Wnt7b expressed by hypertrophic chondrocytes is a stimulatory factor for endochondral ossification that is regulated by Smad4 activity.

Endochondral ossification contributes to longitudinal skeletal growth. Osteoblasts, which are bone-forming cells, appear close to terminally differentiated hypertrophic chondrocytes during endochondral ossification. We established mice with conditional knockout (cKO) of Smad4, an essential co-activator for transforming growth factor ß family signaling. The mice showed a marked increase in bone volume in the metaphysis as a result of increased bone formation by osteoblasts, in which ß-catenin, an effector of canonical Wnt signaling, accumulated. We identified Wnt7b as a factor with increased expression in growth plate cartilage in Smad4 cKO mice. Wnt7b mRNA was expressed in differentiated chondrocytes and suppressed by BMP4 stimulation. Ablation of Wnt7b blunted the increase in bone in adult Smad4 cKO mice and reduced skeletal growth in juvenile mice. Overall, we conclude that Wnt7b is a crucial factor secreted from hypertrophic chondrocytes to initiate endochondral ossification. These results suggest that Smad4-dependent BMP signaling regulates the Wnt7b-ß-catenin axis during endochondral ossification.

A blocking monoclonal antibody reveals dimerization of intracellular domains of ALK2 associated with genetic disorders.

Mutations in activin receptor-like kinase 2 (ALK2) can cause the pathological osteogenic signaling seen in some patients with fibrodysplasia ossificans progressiva and other conditions such as diffuse intrinsic pontine glioma. Here, we report that intracellular domain of wild-type ALK2 readily dimerizes in response to BMP7 binding to drive osteogenic signaling. This osteogenic signaling is pathologically triggered by heterotetramers of type II receptor kinases and ALK2 mutant forms, which form intracellular domain dimers in response to activin A binding. We develop a blocking monoclonal antibody, Rm0443, that can suppress ALK2 signaling. We solve the crystal structure of the ALK2 extracellular domain complex with a Fab fragment of Rm0443 and show that Rm0443 induces dimerization of ALK2 extracellular domains in a back-to-back orientation on the cell membrane by binding the residues H64 and F63 on opposite faces of the ligand-binding site. Rm0443 could prevent heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva that carries the human R206H pathogenic mutant.

Two new species of the dwarf centipede genus Nannarrup Foddai, Bonato, Pereira & Minelli, 2003 (Chilopoda, Geophilomorpha, Mecistocephalidae) from Japan.

The genus Nannarrup Foddai, Bonato, Pereira & Minelli, 2003 is a monotypic genus established on the basis of the possibly introduced species N.hoffmani Foddai, Bonato, Pereira & Minelli, 2003, from New York, USA. In the present study, in a field survey conducted throughout Japan, Nannarrup-like specimens were collected from Honshu, Shikoku, and Kyushu. These specimens clearly showed the diagnostic characteristics of the genus but were morphologically distinct from N.hoffmani. Furthermore, morphological analysis and DNA barcoding revealed that these specimens could be assigned to two distinct undescribed species. On the basis of these results, N.innuptus Tsukamoto, sp. nov. and N.oyamensis Tsukamoto, sp. nov. are described. The three Nannarrup species can be distinguished from each other on the basis of the following combination of characteristics: presence or absence of a pair of smooth or weakly areolate areas along the posterior part of the paraclypeal sutures; the width-to-length ratio of the denticle on the trochanteroprefemur; the pigmentation of the denticle on the tarsungulum. Moreover, the field survey resulted in the collection of exclusively female specimens of N.innuptus Tsukamoto, sp. nov., which shows the possibility of parthenogenesis of this species.

Accumulated Knowledge of Activin Receptor-Like Kinase 2 (ALK2)/Activin A Receptor, Type 1 (ACVR1) as a Target for Human Disorders.

Activin receptor-like kinase 2 (ALK2), also known as Activin A receptor type 1 (ACVR1), is a transmembrane kinase receptor for members of the transforming growth factor-ß family. Wild-type ALK2/ACVR1 transduces osteogenic signaling in response to ligand binding. Fifteen years ago, a gain-of-function mutation in the ALK2/ACVR1 gene was detected in patients with the genetic disorder fibro-dysplasia ossificans progressiva, which is characterized by heterotopic ossification in soft tissues. Additional disorders, such as diffuse intrinsic pontin glioma, diffuse idiopathic skeletal hyperostosis, primary focal hyperhidrosis, and congenital heart defects, have also been found to be associated with ALK2/ACVR1. These findings further expand in vitro and in vivo model system research and promote our understanding of the molecular mechanisms of the pathogenesis and development of novel therapeutics and diagnosis for disorders associated with ALK2/ACVR1. Through aggressive efforts, some of the disorders associated with ALK2/ACVR1 will be overcome in the near future.

