研究者詳細 - 伊藤　美佳子

Fluoxetine ameliorates cartilage degradation in osteoarthritis by inhibiting Wnt/β-catenin signaling 査読有り Open Access

Miyamoto, K; Ohkawara, B; Ito, M; Masuda, A; Hirakawa, A; Sakai, T; Hiraiwa, H; Hamada, T; Ishiguro, N; Ohno, K

PLOS ONE 12 巻 ( 9 ) 頁： e0184388 - - 2017年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plos One

Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.

DOI： 10.1371/journal.pone.0184388

SRSF1 suppresses selection of intron-distal 5′ splice site of DOK7 intron 4 to generate functional full-length Dok-7 protein 査読有り Open Access

Bin Ahsan, K; Masuda, A; Rahman, MA; Takeda, J; Nazim, M; Ohkawara, B; Ito, M; Ohno, K

SCIENTIFIC REPORTS 7 巻 ( 1 ) 頁： 10446 - - 2017年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Dok-7 is a non-catalytic adaptor protein that facilitates agrin-induced clustering of acetylcholine receptors (AChR) at the neuromuscular junction. Alternative selection of 5′ splice sites (SSs) of DOK7 intron 4 generates canonical and frame-shifted transcripts. We found that the canonical full-length Dok-7 enhanced AChR clustering, whereas the truncated Dok-7 did not. We identified a splicing cis-element close to the 3′ end of exon 4 by block-scanning mutagenesis. RNA affinity purification and mass spectrometry revealed that SRSF1 binds to the cis-element. Knocking down of SRSF1 enhanced selection of the intron-distal 5′ SS of DOK7 intron 4, whereas MS2-mediated artificial tethering of SRSF1 to the identified cis-element suppressed it. Isolation of an early spliceosomal complex revealed that SRSF1 inhibited association of U1 snRNP to the intron-distal 5′ SS, and rather enhanced association of U1 snRNP to the intron-proximal 5′ SS, which led to upregulation of the canonical DOK7 transcript. Integrated global analysis of CLIP-seq and RNA-seq also indicated that binding of SRSF1 immediately upstream to two competing 5′ SSs suppresses selection of the intron-distal 5′ SS in hundreds of human genes. We demonstrate that SRSF1 critically regulates alternative selection of adjacently placed 5′ SSs by modulating binding of U1 snRNP.

DOI： 10.1038/s41598-017-11036-z

Wnt/β-catenin signaling suppresses expressions of Scx, Mkx, and Tnmd in tendon-derived cells 査読有り Open Access

Kishimoto, Y; Ohkawara, B; Sakai, T; Ito, M; Masuda, A; Ishiguro, N; Shukunami, C; Docheva, D; Ohno, K

PLOS ONE 12 巻 ( 7 ) 頁： e0182051 2017年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plos One

After tendon injuries, biomechanical properties of the injured tendon are not fully recovered in most cases. Modulation of signaling pathways, which are involved in tendon development and tendon repair, is one of attractive modalities to facilitate proper regeneration of the injured tendon. The roles of TGF-β signaling in tendon homeostasis and tendon development have been elucidated. In contrast, the roles of Wnt/β-catenin signaling in tendon remain mostly elusive. We found that the number of β-catenin-positive cells was increased at the injured site, suggesting involvement of Wnt/β-catenin signaling in tendon healing. Activation of Wnt/β-catenin signaling suppressed expressions of tenogenic genes of Scx, Mkx, and Tnmd in rat tendon-derived cells (TDCs) isolated from the Achilles tendons of 6-week old rats. Additionally, activation of Wnt/β-catenin reduced the amounts of Smad2 and Smad3, which are intracellular mediators for TGF-β signaling, and antagonized upregulation of Scx induced by TGF-β signaling in TDCs. We found that Wnt/β-catenin decreased Mkx and Tnmd expressions without suppressing Scx expression in Scx-programmed tendon progenitors. Our studies suggest that Wnt/β-catenin signaling is a repressor for tenogenic gene expressions.

DOI： 10.1371/journal.pone.0182051

Protein-Anchoring Therapy of Biglycan for Mdx Mouse Model of Duchenne Muscular Dystrophy 査読有り

Ito, M; Ehara, Y; Li, J; Inada, K; Ohno, K

HUMAN GENE THERAPY 28 巻 ( 5 ) 頁： 428 - 436 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Gene Therapy

