Nogo-A is critical for pro-inflammatory gene regulation in myocytes and macrophages

Abstract Background: Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, the role of Nogo-A in inflammatory mechanisms remains unclear. Therefore, in this study, we used Nogo-knockout (KO) mice to explore its potential role in the inflammatory process. Here, we investigated whether Nogo-A affects the inflammatory process through transcription factor C/EBP homologous protein (CHOP). Results: Our results demonstrated that Nogo-A, CHOP, and pro-inflammatory factors were activated in the following: notexin-induced muscle injury, in human Duchenne muscular dystrophy (DMD) patients, in dystrophin-deficient (mdx) mice, in differentiated C2C12 myoblast cells, and in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDM). Moreover, we found that Nogo-KO BMDM exhibited lower migratory ability compared with wild type (WT) BMDM after LPS treatment. Conclusion: Our data demonstrated that the Nogo-A-CHOP signaling pathway regulated the inflammatory process in notexin-induced injured muscle, in mdx mice, in DMD patients, in differentiated C2C12 cells, and in LPS-stimulated BMDM. Taken together, these results suggest that Nogo-A plays a vital role in inflammatory processes, which resembles the pathological mechanisms observed in the CNS.

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Osteocalcin Induces Proliferation via Positive Activation of the PI3K/Akt, P38 MAPK Pathways and Promotes Differentiation Through Activation of the GPRC6A-ERK1/2 Pathway in C2C12 Myoblast Cells

Cellular Physiology and Biochemistry ◽

10.1159/000481752 ◽

2017 ◽

Vol 43 (3) ◽

pp. 1100-1112 ◽

Cited By ~ 22

Author(s):

Suifeng Liu ◽

Feng Gao ◽

Lei Wen ◽

Min Ouyang ◽

Yi Wang ◽

...

Keyword(s):

Cell Proliferation ◽

P38 Mapk ◽

C2c12 Cell ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

C2c12 Myoblasts ◽

Mapk Phosphorylation ◽

Mapk Pathways ◽

Myoblast Cells

Background/Aims: Sarcopenia is characterized by an age-related decline in skeletal muscle plus low muscle strength and/or physical performance. Despite the clinical significance of sarcopenia, the molecular pathways underlying sarcopenia remain elusive. The recent demonstration that undercarboxylated osteocalcin (ucOC) favours muscle function related to insulin sensitivity and glucose metabolism raises the question of whether this hormone may also regulate muscle mass. The present study explored the promotive effects of ucOC in proliferation and differentiation processes of C2C12 myoblasts as well as the possible signalling pathways involved. Methods: The effects of exogenous ucOC on C2C12 myoblasts proliferation were assessed using CCK8 and immunohistological staining assays. C2C12 cells were pretreated with PI3K/Akt or P38 MAPK inhibitors to investigate the possible involvement of the PI3K/Akt and P38 MAPK pathways in proliferation. The levels of Akt, phosphorylated-Akt (p-Akt), P38, and phosphorylated-P38 (p-P38) were measured by Western Blotting. The effects of ucOC on myoblast differentiation were quantified by morphological analysis. A silencing experiment was conducted in which the expression of GPRC6A in C2C12 myoblasts was modified. The expression of GPRC6A, myosin heavy chain (MyHC) and the related ERK1/2 signalling pathway in C2C12 myoblasts were monitored by qRT-PCR and Western Blotting. Results: We showed that treatment with exogenous ucOC stimulated the priming of C2C12 myoblasts proliferation. Inhibition of Akt phosphorylation by wortmannin or inhibition of P38 MAPK phosphorylation by SB203580 decreased C2C12 cell proliferation. Wortmannin also reduced P38 MAPK phosphorylation, whereas SB203580 did not affect Akt activation. Furthermore, ucOC promoted C2C12 myoblast differentiation. Inhibition of ERK1/2 phosphorylation with U0126 decreased C2C12 cell differentiation. Finally, GPRC6A expression was substantially increased after ucOC treatment of C2C12 cells. GPRC6A silencing inhibited Akt, P38 MAPK phosphorylation in C2C12 cells, and ERK1/2 phosphorylation in C2C12 myotubes; GPRC6A silencing also decreased cell proliferation, decreased cell differentiation, and downregulated MyHC expression. Conclusions: The present data suggest that ucOC induces myoblast proliferation via sequential activation of the PI3K/Akt and p38 MAPK pathways in C2C12 myoblast cells. Moreover, ucOC enhances myogenic differentiation via a mechanism involving GPRC6A-ERK1/2 signalling.

