PGC-1α is associated with C2C12 Myoblast differentiation

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.

Download Full-text

PUFA Treatment Affects C2C12 Myocyte Differentiation, Myogenesis Related Genes and Energy Metabolism

Genes ◽

10.3390/genes12020192 ◽

2021 ◽

Vol 12 (2) ◽

pp. 192

Author(s):

Marua Abu Risha ◽

Puntita Siengdee ◽

Dirk Dannenberger ◽

Klaus Wimmers ◽

Siriluck Ponsuksili

Keyword(s):

Energy Metabolism ◽

Wnt Signaling ◽

Cell Fate ◽

Early Stage ◽

Mrna Level ◽

Wnt Signaling Pathway ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Mrna Levels ◽

Dependent Manner

Polyunsaturated fatty acids (PUFAs) are the main components of cell membrane affecting its fluidity, signaling processes and play a vital role in muscle cell development. The effects of docosahexaenoic acid (DHA) on myogenesis are well known, while the effects of arachidonic acid (AA) are largely unclear. The purpose of this study is to evaluate the effect of two PUFAs (DHA and AA) on cell fate during myogenic processes, Wnt signaling and energy metabolism by using the C2C12 cells. The cells were treated with different concentrations of AA or DHA for 48 h during the differentiation period. PUFA treatment increased mRNA level of myogenic factor 5 (Myf5), which is involved in early stage of myoblast proliferation. Additionally, PUFA treatment prevented myoblast differentiation, indicated by decreased myotube fusion index and differentiation index in parallel with reduced mRNA levels of myogenin (MyoG). After PUFA withdrawal, some changes in cell morphology and myosin heavy chain mRNA levels were still observed. Expression of genes associated with Wnt signaling pathway, and energy metabolism changed in PUFA treatment in a dose and time dependent manner. Our data suggests that PUFAs affect the transition of C2C12 cells from proliferation to differentiation phase by prolonging proliferation and preventing differentiation.

Download Full-text

Fer1L5, a Dysferlin Homologue Present in Vesicles and Involved in C2C12 Myoblast Fusion and Membrane Repair

Biology ◽

10.3390/biology9110386 ◽

2020 ◽

Vol 9 (11) ◽

pp. 386

Author(s):

R. Usha Kalyani ◽

K. Perinbam ◽

P. Jeyanthi ◽

Naif Abdullah Al-Dhabi ◽

Mariadhas Valan Arasu ◽

...

Keyword(s):

Myoblast Fusion ◽

C2c12 Cells ◽

Muscle Membrane ◽

C2c12 Myoblast ◽

Related Protein ◽

Membrane Repair ◽

Ionic Detergent ◽

Biochemical Fractionation ◽

Two Stages

Fer1L5 is a dysferlin and myoferlin related protein, which has been predicted to have a role in vesicle trafficking and muscle membrane fusion events. Mutations in dysferlin and otoferlin genes cause heredity diseases: muscular dystrophy and deafness in humans, respectively. Dysferlin is implicated in membrane repair. Myoferlin has a role in myogenesis. In this study, we investigated the role of the Fer1L5 protein during myoblast fusion and membrane repair. To study the functions of Fer1L5 we used confocal microscopy, biochemical fractionation, Western blot analysis and multiphoton laser wounding assay. By immunolabelling, Fer1L5 was detected in vesicular structures. By biochemical fractionation Fer1L5 was observed in low density vesicles. Our studies show that the membranes of Fer1L5 vesicles are non-resistant to non-ionic detergent. Partial co-staining of Fer1L5 with other two ferlin vesicles, respectively, was observed. Fer1L5 expression was highly detected at the fusion sites of two apposed C2C12 myoblast membranes and its expression level gradually increased at D2 and reached a maximum at day 4 before decreasing during further differentiation. Our studies showed that Fer1L5 has fusion defects during myoblast fusion and impaired membrane repair when the C2C12 cultures were incubated with inhibitory Fer1L5 antibodies. In C2C12 cells Fer1L5 vesicles are involved in two stages, the fusion of myoblasts and the formation of large myotubes. Fer1L5 also plays a role in membrane repair.

Download Full-text

Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts.

