[Show abstract][Hide abstract] ABSTRACT: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5′ and 3′ untranslated regions (UTRs).
In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5′UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5′UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5′UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes.
Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.
[Show abstract][Hide abstract] ABSTRACT: A number of anti-angiogenic drugs targeting vascular endothelial growth factor receptors (VEGF-R) have developed and enabled significant advances in cancer therapy including colorectal cancer. However, acquired resistance to the drugs occurs, leading to disease progression, such as invasion and metastasis. How tumors become the resistance and promote their malignancy remains fully uncertain. One of possible mechanisms for the resistance and the progression may be the direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. We investigated here the direct effect of a VEGF-R-targeting agent, regorafenib, which is the first small molecule inhibitor of VEGF-Rs for the treatment of patients with colorectal cancer, on phenotype changes in colon cancer HCT116 cells. Treatment of cells with regorafenib for only 2 days activated cell migration and invasion, while vehicle-treated control cells showed less activity. Intriguingly, chronic exposure to regorafenib for 90 days dramatically increased migration and invasion activities and induced a resistance to hypoxia-induced apoptosis. These results suggest that loss of VEGF signaling in cancer cells may induce the acquired resistance to VEGF/VEGF-R targeting therapy by gaining two major malignant phenotypes, apoptosis resistance and activation of migration/invasion. J. Med. Invest. 62: 195-198, August, 2015.
Full-text · Article · Sep 2015 · The Journal of Medical Investigation
[Show abstract][Hide abstract] ABSTRACT: Background:
In recent years, the number of bedridden people is rapidly increasing due to aging or lack of exercise in Japan. This problem is becoming more serious, since there is no countermeasure against it. In the present study, we designed to investigate whether dietary proteins, especially soy, had beneficial effects on skeletal muscle in 59 volunteers with various physical activities.
We subjected 59 volunteers with various physical activities to meal intervention examination. Persons with low and high physical activities were divided into two dietary groups, the casein diet group and the soy diet group. They ate daily meals supplemented with 7.8 g of powdered casein or soy protein isolate every day for 30 days. Bedridden patients in hospitals were further divided into three dietary groups: the no supplementation diet group, the casein diet group and the soy diet group. They were also subjected to a blood test, a urinalysis, magnetic resonance imaging analysis and muscle strength test of the knee before and after the meal intervention study.
Thirty-day soy protein supplementation significantly increased skeletal muscle volume in participants with low physical activity, compared with 30-day casein protein supplementation. Both casein and soy protein supplementation increased the volume of quadriceps femoris muscle in bedridden patients. Consistently, soy protein significantly increased their extension power of the knee, compared with casein protein. Although casein protein increased skeletal muscle volume more than soy protein in bedridden patients, their muscle strength changes by soy protein supplementation were bigger than those by casein protein supplementation.
The supplementation of soy protein would be one of the effective foods which prevent the skeletal muscle atrophy caused by immobilization or unloading. J. Med. Invest. 62: 177-183, August, 2015.
Full-text · Article · Sep 2015 · The Journal of Medical Investigation
[Show abstract][Hide abstract] ABSTRACT: To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcino-genesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling.
Full-text · Article · Aug 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: To elucidate a potential anabolic role of heat shock proteins (HSPs) in myofiber preservation, we assessed the effect of HSP70 gene silencing versus its overexpression on skeletal muscle atrophy or rescue. HSP72 gene expression was silenced by pre-treatment with HSP72 siRNA in cultured rat L6 myotubes, and the pro-anabolic effect of HSPs was examined in the absence or presence of the HSP inducer celastrol (CEL). Compared to the negative control (NC), both nuclear accumulation and phosphorylation of heat shock transcription factor 1 remained high under the 6-h treatment of CEL. The HSP72 siRNA treatment significantly decreased HSP72 mRNA and protein expression and myotube diameter. CEL treatment, however, markedly increased the HSP72 expression and rendered the myotube size recovered to the NC level even in the siRNA-treated cells. Moreover, the HSP72 siRNA upregulated forkhead box O3 (FoxO3) expression in the nucleus while CEL increased p-FoxO3 exclusively in the cytoplasm, thus leaving the p-FoxO3/FoxO3 balanced to the NC level by siRNA + CEL treatment. The atrophic effect of HSP72 siRNA was consistent with the upregulation of atrogin-1 and proteasome activity but CEL treatment abrogated such effect by activation of Akt1, ribosomal S6 kinase (S6K) and extracellular signal-regulated kinase 1/2 (ERK1/2), irrespective of HSP72 silencing. These results suggest that CEL-mediated overexpression of HSP72 overcomes the atrophic effect of HSP72 gene silencing via both enhancement of FoxO3 phosphorylation and activation of Akt1-ERK1/2 signaling pathway.