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Resveratrol Decreases Oxidative Stress by Restoring Mitophagy and Improves the Pathophysiology of Dystrophin-Deficient mdx Mice

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/9179270 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 21

Author(s):

Rio Sebori ◽

Atsushi Kuno ◽

Ryusuke Hosoda ◽

Takashi Hayashi ◽

Yoshiyuki Horio

Keyword(s):

Creatine Kinase ◽

Therapeutic Agent ◽

Serum Creatine Kinase ◽

Physical Activities ◽

Mdx Mice ◽

C2c12 Myoblast ◽

Autophagic Flux ◽

Oxygen Species ◽

Central Nuclei ◽

Myoblast Cells

We previously showed that treatment with resveratrol (3,5,4′-trihydroxy-trans-stilbene), an activator of the NAD+-dependent deacetylase SIRT1 at 4 g/kg food for 32 weeks, significantly decreased the muscular reactive oxygen species (ROS) levels and ameliorated the pathology of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Here, we treated mdx mice with various doses of resveratrol (0.04, 0.4, and 4 g/kg food) for 56 weeks and examined the effects on serum creatine kinase levels and physical activities. Because resveratrol promotes autophagy, we also investigated whether autophagy including mitochondrial autophagy (mitophagy) is involved in resveratrol’s effects. Autophagy/mitophagy-related genes and autophagic flux were downregulated in the muscle of mdx mice, and these phenomena were reversed by resveratrol with significant ROS reduction. Resveratrol at 4 g/kg food reduced the number of immature myofibers containing central nuclei and fine fibers < 400 μm2 and increased that of thicker myofibers in the quadriceps, suggesting that resveratrol decreased myofiber wasting and promoted muscular maturation. Accordingly, resveratrol at 0.4 g/kg food reduced the creatine kinase levels to one-third of those in untreated mdx mice and significantly increased the animals’ physical activities. In C2C12 myoblast cells, resveratrol promoted mitophagy and eliminated mitochondria containing high superoxide levels. The clearance of damaged mitochondria and ROS reduction by resveratrol was completely suppressed by an autophagy inhibitor (chloroquine) and by knocking down Atg5 or Pink1, essential genes for autophagy and mitophagy, respectively. Thus, resveratrol is a potential therapeutic agent for DMD, and the clearance of damaged mitochondria probably contributes to its action.

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LOW GLUCOSE ENHANCES THE CYTOPROTECTIVE EFFECT OF METFORMIN AGAINST DOXORUBICIN-INDUCED CYTOTOXICITY

Innovation in Aging ◽

10.1093/geroni/igz038.3486 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S961-S961

Author(s):

Fathima Ameer ◽

Xiaomin Zhang ◽

Gohar Azhar ◽

Yingni Che ◽

Jeanne Wei

Keyword(s):

