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1.  A functional polymorphism in the epidermal growth factor gene predicts hepatocellular carcinoma risk in Japanese hepatitis C patients 
OncoTargets and therapy  2013;6:1805-1812.
A single nucleotide polymorphism (SNP) in the epidermal growth factor (EGF) gene (rs4444903) has been associated with increased risk of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the relationship between the EGF SNP genotype and the development and prognosis of HCC, in a Japanese population.
Restriction fragment-length polymorphism was used to determine the presence of the EGF SNP genotype in 498 patients, including 208 patients with HCC. The level of EGF messenger ribonucleic acid (mRNA) expression in cancerous tissues was measured by quantitative reverse transcription polymerase chain reaction. The correlation between the EGF SNP genotype and prognosis was statistically analyzed in the patients with HCC.
The proportion of the A/A, A/G, and G/G genotypes were 5.3%, 42.8%, and 51.9%, respectively, in the patients with HCC, whereas in those without HCC, they were 8.6%, 35.9%, and 55.5%, respectively, revealing that the odds ratio (OR) of developing HCC was higher in patients with a G allele (OR =1.94, P=0.080 for A/G patients and OR =1.52, P=0.261 for G/G patients, as compared with A/A patients). In particular, when the analysis was limited to the 363 patients with hepatitis C, the OR for developing HCC was 3.54 (P=0.014) for A/G patients and was 2.85 (P=0.042) for G/G patients, as compared with A/A patients. Tumoral EGF mRNA expression in G/G patients was significantly higher than that in A/A patients (P=0.033). No statistically significant differences were observed between the EGF SNP genotype and diseasefree or overall survival.
The EGF SNP genotype might be associated with a risk for the development of HCC in Japanese patients but not with prognosis. Of note, the association is significantly stronger in patients with hepatitis C, which is the main risk factor for HCC in Japan.
PMCID: PMC3865974  PMID: 24363559
epidermal growth factor; functional polymorphism; hepatocellular carcinoma; hepatitis C; hepatocarcinogenesis
2.  Dynamin 3: a new candidate tumor suppressor gene in hepatocellular carcinoma detected by triple combination array analysis 
OncoTargets and therapy  2013;6:1417-1424.
To identify genes associated with hepatocellular carcinoma (HCC) pathogenesis, we developed a triple combination array strategy comprising methylation, gene expression, and single nucleotide polymorphism (SNP) array analysis.
Surgical specimens obtained from a 68-year-old female HCC patient were analyzed by triple combination array, and identified Dynamin 3 (DNM3) as a candidate tumor suppressor gene in HCC. Subsequently, samples from 48 HCC patients were evaluated for DNM3 methylation and expression status using methylation specific polymerase chain reaction (PCR; MSP) and semi-quantitative reverse transcriptase (RT)-PCR, respectively. The relationship between clinicopathological factors and DNM3 methylation status was also investigated.
DNM3 was shown to be hypermethylated (methylation value 0.879, range 0–1.0) in cancer tissue compared with adjacent normal tissue (0.213) by methylation array in the 68-year-old female patient. Expression arrays revealed decreased expression of DNM3 in cancerous tissue. SNP arrays revealed that the copy number of chromosome 1q24.3, in which DNM3 resides, was normal. MSP revealed hypermethylation of the DNM3 promoter region in 33 of 48 tumor samples. A trend toward decreased DNM3 expression was observed in patients with DNM3 promoter methylation (P = 0.189). Furthermore, patients with reduced expression of DNM3 in tumor tissues exhibited worse prognosis with decreased disease specific survival compared to patients without decreased expression (P = 0.014).
The present study indicates that a triple combination array strategy is an effective method to detect novel genes related to HCC. We propose that DNM3 is a tumor suppressor gene in HCC.
