Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway

BackgroundMyogenesis is a complex process that requires optimal outside–in substrate–cell signaling. Calponin 3 (CNN3) plays an important role in regulating myogenic differentiation and muscle regeneration; however, the precise function of CNN3 in myogenesis regulation remains poorly understood. Here, we investigated the role of CNN3 in a knockdown model in the mouse muscle cell line C2C12.MethodsMyoblast proliferation, migration, differentiation, fusion, and protein synthesis were examined in CNN3 knockdown C2C12 mouse muscle cells. Involvement of the mTOR pathway in CNN3 signaling was explored by treating cells with the mTOR activator MHY1485. The regulatory mechanisms of CNN3 in myogenesis were further examined by RNA sequencing and subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA).ResultsDuring proliferation, CNN3 knockdown caused a decrease in cell proliferation and migration. During differentiation, CNN3 knockdown inhibited myogenic differentiation, fusion, and protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways; this effect was reversed by MHY1485 treatment. Finally, KEGG and GSEA indicated that the NOD-like receptor signaling pathway is affected in CNN3 knockdown cell lines.ConclusionCNN3 may promote C2C12 cell growth by regulating AKT/mTOR and AMPK/mTOR signaling. The KEGG and GSEA indicated that inhibiting CNN3 may activate several pathways, including the NOD-like receptor pathway and pathways involved in necroptosis, apoptosis, and inflammation.

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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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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.

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Biomechanical signals upregulate myogenic gene induction in the presence or absence of inflammation

AJP Cell Physiology ◽

10.1152/ajpcell.00594.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. C267-C276 ◽

Cited By ~ 35

Author(s):

Ravi Chandran ◽

Thomas J. Knobloch ◽

Mirela Anghelina ◽

Sudha Agarwal

Keyword(s):

Cell Differentiation ◽

Muscle Cell ◽

Kinase Inhibitor ◽

Cell Damage ◽

Myogenic Differentiation ◽

C2c12 Cell ◽

Gene Induction ◽

C2c12 Cells ◽

Proinflammatory Mediators ◽

Tnf Α

Inflammation of the muscle invariably leads to muscle cell damage and impaired regeneration. Biomechanical signals play a vital role in the regulation of myogenesis in healthy and inflamed muscle. We hypothesized that biomechanical signals counteract the actions of proinflammatory mediators and upregulate the basic helix-loop-helix and MADS box transcription enhancer factor 2 (MEF2) families of transcription factors, leading to increased myogenesis in inflamed muscle cells. For this purpose, C2C12 cells plated on collagenized silastic membranes were subjected to equibiaxial cyclic tensile strain (CTS) in the presence or absence of TNF-α, and the myogenic gene induction was examined over a period of 72 h. Exposure of cells to CTS resulted in a significant upregulation of mRNA expressions and synthesis of myogenic regulatory factors, MYOD1, myogenin (MYOG), MEF2A, and cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) as well as muscle structural proteins like myosin heavy chain (MYHC) isoforms (MYH1, MYH2, and MYH4) and α-tropomyosin (TPM1), eventually leading to an increase in myotube formation. Contrarily, TNF-α suppressed the expression of all of the above differentiation-inducing factors in C2C12 cells. Further results revealed that simultaneous exposure of C2C12 cells to CTS and TNF-α abrogated the TNF-α-mediated downregulation of myogenic differentiation. In fact, the mRNA expression and protein synthesis of all myogenic factors ( Myod1, Myog, Mef2a, Cdkn1a, Myh1, Myh2, Myh4, and Tpm1) were increased in stretched C2C12 cells despite the sustained presence of TNF-α. These results demonstrate that mechanotransduction regulates multiple signaling molecules involved in C2C12 cell differentiation. On one hand, these signals are potent transducers of myotube phenotype in myoblasts; on the other, these signals counteract catabolic actions of proinflammatory cytokines like TNF-α and allow the expression of myogenic genes to upregulate muscle cell differentiation.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0612 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1469-1480 ◽

Cited By ~ 37

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”.

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Fibroblast Growth Factor 21 Promotes C2C12 Cells Myogenic Differentiation by Enhancing Cell Cycle Exit

BioMed Research International ◽

10.1155/2017/1648715 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Xinyi Liu ◽

Yongliang Wang ◽

Shuhong Zhao ◽

Xinyun Li

Keyword(s):

Cell Cycle ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Myogenic Differentiation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Fibroblast Growth Factor 21 ◽

Fibroblast Growth ◽

Cell Cycle Exit ◽

Protein Functions

Fibroblast growth factor 21 (FGF21), a secretion protein, functions as a pivotal regulator of energy metabolism and is being considered as a therapeutic candidate in metabolic syndromes. However, the roles of FGF21 in myogenic differentiation and cell cycle remain obscure. In this study, we investigated the function of FGF21 in myogenesis and cell cycle exit using C2C12 cell line. Our data showed that the expression of myogenic genes as well as cell cycle exit genes was increased after FGF21 overexpression, and FGF21 overexpression induces cell cycle arrest. Moreover, cell cycle genes were decreased in FGF21 overexpression cells while they were increased in FGF21 knockdown cells. Further, FGF21/P53/p21/Cyclin-CDK has been suggested as the key pathway for cell cycle exit mediated by FGF21 in C2C12 cells. Also, we deduce that FGF21 promotes the initiation of myogenic differentiation mainly through enhancing cell cycle exit of C2C12 cells. Taken together, our results demonstrated that FGF21 promotes cell cycle exit and enhances myogenic differentiation of C2C12 cells. This study provided new evidence that FGF21 promotes myogenic differentiation, which could be useful for better understanding the roles of FGF21 in myogenesis.