The Journal of Cell Biology ◽

10.1083/jcb.132.1.181 ◽

1996 ◽

Vol 132 (1) ◽

pp. 181-193 ◽

Cited By ~ 60

Author(s):

S Yoshida ◽

A Fujisawa-Sehara ◽

T Taki ◽

K Arai ◽

Y Nabeshima

Keyword(s):

Lysophosphatidic Acid ◽

Intracellular Signaling ◽

Myogenic Differentiation ◽

Mrna Level ◽

Biological Significance ◽

Cell Contact ◽

C2c12 Myoblast ◽

Bhlh Proteins ◽

Cell Cell

Myogenic cells provide excellent in vitro models for studying the cell growth and differentiation. In this study we report that lysophosphatidic acid (LPA), a bioactive phospholipid contained in serum, stimulates the growth and inhibits the differentiation of mouse C2C12 myoblast cells, in a distinct manner from basic fibroblast growth factor (bFGF) whose mitotic and anti-differentiation actions have been well investigated. These actions of LPA were both blocked by pertussis toxin, suggesting the involvement of Gi class of G proteins, whereas bFGF acts through receptor tyrosine kinases. Detailed analysis revealed that LPA and bFGF act differently in regulating the myogenic basic helix-loop-helix (bHLH) proteins, the key players in myogenic differentiation process. LPA stimulates the proliferation of undifferentiated myoblasts allowing the continued expression of MyoD, but in contrast, bFGF does so with the MyoD expression suppressed at the mRNA level. Both compounds maintain the myf-5 expression, and suppress the myogenin expression. In addition, while LPA did not inhibit cell-cell contact-induced differentiation, bFGF strongly inhibited this process. Furthermore, LPA and bFGF act cooperatively in their mitogenic and anti-differentiation abilities. These findings indicate that LPA and bFGF differently stimulate intracellular signaling pathways, resulting in proliferating myoblasts each bearing a distinct expression pattern of myogenic bHLH proteins and distinct differentiation potentials in response to cell-cell contact, and illustrate the biological significance of Gi-mediated and tyrosine kinase-mediated signals.

Download Full-text

Abstract 544: Strenuous Exercise and Quercetin Intake Differentially Modulate PCSK9 and ANGPLT4 in Normal C57BL6 Mice

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.544 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Halleh Mahini ◽

Dhuha Alsayrafi ◽

Tong Wu ◽

Mahdi Garelnabi

Keyword(s):

Lipid Metabolism ◽

Normal Mouse ◽

Mrna Level ◽

Exercise Group ◽

Strenuous Exercise ◽

Mrna Levels ◽

Moderate Exercise ◽

Pcsk9 Gene ◽

Lipids Metabolism

Introduction: We previously reported that intake of quercetin during moderate exercise modulate lipid metabolism in LDLr -/- C57BL6 mice. The current study investigates the role of strenuous exercise and quercetin on lipids metabolism. Study Design: 40 mice were divided into four groups (10 each). These groups are as follows: Control mice, left untreated; control quercetin group, orally supplied with 100 μg/day of quercetin without exercising; exercise group without quercetin, and exercise group with quercetin supplements. The exercise groups were run on a treadmill for 30 minutes, 20-30m/m/ 5 days/week for two month. All animals were on normal mouse chow, at the end of the two month treatment, tissues were collected and expressions of genes associated lipid metabolism were analyzed and the proteins Western Blotting were determined. Results: PCSK9 mRNA level was significantly up-regulated as the result of combination of exercise and quercetin intake (p< 0.05) or quercetin alone. ANGPLT3 mRNA level did not show any significant changes as a result of exercise or quercetin. However, ANGPLT4 mRNA levels significantly down-regulated with the combination after 8 weeks of exercise and quercetin intake compared to both control and Exercise (p < 0.05). ANGPLT4 also decreased with quercetin intake; however this change is not significant. There was a slight change in ANGPLT 4 levels in the exercise group. Conclusion: The combination of strenuous exercise and quercetin intake did not show any positive affect on LDL (plasma LDL levels were measured; however not presented above), this was also reflected by the upregulation of the PCSK9 gene expression. Lipoprotein related genes differentially modulated with the strenuous exercise and quercetin intake. This data suggest that the combination of strenuous exercise and quercetin intake unfavorably impact LDL associated PCSK9 gene; however differentially affect ANGPLT4 levels with the exercise or the combination.