PMCID: PMC3797647  PMID: 24143113
DNM3; hepatocellular carcinoma; methylation; triple combination array
3.  Delayed Development of Pulmonary Hemorrhage in a Patient with Positive Circulating Anti-Neutrophil Cytoplasmic Antibody: A Clinical Dilemma 
Detection of circulating anti-neutrophil cytoplasmic antibody (ANCA) provides a powerful clue in the diagnosis of vasculitis, but the clinical interpretation of the results is difficult in some cases. Here, we describe the case of a 65-year-old man who underwent hemodialysis due to focal segmental glomerulosclerosis and abruptly developed hemoptysis 14 years after a renal biopsy. At the time of the biopsy, computed tomography (CT) showed interstitial shadows in the lungs and pleural thickening, indicating pneumoconiosis that was accompanied by tuberculosis. Circulating myeloperoxidase-ANCA (10.5–32.5 U/ml) was subsequently noted, but the significance of this observation was unclear due to the preexisting disorders in the lungs and kidneys. Potent immunosuppressive therapies were avoided because of the pulmonary lesions and decreased renal function. There were few changes noted on follow-up CT, but infiltrative shadows emerged in the bilateral lungs, consistent with hemoptysis. The hemorrhagic shadows completely disappeared shortly after initiation of steroid therapy, with normalization of the serum ANCA level. Herein, we report this case, with an emphasis on the clinical dilemma faced in deciding the appropriate treatment. The findings in the case provide deep insights into clinical management of ANCA-positive patients.
PMCID: PMC3806712  PMID: 24163688
ANCA-related nephritis and vasculitis; Pulmonary hemorrhage; Focal and segmental glomerulosclerosis; Tuberculosis
4.  Prednisolone improves walking in Japanese Duchenne muscular dystrophy patients 
Journal of Neurology  2013;260:3023-3029.
We evaluated the long-term efficacy of prednisolone (PSL) therapy for prolonging ambulation in Japanese patients with genetically confirmed Duchenne muscular dystrophy (DMD). There were clinical trials have shown a short-term positive effect of high-dose and daily PSL on ambulation, whereas a few study showed a long-term effect. Especially in Japan, “real-life” observation was lacking. We utilized the national registry of muscular dystrophy in Japan for our retrospective study. We compared the age at loss of ambulation (LOA) between patients in PSL group and those in without-PSL group. Out of 791 patients’ in the Remudy DMD/BMD registry from July 2009 to June 2012, 560 were matched with inclusion criteria. Of the 560, all were genetically confirmed DMD patients, 245 (43.8 %) of whom were treated with PSL and 315 (56.2 %) without PSL. There was no difference between the two groups regarding their mutational profile. The age at LOA was significantly greater (11 month on average) in the PSL group than in the without-PSL group (median, 132 vs. 121 months; p = 0.0002). Although strictly controlled clinical trials have shown that corticosteroid therapies achieved a marked improvement in ambulation, discontinuation of the drug due to intolerable side effects led to exclusion of clinical trial participants, which is considered as unavoidable. In our study, patients were not excluded from the PSL group, even if they discontinued the medication shortly after starting it. The results of our study may provide evidence to formulate recommendations and provide a basis for realistic expectations for PSL treatment of DMD patients in Japan, even there are certain limitations due to the retrospectively captured data in the registry.
PMCID: PMC3843366  PMID: 24057148
Duchenne muscular dystrophy; Prednisolone; Walking; National registry; Natural history
5.  Detection of doublecortin domain-containing 2 (DCDC2), a new candidate tumor suppressor gene of hepatocellular carcinoma, by triple combination array analysis 
To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis.
A surgical specimen obtained from a 68-year-old female HCC patient was analyzed by triple combination array, which identified doublecortin domain-containing 2 (DCDC2) as a candidate tumor suppressor gene of HCC. Subsequently, samples from 48 HCC patients were evaluated for their DCDC2 methylation and expression status using methylation specific PCR (MSP) and semi-quantitative reverse transcriptase (RT) PCR, respectively. Then, we investigated the relationship between clinicopathological factors and methylation status of DCDC2.
DCDC2 was revealed to be hypermethylated (methylation value 0.846, range 0–1.0) in cancer tissue, compared with adjacent normal tissue (0.212) by methylation array in the 68-year-old female patient. Expression array showed decreased expression of DCDC2 in cancerous tissue. SNP array showed that the copy number of chromosome 6p22.1, in which DCDC2 resides, was normal. MSP revealed hypermethylation of the promoter region of DCDC2 in 41 of the tumor samples. DCDC2 expression was significantly decreased in the cases with methylation (P = 0.048). Furthermore, the methylated cases revealed worse prognosis for overall survival than unmethylated cases (P = 0.048).