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6-Bromoindirubin-3′-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice

Scientific Reports ◽

10.1038/s41598-019-54574-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Elvira Ragozzino ◽

Mariarita Brancaccio ◽

Antonella Di Costanzo ◽

Francesco Scalabrì ◽

Gennaro Andolfi ◽

...

Keyword(s):

Skeletal Muscle ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Skeletal Muscle Differentiation ◽

Myoblast Proliferation ◽

Enhanced Expression ◽

Pharmacologically Active

AbstractDystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Thus, new drugs for restoring skeletal muscle deterioration are critically needed. To identify new and alternative compounds with a functional role in skeletal muscle myogenesis, we screened a library of pharmacologically active compounds and selected the small molecule 6-bromoindirubin-3′-oxime (BIO) as an inhibitor of myoblast proliferation. Using C2C12 cells, we examined BIO’s effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from cell proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO is able to promote myogenic differentiation in damaged myotubes in-vitro by enriching the population of newly formed skeletal muscle myotubes. Moreover, in-vivo experiments in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers number. Additionally, we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was observed in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was observed in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was observed upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a positive modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.

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MicroRNA-139-5p regulates C2C12 cell myogenesis through blocking Wnt/β-catenin signaling pathway

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0079 ◽

2015 ◽

Vol 93 (1) ◽

pp. 8-15 ◽

Cited By ~ 19

Author(s):

Lin Mi ◽

Youlei Li ◽

Qiangling Zhang ◽

Chen Zhao ◽

Ying Peng ◽

...

Keyword(s):

Signaling Pathway ◽

Molecular Mechanisms ◽

Glycogen Synthase ◽

C2c12 Cell ◽

Negative Regulator ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

Myoblast Proliferation ◽

Gsk 3Β

MicroRNAs (miRNAs) are novel and potent regulators in myogenesis. However, the molecular mechanisms that many miRNAs regulate myoblast proliferation and differentiation which are largely unknown. Here, we found that miR-139-5p increased during C2C12 myoblast proliferation, while presenting an inverse trend during C2C12 myoblast differentiation. Flow cytometry and EdU incorporation assay showed that miR-139-5p slowed down the growth of C2C12 cells. Additional study demonstrated that ectopic introduction of miR-139-5p into C2C12 cells blocked myoblast differentiation. Importantly, we demonstrated for the first time that Wnt1, which is associated with the Wnt/β-catenin signaling pathway, was a direct target of miR-139-5p. Moreover, we found that the expression level of Wnt1 was suppressed significantly (p < 0.01) by miR-139-5p, which triggered inhibition of Wnt/β-catenin signaling through upregulation of glycogen synthase kinase 3 beta (GSK-3β; p < 0.05) and downregulation of p-GSK-3β (p < 0.01), β-catenin (p < 0.05), and nuclear β-catenin (p < 0.01). Taken together, these results suggest that miR-139-5p is an important negative regulator in myogenesis through blocking the Wnt1-mediated Wnt/β-catenin signaling pathway.

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Expression of DDIT4 Is Correlated with NOTCH1 and High Molecular Risk in Acute Myeloid Leukemias

Blood ◽

10.1182/blood.v128.22.5254.5254 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5254-5254 ◽

Cited By ~ 2

Author(s):

