BVES is a novel interactor of ANO5 and regulates myoblast differentiation.
Ontology highlight
ABSTRACT: 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.
SUBMITTER: Li H
PROVIDER: S-EPMC8715634 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Rhabdomyosarcoma (RMS) is a predominantly pediatric soft-tissue cancer where the tumor cells exhibit characteristics of the developing skeletal muscle, and the two most common sub-types are embryonal and alveolar RMS. Elevated activation of the receptor tyrosine kinase (RTK) MET is frequent in RMS and is thought to cause increased tumor metastasis and lack of differentiation. However, the reasons underlying dysregulated MET expression and activation in RMS are not well understood. Therefore, we explored the role of Sprouty 2 (SPRY2), a modulator of RTK signaling, in regulating MET. We identify SPRY2 as a novel MET interactor that colocalizes with and binds MET in both embryonal and alveolar RMS. We find that depletion of SPRY2 leads to MET degradation, resulting in reduced migratory and clonogenic potential, and induction of differentiation in both embryonal and alveolar RMS, outcomes that are identical to depletion of MET. Activation of the ERK/MAPK pathway, known to be crucial for regulating cell migration and whose inhibition is required for myogenic differentiation, was downregulated upon depletion of MET or SPRY2. This provides a direct connection to the decreased migration and induction of differentiation upon depletion of MET or SPRY2. Thus, these data indicate that SPRY2 interacts with MET and stabilizes it in order to maintain signaling downstream of MET, which keeps the ERK/MAPK pathway active, resulting in metastatic potential and inhibition of differentiation in RMS. Our results identify a novel mechanism by which MET signaling is stabilized in RMS, and is a potential target for therapeutic intervention in RMS.
| S-EPMC5833614 | biostudies-literature
Project description:BackgroundType 2 diabetes mellitus is a global health problem. It often leads to a decline in the differentiation capacity of myoblasts and progressive loss of muscle mass, which in turn results in deterioration of skeletal muscle function. However, effective therapies against skeletal muscle diseases are unavailable.MethodsSkeletal muscle mass and differentiation ability were determined in db/+ and db/db mice. Transcriptomics and metabolomics approaches were used to explore the genetic mechanism regulating myoblast differentiation in C2C12 myoblasts.ResultsIn this study, the relatively uncharacterized solute carrier family gene Slc2a6 was found significantly up-regulated during myogenic differentiation and down-regulated during diabetes-induced muscle atrophy. Moreover, RNAi of Slc2a6 impaired the differentiation and myotube formation of C2C12 myoblasts. Both metabolomics and RNA-seq analyses showed that the significantly differentially expressed genes (e.g., LDHB) and metabolites (e.g., Lactate) during the myogenic differentiation of C2C12 myoblasts post-Slc2a6-RNAi were enriched in the glycolysis pathway. Furthermore, we show that Slc2a6 regulates the myogenic differentiation of C2C12 myoblasts partly through the glycolysis pathway by targeting LDHB, which affects lactic acid accumulation.ConclusionOur study broadens the understanding of myogenic differentiation and offers the Slc2a6-LDHB axis as a potential therapeutic target for the treatment of diabetes-associated muscle atrophy. Video abstract.
| S-EPMC9290262 | biostudies-literature
Project description:Enhanced intracellular Ca2+ signaling by stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) is required for skeletal muscle differentiation. However, the contribution of STIM2, STIM1's analogue protein, on muscle cell differentiation has not been clearly elucidated. The present study aimed to explore the contribution of STIM2-mediated SOCE on C2C12 myoblast differentiation.Changes in STIM2 expression level (reverse transcription-polymerase chain reaction and Western blotting) and SOCE activity ([Ca2+]i measurement) were measured during 3 days of in vitro differentiation of C2C12 skeletal myoblast. Transcriptional regulation of STIM2 by nuclear factor of activated T cells, cytoplasmic (NFATc) overexpression was observed, and the effect of STIM2 knockdown on NFAT transcriptional activity (luciferase assay) and myoblast differentiation was quantified.Increase of STIM2 protein level and enhanced SOCE activity were observed in differentiating myoblasts. Treatment with a SOCE blocker (2-APB) inhibited the differentiation. Overexpression of NFATc1 increased STIM2 expression and SOCE activity. Knockdown of STIM2 decreased NFAT transcriptional activity, SOCE activity, and differentiation of C2C12 myoblast.It is suggested that STIM2-activated SOCE controls C2C12 myoblast differentiation.
| S-EPMC5481796 | biostudies-other
Project description:Oncostatin M (OSM) is a cytokine of the interleukin-6 family and plays important roles during inflammation. However, its roles in myoblast differentiation and muscle regeneration remain unexplored. We show here that OSM potently inhibited myoblast differentiation mainly by activating the JAK1/STAT1/STAT3 pathway. OSM downregulated myocyte enhancer-binding factor 2A (MEF2A), upregulated the expression of Id1 and Id2, and inhibited the transcriptional activity of MyoD and MEF2. In addition, OSM also enhanced the expression of STAT3 and OSM receptor, which constituted a positive feedback loop to further amplify OSM-induced signaling. Moreover, we found that STAT1 physically associated with MEF2 and repressed its transcriptional activity, which could account for the OSM-mediated repression of MEF2. Although undetectable in normal muscles in vivo, OSM was rapidly induced on muscle injury and then promptly downregulated just before the majority of myoblasts differentiate. Prolonged expression of OSM in muscles compromised the regeneration process without affecting myoblast proliferation, suggesting that OSM functions to prevent proliferating myoblasts from premature differentiation during the early phase of muscle regeneration.