Mitochondrial Fission ◽

Pegylated Liposomal Doxorubicin ◽

C2c12 Cells ◽

C2c12 Myoblast ◽

Protective Effects ◽

Gene Expressions ◽

Biochemical Alterations ◽

Low Glucose ◽

Pre Treatment ◽

Myoblast Cells

Abstract Metformin, an oral anti-diabetic drug, is currently being investigated for its anti-aging properties and has also been used as adjunct therapy in cancer. Cancer is a disease of aging. Type 2 diabetes is also prevalent in older adults. We wanted to test the hypothesis that metformin could protect normal cells during chemotherapy treatment under different glucose conditions. We used C2C12 myoblast cells to study cellular bioenergetics, variations in gene expressions, and biochemical alterations induced by metformin and pegylated liposomal doxorubicin (L-Doxo) under low glucose (2.7 mM or 50 mg/dL) and normal physiologic glucose (5.5 mM, or 100 mg/dL) conditions. Using confocal microscopy, we noted that treatment of C2C12 cells with 30 µg/mL L-Doxo under low glucose and normal physiologic glucose conditions induced cellular defects. Furthermore, L-Doxo treatment dysregulated the expression of mitochondrial fission and fusion genes, which may influence transformation of the network’s connectivity. L-Doxo treatment significantly reduced mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). However, pre-treatment with 100 nM metformin provided protection against L-Doxo-induced damage and increased cell viability and ATP levels in cells even under low glucose conditions. Our data provide further evidence by which low dose metformin exerts protective effects against L-Doxo, a chemotherapeutic drug, under low glucose conditions. Metformin appears to act via AMPKα, Raptor, and SRF, and has significant cellular protective effects that may be useful in cancer and/or aging.

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Platelet-poor plasma of athletes is a potent inducer of myogenic differentiation of C2C12 myoblasts

Veterinarski glasnik ◽

10.2298/vetgl190414019m ◽

2020 ◽

Vol 74 (1) ◽

pp. 18-33

Author(s):

Irina Maslovaric ◽

Vesna Ilic ◽

Ana Stancic ◽

Juan Santibanez ◽

Drenka Trivanovic ◽

...

Keyword(s):

Myogenic Differentiation ◽

Platelet Rich Plasma ◽

C2c12 Cell ◽

C2c12 Cells ◽

Football Players ◽

C2c12 Myoblast ◽

Potent Inducer ◽

Gelatinase Activity ◽

Platelet Poor Plasma ◽

Myoblast Cells

Introduction. Blood products, i.e. platelet rich plasma (PRP), leukocyte-poor plasma (PRP) and platelet poor plasma (PPP), have previously been used to improve muscle regeneration. In this study, six months? frozen-stored PPP of individuals who practiced different types of physical exercise was analysed; it could steer mouse C2C12 myoblast cells towards proliferation, migration and myogenic differentiation, and it could affect the morphology/shape of myotubes. Materials and Methods. PPP of male Olympic weightlifters, football players and professional folk dancers, aged 15-19, was collected 12 h post-training and stored for 6 months at -20?C. C2C12 cell proliferation was assessed by MTT test, motility by scratch assay, myogenic differentiation by myotube formation and gelatinase activity by gel-zymography. Results and Conclusions. PPP induced proliferation and migration of C2C12 cells. Proliferative capacity was as follows: weightlifters > dancers > football players; mean migratory capacity was: weightlifters = dancers > football players. PPP induced formation of myotubes; significant inter-individual variations were detected: PPP from weightlifters induced formation of round myotubes, and PPP from football players and dancers induced formation of elongated myotubes. The mean myotube area was as follows: football players > dancers > weightlifters. PPP gelatinolytic activity was observed; it was negatively correlated with C2C12 myoblast proliferation. These results provide general but distinct evidence that PPP of individuals practicing certain types of exercise can specifically modify myoblast morphology/function. This is significant for explaining physiological responses and adaptations to exercise. In conclusion, long-term, frozen-stored PPP preserves its potential to modify myoblast morphology and function.