Joseph A Pinto ◽

Leny Bravo ◽

Luis A. Chirinos ◽

Carlos E Vigil

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Meta Analysis ◽

Enrichment Analysis ◽

Mtor Pathway ◽

Gene Set Enrichment Analysis ◽

Stem Cell Markers ◽

Entire Cohort ◽

Molecular Abnormalities ◽

Acute Myeloid

Abstract In a meta-analysis, our team identified previously that DDIT4 expression is related with a worse outcome of several cancer types, including acute myeloid leukemia (AML). DDIT4 gene product is a repressor of mTOR activity and recent findings in the triple negative breast cancer model suggest that mTOR pathway inhibition lead to the enrichment of cancer stem cells, explaining indirectly the relationship between DDIT4 and the outcome and suggesting that a high DDIT4 expression could be related with expression of stem-cells markers, such as NOTCH1. We evaluated 200 AML patients from the TCGA dataset. Clinical features and level 2 data of gene expression were retrieved from the website cbioportal.org for mTOR pathway and stem cell markers including ALDH genes. Gene Set Enrichment Analysis (GSEA) for biological processes was done comparing patients with high risk vs low risk determined by molecular abnormalities. GSEA analysis found not differences in the mTOR pathway. DDIT4 is not included in none gene ontology datasets. NOTCH1 and DDIT4 expression was enriched in patients with high molecular risk and statistical differences were found mainly between good and poor prognostic groups (score -0.45 and -0.46, respectively). We next compared the correlation between DDIT4 and NOTCH1 in the entire cohort. The correlation was significant (R2=0.121; P<0.0001). NOTCH1 was over expressed in the M0 FAB subtype while the lowest expression was found in M3, M4, M5 and M7 subtypes. This work shows the relevance of DDIT4 and NOTCH1 in the biology of AML and his association with most undifferentiated subtypes. Association between DDIT4 with NOTCH1 should be evaluated with the intention of explore therapeutic opportunities. Disclosures No relevant conflicts of interest to declare.

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Palmitic Acid-Induced miR-429-3p Impairs Myoblast Differentiation by Downregulating CFL2

International Journal of Molecular Sciences ◽

10.3390/ijms222010972 ◽

2021 ◽

Vol 22 (20) ◽

pp. 10972

Author(s):

Mai Thi Nguyen ◽

Kyung-Ho Min ◽

Wan Lee

Keyword(s):

Palmitic Acid ◽

Cell Cycle Progression ◽

Myogenic Differentiation ◽

Saturated Fatty Acids ◽

Critical Role ◽

C2c12 Cells ◽

Actin Dynamics ◽

Myoblast Differentiation ◽

Skeletal Myogenesis ◽

Myoblast Proliferation

MicroRNAs are known to play a critical role in skeletal myogenesis and maintenance, and cofilin-2 (CFL2) is necessary for actin cytoskeleton dynamics and myogenic differentiation. Nonetheless, target molecules and the modes of action of miRNAs, especially those responsible for the inhibitory mechanism on the myogenesis by saturated fatty acids (SFA) or obesity, still remain unclear. Here, we reported the role played by miR-429-3p on CFL2 expression, actin filament dynamics, myoblast proliferation, and myogenic differentiation in C2C12 cells. Palmitic acid (PA), the most abundant SFA in diet, inhibited the myogenic differentiation of myoblasts, accompanied by CFL2 reduction and miR-429-3p induction. Interestingly, miR-429-3p suppressed the expression of CFL2 by targeting the 3′UTR of CFL2 mRNA directly. Transfection of miR-429-3p mimic in myoblasts increased F-actin formation and augmented nuclear YAP level, thereby promoting cell cycle progression and myoblast proliferation. Moreover, miR-429-3p mimic drastically suppressed the expressions of myogenic factors, such as MyoD, MyoG, and MyHC, and impaired myogenic differentiation of C2C12 cells. Therefore, this study unveiled the crucial role of miR-429-3p in myogenic differentiation through the suppression of CFL2 and provided implications of SFA-induced miRNA in the regulation of actin dynamics and skeletal myogenesis.

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Syncytin-1 Expression is Increased in Skeletal Muscle of Humans with Obesity and is Inversely Correlated to Muscle Protein Synthesis

10.21203/rs.3.rs-147911/v1 ◽

2021 ◽

Author(s):

Jayachandran Ravichandran ◽

Lori R Roust ◽

Christos Katsanos

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Protein Metabolism ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

C2c12 Cell ◽

C2c12 Cells ◽

Anabolic Hormone ◽

Lower Protein ◽

Potential Link

Abstract Background: Various pathophysiological conditions alter protein metabolism in skeletal muscle, with obesity being one of them. Obesity impairs regeneration of skeletal muscle, and the same biological mechanism(s) may adversely affect protein metabolism in the muscle of these individuals. Methods: We used C2C12 cell line to evaluate the effects of the anabolic hormone insulin on the expression of protein syncytin-1, which regulates regeneration of muscle, and in the presence of fatty acids whose metabolism is altered in obesity. We used muscle biopsy samples from obese humans with lower muscle protein synthesis and lean controls to evaluate expression of syncytin-1 in obesity and its correlation with protein synthesis in muscle. Results: Insulin upregulated syncytin-1 expression in C2C12 cells and this response was impaired in the presence of the fatty acid palmitate, but not oleate. Expression of the protein 4E-BP1, which signals increase in protein synthesis in muscle, showed response similar to that of syncytin-1. Humans with obesity characterized by lower muscle protein synthesis had higher expression of syncytin-1 in muscle compared to lean humans (P < 0.01). The rate of synthesis of protein in skeletal muscle across humans subjects correlated inversely (r = -0.51; P = 0.03) with the expression of syncytin-1 in muscle. Conclusions: Our studies provide novel insights in the regulation of syncytin-1 in skeletal muscle, and describe potential link between syncytin-1 expression and protein metabolism in skeletal muscle of humans. Altered syncytin-1 expression in muscle may mediate lower protein turnover in muscle of humans with obesity.

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