Inhibition of miR338 rescues cleidocranial dysplasia in Runx2 mutant mice partially via the Hif1a-Vegfa axis.

Haploinsufficiency of Runt-related transcription factor-2 (RUNX2) is responsible for cleidocranial dysplasia (CCD), a rare hereditary disease with a range of defects, including delayed closure of the cranial sutures and short stature. Symptom-based treatments, such as a combined surgical-orthodontic approach, are commonly used to treat CCD patients. However, there have been few reports of treatments based on Runx2-specific regulation targeting dwarfism symptoms. Previously, we found that the miR338 cluster, a potential diagnostic and therapeutic target for postmenopausal osteoporosis, could directly target Runx2 during osteoblast differentiation in vitro. Here, we generated miR338-/-;Runx2+/- mice to investigate whether inhibition of miR338 could rescue CCD defects caused by Runx2 mutation in vivo. We found that the dwarfism phenotype caused by Runx2 haploinsufficiency was recovered in miR338-/-;Runx2+/- mice, with complete bone density restoration and quicker closure of fontanels. Single-cell RNA-seq analysis revealed that knockout of miR338 specifically rescued the osteoblast lineage priming ability of bone marrow stromal cells in Runx2+/- femurs, which was further confirmed by Osterix-specific conditional knockout of miR338 in Runx2+/- mice (OsxCre; miR338 fl/fl;Runx2+/-). Mechanistically, ablation of the miR338 cluster in Runx2+/- femurs directly rescued the Hif1a-Vegfa pathway in Runx2+/- osteoblasts, as proven by gene expression profiles and ChIP and Re-ChIP assays. Collectively, our data revealed the genetic interaction between Runx2 and the miR338 cluster during osteoblast differentiation and implied that the miR338 cluster could be a potential therapeutic target for CCD.

The ERK MAPK Pathway Is Essential for Skeletal Development and Homeostasis.

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that function as key signal transducers of a wide spectrum of extracellular stimuli, including growth factors and pro-inflammatory cytokines. Dysregulation of the extracellular signal-regulated kinase (ERK) MAPK pathway is associated with human skeletal abnormalities including Noonan syndrome, neurofibromatosis type 1, and cardiofaciocutaneous syndrome. Here, we demonstrate that ERK activation in osteoprogenitors is required for bone formation during skeletal development and homeostasis. Deletion of Mek1 and Mek2, kinases upstream of ERK MAPK, in osteoprogenitors (Mek1OsxMek2-/-), resulted in severe osteopenia and cleidocranial dysplasia (CCD), similar to that seen in humans and mice with impaired RUNX2 function. Additionally, tamoxifen-induced deletion of Mek1 and Mek2 in osteoprogenitors in adult mice (Mek1Osx-ERTMek2-/-) significantly reduced bone mass. Mechanistically, this corresponded to decreased activation of osteoblast master regulators, including RUNX2, ATF4, and ß-catenin. Finally, we identified potential regulators of osteoblast differentiation in the ERK MAPK pathway using unbiased phospho-mass spectrometry. These observations demonstrate essential roles of ERK activation in osteogenesis and bone formation.

Runx2 regulates cranial suture closure by inducing hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in suture mesenchymal cells.

