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.

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.

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.

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.

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.

Contribution of impaired myofibril and ryanodine receptor function to prolonged low-frequency force depression after in situ stimulation in rat skeletal muscle.

The aim of this study was to examine whether prolonged low-frequency force depression (PLFFD) that occurs in situ is the result of decreased myofibrillar Ca(2+) sensitivity and/or reduced sarcoplasmic reticulum (SR) Ca(2+) release. Intact rat gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected 30 min following the cessation of stimulation. Skinned fibre and whole muscle analyses were performed in the superficial region composed exclusively of type IIB fibres. Fatiguing stimulation significantly reduced the ratio of force at low frequency to that at high frequency to 65% in skinned fibres (1 vs. 50 Hz) and 73% in whole muscles (20 vs. 100 Hz). In order to evaluate changes in myofibrillar Ca(2+) sensitivity and ryanodine receptor caffeine sensitivity, skinned fibres were activated in Ca(2+)- and caffeine-containing solutions, respectively. Skinned fibres from fatigued muscles displayed decreased caffeine sensitivity together with increased myofibrillar Ca(2+) sensitivity. Treatment with 2,2'-dithiodipyridine and reduced glutathione induced a smaller increase in myofibrillar Ca(2+)sensitivity in fatigued than in rested fibres. In fatigued muscles, S-glutathionylation of troponin I was increased and submaximal SR Ca(2+) release, induced by 4-chloro-m-cresol, was decreased. These findings suggest that in the early stage of PLFFD that occurs in fast-twitch muscles of exercising animals and humans, S-glutathionylation of troponin I may attenuate PLFFD by increasing myofibrillar Ca(2+) sensitivity and that under such a circumstance, PLFFD may be ascribable to failure of SR Ca(2+) release.

The effects of eccentric contraction on myofibrillar proteins in rat skeletal muscle.

The present study investigated the effects of eccentric muscle contractions (ECC) on the content of myofibrillar proteins (my-proteins) and the catalytic activity of myofibrillar ATPase (my-ATPase) in skeletal muscles. Rat extensor digitorum longus and tibialis anterior muscles were exposed to 200-repeated ECC or isometric contractions (ISC) and used for measures of force output and for biochemical analyses, respectively. Whereas in ISC-treated muscles, full restoration of tetanic force was attained after 2 days of recovery, force developed by ECC-treated muscles remained depressed (P < 0.05) after 6 days. The total my-protein content and the relative content of myosin heavy chain (MHC) in total my-proteins were unaltered during 4 days of recovery after ECC, but fell (P < 0.05) to 55.9 and 63.4% after 6 days of recovery, respectively. my-ATPase activity expressed on a my-protein weight basis was unaltered immediately after ECC. However, it decreased (P < 0.05) to 75.3, 45.3, and 49.3% after 2, 4 and 6 days of recovery, respectively. Total maximal calpain activity measured at 5 mM Ca(2+) was significantly augmented (P < 0.05) after 2 days of recovery, reaching a level of threefold higher after 6 days. These alterations were specific for ECC and not observed for ISC. These results suggest that depressions in my-ATPase activity contribute to ECC-induced decreases in force and power which can take a number of days to recover.