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Muscle regeneration after sciatic nerve transection is enhanced in RFP knockout mice muscles. A, The expression of skeletal embryonic myosin heavy chain (MHCemb) in muscle fibers was increased in RFP knockout mice (lower panel). B, More nuclei were localized in the center of the muscles in RFP knockout mice. Arrowheads indicate the nuclei that are centrally localized in the muscle fiber. C, Quantification results of muscle regeneration as determined by counting the centrally localized nucleus. D, MyoD expression was increased in RFP knockout mice muscle 14 days after nerve injury. Note that MyoD-positive nuclei were more abundant in the knockout mice muscle. E, Sciatic nerve-transfection-induced reduction of MyoD protein amounts were blunted in RFP knockout mice muscles.
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Skeletal muscle atrophy results from the net loss of muscular proteins and organelles and is caused by pathologic conditions such as nerve injury, immobilization, cancer, and other metabolic diseases. Recently, ubiquitination-mediated degradation of skeletal-muscle-specific transcription factors was shown to be involved in muscle atrophy, although...
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... next checked whether skeletal muscle regeneration was increased in the RFP knockout mice. Expression of skeletal embryonic myosin heavy chain (MHCemb), a regeneration marker [41], was increased in RFP knockout mice (Fig. 6A). Central localization of the nucleus in muscle fiber cross section is an alternative hallmark of muscle regeneration [42,43]. As shown in Fig. 6B, the number of central redistribution of the nucleus (red arrowheads) was much greater in RFP knockout mice, which was quantified in Fig. 6C. The expression of MyoD was examined in the ...
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... skeletal muscle regeneration was increased in the RFP knockout mice. Expression of skeletal embryonic myosin heavy chain (MHCemb), a regeneration marker [41], was increased in RFP knockout mice (Fig. 6A). Central localization of the nucleus in muscle fiber cross section is an alternative hallmark of muscle regeneration [42,43]. As shown in Fig. 6B, the number of central redistribution of the nucleus (red arrowheads) was much greater in RFP knockout mice, which was quantified in Fig. 6C. The expression of MyoD was examined in the cross-sections of leg muscle obtained from those mice (Fig. 6D). MyoD-positive nucleus was more abundant in regenerating muscle fibers in RFP knockout ...
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... marker [41], was increased in RFP knockout mice (Fig. 6A). Central localization of the nucleus in muscle fiber cross section is an alternative hallmark of muscle regeneration [42,43]. As shown in Fig. 6B, the number of central redistribution of the nucleus (red arrowheads) was much greater in RFP knockout mice, which was quantified in Fig. 6C. The expression of MyoD was examined in the cross-sections of leg muscle obtained from those mice (Fig. 6D). MyoD-positive nucleus was more abundant in regenerating muscle fibers in RFP knockout mice (lower panel) than those in wild type mice (upper panel). The expression of MyoD was quantified by immunoblot analysis. MyoD expression ...
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... in muscle fiber cross section is an alternative hallmark of muscle regeneration [42,43]. As shown in Fig. 6B, the number of central redistribution of the nucleus (red arrowheads) was much greater in RFP knockout mice, which was quantified in Fig. 6C. The expression of MyoD was examined in the cross-sections of leg muscle obtained from those mice (Fig. 6D). MyoD-positive nucleus was more abundant in regenerating muscle fibers in RFP knockout mice (lower panel) than those in wild type mice (upper panel). The expression of MyoD was quantified by immunoblot analysis. MyoD expression was reduced by sciatic nerve transection in wild-type mice (Fig. 6E, 1st versus 2nd columns), which was ...
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... of leg muscle obtained from those mice (Fig. 6D). MyoD-positive nucleus was more abundant in regenerating muscle fibers in RFP knockout mice (lower panel) than those in wild type mice (upper panel). The expression of MyoD was quantified by immunoblot analysis. MyoD expression was reduced by sciatic nerve transection in wild-type mice (Fig. 6E, 1st versus 2nd columns), which was completely abolished in RFP knockout mice (3rd versus 4th columns). These results suggest that RFP may participate in skeletal muscle atrophy by induction of the polyubiquitination of ...
Citations
... So, we speculate that UII may have other mechanisms triggering skeletal muscle atrophy such as inhibiting Fxo03A phosphorylation, enhancing expression of the E3 ubiquitin ligases (MuRF1 and MAFbx/atrogin1) under CRF circumstances. What's more, Joung and his colleagues [14] found that ret finger protein (RFP), also known as TRIM27, works as an E3 ligase in Pax7-induced degradation of Myod1 protein but not mRNA. Therefore,in the current study, we will explore whether ubiquitin-proteasome system and satellite cells are involved in skeletal muscle atrophy induced by UII. ...