Cleidocranial dysplasia (CCD, #119600), which is characterized by hypoplastic clavicles, open fontanelles, supernumerary teeth and a short stature, is caused by heterozygous mutations in RUNX2. However, it currently remains unclear why suture closure is severely impaired in CCD patients. The closure of posterior frontal (PF) and sagittal (SAG) sutures was completely interrupted in Runx2+/- mice, and the proliferation of suture mesenchymal cells and their condensation were less than those in wild-type mice. To elucidate the underlying molecular mechanisms, differentially expressed genes between wild-type and Runx2+/- PF and SAG sutures were identified by microarray and real-time reverse transcription polymerase chain reaction analyses. The expression of hedgehog, Fgf, Wnt and Pthlh signaling pathway genes, including Gli1, Ptch1, Ihh, Fgfr2, Fgfr3, Tcf7, Wnt10b and Pth1r, which were directly regulated by Runx2, was reduced in the sutures, but not the calvarial bone tissues of Runx2+/- mice. Bone formation and suture closure were enhanced in an organ culture of Runx2+/- calvariae with ligands or agonists of hedgehog, Fgf, Wnt and Pthlh signaling, while they were suppressed and suture mesenchymal cell proliferation was decreased in an organ culture of wild-type calvariae with their antagonists. These results indicate that more than a half dosage of Runx2 is required for the proliferation of suture mesenchymal cells, their condensation and commitment to osteoblast-lineage cells, and the induction of hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in sutures, but not in calvarial bone tissues, and also that the activation of hedgehog, Fgf, Wnt and Pthlh signaling pathways is necessary for suture closure.

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

mTOR/Raptor signaling is critical for skeletogenesis in mice through the regulation of Runx2 expression.

The mammalian target of rapamycin (mTOR)/regulatory-associated protein of mTOR (Raptor) pathway transmits and integrates different signals including growth factors, nutrients, and energy metabolism. Nearly all these signals have been found to play roles in skeletal biology. However, the contribution of mTOR/Raptor to osteoblast biology in vivo remains to be elucidated as the conclusions of recent studies are controversial. Here we report that mice with a deficiency of either mTOR or Raptor in preosteoblasts exhibited clavicular hypoplasia and delayed fontanelle fusion, similar to those found in human patients with cleidocranial dysplasia (CCD) haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2) or those identified in Runx2+/- mice. Mechanistic analysis revealed that the mTOR-Raptor-S6K1 axis regulates Runx2 expression through phosphorylation of estrogen receptor α, which binds to Distal-less homeobox 5 (DLX5) and augments the activity of Runx2 enhancer. Moreover, heterozygous mutation of raptor in osteoblasts aggravates the bone defects observed in Runx2+/- mice, indicating a genetic interaction between Raptor and Runx2. Collectively, these findings reveal that mTOR/Raptor signaling is essential for bone formation in vivo through the regulation of Runx2 expression. These results also suggest that a selective mTOR/Raptor antagonist, which has been developed for treatment of many diseases, may have the side effect of causing bone loss.

Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced ß-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146-/- mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate ß-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development.

An HDAC Inhibitor, Entinostat/MS-275, Partially Prevents Delayed Cranial Suture Closure in Heterozygous Runx2 Null Mice.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder caused by mutations in RUNX2, coding a key transcription factor of early osteogenesis. CCD patients suffer from developmental defects in cranial bones. Despite numerous investigations and clinical approaches, no therapeutic strategy has been suggested to prevent CCD. Here, we show that fetal administration of Entinostat/MS-275, a class I histone deacetylase (HDAC)-specific inhibitor, partially prevents delayed closure of cranial sutures in Runx2+/- mice strain of C57BL/6J by two mechanisms: 1) posttranslational acetylation of Runx2 protein, which stabilized the protein and activated its transcriptional activity; and 2) epigenetic regulation of Runx2 and other bone marker genes. Moreover, we show that MS-275 stimulates osteoblast proliferation effectively both in vivo and in vitro, suggesting that delayed skeletal development in CCD is closely related to the decreased number of progenitor cells as well as the delayed osteogenic differentiation. These findings provide the potential benefits of the therapeutic strategy using MS-275 to prevent CCD. © 2017 American Society for Bone and Mineral Research.

RUNX2 mutation impairs bone remodelling of dental follicle cells and periodontal ligament cells in patients with cleidocranial dysplasia.