Download Full-text

The role of the α7nAChR-mediated cholinergic anti-inflammatory pathway in Hirschsprung associated enterocolitis

10.21203/rs.3.rs-17106/v1 ◽

2020 ◽

Author(s):

Feng Chen ◽

Xiaoyu Wei ◽

Xiaohua Chen ◽

Lei Xiang ◽

Xinyao Meng ◽

...

Keyword(s):

Western Blotting ◽

Mrna Level ◽

Underlying Mechanism ◽

Mrna Levels ◽

Wild Type ◽

Inflammatory Pathway ◽

Anti Inflammatory ◽

Phosphorylated Stat3 ◽

Morphology Changes

Abstract Background To investigate the role and the underlying mechanism of the α7nAChR-mediated cholinergic anti-inflammatory pathway in the pathogenesis of Hirschsprung(HSCR) associated enterocolitis（HAEC）. Methods Experimental group:twenty-one-day-old Ednrb-/- mice were selected (n=10), with comparable-age wild type(Ednrb+/+) mice controls (n=10). Intestinal samples were collected. The experimental colons were divided into narrow and dilated segments according to morphology changes. The control colons were divided into distal and proximal segments.Colon HE staining was used to judge HAEC.Acetylcholine levels in colon was measured using enzyme-linked immunosorbent assays. Detected phosphorylated Jak2 (p-Jak2), Jak2, phosphorylated Stat3 (p-Stat3), Stat3, phosphorylated IκBα (p-IκBα) and IκBα were studied by Western blotting; mRNA levels of Jak2, Stat3, and IκBα were detected by RT-qPCR. Results Colon HE staining indicated that HAEC mainly occured in the dilated segments of HSCR mice (Ednrb-/- mice) (EDNRB-P).Acetylcholine content in EDNRB-P was significantly lower than that in the narrow segments (EDNRB-D) (P<0.05). Western blotting showed that the Jak2, p-Jak2, Stat3 and p-Stat3 levels in EDNRB-D were significantly higher than those in EDNRB-P (P<0.05). The p-IκBα and IκBα levels in EDNRB-P were significantly higher than those in EDNRB-D(P<0.05). The mRNA levels of Jak2 and Stat3 in EDNRB-D were higher than those in EDNRB-P, but the IκBα mRNA level was significantly lower than that in EDNRB-P (P<0.05). Conclusions During HAEC, the inflammation in the dilated segment was more severe ,while in the narrow segment there was no obvious inflammatory reaction and the content of acetylcholine was higher, which was associated with the α7nAChR-mediated cholinergic anti-inflammatory pathway.

Download Full-text

Dermatopontin in Skeletal Muscle Extracellular Matrix Regulates Myogenesis

Cells ◽

10.3390/cells8040332 ◽

2019 ◽

Vol 8 (4) ◽

pp. 332 ◽

Cited By ~ 8

Author(s):

Kim ◽

Ahmad ◽

Shaikh ◽

Jan ◽

Seo ◽

...

Keyword(s):

Extracellular Matrix ◽

In Silico ◽

State Of The Art ◽

Myogenic Differentiation ◽

3D Structure ◽

Inhibitory Effect ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Protein Protein Interaction

Dermatopontin (DPT) is an extensively distributed non-collagenous component of the extracellular matrix predominantly found in the dermis of the skin, and consequently expressed in several tissues. In this study, we explored the role of DPT in myogenesis and perceived that it enhances the cell adhesion, reduces the cell proliferation and promotes the myoblast differentiation in C2C12 cells. Our results reveal an inhibitory effect with fibronectin (FN) in myoblast differentiation. We also observed that DPT and fibromodulin (FMOD) regulate positively to each other and promote myogenic differentiation. We further predicted the 3D structure of DPT, which is as yet unknown, and validated it using state-of-the-art in silico tools. Furthermore, we explored the in-silico protein-protein interaction between DPT-FMOD, DPT-FN, and FMOD-FN, and perceived that the interaction between FMOD-FN is more robust than DPT-FMOD and DPT-FN. Taken together, our findings have determined the role of DPT at different stages of the myogenic process.