The present study indicates that triple combination array is an effective method to detect novel genes related to HCC. We propose that DCDC2 is a tumor suppressor gene of HCC.
PMCID: PMC3847884  PMID: 24034596
DCDC2; Hepatocellular carcinoma; Methylation; Triple combination array
6.  Initial Pulmonary Respiration Causes Massive Diaphragm Damage and Hyper-CKemia in Duchenne Muscular Dystrophy Dog 
Scientific Reports  2013;3:2183.
The molecular mechanism of muscle degeneration in a lethal muscle disorder Duchene muscular dystrophy (DMD) has not been fully elucidated. The dystrophic dog, a model of DMD, shows a high mortality rate with a marked increase in serum creatine kinase (CK) levels in the neonatal period. By measuring serum CK levels in cord and venous blood, we found initial pulmonary respiration resulted in massive diaphragm damage in the neonates and thereby lead to the high serum CK levels. Furthermore, molecular biological techniques revealed that osteopontin was prominently upregulated in the dystrophic diaphragm prior to the respiration, and that immediate-early genes (c-fos and egr-1) and inflammation/immune response genes (IL-6, IL-8, COX-2, and selectin E) were distinctly overexpressed after the damage by the respiration. Hence, we segregated dystrophic phases at the molecular level before and after mechanical damage. These molecules could be biomarkers of muscle damage and potential targets in pharmaceutical therapies.
PMCID: PMC3711052  PMID: 23851606
7.  Manumycin A corrects aberrant splicing of Clcn1 in myotonic dystrophy type 1 (DM1) mice 
Scientific Reports  2013;3:2142.
Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.
PMCID: PMC3701899  PMID: 23828222
8.  Current Challenges and Future Directions in Recombinant AAV-Mediated Gene Therapy of Duchenne Muscular Dystrophy 
Pharmaceuticals  2013;6(7):813-836.
Various characteristics of adeno-associated virus (AAV)-based vectors with long-term safe expression have made it an exciting transduction tool for clinical gene therapy of Duchenne muscular dystrophy (DMD). Although host immune reactions against the vector as well as transgene products were detected in some instances of the clinical studies, there have been promising observations. Methods of producing AAV vectors for considerable in vivo experimentation and clinical investigations have been developed and a number of studies with AAV vector-mediated muscle transduction were attempted. Notably, an intravenous limb perfusion transduction technique enables extensive transgene expression in the skeletal muscles without noticeable adverse events. Furthermore, cardiac transduction by the rAAV9-microdystrophin would be promising to prevent development of cardiac dysfunction. Recent achievements in transduction technology suggest that long-term transgene expression with therapeutic benefits in DMD treatment would be achieved by the rAAV-mediated transduction strategy with an adequate regimen to regulate host immune response.
PMCID: PMC3816704  PMID: 24276316
DMD; AAV; immune response
9.  Capsaicin mimics mechanical load-induced intracellular signaling events 
Channels  2013;7(3):221-224.
Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca2+]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca2+]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.
PMCID: PMC3710350  PMID: 23584166
skeletal muscle; muscle hypertrophy; calcium signaling; TRPV1; capsaicin; mTOR
11.  Extensive and Prolonged Restoration of Dystrophin Expression with Vivo-Morpholino-Mediated Multiple Exon Skipping in Dystrophic Dogs 
Nucleic Acid Therapeutics  2012;22(5):306-315.
Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6–8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.
PMCID: PMC3464409  PMID: 22888777
12.  Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy 
Nature  2011;478(7367):127-131.
Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene1-3. In FCMD, the SVA insertion occurs in the 3′-untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant mRNA splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in FCMD patients. This region spans part of the 3′ end of the fukutin coding region, proximal part of the 3′ UTR, and the SVA insertion. Correspondingly, fukutin mRNA transcripts in FCMD patients and SVA knock-in (KI) model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin C-terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer, and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in FCMD patient cells and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia4, neutral lipid storage disease5) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known6-8, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.
PMCID: PMC3412178  PMID: 21979053
13.  Plant dehydrins and stress tolerance 
Plant Signaling & Behavior  2011;6(10):1503-1509.
Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic a-helix is especially important since it has been found in all dehydrins. Since more than 20 y, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made toward the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.