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2-D08 treatment regulates C2C12 myoblast proliferation and differentiation via the Erk1/2 and proteasome signaling pathways

Journal of Muscle Research and Cell Motility ◽

10.1007/s10974-021-09605-x ◽

2021 ◽

Author(s):

Hyunju Liu ◽

Su-Mi Lee ◽

Hosouk Joung

Keyword(s):

Myogenic Differentiation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Covalent Attachment ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

Long Term Effects ◽

Proliferation And Differentiation ◽

Myoblast Proliferation ◽

Myoblast Cells

AbstractSUMOylation is one of the post-translational modifications that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) to the substrate. SUMOylation regulates multiple biological processes, including myoblast proliferation, differentiation, and apoptosis. 2-D08 is a synthetically available flavone, which acts as a potent cell-permeable SUMOylation inhibitor. Its mechanism of action involves preventing the transfer of SUMO from the E2 thioester to the substrate without influencing SUMO-activating enzyme E1 (SAE-1/2) or E2 Ubc9-SUMO thioester formation. However, both the effects and mechanisms of 2-D08 on C2C12 myoblast cells remain unclear. In the present study, we found that treatment with 2-D08 inhibits C2C12 cell proliferation and differentiation. We confirmed that 2-D08 significantly hampers the viability of C2C12 cells. Additionally, it inhibited myogenic differentiation, decreasing myosin heavy chain (MHC), MyoD, and myogenin expression. Furthermore, we confirmed that 2-D08-mediated anti-myogenic effects impair myoblast differentiation and myotube formation, reducing the number of MHC-positive C2C12 cells. In addition, we found that 2-D08 induces the activation of ErK1/2 and the degradation of MyoD and myogenin in C2C12 cells. Taken together, these results indicated that 2-D08 treatment results in the deregulated proliferation and differentiation of myoblasts. However, further research is needed to investigate the long-term effects of 2-D08 on skeletal muscles.

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Transcriptional Regulation of the Murine Urokinase-type Plasminogen Activator Gene in Skeletal Myoblasts

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614569 ◽

1999 ◽

Vol 81 (05) ◽

pp. 767-774 ◽

Cited By ~ 9

Author(s):

Francesc Miralles ◽

Inés Ibáñez-Tallon ◽

Maribel Parra ◽

Massimo Crippa ◽

Francesco Blasi ◽

...

Keyword(s):

Plasminogen Activator ◽

C2c12 Cells ◽

Regulatory Function ◽

C2c12 Myoblast ◽

Mobility Shift ◽

Nuclear Extracts ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Myoblast Cells

SummaryWe have previously shown that urokinase-type plasminogen activator (uPA) is highly expressed in murine C2C12 myoblasts and that antibodies against uPA are able to block both myoblast fusion and differentiation. Here we show the characterization of cis-acting elements in the mouse uPA promoter in vitro which are involved in uPA gene expression in C2C12 myoblast cells. DNase I hypersensitive (HS) site analysis revealed the presence of three HS sites in myoblasts. Deletion analysis of stably transfected uPA-promoter constructs revealed that at least two of the three HS sites accounted for the high transcriptional expression in C2C12 cells. One was located at -2.4 kb and corresponded to a known PEA3/AP1A element and the other one was located at -4.9 kb and contained a CArG box and a CRE element. So far, no regulatory function had been assigned to this CRE/CArG element. Both HS sites alone were able to activate transcription of a heterologous promoter and showed a cooperative effect when placed together. Electrophoretic mobility-shift assays using myoblast nuclear extracts and specific antibodies demonstrated that cJun, JunD and ATF2 bound to the PEA3/AP1A element, whereas the CRE/CArG element bound SRF. Altogether, these results suggest that high uPA expression in myoblasts is dependent on the cooperation of two regulatory sites in the uPA promoter.

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Curcumin Ameliorated Oxidative Stress and Inflammation-Related Muscle Disorders in C2C12 Myoblast Cells

Antioxidants ◽

10.3390/antiox10030476 ◽

2021 ◽

Vol 10 (3) ◽

pp. 476

Author(s):

Da-Yeon Lee ◽

Yoon-Seok Chun ◽

Jong-Kyu Kim ◽

Jeong-Ok Lee ◽

Young-Joon Lee ◽

...