Download Full-text

Effect of GABA-T on Reproductive Function in Female Rats

Animals ◽

10.3390/ani10040567 ◽

2020 ◽

Vol 10 (4) ◽

pp. 567

Author(s):

Wenyu Si ◽

Hailing Li ◽

Tiezhu Kang ◽

Jing Ye ◽

Zhiqiu Yao ◽

...

Keyword(s):

Mrna Level ◽

Reproductive Function ◽

Female Rats ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Lactation Performance ◽

Irreversible Inhibitor ◽

Adult Rats ◽

Expression Of Genes

This study explored the role of γ-aminobutyric acid transaminase (GABA-T) in the puberty and reproductive performance of female rats. Immunofluorescence technique, quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution of GABA-T and the expression of genes and hormones in female rats, respectively. The results showed that GABA-T was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and periventricular nucleus (PeN) of the hypothalamus, and in the adenohypophysis, ovarian granulosa cells and oocytes. Abat mRNA level at 28 d was lowest in the hypothalamus and the pituitary; at puberty, it was lowest in the ovary. Abat mRNA level was highest in adults in the hypothalamus; at infancy and puberty, it was highest in the pituitary; and at 21 d it was highest in the ovary. After vigabatrin (GABA-T irreversible inhibitor) was added to hypothalamus cells, the levels of Abat mRNA and Rfrp-3 mRNA were significantly reduced, but Gnrh mRNA increased at the dose of 25 and 50 μg/mL; Kiss1 mRNA was significantly increased but Gabbr1 mRNA was reduced at the 50 μg/mL dose. In prepubertal rats injected with vigabatrin, puberty onset was delayed. Abat mRNA, Kiss1 mRNA and Gnrh mRNA levels were significantly reduced, but Rfrp-3 mRNA level increased in the hypothalamus. Vigabatrin reduced the concentrations of GABA-T, luteinizing hormone (LH) and progesterone (P4), and the ovarian index. Lactation performance was reduced in adult rats with vigabatrin treatment. Four hours after vigabatrin injection, the concentrations of GABA-T and LH were significantly reduced in adult and 25 d rats, but follicle-stimulating hormone (FSH) increased in 25 d rats. In conclusion, GABA-T affects the reproductive function of female rats by regulating the levels of Gnrh, Kiss1 and Rfrp-3 in the hypothalamus as well as the concentrations of LH and P4.

Download Full-text

Role of Osteoglycin in the Linkage between Muscle and Bone

Journal of Biological Chemistry ◽

10.1074/jbc.m111.292193 ◽

2012 ◽

Vol 287 (15) ◽

pp. 11616-11628 ◽

Cited By ~ 72

Author(s):

Ken-ichiro Tanaka ◽

Erika Matsumoto ◽

Yoshiko Higashimaki ◽

Takenobu Katagiri ◽

Toshitsugu Sugimoto ◽

...

Keyword(s):

Transcriptional Activity ◽

Type I Collagen ◽

C2c12 Cells ◽

Type I ◽

Mrna Levels ◽

Humoral Factors ◽

Anabolic Activity ◽

Morphogenetic Protein ◽

Muscle Tissues

The interaction between muscle tissues and bone metabolism is incompletely understood. We hypothesized that there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We, therefore, performed comparative DNA microarray analysis between mouse myoblastic C2C12 cells transfected with either stable empty vector or ALK2 (R206H), the mutation that constitutively activates the bone morphogenetic protein (BMP) receptor, to search for muscle-derived bone anabolic factors. Twenty-five genes whose expression was decreased to <1/4, were identified; these included osteoglycin (OGN). Stable overexpression of OGN significantly decreased the levels of Runx2 and Osterix mRNA compared with those in cells transfected with vector alone in MC3T3-E1 cells. On the other hand, it significantly enhanced the levels of alkaline phosphatase (ALP), type I collagen (Col1), and osteocalcin (OCN) mRNA as well as β-catenin and mineralization. A reduction in endogenous OGN level showed the opposite effects to those of OGN overexpression in MC3T3-E1 and mouse calvarial osteoblastic cells. Transient OGN overexpression significantly suppressed the levels of Runx2, Osterix, ALP, Col1, and OCN mRNA induced by BMP-2 in C2C12 cells. The conditioned medium from OGN-overexpressed and OGN-suppressed myoblastic cells enhanced and decreased, respectively, the levels of ALP, Col1, and β-catenin in MC3T3-E1 cells. Moreover, OGN increased Smad3/4-responsive transcriptional activity as well as Col1 mRNA levels independently of endogenous TGF-β in these cells. In conclusion, this study suggests that OGN may be a crucial humoral bone anabolic factor that is produced by muscle tissues.