A new amphibious species of the genus emScolopendra/em Linnaeus, 1758 (Scolopendromorpha, Scolopendridae) from the Ryukyu Archipelago and Taiwan.

In Japan and Taiwan, five valid species of the genus Scolopendra Linnaeus, 1758 have been described: S. morsitans Linnaeus, 1758, S. subspinipes Leach, 1816, S. mutilans Koch, 1878, S. japonica Koch, 1878, and S. multidens Newport, 1844. Recently, an undetermined species was found in the Ryukyu Archipelago and Taiwan. Using molecular phylogenetic analyses with mitochondrial COI and 16S rRNA and nuclear 28S rRNA and 18S rRNA genes as well as conventional morphological examination, we successfully discriminated this sixth species as an independent lineage from S. subspinipes, S. mutilans, and other named congeners from East and Southeast Asia. Therefore, the species was described as S. alcyona Tsukamoto Shimano, sp. nov. Several situational evidences suggest that this species prefers streamside environments and exhibits amphibious behavior.

Design of primers for direct sequencing of nine coding exons in the human ACVR1 gene.

The human ACVR1 gene encodes a transmembrane protein consisting of 509 amino acids called activin A receptor, type I (ACVR1) or activin receptor-like kinase 2 (ALK2) and has nine coding exons. The ALK2 protein functions as a signaling receptor for ligands of the transforming growth factor-ß family. In the human ACVR1 gene, approximately 20 types of heterozygotic mutations in the coding exons have been associated with congenital disorders and somatic cancer, such as fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma, diffuse idiopathic skeletal hyperostosis and some congenital heart disorders. In the present study, we designed primers for direct sequencing of the nine coding exons in the human ACVR1 gene. The reliability of the primers was examined by PCR and DNA sequencing using genomic DNA prepared from peripheral blood or swab samples of three patients with FOP who had different mutations in the ACVR1 gene. A single nucleotide heterozygotic mutation was identified in each genomic sample without additional mutations in other regions. Therefore, the primers designed for the nine coding exons of the ACVR1 gene could be useful for the genetic diagnosis of patients who may have disorders associated with mutations in the ACVR1 gene.

Functional characterization of a unique mutant of ALK2, p.K400E, that is associated with a skeletal disorder, diffuse idiopathic skeletal hyperostosis.

Bone morphogenetic protein (BMP) signaling regulates the physiological and pathological development of skeletal tissues. Activin receptor-like kinase 2 (ALK2) is a BMP type I transmembrane serine/threonine kinase receptor. Recently, a p.K400E mutation was found in ALK2 in a patient with diffuse idiopathic skeletal hyperostosis (DISH), which is a disorder characterized by calcification and ossification of spinal ligaments and entheses. We report here the functional characterization of ALK2 p.K400E in vitro. Cells overexpressing ALK2 p.K400E activated BMP signaling in response to osteogenic BMP ligands. However, ALK2 p.K400E was not activated by a nonosteogenic ligand, Activin A. BMP signaling through ALK2 p.K400E was further enhanced by the coexpression of a BMP type II receptor. The type II receptor increased the phosphorylation level of ALK2 p.K400E, suggesting that ALK2 p.K400E is a hypersensitive mutant to the BMP type II receptor kinases. Our findings suggest that pathological calcification and ossification in DISH are caused by overactivated BMP signaling through ALK2 p.K400E enhanced by type II receptors in response to osteogenic BMPs rather than Activin A.

Smad4-dependent transforming growth factor-ß family signaling regulates the differentiation of dental epithelial cells in adult mouse incisors.

Teeth consist of two major tissues, enamel and dentin, which are formed during development by epithelial and mesenchymal cells, respectively. Rodent incisors are useful experimental models for studying the molecular mechanisms of tooth formation because they are simultaneously growing in not only embryos but also adults. Members of the transforming growth factor-ß (TGF-ß) family regulate epithelial-mesenchymal interactions through an essential coactivator, Smad4. In the present study, we established Smad4 conditional knockout (cKO) mice and examined phenotypes in adult incisors. Smad4 cKO mice died with severe anemia within one month. Phosphorylated Smad1/5/9 and Smad2/3 were detected in epithelial cells in both control and Smad4 cKO mice. Disorganized and hypoplastic epithelial cells, such as ameloblasts, were observed in Smad4 cKO mice. Moreover, alkaline phosphatase expression and iron accumulation were reduced in dental epithelial cells in Smad4 cKO mice. These findings suggest that TGF-ß family signaling through Smad4 is required for the differentiation and functions of dental epithelial cells in adult mouse incisors.