| S-EPMC3193431 | biostudies-literature
Project description:Myostatin, a member of the transforming growth factor-? superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the ?-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.
| S-EPMC3129832 | biostudies-other
Project description:Skeletal muscles are dynamic tissues that possess regenerative abilities, which require multiple processes and regulatory factors. Ubiquitin C-Terminal Hydrolase L1 (UCHL1), which is primarily expressed in neuronal tissues, was upregulated in skeletal muscles in disease conditions but its functional role in skeletal muscles is unknown. Using mouse myoblast cells C2C12 as an in vitro model, this study reported that UCHL1 elicits different regulation in myoblast cell proliferation and differentiation. We first observed that UCHL1 protein level was continuously declined during cell differentiation. Gene knockdown of UCHL1 by siRNA resulted in a significant decrease in cell proliferation but marked acceleration of cell differentiation and myotube formation. Meanwhile, UCHL1 gene knockdown upregulated myogenic factors myoD and Myogenin (MyoG). In mice, UCHL1 was significantly upregulated in denervated skeletal muscle. Overall, these novel data suggest that UCHL1 may play a role in myogenesis by promoting myoblast proliferation and inhibiting differentiation.
| S-EPMC5584584 | biostudies-literature
Project description:Myogenic differentiation factor (MyoD) is a master transcription factor in muscle development and differentiation. Although several long non-coding RNAs (lncRNAs) linked to MyoD have been found to influence muscle development, the functions of many lncRNAs have not been explored. Here we utilized lncRNA and mRNA microarray analysis to identify potential lncRNAs regulated by MyoD in muscle cells. A total of 997 differentially expressed lncRNAs (335 up-regulated and 662 down-regulated) and 1,817 differentially expressed mRNAs (148 up-regulated and 1,669 down-regulated) were identified after MyoD knockdown in C2C12 cells. Functional predictions suggested that most lncRNAs are involved in the biological pathways related to muscle differentiation and cell cycle with co-expressed genes. To gain further insight into the MyoD-mediated lncRNA expression in muscle differentiation, tissue expression profiles and MyoD overexpression were performed, and we found one of the candidate lncRNAs-AK143003 was significantly regulated by MyoD. Further analyses showed its noncoding ability and cytoplasmic localisation. Silencing of AK143003 stimulated the accumulation of myogenic marker genes, whereas AK143003 overexpression led to their decreased synthesis. This study identified a multitude of MyoD-mediated lncRNAs for further investigation and identified a novel lncRNA, lnc-AK143003, which plays a role in controlling muscle differentiation.
| S-EPMC5460278 | biostudies-literature
Project description:Cytokines are cell-secreted signaling molecules that modulate various cellular functions, with the best-characterized roles in immune responses. The expression of numerous cytokines in skeletal muscle tissues and muscle cells has been reported, but their function in skeletal myogenesis, the formation of skeletal muscle, has been largely underexplored. To systematically examine the potential roles of cytokines in skeletal myogenesis, we undertook an RNAi screen of 134 mouse cytokine genes for their involvement in the differentiation of C2C12 myoblasts. Our results have uncovered 29 cytokines as strong candidates for novel myogenic regulators, potentially conferring positive and negative regulation at distinct stages of myogenesis. These candidates represent a diverse collection of cytokine families, including interleukins, TNF-related factors, and chemokines. Our findings suggest the fundamental importance of cytokines in the cell-autonomous regulation of myoblast differentiation, and may facilitate future identification of novel therapeutic targets for improving muscle regeneration and growth in health and diseases.
| S-EPMC3699544 | biostudies-literature
Project description:The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the alpha subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1alpha by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-kappaB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IkappaBalpha. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-kappaB activation induced by overexpressed TRAF2 or IkappaB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-kappaB. These data suggest that EGLN3 is a negative regulator of NF-kappaB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-kappaB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent.
| S-EPMC2838314 | biostudies-literature
Project description:Myocardin-related transcription factor-A/serum response factor (MRTF-A/SRF), a well-known transcriptional programme, has been proposed to play crucial roles in skeletal muscle development and function. However, whether MRTF-A participates in muscle regeneration and the molecular mechanisms are not completely understood. Here, we show that MRTF-A levels are highly correlated with myogenic genes using a RNA-seq assay, which reveal that MRTF-A knockdown in C2C12 cells significantly reduces PAX7 expression. Subsequent in vitro and in vivo data show that MRTF-A and PAX7 present identical expression patterns during myoblast differentiation and CTX-induced muscle injury and repair. Remarkably, MRTF-A overexpression promotes myoblast proliferation, while inhibiting cell differentiation and the expression of MyoD and MyoG. MRTF-A loss of function produces the opposite effect. Moreover, mice with lentivirus (MRTF-A) injection possesses more PAX7+ satellite cells, but less differentiating MyoD+ and MyoG+ cells, leading subsequently to diminished muscle regeneration. Our mechanistic results reveal that MRTF-A contributes to PAX7-mediated myoblast self-renewal, proliferation, and differentiation by binding to its distal CArG box. Overall, we propose that MRTF-A functions as a novel PAX7 regulator upon myoblast commitment to differentiation, which could provide pathways for dictating muscle stem cell fate and open new avenues to explore stem cell-based therapy for muscle degenerative diseases.
| S-EPMC8435411 | biostudies-literature