RUNX2 is an important osteo-specific factor with crucial functions in bone formation and remodelling as well as resorption of teeth. Heterozygous mutation of RUNX2 can cause cleidocranial dysplasia (CCD), a systemic disease with extensive skeletal dysplasia and abnormality of tooth growth. In our study, dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) were isolated, cultured and identified from one patient with CCD and compared with normal controls. This CCD patient was confirmed to have a heterozygous frameshift mutation of RUNX2 (c.514delT, p.Ser172fs) in the previous study. The results showed that the proliferation abilities of DFCs and PDLCs were both disturbed by the RUNX2 mutation in the CCD patient compared with the normal control. A co-culture system of these cells with human peripheral blood mononuclear cells was then used to investigate the effect of RUNX2 mutation on osteoclastogenesis. We found that the RUNX2 mutation in CCD reduced the expression of osteoclast-related genes, such as RUNX2, CTR, CTSK, RANKL and OPG The ability of osteoclastogenesis in DFCs and PDLCs detected by tartrate-resistant acid phosphatase staining in the co-culture system was also reduced by the RUNX2 mutation compared with the normal control. These outcomes indicate that the RUNX2 mutation disturbs the modulatory effects of DFCs and PDLCs on the differentiation of osteoclasts and osteoblasts, thereby interfering with bone remodelling. These effects may contribute in part to the pathological manifestations of retention of primary teeth and delayed eruption of permanent teeth in patients with CCD.

Generation of cleidocranial dysplasia-specific human induced pluripotent stem cells in completely serum-, feeder-, and integration-free culture.

Human pluripotent stem cells hold great promise for their practical and scientific potentials. To improve understanding of self-renewal and differentiation, we previously reported a defined serum-free medium hESF9 could generate and maintain human induced pluripotent stem cells (iPSCs) in serum- and feeder-free culture conditions using retroviral vectors. To avoid the unpredictable side effects associated with retrovirus integration, we report here the successful generation of hiPSCs from dental pulp cells with a non-integrating replication-defective and persistent Sendai virus (SeVdp) vector expressing four key reprogramming genes. We found that hESF9 medium in combination with fibronectin are effective for generating and maintaining hiPSCs with SeVdp (KOSM). Using this system, pluripotent and self-renewing hiPSCs could be easily and stably generated and propagated. With this system, we successfully generated hiPSCs from cleidocranial dysplasia (CCD) caused by a heterozygous germ-line mutation of runt-related protein2 (RUNX2), which has an important role in the differentiation of osteoblasts and maturation of chondrocytes. This is the first report of the establishment of CCD-specific iPSCs. The cartilage in the teratomas of CCD-iPSCs showed abnormalities. These CCD-iPSCs would be beneficial to clarify the molecular mechanism and for development of medical applications. Moreover, it brings new pathophysiological role of RUNX2 in the differentiation of the human chondrocytes and osteocytes.

Rescue of neurodegeneration in the Fig4 null mouse by a catalytically inactive FIG4 transgene.

The lipid phosphatase FIG4 is a subunit of the protein complex that regulates biosynthesis of the signaling lipid PI(3,5)P2. Mutations of FIG4 result in juvenile lethality and spongiform neurodegeneration in the mouse, and are responsible for the human disorders Charcot-Marie-Tooth disease, Yunis-Varon syndrome and polymicrogyria with seizures. We previously demonstrated that conditional expression of a wild-type FIG4 transgene in neurons is sufficient to rescue most of the abnormalities of Fig4 null mice, including juvenile lethality and extensive neurodegeneration. To evaluate the contribution of the phosphatase activity to the in vivo function of Fig4, we introduced the mutation p.Cys486Ser into the Sac phosphatase active-site motif CX5RT. Transfection of the Fig4(Cys486Ser) cDNA into cultured Fig4(-/-) fibroblasts was effective in preventing vacuolization. The neuronal expression of an NSE-Fig4(Cys486Ser) transgene in vivo prevented the neonatal neurodegeneration and juvenile lethality seen in Fig4 null mice. These observations demonstrate that the catalytically inactive FIG4 protein provides significant function, possibly by stabilization of the PI(3,5)P2 biosynthetic complex and/or localization of the complex to endolysosomal vesicles. Despite this partial rescue, later in life the NSE-Fig4(Cys486Ser) transgenic mice display significant abnormalities that include hydrocephalus, defective myelination and reduced lifespan. The late onset phenotype of the NSE-Fig4(Cys486Ser) transgenic mice demonstrates that the phosphatase activity of FIG4 has an essential role in vivo.

