The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

Tooth transplantation and replantation: Biological insights towards therapeutic improvements.

Transplantation and replantation of teeth are effective therapeutic approaches for tooth repositioning and avulsion, respectively. Transplantation involves transplanting an extracted tooth from the original site into another site, regenerating tissue including the periodontal ligament (PDL) and alveolar bone, around the transplanted tooth. Replantation places the avulsed tooth back to its original site, regenerating functional periodontal tissue. In clinical settings, transplantation and replantation result in favorable outcomes with regenerated PDL tissue in many cases. However, they often result in poor outcomes with two major complications: tooth ankylosis and root resorption. In tooth ankylosis, the root surface and alveolar bone are fused, reducing the PDL tissue between them. The root is subjected to remodeling processes and is partially replaced by bone. In severe cases, the resorbed root is completely replaced by bone tissue, which is called as "replacement resorption." Resorption is sometimes accompanied by infection-mediated inflammation. The molecular mechanisms of ankylosis and root resorption remain unclear, although some signaling mechanisms have been proposed. In this mini-review, we summarized the biological basis of repair mechanisms of tissues in transplantation and replantation and the pathogenesis of their healing failure. We also discussed possible therapeutic interventions to improve treatment success rates.

Profilin-1 negatively controls osteoclast migration by suppressing the protrusive structures based on branched actin filaments.

BACKGROUND: Profilin-1 (Pfn1), an evolutionarily conserved actin-binding protein, is an important regulator of the cytoskeleton. We previously reported the osteoclast-specific Pfn1-conditional knockout (cKO) mice had postnatal osteolytic phenotype with craniofacial and long-bone deformities associated with increased migration of cultured osteoclasts. We hypothesized the increased cellular processes structured with branched actin filaments may underlies the mechanism of increased bone resorption in these mutant mice. MATERIALS AND METHODS: The morphological structure and cell migration of the cultured osteoclasts were analyzed using fluorescent microscopy and time-lapse image capturing. Fractional migration distances, as well as the index of protrusive structures (%-PB) that evaluates relative border length of the protrusion were compared between the cells from control and Pfn1-cKO mice. RESULTS: Time-lapse image analysis showed that %-PB was significantly larger in Pfn1-cKO osteoclasts. In addition, the fractional migration distance was positively correlated with the index. When the branched actin filament organization was suppressed by chemical inhibitors, the osteoclast migration was declined. Importantly, the suppression was more extensive in Pfn1-cKO than in control osteoclasts. CONCLUSION: Our results indicated the causative involvement of the increased branched actin filament formation at least in part for their excessive migration. Our findings provide a mechanistic rationale for testing novel therapeutic approaches targeting branched actin filaments in osteolytic disorders.

Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.

The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues. However, partial reprogramming and genetic instabilities in iPSCs could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

Essential roles of G9a in cell proliferation and differentiation during tooth development.

Teeth develop through interactions between epithelial and mesenchymal tissues mediated by a signaling network comprised of growth factors and transcription factors. However, little is known about how epigenetic modifiers affect signaling pathways and thereby regulate tooth formation. We previously reported that the histone 3 lysine 9 (H3K9) methyltransferase (MTase) G9a is specifically enriched in the tooth mesenchyme during mouse development. In this study, we investigated the functions of G9a in tooth development using G9a conditional knockout (KO) mice. We used Sox9-Cre mice to delete G9a in the tooth mesenchyme because Sox9 is highly expressed in the mesenchyme derived from the cranial neural crest. Immunohistochemical analyses revealed that G9a expression was significantly decreased in the mesenchyme of Sox9-Cre;G9afl/fl (G9a cKO) mice compared with that in Sox9-Cre;G9a fl/+(control) mice. Protein levels of the G9a substrate H3K9me2 were also decreased in the tooth mesenchyme. G9a cKO mice showed smaller tooth germ after embryonic day (E) 16.5 and E17.5, but not at E15.5. The developing cusp tips, which were visible in control mice, were absent in G9a cKO mice at E17.5. At 3 weeks after birth, small first molars with smaller cusps and unseparated roots were formed. Organ culture of tooth germs derived from E15.5 cKO mouse embryos showed impaired tooth development, suggesting that tooth development per se is affected independently of skull development. BrdU labeling experiments revealed that the proliferation rates were decreased in the mesenchyme in G9a cKO mice at E17.5. In addition, the proliferation rates in the tooth inner enamel epithelium were also decreased. In situ hybridization revealed altered localization of genes associated with tooth development. In cKO mice, intensively localized expression of mRNAs encoding bone morphogenic protein (Bmp2 and Bmp4) was observed in the tooth mesenchyme at E17.5, similar to the expression patterns observed in control mice at E15.5. Localization of Shh and related signaling components, including Gli1, Ptch1, and Ptch2, in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21, both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development.

BMP-2 Enhances Lgr4 Gene Expression in Osteoblastic Cells.