Keyword(s):

Oxidative Stress ◽

Radical Scavenging ◽

C2c12 Cells ◽

C2c12 Myoblast ◽

Related Factor ◽

Dependent Manner ◽

Quinone Oxidoreductase ◽

Muscle Disorders ◽

Myoblast Cells ◽

Dose Dependent

The purpose of the current study was to investigate antioxidant and anti-inflammatory effects of spray dry powder containing 40% curcumin (CM-SD) in C2C12 myoblast cells. CM-SD increased DPPH radical scavenging activity in a dose-dependent manner, and up to 30 μg/mL of CM-SD did not express cytotoxicity in C2C12 cells. Exposure to hydrogen peroxide (H2O2) drastically decreased the viability of C2C12 cells, but pre-treatment of CM-SD significantly increased the cell viability (p < 0.01). CM-SD significantly transactivated the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent luciferase activity in a dose-dependent manner and enhanced the levels of heme oxygenase (HO)-1, glutamate cysteine ligase catalytic subunit (GCLC), and NAD(P)H-dependent quinone oxidoreductase (NQO)-1. CM-SD also significantly reduced reactive oxygen species (ROS) production and lipid peroxidation and restored glutathione (GSH) depletion in H2O2-treated C2C12 cells. Moreover, CM-SD significantly reduced lipopolysaccharides (LPS)-mediated interleukin (IL)-6 production in the conditioned medium. Results from the current study suggest that CM-SD could be a useful candidate against oxidative stress and inflammation-related muscle disorders.

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A Study of C2C12 Myoblast Bioenergetics in Response to the CCG-1423 Rho A Inhibitor

Innovation in Aging ◽

10.1093/geroni/igab046.2567 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. 689-689

Author(s):

Bachkhoa Nguyen ◽

Fathima Ameer ◽

Jasmine Crane ◽

Gohar Azhar ◽

Xiaomin Zhang ◽

...

Keyword(s):

Gene Expression ◽

Oxygen Consumption ◽

Mitochondrial Function ◽

Consumption Rate ◽

C2c12 Cells ◽

C2c12 Myoblast ◽

Dependent Manner ◽

Mitochondrial Oxygen Consumption ◽

Myoblast Cells ◽

Dose Dependent

Abstract CCG-1423 is a Rho A pathway inhibitor which has been reported to inhibit Rho/SRF-mediated transcriptional regulation. SRF and SRF cofactors, which include ternary complex factors (TCFs) and myocardin-related transcription factor (MRTF), regulate various cellular functions. The Rho/SRF signaling pathway also regulates the sirtuin 2 (SIRT2) gene that contains a classic serum response element (SRE) sequence. Current research on CCG-1423 focuses on gene expression levels of SRF in response to CCG-1423 and how SRF levels affect the cells; the studies are focused on cell morphology, migration, viability/reproduction, and overall function. The pathways of this inhibitor have yet to be fully elucidated, but several have been suggested with good evidence. Our goal is to study the effect of CCG-1423 on mitochondrial function and gene expression of cells. In this work C2C12 myoblast cells have been used as an in-vitro model to study cellular bioenergetics and variations in gene expressions induced by CCG-1423. The effect of CCG-1423 on mitochondrial function was determined by measuring the mitochondrial oxygen consumption rate and glycolysis rate after treating C2C12 cells with varying concentrations of CCG-1423 overnight. In C2C12 myoblast cells, CCG-1423 treatment significantly reduced mitochondrial oxygen consumption rate (OCR) in a dose-dependent manner. However, treatment of C2C12 cells with CCG-1423 overnight increased the extracellular acidification rate (ECAR) in a dose-dependent manner. By indicating that CCG-1423 represses mitochondrial respiration via the Rho-SRF signaling pathway, the results of this study may enable a better understanding of the bioenergetics of the cell in the aging body.