... Julie found that in cultured myotubes undergoing atrophy, MAFbx nuclear translocation is tightly linked to Myod1 degradation in muscle cells, and Myod1 down-regulation is crucial for muscle loss [24]. Joung and his colleagues [14] found that ret finger protein (RFP), also known as TRIM27, works as an E3 ligase in Pax7-induced degradation of Myod1 protein but not mRNA. Therefore, the ubiquitin-proteasome pathway causes the atrophy of skeletal muscle not only by degrading proteins in the formed skeletal muscles but also by inhibiting satellite cells differentiation. ...
Skeletal muscle wasting and atrophy is highly prevalent in chronic renal failure (CRF) and increases the risk of mortality. According to our previous study, we speculate that urotensin II (UII) can induce skeletal muscle atrophy by upregulating ubiquitin–proteasome system(UPS) in CRF. C2C12 mouse myoblast cells were differentiated into myotubes, and myotubes were exposed to different concentrations of UII. Myotube diameters, myosin heavy chain(MHC), p-Fxo03A, skeletal muscle-specific E3 ubiquitin ligases such as muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx/atrogin1) were detected. Three animal models (the sham operation mice as normal control (NC) group, wild-type C57BL/6 mice with 5/6 nephrectomy (WT CRF) group, UII receptor gene knock out (UT KO) mice with 5/6 nephrectomy (UT KO CRF) group) were designed. Cross-sectional area (CSA) of skeletal muscle tissues in three animal models were measured, and western blot detected protein of UII, p-Fxo03A, MAFbx and MuRF1, and immunofluorescence assays explored the satellite cell marker of Myod1 and Pax7, and PCR arrays detected the muscle protein degradation genes, protein synthesis genes and the genes which were involved in muscle components. UII could decrease mouse myotube diameters, and upregulate dephosphorylated Fxo03A protein. MAFbx and MuRF1 were higher in WT CRF group than that in NC group, but after UII receptor gene was knocked out (UT KO CRF), their expressions were downregulated. UII could inhibit the expression of Myod1 but not Pax7 in animal study. We first demonstrate that skeletal muscle atrophy induced by UII associated with upregulating ubiquitin–proteasome system and inhibiting the differentiation of satellite cells in CRF mice.
... The present results also demonstrated that ubiquitination levels of PDCD4 were upregulated after TRIM27 overexpression, while TRIM27 knockdown inhibited the ubiquitination level of PDCD4, indicating that TRIM27 can play a role in promoting the ubiquitination of PDCD4. It has been reported that RFP (TRIM27) physically interacts with Pax7 and serves as an E3 ligase to induce ubiquitin-proteasome-dependent degradation of MyoD in muscle atrophy, which results in resistance of muscles to denervation injury (35). Cai et al (26) reported that TRIM27 functioned as an E3 ligase and mediated lysine 48 polyubiquitination of PI3KC2β, leading to a decrease in PI3K enzyme activity. ...
Programmed cell death 4 (PDCD4) is regarded as an important tumor suppressor that is lowly expressed or deleted in numerous human types of cancer, including ovarian and endometrial cancer. Tripartite motif‑containing 27 (TRIM27) is closely related to the occurrence and development of tumors and is highly expressed in numerous types of cancer such as ovarian and endometrial cancer. PDCD4 can be degraded through ubiquitination, while TRIM27 has the E3 ubiquitin ligase activity. However, whether TRIM27 may regulate the expression of PDCD4 by ubiquitination effect remains unclear. In the present study, the expression of PDCD4 and TRIM27 in different ovarian and endometrial cancer cell lines was detected by reverse transcription‑quantitative PCR (RT‑qPCR), western blotting and immunocytochemistry. The impact of TRIM27 overexpression and knockdown on PDCD4 expression and the effective mechanism of TRIM27 regulating PDCD4 expression were also investigated in vitro by RT‑qPCR, western blotting, co‑immunoprecipitation assay, Transwell migration and Matrigel invasion assays. The results showed that the expression of TRIM27 and PDCD4 had a negative association at the protein level, and the distribution of TRIM27 and PDCD4 proteins had a phenomenon of co‑localization in different ovarian and endometrial cancer cell lines. TRIM27 promoted the degradation of PDCD4 through the ubiquitin‑proteasome pathway. To sum up, TRIM27 could increase the migration and invasion of ovarian and endometrial cancer cells by promoting the ubiquitination and degradation of PDCD4. The present findings may provide a new target for the treatment of ovarian and endometrial cancer.