PMCID: PMC3256378  PMID: 21897131
abiotic stress; dehydration stress; drought; cold acclimation; freezing tolerance; LEA proteins; dehydrins
14.  Reprogramming efficiency and quality of induced Pluripotent Stem Cells (iPSCs) generated from muscle-derived fibroblasts of mdx mice at different ages 
PLoS Currents  2011;3:RRN1274.
Induced pluripotent stem cells (iPSCs) hold promise as a potential treatment for Duchenne muscular dystrophy (DMD). To determine the impact of the donor’s age on reprogramming, we generated iPSCs from muscle-derived fibroblasts (MuFs) of mdx mice aged 6 weeks, 6 months, and 14 months. MuFs from 14-month-old mdx mice showed lower proliferative activity and lower reprogramming efficiency, compared with those from younger mdx mice. Furthermore, iPSCs derived from 14-month-old mdx mice (14m-MuF-iPSCs) gradually lost Nanog expression, and regressed in conventional ES medium during passages. Interestingly, inhibition of TGF-β signaling and BMP signaling stabilized Nanog expression and promoted self-renewal of 14m-MuF-iPSCs. Finally, rescued mdx-derived iPSCs efficiently differentiated into the skeletal muscle lineage.
PMCID: PMC3203521  PMID: 22101343
15.  Identification of Muscle-Specific MicroRNAs in Serum of Muscular Dystrophy Animal Models: Promising Novel Blood-Based Markers for Muscular Dystrophy 
PLoS ONE  2011;6(3):e18388.
Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMDJ), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMDJ. Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy.
PMCID: PMC3068182  PMID: 21479190
16.  Mammalian Models of Duchenne Muscular Dystrophy: Pathological Characteristics and Therapeutic Applications 
Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder characterized by muscle wasting which is caused by mutations in the DMD gene. The DMD gene encodes the sarcolemmal protein dystrophin, and loss of dystrophin causes muscle degeneration and necrosis. Thus far, therapies for this disorder are unavailable. However, various therapeutic trials based on gene therapy, exon skipping, cell therapy, read through therapy, or pharmaceutical agents have been conducted extensively. In the development of therapy as well as elucidation of pathogenesis in DMD, appropriate animal models are needed. Various animal models of DMD have been identified, and mammalian (murine, canine, and feline) models are indispensable for the examination of the mechanisms of pathogenesis and the development of therapies. Here, we review the pathological features of DMD and therapeutic applications, especially of exon skipping using antisense oligonucleotides and gene therapies using viral vectors in murine and canine models of DMD.
PMCID: PMC3022202  PMID: 21274260
17.  Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient 
PLoS ONE  2010;5(8):e12239.
Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMDJ) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells.
Methodology/Principal Findings
We converted fibroblasts of CXMDJ and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMDJ and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.
Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.
PMCID: PMC2923599  PMID: 20805873
18.  Progress in muscular dystrophy research with special emphasis on gene therapy 
Duchenne muscular dystrophy (DMD) is an X-linked, progressive muscle-wasting disease caused by mutations in the DMD gene. Since the disease was described by physicians in the 19th century, information about the subject has been accumulated. One author (Sugita) was one of the coworkers who first reported that the serum creatine kinase (CK) level is elevated in progressive muscular dystrophy patients. Even 50 years after that first report, an elevated serum CK level is still the most useful marker in the diagnosis of DMD, a sensitive index of the state of skeletal muscle, and useful to evaluate therapeutic effects. In the latter half of this article, we describe recent progress in the therapy of DMD, with an emphasis on gene therapies, particularly exon skipping.
PMCID: PMC3066538  PMID: 20689232
Duchenne muscular dystrophy; dystrophin; exon skipping; out-of-frame mutation; clinical trial; antisense oligonucleotides
19.  Ubiquitin Ligase Cbl-b Is a Negative Regulator for Insulin-Like Growth Factor 1 Signaling during Muscle Atrophy Caused by Unloading▿ † 
Molecular and Cellular Biology  2009;29(17):4798-4811.
Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine608-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.