Fibrodysplasia ossificans progressiva: Review and research activities in Japan.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic skeletal disorder manifesting progressive heterotopic ossification (HO) and congenital malformation of the great toes. Since 2007, we have conducted research on FOP. Here, we review the findings on FOP published to date, including the results of our research. Epidemiological studies in Japan have indicated that FOP has nearly the same prevalence in Japan as in the rest of the world. Basic research on its pathoetiology has progressed rapidly since the identification of the causal gene in 2006. Clinical and radiological findings have been thoroughly researched, including early radiological signs, and diagnostic criteria were established, designating FOP as an intractable disease in Japan. In patients with FOP, the progression of HO is associated with numerous disabilities, often manifesting in vicious cycles that can lead to early mortality. Through cross-sectional and short-term longitudinal studies, we have explored patient education, quality of life, and activities of daily living among Japanese patients. The management of FOP requires education of patients and caregivers, the use of medications to settle inflammation and flare-ups, instructions to ensure proper oral care, and other compensatory approaches that aid in rehabilitation. An avoidance of medical intervention, which may cause HO to progress, is also important. The advent of new drugs to prevent HO could have clinical benefit.

Dual usage of a stage-specific fluorescent reporter system based on a helper-dependent adenoviral vector to visualize osteogenic differentiation.

We developed a reporter system that can be used in a dual manner in visualizing mature osteoblast formation. The system is based on a helper-dependent adenoviral vector (HDAdV), in which a fluorescent protein, Venus, is expressed under the control of the 19-kb human osteocalcin (OC) genomic locus. By infecting human and murine primary osteoblast (POB) cultures with this reporter vector, the cells forming bone-like nodules were specifically visualized by the reporter. In addition, the same vector was utilized to efficiently knock-in the reporter into the endogenous OC gene of human induced pluripotent stem cells (iPSCs), by homologous recombination. Neural crest-like cells (NCLCs) derived from the knock-in reporter iPSCs were differentiated into osteoblasts forming bone-like nodules and could be visualized by the expression of the fluorescent reporter. Living mature osteoblasts were then isolated from the murine mixed POB culture by fluorescence-activated cell sorting (FACS), and their mRNA expression profile was analyzed. Our study presents unique utility of reporter HDAdVs in stem cell biology and related applications.

A new species of the genus Arrup from a limestone cave in Akiyoshi-dai, Western Japan (Chilopoda, Geophilomorpha, Mecistocephalidae).

Arrupakiyoshiensis Tsukamoto & Shimano, sp. n. is described from a limestone cave, Kagekiyo-ana, in Akiyoshi-dai, one of the largest karst regions in Japan, Yamaguchi prefecture. It is distinguishable from 14 valid named congeners by some unique characteristics including entire areolation on the cephalic pleurite, elongation of distal part of female gonopod, and a tubercle on forcipular segment II. In addition, the 18S rRNA gene sequences of A.akiyoshiensis Tsukamoto & Shimano, sp. n. and A.ishiianus, one of the most morphologically similar species, differed by four bp out of 1821 bp. The fact that only troglobionts and troglophilic species are found in the collection site suggests that this new species might be a cave-dweller.

Discovery of Heterotopic Bone-Inducing Activity in Hard Tissues and the TGF-ß Superfamily.

Bone is a unique organ because it can be experimentally induced in soft tissues by implanting a single growth factor, bone morphogenetic protein (BMP). Heterotopic bone-inducing activity was found in demineralized bone matrix in 1965. The characterization of this activity in bone enabled the purification and molecular cloning of BMPs and showed that they are members of the transforming growth factor-ß (TGF-ß) superfamily. Assay systems developed for this bone-inducing activity revealed the molecular mechanisms of the intracellular signaling of members of the superfamily, including BMPs. Moreover, they are being applied to elucidate molecular mechanisms and to develop novel therapeutics for a disease caused by an abnormality in BMP signaling.

Recent Topics in Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease that is characterized by the formation of heterotopic bone tissues in soft tissues, such as skeletal muscle, ligament, and tendon. It is difficult to remove such heterotopic bones via internal medicine or invasive procedures. The identification of activin A receptor, type I (ACVR1)/ALK2 gene mutations associated with FOP has allowed the genetic diagnosis of FOP. The ACVR1/ALK2 gene encodes the ALK2 protein, which is a transmembrane kinase receptor in the transforming growth factor-ß family. The relevant mutations activate intracellular signaling in vitro and induce heterotopic bone formation in vivo. Activin A is a potential ligand that activates mutant ALK2 but not wild-type ALK2. Various types of small chemical and biological inhibitors of ALK2 signaling have been developed to establish treatments for FOP. Some of these are in clinical trials in patients with FOP.

Effects of FKBP12 and type II BMP receptors on signal transduction by ALK2 activating mutations associated with genetic disorders.

