scholarly journals Reduction of Real-Time Imaging of M1 Macrophage Chemotaxis toward Damaged Muscle Cells is PI3K-Dependent

Antioxidants ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 138 ◽  
Author(s):  
Hiromi Yano ◽  
Masataka Uchida ◽  
Tatsuya Saito ◽  
Takafumi Aoki ◽  
Michael Kremenik ◽  
...  
Keyword(s):  
Real Time ◽  
C2c12 Cell ◽  
Muscle Cells ◽  
Chemotactic Activity ◽  
C2c12 Cells ◽  
Migration Behavior ◽  
M1 Macrophage ◽  
J774 Cells ◽  
Phosphoinositide 3 Kinase

Macrophages migrate and invade into damaged muscle rapidly and are important for muscle repair and subsequent regeneration. The exact cellular and biological events that cause macrophage migration toward injured muscle are not completely understood. In this study, the effect of macrophage differentiation on the chemotactic capability to invade local damaged muscle was investigated using an in vitro model of muscle injury. We used C2C12 cell myoblasts and J774 cell macrophages, and the “killed-C2C12” cells were combined with live C2C12 cells as a partially damaged muscle model. The cultured J774 cells, with or without lipopolysaccharide (LPS), were treated with Ly294002 (Ly), which is an inhibitor of phosphoinositide 3-kinase (PI3K). In order to evaluate the polarization effect of LPS stimulation on J774 cells, expression of cell surface Toll-like receptor 4 (TLR4), CD11c and CCR2, and expression of F-actin intensity, were analyzed by flow cytometry. The real-time horizontal chemotaxis assay of J774 cells was tested using the TAXIScan device. The expressions of TLR4, CD11c, and F-actin intensity in LPS-treated cells were significantly higher than those in Ctrl cells. In LPS-treated cells, the chemotactic activity toward damaged muscle cells completely disappeared. Moreover, the reduced chemotaxis depended far more on directionality than velocity. However, Ly treatment reversed the reduced chemotactic activity of the LPS-treated cells. In addition, cell-adhesion and F-actin intensity, but not CCR2 expression, in LPS-treated cells, was significantly reduced by Ly treatment. Taken together, our results suggest that the PI3K/Akt activation state drives migration behavior towards damaged muscle cells.

CORTISOL CIRCADIAN RHYTHM AND INSULIN RESISTANCE IN MUSCLE: EFFECT OF DOSING AND TIMING OF HYDROCORTISONE EXPOSURE ON INSULIN SENSITIVITY IN SYNCHRONIZED MUSCLE CELLS.

2020 ◽  
Author(s):  
Mariarosaria Negri ◽  
Claudia Pivonello ◽  
Chiara Simeoli ◽  
Gilda Di Gennaro ◽  
Mary Anna Venneri ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Circadian Rhythm ◽  
Insulin Sensitivity ◽  
Circadian Clock ◽  
Insulin Signaling ◽  
Clock Genes ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Circadian Expression

Introduction/Aim: Circadian rhythm disruption is emerging as a risk factor for metabolic disorders and particularly, alterations in clock genes circadian expression have been shown to influence insulin sensitivity. Recently, the reciprocal interplay between the circadian clock machinery and HPA axis has been largely demonstrated: the circadian clock may control the physiological circadian endogenous glucocorticoids secretion and action; glucocorticoids, in turn, are potent regulator of the circadian clock and their inappropriate replacement has been associated with metabolic impairment. The aim of the current study was to investigate in vitro the interaction between the timing-of-the-day exposure to different hydrocortisone (HC) concentrations on muscle insulin sensitivity. Methods: Serum-shock synchronized mouse skeletal muscle C2C12 cells were exposed to different HC concentrations recapitulating the circulating daily physiological cortisol profile (standard cortisol profile), the circulating daily cortisol profile that reached in adrenal insufficient (AI) patients treated with once-daily MR-HC (flat cortisol profile) and treated with thrice-daily of conventional IR-HC (steep cortisol profile). The 24 hrs spontaneous oscillation of the clock genes in synchronized C2C12 cells was used to align the timing for in vitro HC exposure (Bmal1 acrophase, midphase and bathyphase) with the reference times of cortisol peaks in AI treated with IR-HC (8 am, 1 pm, 6 pm). A panel of 84 insulin sensitivity related genes and intracellular insulin signaling proteins were analyzed by RT-qPCR and western blot, respectively. Results: Only the steep profile, characterized by a higher HC exposure during Bmal1 bathyphase, produced significant downregulation in 21 insulin sensitivity-related genes. Among these, Insr, Irs1, Irs2, Pi3kca and Adipor2 were downregulated when compared the flat to the standard or steep profile. Reduced intracellular IRS1 Tyr608, AKT Ser473, AMPK Thr172 and ACC Ser79 phosphorylations were also observed. Conclusions: The current study demonstrated that is late-in-the-day cortisol exposure that modulates insulin sensitivity-related genes expression and intracellular insulin signaling in skeletal muscle cells.