PMCID: PMC2725709  PMID: 19546233
20.  CCN3 and bone marrow cells 
CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells. CCN3 expression in bone marrow has been denied by several investigations, but we found CCN3 positive stromal and hematopoietic cells at bone extremities with a new antibody although they are a very few populations. We investigated the expression pattern of CCN3 in the cultured bone marrow derived mesenchymal stem cells and found its preference for osteogenic differentiation. From the analyses of in vitro experiment using an osteogenic mesenchymal stem cell line, Kusa-A1, we found that CCN3 downregulates osteogenesis by two different pathways; suppression of BMP and stimulation of Notch. Secreted CCN3 from Kusa cells inhibited the differentiation of osteoblasts in separate culture, which indicates the paracrine manner of CCN3 activity. CCN3 may also affect the extracellular environment of the niche for hematopoietic stem cells.
PMCID: PMC2721088  PMID: 19626464
Nov; Notch; Stem cells; Niche; Hematopoiesis; Osteogenesis
21.  CCN3 and bone marrow cells 
CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells. CCN3 expression in bone marrow has been denied by several investigations, but we found CCN3 positive stromal and hematopoietic cells at bone extremities with a new antibody although they are a very few populations. We investigated the expression pattern of CCN3 in the cultured bone marrow derived mesenchymal stem cells and found its preference for osteogenic differentiation. From the analyses of in vitro experiment using an osteogenic mesenchymal stem cell line, Kusa-A1, we found that CCN3 downregulates osteogenesis by two different pathways; suppression of BMP and stimulation of Notch. Secreted CCN3 from Kusa cells inhibited the differentiation of osteoblasts in separate culture, which indicates the paracrine manner of CCN3 activity. CCN3 may also affect the extracellular environment of the niche for hematopoietic stem cells.
PMCID: PMC2721088  PMID: 19626464
Nov; Notch; Stem cells; Niche; Hematopoiesis; Osteogenesis
22.  Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy 
Human Molecular Genetics  2008;18(4):621-631.
Hypoglycosylation and reduced laminin-binding activity of α-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated α-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact α-dystroglycan, and solid-phase assays determined laminin binding levels to be ∼50% of normal. In contrast, intact α-dystroglycan is undetectable in the dystrophic Largemyd mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact α-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of α-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of α-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of α-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.
PMCID: PMC2638827  PMID: 19017726
23.  NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS 
The Journal of Clinical Investigation  2007;117(9):2468-2476.
Forkhead box O (Foxo) transcription factors induce muscle atrophy by upregulating the muscle-specific E3 ubiquitin ligases MuRF-1 and atrogin-1/MAFbx, but other than Akt, the upstream regulators of Foxos during muscle atrophy are largely unknown. To examine the involvement of the dystrophin glycoprotein complex (DGC) in regulation of Foxo activities and muscle atrophy, we analyzed the expression of DGC members during tail suspension, a model of unloading-induced muscle atrophy. Among several DGC members, only neuronal NOS (nNOS) quickly dislocated from the sarcolemma to the cytoplasm during tail suspension. Electron paramagnetic resonance spectrometry revealed production of NO in atrophying muscle. nNOS-null mice showed much milder muscle atrophy after tail suspension than did wild-type mice. Importantly, nuclear accumulation of dephosphorylated Foxo3a was not evident in nNOS-null muscle, and neither MuRF-1 nor atrogin-1/MAFbx were upregulated during tail suspension. Furthermore, an nNOS-specific inhibitor, 7-nitroindazole, significantly prevented suspension-induced muscle atrophy. The NF-κB pathway was activated in both wild-type and nNOS-null muscle during tail suspension. We also show that nNOS was involved in the mechanism of denervation-induced atrophy. We conclude that nNOS/NO mediates muscle atrophy via regulation of Foxo transcription factors and is a new therapeutic target for disuse-induced muscle atrophy.
PMCID: PMC1952622  PMID: 17786240
24.  Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo 
The Journal of Cell Biology  2007;176(3):329-341.
Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein–positive CSPs were intravenously infused into adult rats, many more (∼12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.
PMCID: PMC2063959  PMID: 17261849
25.  Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ) 
Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMDJ) model, a suitable animal model for Duchenne muscular dystrophy.
We used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMDJ dogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and in situ zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR.
In CXMDJ skeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMDJ muscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMDJ muscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMDJ did not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMDJ dogs compared to the levels observed in normal controls.
MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.
PMCID: PMC1929071  PMID: 17598883

Results 1-25 (31)