Various substitution mutations in ALK2, a transmembrane serine/threonine kinase receptor for bone morphogenetic proteins (BMPs), have been identified in patients with genetic disorders such as fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma (DIPG) and heart defects. In this study, we characterized the ALK2 mutants R258G, G328V and F246Y, which were identified in patients with severe FOP, DIPG and unusual hereditary skeletal dysplasia, respectively. Both R258G and G328V were gain-of-function mutations, but F246Y was equivalent to wild-type ALK2. We also examined the effect of the suppressor FKBP12 on the signal transduction of a further 14 ALK2 mutations associated with FOP and/or DIPG. To varying extents FKBP12 over-expression suppressed the basal signaling induced by thirteen of the ALK2 mutants, whereas PF197-8L was uniquely resistant. In the PF197-8L mutant, the modelled ALK2 residue L197 induced a steric clash with the D36 residue in FKBP12 and dissociated their interaction. The co-expression of BMP type II receptors or stimulation with ligands relieved the suppression by FKBP12 by disrupting the interaction between mutant ALK2 and FKBP12. Taken together, FKBP12 binds to and suppresses mutant ALK2 proteins associated with FOP and DIPG, except for PF197-8L.

Heterotopic bone induction via BMP signaling: Potential therapeutic targets for fibrodysplasia ossificans progressiva.

More than 50years ago, Marshal M. Urist detected "heterotopic bone-inducing activity" in demineralized bone matrix. This unique activity was referred to as "bone morphogenetic protein (BMP)" because it was sensitive to trypsin digestion. Purification of the bone-inducing activity from demineralized bone matrix using a bone-inducing assay in vivo indicated that the original "BMP" consisted of a mixture of new members of the transforming growth factor-ß (TGF-ß) family. The establishment of new in vitro assay systems that reflect the bone-inducing activity of BMPs in vivo have revealed the functional receptors and downstream effectors of BMPs. Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by progressive heterotopic bone formation in soft tissues similar to the event induced by the transplantation of BMPs in skeletal muscle. In patients with FOP, genetic mutations have been identified in the ACVR1 gene, which encodes the BMP receptor ALK2. The mutations in ALK2 associated with FOP are hypersensitive to type II receptor kinases. Recently, activin A, a non-osteogenic member of the TGF-ß family, was identified as the ligand of the mutant ALK2 in FOP, and various types of signaling inhibitors for mutant ALK2 are currently under development to establish effective treatments for FOP.

Mutant activin-like kinase 2 in fibrodysplasia ossificans progressiva are activated via T203 by BMP type II receptors.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by progressive heterotopic ossification in soft tissues, such as the skeletal muscles. FOP has been shown to be caused by gain-of-function mutations in activin receptor-like kinase (ALK)-2, which is a type I receptor for bone morphogenetic proteins (BMPs). In the present study, we examined the molecular mechanisms that underlie the activation of intracellular signaling by mutant ALK2. Mutant ALK2 from FOP patients enhanced the activation of intracellular signaling by type II BMP receptors, such as BMPR-II and activin receptor, type II B, whereas that from heart disease patients did not. This enhancement was dependent on the kinase activity of the type II receptors. Substitution mutations at all nine serine and threonine residues in the ALK2 glycine- and serine-rich domain simultaneously inhibited this enhancement by the type II receptors. Of the nine serine and threonine residues in ALK2, T203 was found to be critical for the enhancement by type II receptors. The T203 residue was conserved in all of the BMP type I receptors, and these residues were essential for intracellular signal transduction in response to ligand stimulation. The phosphorylation levels of the mutant ALK2 related to FOP were higher than those of wild-type ALK2 and were further increased by the presence of type II receptors. The phosphorylation levels of ALK2 were greatly reduced in mutants carrying a mutation at T203, even in the presence of type II receptors. These findings suggest that the mutant ALK2 related to FOP is enhanced by BMP type II receptors via the T203-regulated phosphorylation of ALK2.

Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-ß1 (TGF-ß1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.

Expression of TLE3 by bone marrow stromal cells is regulated by canonical Wnt signaling.

Transducing-like enhancer of split 3 (TLE3), one of the Groucho/TLE family members, targets Runx2 transcription and suppresses osteoblast differentiation in bone marrow stromal cells (BMSCs). Here, we identify Wnt responsive elements of the TLE3 promoter region through comparative genomic and functional analyses and show that expression of TLE3 is increased by Wnt signaling, which is important for osteoblast differentiation. We also demonstrated that TLE3 is able to suppress canonical Wnt signaling in BMSCs. Taken together, our data suggest that induction of TLE3 by Wnt signaling is part of a negative feedback loop active during osteoblast differentiation.