scholarly journals Diverse Action of Selected Statins on Skeletal Muscle Cells—An Attempt to Explain the Protective Effect of Geranylgeraniol (GGOH) in Statin-Associated Myopathy (SAM)

2019 ◽  
Vol 8 (5) ◽  
pp. 694 ◽  
Author(s):  
Anna Jaśkiewicz ◽  
Beata Pająk ◽  
Magdalena Łabieniec-Watała ◽  
Clara De Palma ◽  
Arkadiusz Orzechowski
Keyword(s):  
Skeletal Muscle ◽  
Protein Expression ◽  
Cell Viability ◽  
Mitochondrial Respiration ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
C2c12 Cell ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Calpain Inhibitor

The present study is centered on molecular mechanisms of the cytoprotective effect of geranylgeraniol (GGOH) in skeletal muscle harmed by statin-associated myopathy (SAM). GGOH via autophagy induction was purportedly assumed to prevent skeletal muscle viability impaired by statins, atorvastatin (ATR) or simvastatin (SIM). The C2C12 cell line was used as the ‘in vitro’ model of muscle cells at different stages of muscle formation, and the effect of ATR or SIM on the cell viability, protein expression and mitochondrial respiration were tested. Autophagy seems to be important for the differentiation of muscle cells; however, it did not participate in the observed GGOH cytoprotective effects. We showed that ATR- and SIM-dependent loss in cell viability was reversed by GGOH co-treatment, although GGOH did not reverse the ATR-induced drop in the cytochrome c oxidase protein expression level. It has been unambiguously revealed that the mitochondria of C2C12 cells are not sensitive to SIM, although ATR effectively inhibits mitochondrial respiration. GGOH restored proper mitochondria functioning. Apoptosis might, to some extent, explain the lower viability of statin-treated myotubes as the pan-caspase inhibitor, N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK), partly reversed ATR- or SIM-induced cytotoxic effects; however, it does not do so in conjunction with caspase-3. It appears that the calpain inhibitor, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLM), restored the viability that was reduced by ATR and SIM (p < 0.001). GGOH prevents SAM, in part, as a consequence of a caspase-3 independent pathway, probably by calpain system inactivation.

Gene Expression of C2C12 Cells on Calcium Phosphate Ceramic In Vitro

2005 ◽  
Vol 288-289 ◽  
pp. 265-268 ◽  
Author(s):  
Yan Fei Tan ◽  
Ling Li Zhang ◽  
Xin Lai He ◽  
Wei Qiang Xiao ◽  
Hong Song Fan ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Type I Collagen ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Type I ◽  
Calcium Phosphate Ceramic ◽  
Mtt Method ◽  
Difference Expression

The osteoinduction of Calcium Phosphate (CaP) had been proved and generally been investigated by in vivo implantation. However, the mechanism of the osteoinductivity was not clear and it was difficult to judge the osteoinductivity in vitro. In this study, Mouse C2C12 cell line, a kind of myoblast precursor cell, was employed to co-culture with CaP. The induction of cell differentiation by materials was tested by MTT method, fluorescence observation, especially the mRNA expression of Osteocalcin, Type I collagen and Fibronectin by RT-PCR. It was founded that C2C12 cells could be induced to expression osteocalcin when growth on the surface of the HA/TCP ceramics. At the same time, the ceramics with different composition and sintering temperature seemed to induce difference expression level of the related genes. The results proved that phase composition was one of the most important factors in the regulation of bone-related genes. This study provided a potential model to evaluate the osteoinductivity of CaP ceramics in vitro.

