Caspase-1-Dependent Pyroptosis Mediates Adjuvant Activity of Platycodin D as an Adjuvant for Intramuscular Vaccines

Platycodin D (PD) is a potent adjuvant with dual Th1 and Th2 potentiating activity, but its mechanisms of action remain unclear. Here, the C2C12 myoblast cell line and mice were used as in vitro and in vivo models to identify potential signaling pathways involved in the adjuvant activity of PD. PD induced a transient cytotoxicity and inflammatory response in the C2C12 cells and in mouse quadricep muscles. A comparative analysis of microarray data revealed that PD induced similar gene expression profiles in the C2C12 cells and in the quadricep muscles, and triggered rapid regulation of death, immune, and inflammation-related genes, both in vivo and in vitro. It was further demonstrated that caspase-1-dependent pyroptosis was involved in the PD-induced cytotoxicity and inflammatory response in the C2C12 cells via the Ca2+–c-jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK)–NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. Consistently, the in vivo analysis revealed that a local blockage of NLRP3 and caspase-1 inhibited PD-induced cytokine production and immune cell recruitment at the injection site, and impaired the adjuvant activity of PD on antigen-specific immune responses to model antigen ovalbumin (OVA) in mice. These findings identified the caspase-1-dependent adjuvanticity of PD and expanded the current knowledge on the mechanisms of action of saponin-based adjuvants.

VEGFB Promotes Myoblasts Proliferation and Differentiation through VEGFR1-PI3K/Akt Signaling Pathway

It has been demonstrated that vascular endothelial growth factor B (VEGFB) plays a vital role in regulating vascular biological function. However, the role of VEGFB in regulating skeletal muscle cell proliferation and differentiation remains unclear. Thus, this study aimed to investigate the effects of VEGFB on C2C12 myoblast proliferation and differentiation and to explore the underlying mechanism. For proliferation, VEGFB significantly promoted the proliferation of C2C12 myoblasts with the upregulating expression of cyclin D1 and PCNA. Meanwhile, VEGFB enhanced vascular endothelial growth factor receptor 1 (VEGFR1) expression and activated the PI3K/Akt signaling pathway in a VEGFR1-dependent manner. In addition, the knockdown of VEGFR1 and inhibition of PI3K/Akt totally abolished the promotion of C2C12 proliferation induced by VEGFB, suggesting that VEGFB promoted C2C12 myoblast proliferation through the VEGFR1-PI3K/Akt signaling pathway. Regarding differentiation, VEGFB significantly stimulated the differentiation of C2C12 myoblasts via VEGFR, with elevated expressions of MyoG and MyHC. Furthermore, the knockdown of VEGFR1 rather than NRP1 eliminated the VEGFB-stimulated C2C12 differentiation. Moreover, VEGFB activated the PI3K/Akt/mTOR signaling pathway in a VEGFR1-dependent manner. However, the inhibition of PI3K/Akt/mTOR blocked the promotion of C2C12 myoblasts differentiation induced by VEGFB, indicating the involvement of the PI3K/Akt pathway. To conclude, these findings showed that VEGFB promoted C2C12 myoblast proliferation and differentiation via the VEGFR1-PI3K/Akt signaling pathway, providing new insights into the regulation of skeletal muscle development.

Taurine stimulates protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway

Taurine (Tau) has many profound physiological functions, but its role and molecular mechanism in muscle cells are still not fully understood. In this study, we investigated the role and underlying molecular mechanism of Tau on protein synthesis and proliferation of C2C12 myoblast cells. Cells were treated with Tau (0, 60, 120, 180 and 240 μM) for 24 h. Tau dose-dependently promoted protein synthesis, cell proliferation, mTOR phosphorylation, and also AT-rich interaction domain 4B (ARID4B) expression, with the best stimulatory effects at 120 μM. LY 294002 treatment showed that Tau promoted ARID4B expression in a PI3K-dependent manner. ARID4B knockdown (by siRNA transfection for 24 h) prevented Tau from stimulating protein synthesis and cell proliferation, whereas ARID4B gene activation (using the CRISPR/dCas9 technology) had stimulatory effects. ARID4B knockdown abolished Tau signaling to mRNA expression and protein phosphorylation of mTOR, whereas ARID4B gene activation had stimulatory effects. ChIP-PCR identified that all of ARID4B, H3K27ac and H3K27me3 bound to the -4368∼-4591 bp site in the mTOR promoter, and ChIP-qPCR further detected that Tau stimulated ARID4B binding to this site. ARID4B knockdown or gene activation did not affect H3K27me3 binding to the mTOR promoter, but decreased or increased H3K27ac binding, respectively. Furthermore, ARID4B knockdown abolished the stimulation of Tau on H3K27ac binding to the mTOR promoter. In summary, these data uncover that Tau promotes protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway, providing a deep understanding how Tau regulates anabolism in muscle cells.