Download Full-text

Doxycycline Inhibits IL-17-Stimulated MMP-9 Expression by Downregulating ERK1/2 Activation: Implications in Myogenic Differentiation

Mediators of Inflammation ◽

10.1155/2016/2939658 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Hristina Obradović ◽

Jelena Krstić ◽

Tamara Kukolj ◽

Drenka Trivanović ◽

Ivana Okić Đorđević ◽

...

Keyword(s):

Inflammatory Diseases ◽

Myogenic Differentiation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Interleukin 17 ◽

Myoblast Cells ◽

Pathological Consequence ◽

Muscle Remodeling

Interleukin 17 (IL-17) is a cytokine with pleiotropic effects associated with several inflammatory diseases. Although elevated levels of IL-17 have been described in inflammatory myopathies, its role in muscle remodeling and regeneration is still unknown. Excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting. In this study, the role of IL-17 on the expression of matrix metalloproteinase- (MMP-) 9 in myoblast cells was investigated. The expression of MMP-9 after IL-17 treatment was analyzed in mouse myoblasts C2C12 cell line. The increase in MMP-9 production by IL-17 was concomitant with its capacity to inhibit myogenic differentiation of C2C12 cells. Doxycycline (Doxy) treatment protected the myogenic capacity of myoblasts from IL-17 inhibition and, moreover, increased myotubes hypertrophy. Doxy blocked the capacity of IL-17 to stimulate MMP-9 production by regulating IL-17-induced ERK1/2 MAPK activation. Our results imply that MMP-9 mediates IL-17’s capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that Doxy can modulate myoblast response to inflammatory induction by IL-17.

Download Full-text

Role of α2-macroglobulin in fever and cytokine responses induced by lipopolysaccharide in mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00746.2001 ◽

2002 ◽

Vol 283 (1) ◽

pp. R218-R226 ◽

Cited By ~ 24

Author(s):

Alexander V. Gourine ◽

Valery N. Gourine ◽

Yohannes Tesfaigzi ◽

Nathalie Caluwaerts ◽

Fred Van Leuven ◽

...

Keyword(s):

Mrna Level ◽

Physiological Role ◽

Mrna Levels ◽

Wild Type ◽

Cytokine Responses ◽

Α2 Macroglobulin ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

α2-Macroglobulin (α2M) is not only a proteinase inhibitor in mammals, but it is also a specific cytokine carrier that binds pro- and anti-inflammatory cytokines implicated in fever, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). To define the role of α2M in regulation of febrile and cytokine responses, wild-type mice and mice deficient in α2M (α2M −/−) were injected with lipopolysaccharide (LPS). Changes in body temperature as well as plasma levels of IL-1β, IL-6, and TNF-α and hepatic TNF-α mRNA level during fever in α2M −/− mice were compared with those in wild-type control mice. The α2M −/− mice developed a short-term markedly attenuated (ANOVA, P < 0.05) fever in response to LPS (2.5 mg/kg ip) compared with the wild-type mice. At 1.5 h after injection of LPS, the plasma concentration of TNF-α, but not IL-1β or IL-6, was significantly lower (by 58%) in the α2M −/− mice compared with their wild-type controls (ANOVA, P < 0.05). There was no difference in hepatic TNF-α mRNA levels between α2M −/− and wild-type mice 1.5 h after injection of LPS. These data support the hypotheses that 1) α2M is important for the normal development of LPS-induced fever and 2) a putative mechanism of α2M involvement in fever is through the inhibition of TNF-α clearance. These findings indicate a novel physiological role for α2M.

Download Full-text