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.

Critical Roles of NF-κB Signaling Molecules in Bone Metabolism Revealed by Genetic Mutations in Osteopetrosis.

The nuclear factor-κB (NF-κB) transcription factor family consists of five related proteins, RelA (p65), c-Rel, RelB, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). These proteins are important not only for inflammation and the immune response but also for bone metabolism. Activation of NF-κB occurs via the classic and alternative pathways. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, activate the former, and cytokines involved in lymph node formation, such as receptor activator of NF-κB ligand (RANKL) and CD40L, activate the latter. p50 and p52 double-knockout mice revealed severe osteopetrosis due to the total lack of osteoclasts, which are specialized cells for bone resorption. This finding suggests that the activation of NF-κB is required for osteoclast differentiation. The NF-κB signaling pathway is controlled by various regulators, including NF-κB essential modulator (NEMO), which is encoded by the IKBKG gene. In recent years, mutant forms of the IKBKG gene have been reported as causative genes of osteopetrosis, lymphedema, hypohidrotic ectodermal dysplasia, and immunodeficiency (OL-EDA-ID). In addition, a mutation in the RELA gene, encoding RelA, has been reported for the first time in newborns with high neonatal bone mass. Osteopetrosis is characterized by a diffuse increase in bone mass, ranging from a lethal form observed in newborns to an asymptomatic form that appears in adulthood. This review describes the genetic mutations in NF-κB signaling molecules that have been identified in patients with osteopetrosis.

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.

Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva.

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

Inhibitory effects of sesquiterpene lactones from the Indonesian marine sponge Lamellodysidea cf. herbacea on bone morphogenetic protein-induced osteoblastic differentiation.

A new unique sesquiterpene lactone, bicyclolamellolactone A (1), was isolated together with two known monocyclofarnesol-type sesquiterpenes, lamellolactones A (2) and B (3), from the Indonesian marine sponge Lamellodysidea sp. (cf. L. herbacea). The planar structure of 1 was assigned based on its spectroscopic data (1D and 2D NMR, HRESIMS, UV, and IR spectra). The relative and absolute configuration of 1 was determined by comparison of its calculated and experimental electronic circular dichroism spectra in combination with NOESY correlations. Compounds 1-3 inhibited bone morphogenic protein (BMP)-induced alkaline phosphatase activity in mutant BMP receptor-carrying C2C12 cells with IC50 values of 51, 4.6, and 20 µM, respectively.

Screening for Small Molecule Inhibitors of BMP-Induced Osteoblastic Differentiation from Indonesian Marine Invertebrates.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder with heterotopic ossification (HO) in soft tissues. The abnormal activation of bone morphogenetic protein (BMP) signaling by a mutant activin receptor-like kinase-2 (ALK2) leads to the development of HO in FOP patients, and, thus, BMP signaling inhibitors are promising therapeutic applications for FOP. In the present study, we screened extracts of 188 Indonesian marine invertebrates for small molecular inhibitors of BMP-induced alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation in a C2C12 cell line stably expressing ALK2(R206H) (C2C12(R206H) cells), and identified five marine sponges with potent ALP inhibitory activities. The activity-guided purification of an EtOH extract of marine sponge Dysidea sp. (No. 256) resulted in the isolation of dysidenin (1), herbasterol (2), and stellettasterol (3) as active components. Compounds 1-3 inhibited ALP activity in C2C12(R206H) cells with IC50 values of 2.3, 4.3, and 4.2 µM, respectively, without any cytotoxicity, even at 18.4-21.4 µM. The direct effects of BMP signaling examined using the Id1WT4F-luciferase reporter assay showed that compounds 1-3 did not decrease the reporter activity, suggesting that they inhibit the downstream of the Smad transcriptional step in BMP signaling.

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.

A new diketopiperazine-like inhibitor of bone morphogenetic protein-induced osteoblastic differentiation produced by marine-derived Aspergillus sp. BFM-0085.

A new diketopiperazine-like compound, designated protuboxepin K (1), was isolated together with the known structurally related protuboxepin A (2) from culture broth of the marine-derived fungal strain Aspergillus sp. BFM-0085 isolated from a sediment sample of Tokyo Bay. The structure of protuboxepin K was elucidated by spectroscopic data, including 1D and 2D NMR. Compounds 1 and 2 inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity with IC50 values of 4.7 and 25.2 µM, respectively, in mutant BMP receptor-carrying C2C12(R206H) cells.

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.

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.

BMPR2 inhibits activin and BMP signaling via wild-type ALK2.

TGF-ß/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-ß superfamily member, inhibits growth of multiple myeloma cells, but the mechanism for this is unknown. We therefore aimed to clarify how activins affect myeloma cell survival. Activin A activates the transcription factors SMAD2/3 through the ALK4 type 1 receptor, but may also activate SMAD1/5/8 through mutated variants of the type 1 receptor ALK2 (also known as ACVR1). We demonstrate that activin A and B activate SMAD1/5/8 in myeloma cells through endogenous wild-type ALK2. Knockdown of the type 2 receptor BMPR2 strongly potentiated activin A- and activin B-induced activation of SMAD1/5/8 and subsequent cell death. Furthermore, activity of BMP6, BMP7 or BMP9, which may also signal via ALK2, was potentiated by knockdown of BMPR2. Similar results were seen in HepG2 liver carcinoma cells. We propose that BMPR2 inhibits ALK2-mediated signaling by preventing ALK2 from oligomerizing with the type 2 receptors ACVR2A and ACVR2B, which are necessary for activation of ALK2 by activins and several BMPs. In conclusion, BMPR2 could be explored as a possible target for therapy in patients with multiple myeloma.This article has an associated First Person interview with the first author of the paper.

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.