BVES is a novel interactor of ANO5 and regulates myoblast differentiation

Background Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. Results In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Conclusions Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

Low-level laser prevents doxorubicin-induced skeletal muscle atrophy by modulating AMPK/SIRT1/PCG-1α-mediated mitochondrial function, apoptosis and up-regulation of pro-inflammatory responses

Background Doxorubicin (Dox) is a widely used anthracycline drug to treat cancer, yet numerous adverse effects influencing different organs may offset the treatment outcome, which in turn affects the patient’s quality of life. Low-level lasers (LLLs) have resulted in several novel indications in addition to traditional orthopedic conditions, such as increased fatigue resistance and muscle strength. However, the mechanisms by which LLL irradiation exerts beneficial effects on muscle atrophy are still largely unknown. Results The present study aimed to test our hypothesis that LLL irradiation protects skeletal muscles against Dox-induced muscle wasting by using both animal and C2C12 myoblast cell models. We established SD rats treated with 4 consecutive Dox injections (12 mg/kg cumulative dose) and C2C12 myoblast cells incubated with 2 μM Dox to explore the protective effects of LLL irradiation. We found that LLL irradiation markedly alleviated Dox-induced muscle wasting in rats. Additionally, LLL irradiation inhibited Dox-induced mitochondrial dysfunction, apoptosis, and oxidative stress via the activation of AMPK and upregulation of SIRT1 with its downstream signaling PGC-1α. These aforementioned beneficial effects of LLL irradiation were reversed by knockdown AMPK, SIRT1, and PGC-1α in C2C12 cells transfected with siRNA and were negated by cotreatment with mitochondrial antioxidant and P38MAPK inhibitor. Therefore, AMPK/SIRT1/PGC-1α pathway activation may represent a new mechanism by which LLL irradiation exerts protection against Dox myotoxicity through preservation of mitochondrial homeostasis and alleviation of oxidative stress and apoptosis. Conclusion Our findings may provide a novel adjuvant intervention that can potentially benefit cancer patients from Dox-induced muscle wasting.

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

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.

Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

Lipid droplet (LD), a multi-functional organelle, is found in most eukaryotic cells. LDs participate in the regulation of many cellular processes including proliferation, stress, and apoptosis. Previous studies showed the athlete’s paradox that trained athletes accumulate LDs in their skeletal muscle. However, the impact of LDs on skeletal muscle and myogenesis is not clear. We discovered that C2C12 myoblast cells containing more LDs formed more multinucleated muscle fibers. We also discovered that LDs promoted cell migration and fusion by promoting actin-filaments remodeling. Mechanistically, two LD-proteins, Acyl-CoA synthetase long chain family member 3 (ACSL3) and lysophosphatidylcholine acyltransferase 1 (LPCAT1), medicated the recruitment of actinin proteins which contributed to actin-filaments formation on the surface of LDs. During remodeling, the actinin proteins on LDs surface translocated to actin-filaments via ARF1/COPI vesicles. Our study demonstrate LDs contribute to cell differentiation, which lead to new insight into the LD function.

­­BVES Is a Novel Interactor of ANO5 and Regulates Myoblast Differentiation

Background: Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. Results: In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins specifically from ANO5-BioID2 samples but not BioID2 fused with ANO6 or MG53, including BVES and POPDC3. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with BVES through its C-terminus. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Conclusions:Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.