Characterisation of novel RUNX2 mutation with alanine tract expansion from Japanese cleidocranial dysplasia patient.

Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant skeletal dysplasia characterised by hypopalstic and/or aplastic clavicles, midface hypoplasia, absent or delayed closure of cranial sutures, moderately short stature, delayed eruption of permanent dentition and supernumerary teeth. The molecular pathogenesis can be explained in about two-thirds of CCD patients by haploinsufficiency of the RUNX2 gene. In our current study, we identified a novel and rare variant of the RUNX2 gene (c.181_189dupGCGGCGGCT) in a Japanese patient with phenotypic features of CCD. The insertion led an alanine tripeptide expansion (+3Ala) in the polyalanine tract. To date, a RUNX2 variant with alanine decapeptide expansion (+10Ala) is the only example of a causative variant of RUNX2 with polyalanine tract expansion to be reported, whilst RUNX2 (+1Ala) has been isolated from the healthy population. Thus, precise analyses of the RUNX2 (+3Ala) variant were needed to clarify whether the tripeptide expanded RUNX2 is a second disease-causing mutant with alanine tract expansion. We therefore investigated the biochemical properties of the mutant RUNX2 (+3Ala), which contains 20 alanine residues in the polyalanine tract. When transfected in COS7 cells, RUNX2 (+3Ala) formed intracellular ubiquitinated aggregates after 24h, and exerted a dominant negative effect in vitro. At 24h after gene transfection, whereas slight reduction was observed in RUNX2 (+10Ala), all of these mutants significantly activated osteoblast-specific element-2, a cis-acting sequence in the promoter of the RUNX2 target gene osteocalcin. The aggregation growth of RUNX2 (+3Ala) was clearly lower and slower than that of RUNX2 (+10Ala). Furthermore, we investigated several other RUNX2 variants with various alanine tract lengths, and found that the threshold for aggregation may be RUNX2 (+3Ala). We conclude that RUNX2 (+3Ala) is the cause of CCD in our current case, and that the accumulation of intracellular aggregates in vitro is related to the length of the alanine tract.

Familial cleidocranial dysplasia misdiagnosed as rickets over three generations.

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia characterized by hypoplastic clavicles, late closure of the fontanels, dental problems and other skeletal features. CCD is caused by mutations, deletions or duplications in runt-related transcription factor 2 (RUNX2), which encodes for a protein essential for osteoblast differentiation and chondrocyte maturation. We describe three familial cases of CCD, misdiagnosed as rickets over three generations. No mutations were detected on standard DNA sequencing of RUNX2, but a novel deletion was identified on quantitative polymerase chain reaction (qPCR) and multiple ligation-dependent probe amplification (MLPA). The present cases indicate that CCD could be misdiagnosed as rickets, leading to inappropriate treatment, and confirm that mutations in RUNX2 are not able to be identified on standard DNA sequencing in all CCD patients, but can be identified on qPCR and MLPA.

Expression of RUNX2 and its signaling partners TCF7, FGFR1/2 in cleidocranial dysplasia.

RUNX2 is a member of the PEBP2/CBF transcription factors family controlling the expression of genes whose products are essential for bone formation. Mutations in the RUNX2 gene may be associated with cleidocranial dysplasia (CCD), a rare skeletal disease characterized by stature aberrations, delayed closure of the cranial sutures, hypoplastic or aplastic clavicles, and multiple dental abnormalities. As RUNX2 is involved in many signaling pathways, we hypothesize that CCD may be associated with their changes. We determined the expression of RUNX2 and its signaling partners TCF7, involved in canonical Wnt signaling, and fibroblast growth factor receptors, FGFR1 and FGFR2 in periodontum of CCD patients and control individuals. We did not observe any differences between the level of RUNX2, TCF7 and FGFR1/2 mRNA, determined by real-time PCR, in CDD patients and controls. Therefore, RUNX2 signaling pathways with their partners TCF7 and FGFR1/2 may not be involved in CCD pathogenesis.