scholarly journals The USP14–NLRC5 pathway inhibits titanium particle–induced osteolysis in mice by suppressing NF-κB and PI3K/AKT activities

2020 ◽  
Vol 295 (20) ◽  
pp. 7018-7032 ◽  
Author(s):  
Guibin Fang ◽  
Yuan Fu ◽  
Shixun Li ◽  
Junxiong Qiu ◽  
Manyuan Kuang ◽  
...  
Keyword(s):  
Macrophage Polarization ◽  
Wear Particle ◽  
Wear Particles ◽  
Titanium Particle ◽  
Pathway Activation ◽  
M1 Macrophage ◽  
Phosphoinositide 3 Kinase ◽  
Factor Α

Total hip arthroplasty (THA) is a widely-used surgical intervention for treating patients with end-stage degenerative and inflammatory osteoarthropathy. However, wear particles from the artificial titanium joint can induce osteolysis, limiting the long-term survivorship of THA. Monocyte/macrophage lineage cells are the key players in the response to wear particles, and the proinflammatory NF-κB and phosphoinositide 3-kinase (PI3K)–AKT Ser/Thr kinase (AKT)-signaling pathways have been shown to be the most important contributors to wear particle–induced osteolysis. In contrast, ubiquitin-specific protease 14 (USP14) specifically removes the polyubiquitin chains from the nucleotide-binding and oligomerization domain (NOD)-like receptor family Caspase recruitment domain (CARD)–containing 5 (NLRC5) and thereby enhances the NLRC5-mediated inhibition of NF-κB signaling. In this study, we aimed to clarify the role of the USP14–NLRC5 pathway in wear particle–induced osteolysis in vitro and in vivo. We found that NLRC5 or USP14 overexpression inhibits titanium particle–induced proinflammatory tumor necrosis factor α (TNFα) production and NF-κB pathway activation, and it also decreases M1 macrophage polarization and PI3K/AKT pathway activation. Of note, NLRC5 and USP14 overexpression attenuated titanium particle–induced cranial osteolysis in mice. In conclusion, the findings of our study indicate that the USP14–NLRC5 pathway inhibits titanium particle–induced osteolysis by suppressing the NF-κB and PI3K/AKT pathways both in vitro and in vivo.

scholarly journals p27Kip1 Acts Downstream of N-Cadherin-mediated Cell Adhesion to Promote Myogenesis beyond Cell Cycle Regulation

2005 ◽  
Vol 16 (3) ◽  
pp. 1469-1480 ◽  
Author(s):  
Graziella Messina ◽  
Cristiana Blasi ◽  
Severina Anna La Rocca ◽  
Monica Pompili ◽  
Attilio Calconi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myogenic Differentiation ◽  
Cell Cycle Control ◽  
C2c12 Cell ◽  
Terminal Differentiation ◽  
Active Role ◽  
C2c12 Cells ◽  
Low Density

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”.

scholarly journals Leukocyte antigen-related inhibition attenuates palmitate-induced insulin resistance in muscle cells

2012 ◽  
Vol 215 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Sattar Gorgani-Firuzjaee ◽  
Salar Bakhtiyari ◽  
Abolfazl Golestani ◽  
Reza Meshkani
Keyword(s):  
Insulin Resistance ◽  
Glucose Uptake ◽  
Cell Line ◽  
C2c12 Cell ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Stable Cell Line ◽  
Protein Levels ◽  
Induce Insulin Resistance ◽  
And Control