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by loss-of-function mutations in DMD encoding dystrophin. No rational therapy is currently available. Utrophin is a paralog of dystrophin and is highly expressed at the neuromuscular junction. In mdx mice, utrophin is naturally upregulated throughout the muscle fibers, which mitigates muscular dystrophy. Protein-anchoring therapy was previously reported, in which a recombinant extracellular matrix (ECM) protein is delivered to and anchored to a specific target using its proprietary binding domains. Being prompted by a report that intramuscular and intraperitoneal injection of an ECM protein, biglycan, upregulates expression of utrophin and ameliorates muscle pathology in mdx mice, protein-anchoring therapy was applied to mdx mice. Recombinant adeno-associated virus serotype 8 (rAAV8) carrying hBGN encoding human biglycan was intravenously injected into 5-week-old mdx mice. The rAAV8-hBGN treatment improved motor deficits and decreased plasma creatine kinase activities. In muscle sections of treated mice, the number of central myonuclei and the distribution of myofiber sizes were improved. The treated mice increased gene expressions of utrophin and β1-syntrophin, as well as protein expressions of biglycan, utrophin, γ-sarcoglycan, dystrobrevin, and α1-syntrophin. The expression of hBGN in the skeletal muscle of the treated mice was 1.34-fold higher than that of the native mouse Bgn (mBgn). The low transduction efficiency and improved motor functions suggest that biglycan expressed in a small number of muscle fibers was likely to have been secreted and anchored to the cell surface throughout the whole muscular fibers. It is proposed that the protein-anchoring strategy can be applied not only to deficiency of an ECM protein as previously reported, but also to augmentation of a naturally induced ECM protein.

DOI： 10.1089/hum.2015.088

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms 査読有り Open Access

Nazim, M; Masuda, A; Rahman, MA; Nasrin, F; Takeda, JI; Ohe, K; Ohkawara, B; Ito, M; Ohno, K

NUCLEIC ACIDS RESEARCH 45 巻 ( 3 ) 頁： 1455 - 1468 2017年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nucleic Acids Research

Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

DOI： 10.1093/nar/gkw823

Agrin-LRP4-MuSK signaling as a therapeutic target for myasthenia gravis and other neuromuscular disorders 査読有り

Ohno, K; Ohkawara, B; Ito, M

EXPERT OPINION ON THERAPEUTIC TARGETS 21 巻 ( 10 ) 頁： 949 - 958 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Expert Opinion on Therapeutic Targets

Introduction: Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including myasthenia gravis, Lambert-Eaton myasthenic syndrome, Isaacs’ syndrome, congenital myasthenic syndromes, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. Except for sarcopenia, all are orphan diseases. In addition, the NMJ signal transduction is impaired by tetanus, botulinum, curare, α-bungarotoxin, conotoxins, organophosphate, sarin, VX, and soman to name a few. Areas covered: This review covers the agrin-LRP4-MuSK signaling pathway, which drives clustering of acetylcholine receptors (AChRs) and ensures efficient signal transduction at the NMJ. We also address diseases caused by autoantibodies against the NMJ molecules and by germline mutations in genes encoding the NMJ molecules. Expert opinion: Representative small compounds to treat the defective NMJ signal transduction are cholinesterase inhibitors, which exert their effects by increasing the amount of acetylcholine at the synaptic space. Another possible therapeutic strategy to enhance the NMJ signal transduction is to increase the number of AChRs, but no currently available drug has this functionality.

DOI： 10.1080/14728222.2017.1369960

Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice 査読有り Open Access

Hasegawa, S; Ito, M; Fukami, M; Hashimoto, M; Hirayama, M; Ohno, K

REDOX REPORT 22 巻 ( 1 ) 頁： 26 - 34 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Report

Objective: Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by a mutation in DMD encoding dystrophin. Oxidative stress accounts for dystrophic muscle pathologies in DMD. We examined the effects of molecular hydrogen in mdx mice, a model animal for DMD. Methods: The pregnant mother started to take supersaturated hydrogen water (>5 ppm) ad libitum from E15.5 up to weaning of the offspring. The mdx mice took supersaturated hydrogen water from weaning until age 10 or 24 weeks when they were sacrificed. Results: Hydrogen water prevented abnormal body mass gain that is commonly observed in mdx mice. Hydrogen improved the spontaneous running distance that was estimated by a counter-equipped running-wheel, and extended the duration on the rota-rod. Plasma creatine kinase activities were decreased by hydrogen at ages 10 and 24 weeks. Hydrogen also decreased the number of central nuclei of muscle fibers at ages 10 and 24 weeks, and immunostaining for nitrotyrosine in gastrocnemius muscle at age 24 weeks. Additionally, hydrogen tended to increase protein expressions of antioxidant glutathione peroxidase 1, as well as anti-apoptotic Bcl-2, in skeletal muscle at age 10 weeks. Discussion: Although molecular mechanisms of the diverse effects of hydrogen remain to be elucidated, hydrogen potentially improves muscular dystrophy in DMD patients.

DOI： 10.1080/13510002.2015.1135580