Full-text · Article · Apr 2015 · Journal of physiology and pharmacology: an official journal of the Polish Physiological Society
[Show abstract][Hide abstract] ABSTRACT: Muscle atrophy is a complex process that occurs as a consequence of various stress events. Muscle atrophy-associated genes (atrogenes) such as atrogin-1/MAFbx and MuRF-1 are induced early in the atrophy process, and the increase in their expression precedes the loss of muscle weight. Although antioxidative nutrients suppress atrogene expression in skeletal muscle cells, the inhibitory effects of flavonoids on inflammation-induced atrogin-1/MAFbx expression have not been clarified. Here, we investigated the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced atrogin-1/MAFbx expression. We examined whether nine flavonoids belonging to six flavonoid categories inhibited atrogin-1/MAFbx expression in mouse C2C12 myotubes. Two major flavones, apigenin and luteolin, displayed potent inhibitory effects on atrogin-1/MAFbx expression. The pretreatment with apigenin and luteolin significantly prevented C2C12 myotube diameter caused by LPS stimulation. Importantly, the pretreatment of LPS-stimulated myoblasts with these flavones significantly inhibited LPS-induced JNK phosphorylation in C2C12 myotubes, resulting in the significant suppression of atrogin-1/MAFbx promoter activity. These results suggest that apigenin and luteolin, prevent LPS-mediated atrogin-1/MAFbx expression through the inhibition of the JNK signaling pathway in C2C12 myotubes. Thus, these flavones, apigenin and luteolin, may be promising agents to prevent LPS-induced muscle atrophy.
Full-text · Article · Apr 2015 · Journal of Nutritional Science and Vitaminology
[Show abstract][Hide abstract] ABSTRACT: VEGF-targeting anti-angiogenic drugs have enabled significant advances in cancer therapy. However, acquired resistance to VEGF-targeting drugs occurs, leading to disease progression. How tumors become the resistance remains fully uncertain. One of possible mechanisms for the resistance may be the direct effect of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGF-R). We investigated here the direct effect of chronic VEGF inhibition on phenotype changes in cancer cells. To chronically inhibit cancer cell-derived VEGF, human colon cancer HCT116 cells were chronically exposed (3 months) to anti-VEGF neutralizing monoclonal antibody (HCT/mAb cells, blockade of VEGF alone) or VEGF-R tyrosine kinase inhibitor foretinib (HCT/fore cells, blockade of all VEGF family). HCT/mAb cells redundantly increased VEGF family member (VEGF, PlGF, VEGF-B, VEGF-R1 and VEGF-R2) and induced a resistance to hypoxia-induced apoptosis. By contrast, HCT/fore cells did not show the redundant increase in VEGF family member, but significantly increased a VEGF-independent pro-angiogenic factor FGF-2. HCT/fore cells showed increased migration and invasion activities in addition to a resistance to hypoxia-induced apoptosis. The resistance to apoptosis was significantly suppressed by inhibition of hypoxia-inducible factor-1α in HCT/mAb cells, but not in HCT/fore cells. These findings suggest that chronic inhibition of VEGF/VEGF-R accelerates malignant phenotypes of colon cancer cells. J. Med. Invest. 62: 75-79, February, 2015.