Palmitate has been shown to induce insulin resistance in skeletal muscle cells. The aim of this study was to investigate the role of the leukocyte common antigen-related (LAR) gene in palmitate-induced insulin resistance in C2C12 cells. A stable C2C12 cell line was generated using LAR short hairpin RNA. The levels of LAR protein and phosphorylation of insulin receptor substrate-1 (IRS1) and Akt were detected by western blot analysis. 2-Deoxyglucose uptake was measured in LAR knockdown and control cells using d-[2-3H]glucose. LAR protein level was decreased by 65% in the stable cell line compared with the control cells. Palmitate (0.5 mM) significantly induced LAR mRNA (65%) and protein levels (40%) in myotubes compared with untreated cells. Palmitate significantly reduced insulin-stimulated glucose uptake in both the control and LAR knockdown cells by 33 and 51% respectively. However, LAR depletion improved insulin-stimulated glucose uptake in myotubes treated with palmitate. Furthermore, the inhibition of LAR prevented palmitate-induced decreases in phosphorylation of IRS1Tyr632 and AktSer473 in C2C12 cells. In conclusion, these results reveal that palmitate induces LAR expression in C2C12 cells. We also provided evidence that the inhibition of LAR attenuates palmitate-induced insulin resistance in myotubes.

scholarly journals Calcium/Calmodulin-Dependent Protein Kinase IV Mediates IFN-γ-Induced Immune Behaviors in Skeletal Muscle Cells

2018 ◽  
Vol 46 (1) ◽  
pp. 351-364 ◽  
Author(s):  
RuiCai Gu ◽  
MaoChao Ding ◽  
DanDan Shi ◽  
Tao Huang ◽  
MengXia Guo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Inflammatory Cytokines ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Ifn Γ ◽  
Pro Inflammatory Cytokines

Background/Aims: Whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a role in regulating immunologic features of muscle cells in inflammatory environment, as it does for immune cells, remains mostly unknown. In this study, we investigated the influence of endogenous CaMKIV on the immunological characteristics of myoblasts and myotubes received IFN-γ stimulation. Methods: C2C12 and murine myogenic precursor cells (MPCs) were cultured and differentiated in vitro, in the presence of pro-inflammatory IFN-γ. CaMKIV shRNA lentivirus transfection was performed to knockdown CaMKIV gene in C2C12 cells. pEGFP-N1-CaMKIV plasmid was delivered into knockout cells for recovering intracellular CaMKIV gene level. CREB1 antagonist KG-501 was used to block CREB signal. qPCR, immunoblot analysis, or immunofluorescence was used to detect mRNA and protein levels of CaMKIV, immuno-molecules, or pro-inflammatory cytokines and chemokines. Co-stimulatory molecules expression was assessed by FACS analysis. Results: IFN-γ induces the expression or up-regulation of MHC-I/II and TLR3, and the up-regulation of CaMKIV level in muscle cells. In contrast, CaMKIV knockdown in myoblasts and myotubes leads to expression inhibition of the above immuno-molecules. As well, CaMKIV knockdown selectively inhibits pro-inflammatory cytokines/chemokines, and co-stimulatory molecules expression in IFN-γ treated myoblasts and myotubes. Finally, CaMKIV knockdown abolishes IFN-γ induced CREB pathway molecules accumulation in differentiated myotubes. Conclusions: CaMKIV can be induced to up-regulate in muscle cells under inflammatory condition, and positively mediates intrinsic immune behaviors of muscle cells triggered by IFN-γ.

scholarly journals A Three-Dimensional Culture Model of Reversibly Quiescent Myogenic Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Salvatore Aguanno ◽  
Claudia Petrelli ◽  
Sara Di Siena ◽  
Luciana De Angelis ◽  
Manuela Pellegrini ◽  
...  
Keyword(s):  
Stem Cells ◽  
C2c12 Cell ◽  
Three Dimensional ◽  
Notch Pathway ◽  
C2c12 Cells ◽  
Quiescent State ◽  
Cellular Interactions ◽  
Culture Model