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PGC-1α is associated with C2C12 Myoblast differentiation

Open Life Sciences ◽

10.2478/s11535-014-0341-y ◽

2014 ◽

Vol 9 (11) ◽

pp. 1030-1036 ◽

Cited By ~ 5

Author(s):

Yaqiu Lin ◽

Yanying Zhao ◽

Ruiwen Li ◽

Jiaqi Gong ◽

Yucai Zheng ◽

...

Keyword(s):

Mrna Level ◽

Biological Significance ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

Mrna Levels ◽

Transfected Cells ◽

Myosin Heavy Chain Isoform ◽

Important Mediator

AbstractPGC-1α has been implicated as an important mediator of functional capacity of skeletal muscle. However, the role of PGC-1α in myoblast differentiation remains unexplored. In the present study, we observed a significant up-regulation of PGC-1α expression during the differentiation of murine C2C12 myoblast. To understand the biological significance of PGC-1α up-regulation in myoblast differentiation, C2C12 cells were transfected with murine PGC-1α cDNA and siRNA targeting PGC-1α, respectively. PGC-1α over-expressing clones fused to form typical myotubes with higher mRNA level of myosin heavy chain isoform I (MyHCI) and lower MyHCIIX. No obvious differentiation was observed in PGC-1α-targeted siRNA-transfected cells with marked decrement of mRNA levels of MyHCI and MyHCIIX. Furthermore, PGC-1α increased the expression of MyoD and MyoG in C2C12 cells, which controlled the commitment of precursor cells to myotubes. These results indicate that PGC-1α is associated with myoblast differentiation and elevates MyoD and MyoG expression levels in C2C12 cells.

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Abstract 204: HAX-1: A Novel Regulator of Energetics and Oxidative Stress in the Heart

Circulation Research ◽

10.1161/res.115.suppl_1.204 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Chi K Lam ◽

Wen Zhao ◽

Wenfeng Cai ◽

Guansheng Liu ◽

Phil Bidwell ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Heart Function ◽

Ischemia Reperfusion ◽

Calcium Handling ◽

Calcium Kinetics ◽

Atp Production ◽

Homologous Protein ◽

Induced Apoptosis ◽

Factor C

Sarcoplasmic reticulum (SR) calcium handling is central not only in the control of heart function during excitation-contraction coupling but also in mitochondrial energetics and apoptosis. Recent studies have identified the anti-apoptotic protein, HS-1 associated protein X-1 (HAX-1) as a novel regulator of SR calcium cycling. Although HAX-1 has been shown to localize to mitochondria in various tissues, we found out that it also localizes to SR through its interaction with phospholamban (PLN) in cardiac muscle. Acute or chronic overexpression of HAX-1 in cardiomyocytes promoted PLN inhibition on the calcium ATPase (SERCA) and decreased cardiomyocyte calcium kinetics and contractile parameters. Accordingly, ablation of HAX-1 significantly enhanced SERCA activity and calcium kinetics. Furthermore, the HAX-1/PLN interaction appeared to also regulate cardiomyocyte survival. Indeed, overexpression of HAX-1 and the associated depressed SR Ca-load attenuated endoplasmic reticulum stress induced apoptosis, as evidenced by reduction of both caspase-12 activation and pro-apoptotic transcription factor C/EBP homologous protein induction during ischemia/reperfusion injury. In addition, the depressed SR Ca-cycling by HAX-1 overexpression was associated with reduced mitochondrial Ca-load as reflected by: a) hyper-phosphorylation of pyruvate dehydrogenase (PDH) and decreases in its activity, to diminish ATP production consistent with the attenuated energetic demand in these hearts; and b) reduced levels of reactive oxygen species, indicating protection from oxidative damage and preserved mitochondrial integrity. These findings suggest that HAX-1 is a key regulator of Ca-cycling, apoptosis and energetics in the heart. Thus, decreases in HAX-1 levels, observed during ischemia/reperfusion injury, may contribute to the deteriorated function and progression to heart failure development.

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