An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.

Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disease characterized by extraskeletal bone formation through endochondral ossification. Patients with FOP harbor point mutations in ACVR1, a type I receptor for BMPs. Although mutated ACVR1 (FOP-ACVR1) has been shown to render hyperactivity in BMP signaling, we and others have uncovered a mechanism by which FOP-ACVR1 mistransduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-ß signaling. Although Activin-A evokes enhanced chondrogenesis in vitro and heterotopic ossification (HO) in vivo, the underlying mechanisms have yet to be revealed. To this end, we developed a high-throughput screening (HTS) system using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) to identify pivotal pathways in enhanced chondrogenesis that are initiated by Activin-A. In a screen of 6,809 small-molecule compounds, we identified mTOR signaling as a critical pathway for the aberrant chondrogenesis of mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs). Two different HO mouse models, an FOP model mouse expressing FOP-ACVR1 and an FOP-iPSC-based HO model mouse, revealed critical roles for mTOR signaling in vivo. Moreover, we identified ENPP2, an enzyme that generates lysophosphatidic acid, as a linker of FOP-ACVR1 and mTOR signaling in chondrogenesis. These results uncovered the crucial role of the Activin-A/FOP-ACVR1/ENPP2/mTOR axis in FOP pathogenesis.

[Application of disease-specific iPS cells for intractable diseases-from pathomechanisms to drug discovery].

Genetic diseases affecting bone and cartilage, which are main components of the locomotive system, are extremely diverse. Even if the causative genes are known, detail pathomechanisms are not yet disclosed in most of them and no effective treatments are established. One of such condition is fibrodysplasia ossificance progressive, which is characterized by systemic ectopoic bone formation and caused by mutations of ACVR1/ALK2 gene encoding one of typeⅠBMP receptors. Using patient-derived iPS cells, we have succeeded to recapitulate the disease in vitro and found a unexpected molecular mechanism that Activin-A induced the BMP signal through mutant receptors. This novel finding provides us with a key to discover drugs for this condition.

Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-ß signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-ß signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.

New Protocol to Optimize iPS Cells for Genome Analysis of Fibrodysplasia Ossificans Progressiva.

Successful in vitro disease-recapitulation using patient-specific induced pluripotent stem cells (iPSCs) requires two fundamental technical issues: appropriate control cells and robust differentiation protocols. To investigate fibrodysplasia ossificans progressiva (FOP), a rare genetic disease leading to extraskeletal bone formation through endochondral ossification, gene-corrected (rescued) iPSC clones (resFOP-iPSC) were generated from patient-derived iPSC (FOP-iPSC) as genetically matched controls, and the stepwise induction method of mesenchymal stromal cells (iMSCs) through neural crest cell (NCC) lineage was used to recapitulate the disease phenotype. FOP-iMSCs possessing enhanced chondrogenic ability were transcriptionally distinguishable from resFOP-iMSCs and activated the SMAD1/5/8 and SMAD2/3 pathways at steady state. Using this method, we identified MMP1 and PAI1 as genes responsible for accelerating the chondrogenesis of FOP-iMSCs. These data indicate that iMSCs through NCC lineage are useful for investigating the molecular mechanism of FOP and corresponding drug discovery.

Screening for adiponectin secretion regulators.

Adiponectin is an adipokine secreted from adipocytes and plays important roles in the suppression of metabolic syndromes that can result in type 2 diabetes, obesity, and atherosclerosis. Adiponectin is a promising drug target because a number of studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Extensive efforts have revealed various adiponectin regulators, such as cytokines, transcription factors, and drugs. Cytokines, such as tumor necrosis factor α, IL-6, and IL-18, downregulate adiponectin production. On the other hand, transcription factors such as peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT-enhancer-binding protein α, and forkhead box O1 (FoxO1) upregulate adiponectin expression, although the activating transcription factor 3 and cAMP response element-binding protein downregulate it. Although a number of therapeutic drugs have been reported as adiponectin secretion regulators, most of them act through PPARγ-dependent mechanisms, leaving PPARγ-derived side effects as a concern. Using high-throughput screening, we have identified PPARγ-independent adiponectin secretion regulators as potential drug candidates with a novel mechanism of action.

High-throughput screening for small-molecule adiponectin secretion modulators.

Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.

Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks.

Achieving a real understanding of animal development obviously requires a comprehensive rather than partial identification of the genes working in each developmental process. Recent decoding of genome sequences will enable us to perform such studies. An ascidian, Ciona intestinalis, one of the animals whose genome has been sequenced, is a chordate sharing a basic body plan with vertebrates, although its genome contains less paralogs than are usually seen in vertebrates. In the present study, we discuss the genomewide approach to networks of developmental genes in Ciona embryos. We focus on transcription factor genes and some major groups of signal transduction genes. These genes are comprehensively listed and examined with regard to their embryonic expression by in situ hybridization (http://ghost.zool.kyoto-u.ac.jp/tfst.html). The results revealed that 74% of the transcription factor genes are expressed maternally and that 56% of the genes are zygotically expressed during embryogenesis. Of these, 34% of the transcription factor genes are expressed both maternally and zygotically. The number of zygotically expressed transcription factor genes increases gradually during embryogenesis. As an example, and taking advantage of this comprehensive description of gene expression profiles, we identified transcription factor genes and signal transduction genes that are expressed at the early gastrula stage and that work downstream of beta-catenin, FoxD and/or Fgf9/16/20. Because these three genes are essential for ascidian endomesoderm specification, transcription factor genes and signal transduction genes involved in each of the downstream processes can be deduced comprehensively using the present approach.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VI. Genes for Wnt, TGFbeta, Hedgehog and JAK/STAT signaling pathways.

Cell-cell interactions play important roles in a variety of developmental processes, and therefore molecules involved in the signaling pathways have been studied extensively. Recently, the draft genome sequence of the basal chordate, Ciona intestinalis, was determined. Here we annotated genes for the signaling pathways of Wnt, transforming growth factor beta (TGFbeta), Hedgehog, and JAK/STAT in the genome of Ciona intestinalis. The Ciona genome contains ten wnt genes, six frizzled genes, four sFRP genes, ten TGFbeta family member genes, five TGFbeta-receptor genes, and five Smad genes; most of the genes were found with less redundancy than in vertebrate genomes. The other genes in the signaling pathways are present as a single copy in the Ciona genome. In addition, all of the identified genes for the signaling pathway, except for a few genes, have EST evidence, and their cDNAs are available from the Ciona intestinalis gene collection. Therefore, Ciona intestinalis may provide an experimental system for exploring the basic genetic cascade associated with the signaling pathways in chordates.

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.