Osteoporosis is one of the most prevalent diseases and the number of patients suffering from this disease is soaring due to the increase in the aged population in the world. The severity of bone loss in osteoporosis is based on the levels of impairment in the balance between bone formation and bone resorption, two arms of the bone metabolism, and bone remodeling. However, determination of bone formation levels is under many layers of control that are as yet fully defined. Bone morphogenetic protein (BMP) plays a key role in regulation of bone formation while its downstream targets are still incompletely understood. Lgr4 gene encodes an orphan receptor and has been identified as a genetic determinant for bone mass in osteoporotic patients. Here, we examine the effects of BMP on the expression of Lgr4 in osteoblastic cells. Lgr4 gene is expressed in an osteoblastic cell line, MC3T3E1 in a time dependent manner during the culture. BMP treatment enhances Lgr4 mRNA expression at least in part via transcriptional event. When Lgr4 mRNA is knocked down, the levels of BMP-induced increase in alkaline phosphatase (Alp) activity and Alp mRNA are suppressed. BMP enhancement of Lgr4 gene expression is suppressed by FGF and reversed by dexamethasone. BMP also enhances Lgr4 expression in primary cultures of calvarial osteoblasts. These data indicate that Lgr4 gene is regulated by BMP and is required for BMP effects on osteoblastic differentiation. J. Cell. Physiol. 231: 887-895, 2016. © 2015 Wiley Periodicals, Inc.

Coordinated expression of H3K9 histone methyltransferases during tooth development in mice.

Tissue-specific gene expression is subjected to epigenetic and genetic regulation. Posttranslational modifications of histone tails alter the accessibility of nuclear proteins to DNA, thus affecting the activity of the regulatory complex of nuclear proteins. Methylation at histone 3 lysine 9 (H3K9) is a crucial modification that affects gene expression and cell differentiation. H3K9 is known to have 0-3 methylation states, and these four methylated states are determined by the expression of sets of histone methyltransferases. During development, teeth are formed through mutual interactions between the mesenchyme and epithelium via a process that is subjected to the epigenetic regulation. In this study, we examined the expression of all H3K9 methyltransferases (H3K9MTases) during mouse tooth development. We found that four H3K9MTases-G9a, Glp, Prdm2, and Suv39h1-were highly expressed in the tooth germ, with expression peaks at around embryonic days 16.5 and 17.5 in mice. Immunohistochemical and in situ hybridization analyses revealed that all four H3K9MTases were enriched in the mesenchyme more than in the epithelium. Substrates of H3K9MTases, H3K9me1, H3K9me2, and H3K9me3 were also enriched in the mesenchyme. Taken together, these data suggested that coordinated expression of four H3K9MTases in the dental mesenchyme might play important roles in tooth development.

H3K9MTase G9a is essential for the differentiation and growth of tenocytes in vitro.

Cell differentiation is controlled by specific transcription factors. The functions and expression levels of these transcription factors are regulated by epigenetic modifications, such as histone modifications and cytosine methylation of the genome. In tendon tissue, tendon-specific transcription factors have been shown to play functional roles in the regulation of tenocyte differentiation. However, the effects of epigenetic modifications on gene expression and differentiation in tenocytes are unclear. In this study, we investigated the epigenetic regulation of tenocyte differentiation, focusing on the enzymes mediating histone 3 lysine 9 (H3K9) methylation. In primary mouse tenocytes, six H3K9 methyltransferase (H3K9MTase) genes, i.e., G9a, G9a-like protein (GLP), PR domain zinc finger protein 2 (PRDM2), SUV39H1, SUV39H2, and SETDB1/ESET were all expressed, with increased mRNA levels observed during tenocyte differentiation. In mouse embryos, G9a and Prdm2 mRNAs were expressed in tenocyte precursor cells, which were overlapped with or were adjacent to cells expressing a tenocyte-specific marker, tenomodulin. Using tenocytes isolated from G9a-flox/flox mice, we deleted G9a by infecting the cells with Cre-expressing adenoviruses. Proliferation of G9a-null tenocytes was significantly decreased compared with that of control cells infected with GFP-expressing adenoviruses. Moreover, the expression levels of tendon transcription factors gene, i.e., Scleraxis (Scx), Mohawk (Mkx), Egr1, Six1, and Six2 were all suppressed in G9a-null tenocytes. The tendon-related genes Col1a1, tenomodulin, and periostin were also downregulated. Consistent with this, Western blot analysis showed that tenomodulin protein expression was significantly suppressed by G9a deletion. These results suggested that expression of the H3K9MTase G9a was essential for the differentiation and growth of tenocytes and that H3K9MTases may play important roles in tendinogenesis.

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method.

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell-cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.

Epigenetic modifier G9a is involved in regulation of mouse tongue development.