Satellite cells (SC) are the stem cells of skeletal muscles. They are quiescent in adult animals but resume proliferation to allow muscle hypertrophy or regeneration after injury. The mechanisms balancing quiescence, self-renewal, and differentiation of SC are difficult to analyze in vivo owing to their complexity and in vitro because the staminal character of SC is lost when they are removed from the niche and is not adequately reproduced in the culture models currently available. To overcome these difficulties, we set up a culture model of the myogenic C2C12 cell line in suspension. When C2C12 cells are cultured in suspension, they enter a state of quiescence and form three-dimensional aggregates (myospheres) that produce the extracellular matrix and express markers of quiescent SC. In the initial phase of culture, a portion of the cells fuses in syncytia and abandons the myospheres. The remaining cells are mononucleated and quiescent but resume proliferation and differentiation when plated in a monolayer. The notch pathway controls the quiescent state of the cells as shown by the fact that its inhibition leads to the resumption of differentiation. Within this context, notch3 appears to play a central role in the activity of this pathway since the expression of notch1 declines soon after aggregation. In summary, the culture model of C2C12 in suspension may be used to study the cellular interactions of muscle stem cells and the pathways controlling SC quiescence entrance and maintenance.

scholarly journals Activated Integrin-Linked Kinase Negatively Regulates Muscle Cell Enhancement Factor 2C in C2C12 Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-9
Author(s):  
Zhenguo Dong ◽  
Wei Pan ◽  
Haiqing Wu ◽  
Dongjun Liu ◽  
Ming Cang
Keyword(s):  
Mrna Expression ◽  
Muscle Cell ◽  
Enhancement Factor ◽  
C2c12 Cell ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Pi3k Activity ◽  
New Ideas ◽  
Integrin Linked Kinase ◽  
Related Proteins

Our previous study reported that muscle cell enhancement factor 2C (MEF2C) was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK) in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors.

FABRICATION OF NOVEL ANTICANCER POLYOXOMETALATE [CoW11O39(CpTi)]7- -CHITOSAN NANO-COMPOSITE, ITS TOXICITY REDUCTION AND SUSTAINED RELEASE

2017 ◽  
Vol 10 (4) ◽  
pp. 278
Author(s):  
Vamangi M Pandya ◽  
Sachin A Joshi
Keyword(s):  
Sustained Release ◽  
Cell Line ◽  
Cell Mass ◽  
C2c12 Cell ◽  
A549 Cells ◽  
In Vitro Cytotoxicity ◽  
Lung Cancer Cell Line ◽  
C2c12 Cells ◽  
Interaction Technique

Objectives: Polyoxometalates (POMs) are proved to be important for applications in medicine and in material science. Here, we representnanocomposite formation of tungsten-containing potent anticancer polyanion, K 6 H [CoW 11 O 39  (CpTi)].13H 2 O (CoW CpTi) with biocompatible ChitosanYC-100 (CSYC100) with the goal to reduce its heavy metal toxicity.Methods: Synthesis of “POM-CSYC100 nanocomposite” was attained without the aid of any cross-linker through electrostatic interaction technique. Nanocomposites were characterized using Fourier transform infrared spectroscopy, dynamic light scattering, transmission electron microscopy, and thermogravimetric analysis. The release profile recorded was slow and sustained at physiological pH. In vitro cytotoxicity assays which show an attribute to reduce the toxicity of these POM were performed on C2C12 (mouse myoblast cell line) and A-549 (lung cancer cell line), which proved the reduced toxicity of nanocomposites as compared to the bare drugs.Results: Sustained release studies showed there was a slow and steady release of CoW CpTi for 11 hrs, with the 98% of collective release at the end. From in vitro cytotoxic assay, it was deduced that CoW 11 11 CpTi -CSYC100 nanocomposite at the concentrations of 1.25 mM, and lower did not exhibit toxic effect on C2C12 cells as 95% total C2C12 cell mass remained viable. While in the case of A549 cells highest 5 mM concentration of bare CoW 11 CpTi is toxic to the cancer cells and after encapsulation cell viability increases from 10% to 55%.Conclusion: Thus, this study has designated the probability of using POM-chitosan nanocomposite for less toxic and effective biomedicinal applications.Keywords: Anticancer, Chitosan, Nanocomposite, In vitro cytotoxicity, Drug release.

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