Full-text · Article · Mar 2015 · The Journal of Medical Investigation
[Show abstract][Hide abstract] ABSTRACT: The central circadian clock in the suprachiasmatic nucleus of the hypothalamus synchronizes peripheral clocks through neural and humoral signals in most mammalian tissues. Here, we analyzed the effects of unilateral sciatic denervation on the expression of circadian clock- and clock-controlled genes in the gastrocnemius muscles of mice twice per day on days 0, 3, 7, 9, 11 and 14 after denervation and six times on each of days 7 and 28 after denervation to assess the regulation mechanism of the circadian clock in skeletal muscle. Sciatic denervation did not affect systemic circadian rhythms since core body temperature (Day 7), corticosterone secretion (Days 7 and 28), and hepatic clock gene expression remained intact (Days 7 and 28). Expression levels of most circadian clock-related genes such as Arntl, Per1, Rora, Nr1d1 and Dbp were reduced in accordance with the extent of muscle atrophy, although circadian Per2 expression was significantly augmented (Day 28). Cosinor analysis revealed that the circadian expression of Arntl (Days 7 and 28) and Dbp (Day 28) was phase advanced in denervated muscle. The mRNA expression of Clock was significantly increased in denervated muscle on Day 3 when the severe atrophy was absent, and it was not affected by atrophic progression for 28 days. Sciatic denervation did not affect the expression of these genes in the contralateral muscle (Days 7 and 28), suggesting that humoral changes were not involved in denervation-induced muscle clock disruption. We then analyzed genome-wide gene expression using microarrays to determine the effects of disrupting the molecular clock in muscle on circadian rhythms at Day 7. Among 478 circadian genes, 313 lost rhythmicity in the denervated muscles. These denervation-sensitive genes included the lipid metabolism-related genes, Nrip1, Bbs1, Ptgis, Acot1, Scd2, Hpgd, Insig1, Dhcr24, Ldlr and Mboat1. Our findings revealed that sciatic denervation disrupts the circadian expression of clock and clock-controlled genes either directly or indirectly via muscle atrophy in the gastrocnemius muscles of mice in a gene-specific manner.
No preview · Article · Mar 2015 · Chronobiology International
[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle atrophy occurs when the rate of protein degradation exceeds that of protein synthesis in various catabolic conditions, such as fasting, disuse, aging, and chronic diseases. Insulin like growth factor-1 (IGF-1) signaling stimulates muscle growth and suppresses muscle protein breakdown. In atrophied muscles, ubiquitin ligase, Cbl-b, increases and stimulates the ubiquitination and degradation of IRS-1, an intermediate in IGF-1 signaling pathway, resulting in IGF-1 resistance. In this study, we evaluated the efficacy of atelocollagen (ATCOL)-transported anti-ubiquitination oligopeptide (Cblin: Cbl-b inhibitor) (consisting of tyrosine phosphorylation domain of IRS-1) in starved C2C12 myotubes. The amount of IRS-1 protein was lower in starved versus unstarved myotubes. The Cblin-ATCOL complex inhibited IRS-1 degradation in a concentration-dependent manner. Myotubes incubated with Cblin-ATCOL complex showed significant resistance to starvation-induced atrophy (p<0.01). Furthermore, Cblin-ATCOL complex significantly inhibited any decrease in Akt phosphorylation (p<0.01) and localization of FOXO3a to the nucleus in starved myotubes. These results suggest that Cblin prevented starvation-induced C2C12 myotube atrophy by maintaining the IGF-1/Akt/FOXO signaling. Therefore, attachment of anti-ubiquitination oligopeptide, Cblin, to ATCOL enhances its delivery to myotubes and could be a potentially effective strategy in the treatment of atrophic myopathies.
No preview · Article · Feb 2015 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine whether osteoactivin attenuated skeletal muscle fibrosis caused by distraction osteogenesis. Tibial osteotomies were performed on wild-type and osteoactivin-transgenic (OA-Tg) mice, and tibiae were distracted for 2 weeks. Ankle plantar flexion torque and the gastrocnemius muscles were analyzed. The amount and area of collagenous tissue and the passive torque were reduced in the OA-Tg group at 8 weeks after osteotomy. Transcript levels of matrix metalloprotease (mmp)-3 and MMP-9 were upregulated, and MMP-3 and MMP-9 proteins were increased in the OA-Tg group. Osteoactivin-mediated increase in MMPs may attenuate skeletal muscle fibrosis.
