH in humans, we analyzed the effect of TH on the osteogenic differentiation of DPSCs, and the in-vivo osteogenesis ability of TH-induced DPSCs, taking advantage of the simple transplantation system using cell-sheet technology. METHODS: DPSCs were obtained from dental pulp of the wisdom teeth of five healthy patients (18-22 years old) and cultured in regular medium and osteogenic medium with or without TH. To evaluate osteogenesis of TH-induced DPSCs in vivo, we transplanted DPSC sheets into mouse calvaria defects. RESULTS: We demonstrated that osteogenic conditions with TH induce the osteogenic differentiation of DPSCs more efficiently than those without TH and those with bone morphogenetic protein-2. However, regular medium with TH did not induce the osteogenic differentiation of DPSCs. TH induced osteogenesis in both DPSCs and BMSCs, although the gene expression pattern in DPSCs differed from that in BMSCs up to 14 days after induction with TH. Furthermore, we succeeded in bone regeneration in vivo using DPSC sheets with TH treatment, without using any scaffolds or growth factors. CONCLUSIONS: Our results demonstrate that TH-induced DPSCs are a useful cell source for bone regenerative medicine, and the transplantation of DPSC sheets treated with TH is a convenient scaffold-free method of bone healing.

Repair of segmental radial defects in dogs using tailor-made titanium mesh cages with plates combined with calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel.

Repair of large segmental defects of long bones are a tremendous challenge that calls for a novel approach to supporting immediate weight bearing and bone regeneration. This study investigated the functional and biological characteristics of a combination of a tailor-made titanium mesh cage with a plate (tTMCP) with tetrapod-shaped alpha tricalcium phosphate granules (TB) and basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) to repair 20-mm segmental radial defects in dogs. The defects were created surgically in 18 adult beagle dogs and treated by implantation of tTMCPs with TB with (TB-gel group) or without (TB group) f-IC gel. Each tTMCP fitted the defect well, and all dogs could bear weight on the affected limb immediately after surgery. Dogs were euthanized 4, 8 and 24 weeks after implantation. Histomorphometry showed greater infiltration of new vessels and higher bone union rate in the TB-gel group than in the TB group. The lamellar bone volume and mineral apposition rate did not differ significantly between the groups, indicating that neovascularization may be the primary effect of f-IC gel on bone regeneration. This combination method which is tTMCP combined with TB and f-IC gel, would be useful for the treatment of segmental long bone defects.

Local administration of a hedgehog agonist accelerates fracture healing in a mouse model.

Bone fracture healing is processed through multiple biological stages including the transition from cartilaginous callus to bony callus formation. Because of its specific, temporal and indispensable functions demonstrated by mouse genetic studies, Hedgehog (Hh) signaling is one of the most potent signaling pathways involved in these processes, but the effect of Hh-signaling activation by small compounds on the repair process had not yet been addressed. Here we examined therapeutic effects of local and one shot-administration of the Hh agonist known as smoothened agonist (SAG) on bone fracture healing in a mouse model. A quantitative analysis with three-dimensional micro-computed tomography showed that SAG administration increased the size of both the cartilaginous callus and bony callus at 14 days after the surgery. A histological analysis showed that SAG administration increased the number of cells expressing a proliferation marker and a chondrocyte marker in cartilaginous callus as well as the cells expressing an osteoblast marker in bony callus. These results indicate that the SAG administration resulted in an enhancement of callus formation during bone fracture healing, which is at least in part mediated by an increase in chondrocyte proliferation in cartilaginous callus and the promotion of bone formation in bony callus. Therapeutic strategies with a SAG-mediated protocol may thus be useful for the treatment of bone fractures.

Distinct Transcriptional Programs Underlie Sox9 Regulation of the Mammalian Chondrocyte.

Sox9 encodes an essential transcriptional regulator of chondrocyte specification and differentiation. When Sox9 nuclear activity was compared with markers of chromatin organization and transcriptional activity in primary chondrocytes, we identified two distinct categories of target association. Class I sites cluster around the transcriptional start sites of highly expressed genes with no chondrocyte-specific signature. Here, Sox9 association reflects protein-protein association with basal transcriptional components. Class II sites highlight evolutionarily conserved active enhancers that direct chondrocyte-related gene activity through the direct binding of Sox9 dimer complexes to DNA. Sox9 binds through sites with sub-optimal binding affinity; the number and grouping of enhancers into super-enhancer clusters likely determines the levels of target gene expression. Interestingly, comparison of Sox9 action in distinct chondrocyte lineages points to similar regulatory strategies. In addition to providing insights into Sox family action, our comprehensive identification of the chondrocyte regulatory genome will facilitate the study of skeletal development and human disease.

Gli1 haploinsufficiency leads to decreased bone mass with an uncoupling of bone metabolism in adult mice.

Hedgehog (Hh) signaling plays important roles in various development processes. This signaling is necessary for osteoblast formation during endochondral ossification. In contrast to the established roles of Hh signaling in embryonic bone formation, evidence of its roles in adult bone homeostasis is not complete. Here we report the involvement of Gli1, a transcriptional activator induced by Hh signaling activation, in postnatal bone homeostasis under physiological and pathological conditions. Skeletal analyses of Gli1+/- adult mice revealed that Gli1 haploinsufficiency caused decreased bone mass with reduced bone formation and accelerated bone resorption, suggesting an uncoupling of bone metabolism. Hh-mediated osteoblast differentiation was largely impaired in cultures of Gli1+/- precursors, and the impairment was rescued by Gli1 expression via adenoviral transduction. In addition, Gli1+/- precursors showed premature differentiation into osteocytes and increased ability to support osteoclastogenesis. When we compared fracture healing between wild-type and Gli1+/- adult mice, we found that the Gli1+/- mice exhibited impaired fracture healing with insufficient soft callus formation. These data suggest that Gli1, acting downstream of Hh signaling, contributes to adult bone metabolism, in which this molecule not only promotes osteoblast differentiation but also represses osteoblast maturation toward osteocytes to maintain normal bone homeostasis.

Bone regeneration by the combined use of tetrapod-shaped calcium phosphate granules with basic fibroblast growth factor-binding ion complex gel in canine segmental radial defects.

The effect of tetrapod-shaped alpha tricalcium phosphate granules (Tetrabones(®) [TB]) in combination with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on bone defect repair was examined. Bilateral segmental defects 20-mm long were created in the radius of 5 dogs, stabilized with a plate and screws and implanted with 1 of the following: TB (TB group), TB and bFGF solution (TB/f group), and TB and f-IC gel (TB/f-IC group). Dogs were euthanized 4 weeks after surgery. Radiographs showed well-placed TB granules in the defects and equal osseous callus formation in all the groups. Histomorphometry revealed that the number of vessels and volume of new bone in the TB/f-IC group were significantly higher than those in the other groups. However, no significant differences in neovascularization and new bone formation were observed between the TB/f and TB groups. Furthermore, no significant difference in the lamellar bone volume or rate of mineral apposition was observed among groups. These results suggest that increased bone formation might have been because of the promotion of neovascularization by the f-IC gel. Therefore, the combinatorial method may provide a suitable scaffold for bone regeneration in large segmental long bone defects.

Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation.

Induced pluripotent stem cells (iPSC) are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC) marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.