OBJECTIVES: The tongue comprises multiple tissues of different embryonic origins, including pharyngeal arch, somite, and cranial neural crest (CNC). However, its developmental regulatory mechanisms, especially those involving epigenetic modifiers, remain poorly understood. This study examined the roles of the epigenetic modifier G9a in murine tongue development. METHODS: We deleted G9a using Sox 9 (SRY-related HMG-box gene 9)-Cre recombinase, which acts in tongue progenitor cells, including CNC-derived cells, to generate G9a conditional knockout (cKO) mice. Histochemical and immunohistochemical analyses were conducted on sections prepared from tongue tissues of control and cKO mice. RESULTS: Cre-dependent LacZ reporter mice, generated by crossing Rosa-LacZ mice with sox9-Cre mice, revealed Cre recombinase activity in the mucosal epithelium and tongue connective tissue of the embryonic tongue. Tongue volume was significantly reduced on embryonic day 17.5 (E17.5) and postnatal day 0 (P0) in cKO mice. Histological sections showed that the lingual mucosal epithelium was thinner in cKO mice. Reduced G9a levels were accompanied by decreased levels of a G9a substrate, dimethylated lysine 9 in histone H3, in the embryonic tongue. BrdU injection at E16.5 revealed reduced numbers of BrdU-positive cells in the mucosal epithelium and underlying connective tissue at E17.5 in cKO mice, indicating suppression of cell proliferation in both tissues. Investigation of keratin 5 and 8 protein localization showed significantly suppressed expression in the lingual mucosal epithelium in cKO mice. CONCLUSIONS: G9a is required for proper proliferation and differentiation of sox9-expressing tongue progenitor cells and is thereby involved in tongue development.

Osteolytic Bone Loss and Skeletal Deformities in a Mouse Model for Early-Onset Paget's Disease of Bone with PFN1 Mutation Are Treatable by Alendronate.

A novel osteolytic disorder due to PFN1 mutation was discovered recently as early-onset Paget's disease of bone (PDB). Bone loss and pain in adult PDB patients have been treated using bisphosphonates. However, therapeutic strategies for this specific disorder have not been established. Here, we evaluated the efficiency of alendronate (ALN) on a mutant mouse line, recapitulating this disorder. Five-week-old conditional osteoclast-specific Pfn1-deficient mice (Pfn1-cKOOCL) and control littermates (33 females and 22 males) were injected with ALN (0.1 mg/kg) or vehicle twice weekly until 8 weeks of age. After euthanizing, bone histomorphometric parameters and skeletal deformities were analyzed using 3D µCT images and histological sections. Three weeks of ALN administration significantly improved bone mass at the distal femur, L3 vertebra, and nose in Pfn1-cKOOCL mice. Histologically increased osteoclasts with expanded distribution in the distal femur were normalized in these mice. Geometric bone shape analysis revealed a partial recovery from the distal femur deformity. A therapeutic dose of ALN from 5 to 8 weeks of age significantly improved systemic bone loss in Pfn1-cKOOCL mice and femoral bone deformity. Our study suggests that preventive treatment of bony deformity in early-onset PDB is feasible.

Improved methods for detection of ß-galactosidase (lacZ) activity in hard tissue.

The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.

Platelet-derived growth factor-BB regenerates functional periodontal ligament in the tooth replantation.

Tooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rß- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear ß-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on ß-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.

The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass by inhibiting bone morphogenetic protein-induced bone formation.

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)-induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2-induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation.

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.

Protein related to DAN and cerberus (PRDC) inhibits osteoblastic differentiation and its suppression promotes osteogenesis in vitro.

Protein related to DAN and cerberus (PRDC) is a secreted protein characterized by a cysteine knot structure, which binds bone morphogenetic proteins (BMPs) and thereby inhibits their binding to BMP receptors. As an extracellular BMP antagonist, PRDC may play critical roles in osteogenesis; however, its expression and function in osteoblastic differentiation have not been determined. Here, we investigated whether PRDC is expressed in osteoblasts and whether it regulates osteogenesis in vitro. PRDC mRNA was found to be expressed in the pre-osteoblasts of embryonic day 18.5 (E18.5) mouse calvariae. PRDC mRNA expression was elevated by treatment with BMP-2 in osteoblastic cells isolated from E18.5 calvariae (pOB cells). Forced expression of PRDC using adenovirus did not affect cell numbers, whereas it suppressed exogenous BMP activity and endogenous levels of phosphorylated Smad1/5/8 protein. Furthermore, PRDC inhibited the expression of bone marker genes and bone-like mineralized matrix deposition in pOB cells. In contrast, the reduction of PRDC expression by siRNA elevated alkaline phosphatase activity, increased endogenous levels of phosphorylated Smad1/5/8 protein, and promoted bone-like mineralized matrix deposition in pOB cells. These results suggest that PRDC expression in osteoblasts suppresses differentiation and that reduction of PRDC expression promotes osteogenesis in vitro. PRDC is accordingly identified as a potential novel therapeutic target for the regulation of bone formation.

Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function.

Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

G9a is involved in the regulation of cranial bone formation through activation of Runx2 function during development.

The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9aflox/flox (fl/fl) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9afl/fl mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.