No preview · Article · Nov 2014 · Journal of Pediatric Orthopaedics B
[Show abstract][Hide abstract] ABSTRACT: Heat shock protein 70 is known to inhibit muscle atrophy in in vitro and in vivo models. The goal of this study was to
investigate whether a herbal compound celastrol (CEL) promoted HSP70 overexpression via activation of heat shock
transcription factor 1 (HSF1) in C2C12 myotubes. The HSP70 expression was increased in a time-dependent manner with
5.8- to 9.1-fold upregulation at 4 h after 1.5 M CEL treatment. Nuclear accumulation of phospho-HSF1 was evident at
2 h over the 6-h CEL treatment. Phosphorylation of Akt1 (the protein anabolic marker) was also increased 3.5- to 6.6-
fold at 2 h while total Akt1 expression was not changed over the CEL treatment. These results imply that CEL has a
potency to inhibit muscle atrophy via HSP70 induction and Akt1 activation. The results will be used for development of a
protocol for a future ISS/JEM onboard experiment.
Full-text · Article · Oct 2014 · Transactions of the Japan Society for Aeronautical and Space Sciences, Aerospace Technology Japan
[Show abstract][Hide abstract] ABSTRACT: Objective: Insulin like growth factor-1 (IGF-1) signaling stimulates muscle growth as well as suppresses muscle protein breakdown. In atrophied muscles, ubiquitin ligase, Cbl-b, increases and it stimulates the ubiquitination and degradation of IRS-1, an intermediate in IGF-1 singnaling pathway, resulting in IGF-1 resistance. In this study, we evaluate the efficacy of atelocollagen (ATCOL) mediated application of anti-ubiquitination oligopeptide that inhibits the interaction between Cbl-b and IRS-1 into starved C2C12 myotubes and denervated tibialis anterior muscle of C57BL/6 wild-type mice.
Method: After serum-starvation for 24 hours, the protein level of IRS-1 and Akt phosphorylation were examined by immunoblotting analysis. The tibialis anterior muscles were dissected 10 days after the denervation for histometric analyses.
Result: Although the amount of IRS-1 protein decreased in starved myotubes, oligopeptide inhibited the degradation of IRS-1 does-dependently. Moreover, oligopeptide significantly inhibited the decrease of Akt phosphorylation in starved myotubes. In addition, sectional area of denervated muscle fibers treated with oligopeptide was significantly larger compared to non-treated denervated muscles.
Conclusion: These results suggest that ATCOL mediated application of anti-ubiquitination oligopeptide could be an effective tool for therapeutic use in muscular atrophy diseases.
[Show abstract][Hide abstract] ABSTRACT: The activation of T cells is known to be accompanied by the temporary down-modulation of the T-cell receptor (TCR)/CD3 complex on the cell surface. Here we established a novel monoclonal antibody, Dow2, that temporarily induces down-modulation of the TCR/CD3 complex in mouse CD4+ T cells without activating T cells. Dow2 recognized the determinant on CD3ε; however, differences were observed in the binding mode between Dow2 and the agonistic anti-CD3ε Ab, 145–2C11. An injection of Dow2 in vivo resulted in T-cell anergy, and prolonged the survival of cardiac allografts without a marked increase in cytokine release. The phosphorylated forms of the signaling proteins PLC-γ1 and LAT in Dow2-induced anergic T cells were markedly decreased upon stimulation. However, the levels of phosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells could be rescued in the presence of the proteasome inhibitor MG-132. These results suggest that proteasome-mediated degradation is involved in hypophosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells. The novel CD3-specific Ab, Dow2, may provide us with a unique tool for inducing immunosuppression. This article is protected by copyright. All rights reserved
No preview · Article · Jun 2014 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Obesity causes type 2 diabetes, atherosclerosis and cardiovascular diseases by inducing systemic insulin resistance. It is now recognized that obesity is related to chronic low-grade inflammation in adipose tissue. Specifically, activated immune cells infiltrate adipose tissue and cause inflammation. There is increasing evidence that activated macrophages accumulate in the hypertrophied adipose tissue of rodents and humans and induce systemic insulin resistance by secreting inflammatory cytokines. Accordingly, a better understanding of the molecular mechanisms underlying macrophage activation in adipose tissue will facilitate the development of new therapeutic strategies. Currently, little is known about the regulation of macrophage activation, although E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl)-b was identified recently as a novel negative regulator of macrophage activation in adipose tissue. Cbl-b, which is a suppressor of T- and B-cell activation, inhibits intracellular signal transduction by targeting some tyrosine kinases. Notably, preventing Cbl-b-mediated macrophage activation improves obesity-induced insulin resistance in mice. c-Cbl is another member of the Cbl family that is associated with insulin resistance in obesity. These reports suggest that Cbl-b and c-Cbl are potential therapeutic targets for treating obesity-induced insulin resistance. In this review, we focus on the importance of Cbl-b in macrophage activation in aging-induced and high-fat diet-induced obesity.