Loss of Tbx1 induces bone phenotypes similar to cleidocranial dysplasia.

T-box transcription factor, TBX1, is the major candidate gene for 22q11.2 deletion syndrome (DiGeorge/ Velo-cardio-facial syndrome) characterized by facial defects, thymus hypoplasia, cardiovascular anomalies and cleft palates. Here, we report that the loss of Tbx1 in mouse (Tbx1(-/-)) results in skeletal abnormalities similar to those of cleidocranial dysplasia (CCD) in humans, which is an autosomal-dominant skeletal disease caused by mutations in RUNX2. Tbx1(-/-) mice display short stature, absence of hyoid bone, failed closure of fontanelle, bifid xiphoid process and hypoplasia of clavicle and zygomatic arch. A cell-type-specific deletion of Tbx1 in osteochondro-progenitor (Tbx1(OPKO)) or mesodermal (Tbx1(MKO)) lineage partially recapitulates the Tbx1(-/-) bone phenotypes. Although Tbx1 expression has not been previously reported in neural crest, inactivation of Tbx1 in the neural crest lineage (Tbx1(NCKO)) leads to an absence of the body of hyoid bone and postnatal lethality, indicating an unanticipated role of Tbx1 in neural crest development. Indeed, Tbx1 is expressed in the neural crest-derived hyoid bone primordium, in addition to mesoderm-derived osteochondral progenitors. Ablation of Tbx1 affected Runx2 expression in calvarial bones and overexpression of Tbx1 induced Runx2 expression in vitro. Taken together, our current studies reveal that Tbx1 is required for mesoderm- and neural crest-derived osteoblast differentiation and normal skeletal development. TBX1 mutation could lead to CCD-like bone phenotypes in human.

Cbfß deletion in mice recapitulates cleidocranial dysplasia and reveals multiple functions of Cbfß required for skeletal development.

The pathogenesis of cleidocranial dysplasia (CCD) as well as the specific role of core binding factor ß (Cbfß) and the Runt-related transcription factor (RUNX)/Cbfß complex in postnatal skeletogenesis remain unclear. We demonstrate that Cbfß ablation in osteoblast precursors, differentiating chondrocytes, osteoblasts, and odontoblasts via Osterix-Cre, results in severe craniofacial dysplasia, skeletal dysplasia, abnormal teeth, and a phenotype recapitulating the clinical features of CCD. Cbfß(f/f)Osterix-Cre mice have fewer proliferative and hypertrophic chondrocytes, fewer osteoblasts, and almost absent trabecular bone, indicating that Cbfß may maintain trabecular bone formation through its function in hypertrophic chondrocytes and osteoblasts. Cbfß(f/f)Collagen, type 1, alpha 1 (Col1α1)-Cre mice show decreased bone mineralization and skeletal deformities, but no radical deformities in teeth, mandibles, or cartilage, indicating that osteoblast lineage-specific ablation of Cbfß results in milder bone defects and less resemblance to CCD. Activating transcription factor 4 (Atf4) and Osterix protein levels in both mutant mice are dramatically reduced. ChIP assays show that Cbfß directly associates with the promoter regions of Atf4 and Osterix. Our data further demonstrate that Cbfß highly up-regulates the expression of Atf4 at the transcriptional regulation level. Overall, our genetic dissection approach revealed that Cbfß plays an indispensable role in postnatal skeletal development and homeostasis in various skeletal cell types, at least partially by up-regulating the expression of Atf4 and Osterix. It also revealed that CCD may result from functional defects of the Runx2/Cbfß heterodimeric complex in various skeletal cells. These insights into the role of Cbfß in postnatal skeletogenesis and CCD pathogenesis may assist in the development of new therapies for CCD and osteoporosis.

Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction.

The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone ß-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form--alone or with polyQ and polyQA--CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2--a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion--and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases.

Pin1-mediated Runx2 modification is critical for skeletal development.

Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/- mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.

Microduplications upstream of MSX2 are associated with a phenocopy of cleidocranial dysplasia.

BACKGROUND: Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder characterised by hypoplastic or absent clavicles, increased head circumference, large fontanels, dental anomalies and short stature. Although CCD is usually caused by mutations leading to haploinsufficiency of RUNX2, the underlying genetic cause remains unresolved in about 25% of cases. METHODS: Array comparative genomic hybridisation was performed to detect copy number variations (CNVs). Identified CNVs were characterised by quantitative PCR and sequencing analyses. The effect of candidate genes on mineralisation was evaluated using viral overexpression in chicken cells. RESULTS: In 2 out of 16 cases, the authors identified microduplications upstream of MSX2 on chromosome 5q35.2. One of the unrelated affected individuals presented with a phenocopy of CCD. In addition to a classical CCD phenotype, the other subject had a complex synpolydactyly of the hands and postaxial polydactyly of the feet which have so far never been reported in association with CCD or CNVs on 5q35.2. The duplications overlap in an ∼219 kb region that contains several highly conserved non-coding elements which are likely to be involved in MSX2 gene regulation. Functional analyses demonstrated that the inhibitory effect of Msx2 overexpression on mineralisation cannot be ameliorated by forced Runx2 expression. CONCLUSIONS: These results indicate that CNVs in non-coding regions can cause developmental defects, and that the resulting phenotype can be distinct from those caused by point mutations within the corresponding gene. Taken together, these findings reveal an additional mechanism for the pathogenesis of CCD, particularly with regard to the regulation of MSX2.

Nell-1, a key functional mediator of Runx2, partially rescues calvarial defects in Runx2(+/-) mice.

Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Through promoter analyses, we have recently identified a new direct transcriptional target of Runx2, Nell-1, a craniosynostosis (CS)-associated molecule with potent osteogenic properties. This study investigated the mechanistic and functional relationship between Nell-1 and Runx2 in regulating osteoblast differentiation. The results showed that spatiotemporal distribution and expression levels of Nell-1 correlated closely with those of endogenous Runx2 during craniofacial development. Phenotypically, cross-mating Nell-1 overexpression transgenic (CMV-Nell-1) mice with Runx2 haploinsufficient (Runx2(+/-)) mice partially rescued the calvarial defects in the cleidocranial dysplasia (CCD)-like phenotype of Runx2(+/-) mice, whereas Nell-1 protein induced mineralization and bone formation in Runx2(+/-) but not Runx2(-/-) calvarial explants. Runx2-mediated osteoblastic gene expression and/or mineralization was severely reduced by Nell-1 siRNA oligos transfection into Runx2(+/+) newborn mouse calvarial cells (NMCCs) or in N-ethyl-N-nitrosourea (ENU)-induced Nell-1(-/-) NMCCs. Meanwhile, Nell-1 overexpression partially rescued osteoblastic gene expression but not mineralization in Runx2 null (Runx2(-/-)) NMCCs. Mechanistically, irrespective of Runx2 genotype, Nell-1 signaling activates ERK1/2 and JNK1 mitogen-activated protein kinase (MAPK) pathways in NMCCs and enhances Runx2 phosphorylation and activity when Runx2 is present. Collectively, these data demonstrate that Nell-1 is a critical downstream Runx2 functional mediator insofar as Runx2-regulated Nell-1 promotes osteoblastic differentiation through, in part, activation of MAPK and enhanced phosphorylation of Runx2, and Runx2 activity is significantly reduced when Nell-1 is blocked or absent.

Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-ß and Runx2 in bone is required for hearing.

Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-ß (TGFß)-responsive pathway that controls osteoblast differentiation. Deregulated TGFß or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/⁻ mice, inhibition of TGFß signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.