No preview · Article · Mar 2014 · Endocrine Journal
[Show abstract][Hide abstract] ABSTRACT: Proinflammatory cytokines are factors that induce ubiquitin-proteasome-dependent proteolysis in skeletal muscle, causing muscle atrophy. Although isoflavones, as potent antioxidative nutrients, have been known to reduce muscle damage during the catabolic state, the non-antioxidant effects of isoflavones against muscle atrophy are not well known. Here we report on the inhibitory effects of isoflavones such as genistein and daidzein on muscle atrophy caused by tumor necrosis factor (TNF)-α treatment. In C2C12 myotubes, TNF-α treatment markedly elevated the expression of the muscle-specific ubiquitin ligase MuRF1, but not of atrogin-1, leading to myotube atrophy. We found that MuRF1 promoter activity was mediated by acetylation of p65, a subunit of NFκB, a downstream target of the TNF-α signaling pathway; increased MuRF1 promoter activity was abolished by SIRT1, which is associated with deacetylation of p65. Of interest, isoflavones induced expression of SIRT1 mRNA and phosphorylation of AMP kinase, which is well known to stimulate SIRT1 expression, although there was no direct effect on SIRT1 activation. Moreover, isoflavones significantly suppressed MuRF1 promoter activity and myotube atrophy induced by TNF-α in C2C12 myotubes. These results suggest that isoflavones suppress myotube atrophy in skeletal muscle cells through activation of SIRT1 signaling. Thus, the efficacy of isoflavones could provide a novel therapeutic approach against inflammation-related muscle atrophy.
Full-text · Article · Sep 2013 · Journal of Nutritional Science and Vitaminology
[Show abstract][Hide abstract] ABSTRACT: Celastrol (CEL) is known as a potent inducer of heat shock protein (HSP) in non-muscle cells and exhibits cytoprotective function and inhibitory effects on proteasome and glucocorticoid receptor activities. To investigate an anti-atrophic effect of CEL on skeletal muscle cells, C2C12 myotubes were treated with 150 μM dexamethasone (DEX) for 24 h and 1.5 μM CEL was added for the last 6 h during the 24h DEX treatment. Compared to the control, the myotube diameter was reduced by a factor of 0.30 by DEX, but CEL treatment almost abrogated the DEX-induced atrophy. CEL treatment also increased expression of HSP72 and phosphorylation of heat shock transcription factor 1 (p-HSF1) 11-fold and 3.4-fold, respectively, as well as accumulation of p-HSF1 in the nucleus. Furthermore, CEL treatment elevated activities of Akt1, p70/S6K and ERK1/2 2.0- to 4.4-fold whereas DEX had no effect on these signaling activities. Inhibition of Akt1 and ERK1/2 pathways by specific inhibitors confirmed CEL-induced anti-atrophic effect. Moreover, DEX-mediated downregulation of FoxO3 phosphorylation and upregulation of MuRF1 expression and proteasome activity were abrogated by CEL treatment. These results demonstrate a novel anti-atrophic function of CEL in muscle cells via both activation of protein anabolic signals and suppression of catabolic signaling activities.
Full-text · Article · Jun 2013 · Archives of Biochemistry and Biophysics