... As shown by Joung and cols. (Joung et al., 2014), the Ret finger protein (RFP/TRIM27) E3 ligase can interact with both Pax7 and MyoD (which is ubiquitinated by RFP), mediating the Pax7-induced downregulation of MyoD. RFP expression in muscle fibers is increased during denervation-induced atrophy, while RFPnull mice exhibited significantly less reduction in muscle mass upon denervation. ...
... RFP expression in muscle fibers is increased during denervation-induced atrophy, while RFPnull mice exhibited significantly less reduction in muscle mass upon denervation. Noteworthy, RFP-null muscles showed increase numbers of MyoD positive nuclei following denervation (Joung et al., 2014). ...
In recent years, the ubiquitin-proteasome system (UPS) has emerged as an important regulator of stem cell function. Here we review recent findings indicating that UPS also plays critical roles in the biology of satellite cells, the muscle stem cell responsible for its maintenance and regeneration. While we focus our attention on the control of key transcriptional regulators of satellite cell function, we briefly discuss early studies suggesting the UPS participates more broadly in the regulation of satellite cell stemness and regenerative capacity.
... Total cellular protein lysates were prepared and western blot analyses were performed as previously described (Joung et al. 2014(Joung et al. , 2018. C2C12 cells were lysed in an RIPA buffer (R2002, Biosesang) supplemented with protease inhibitors (P8340, Sigma-Aldrich) and the protein concentration of the lysate was determined using the Pierce BCA Protein Assay Kit (#23225, ThermoFisher Scientific). ...
... Immunostaining for MHC expression was performed as described previously (Joung et al. 2014(Joung et al. , 2018. C2C12 cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 (T8787-50ML, Sigma-Aldrich) for 5 min at room temperature. ...
SUMOylation 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.
... Both processes are critical in order to ensure myofiber formation/repair and self-renewal of the quiescent SC pool through multiple insults over the organism's life span. Previous work indicates that the ubiquitin-proteasome system (UPS) actively regulates Pax7 and MRF function [6][7][8][9][10][11][12][13], thus controlling the transition between different cellular states during myogenic progression. ...
Satellite cells (SCs) are myogenic progenitors responsible for skeletal muscle regeneration and maintenance. Upon activation, SCs enter a phase of robust proliferation followed by terminal differentiation. Underlying this myogenic progression, the sequential expression of muscle regulatory transcription factors (MRFs) and the downregulation of transcription factor paired box gene 7 (Pax7) are key steps regulating SC fate. In addition to transcriptional regulation, post‐translational control of Pax7 and the MRFs provides another layer of spatiotemporal control to the myogenic process. In this context, previous work showed that Pax7 is ubiquitinated by the E3 ligase neural precursor cell‐expressed developmentally downregulated protein 4 and interacts with several proteins related to the ubiquitin–proteasome system, including the deubiquitinase ubiquitin‐specific protease 7 (USP7). Although USP7 functions in diverse cellular contexts, its role(s) during myogenesis remains poorly explored. Here, we show that USP7 is transiently expressed in adult muscle progenitors, correlating with the onset of myogenin expression, while it is downregulated in newly formed myotubes/myofibers. Acute inhibition of USP7 activity upon muscle injury results in persistent expression of early regeneration markers and a significant reduction in the diameter of regenerating myofibers. At the molecular level, USP7 downregulation or pharmacological inhibition impairs muscle differentiation by affecting myogenin stability. Co‐immunoprecipitation and in vitro activity assays indicate that myogenin is a novel USP7 target for deubiquitination. These results suggest that USP7 regulates SC myogenic progression by enhancing myogenin stability.
... The C2C12 cell line has been previously described. 21 Cells were maintained in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA). Differentiation was induced in Dulbecco's modified Eagle's medium plus 2% horse serum (Hyclone). ...
... Luciferase reporter assay, RNA isolation, quantitative realtime PCR, western blotting and immunoprecipitation analyses These procedures have been previously described. [21][22][23] The primer sequences for quantitative real-time PCR will be provided upon request. The real-time PCR amplification products were confirmed by sequencing. ...
Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.
... The potential TF-binding sites for MyoD, PAX7, MEF3 and CREB motif were mutated with the corresponding primers (Table 1) MyoD, PAX7, CREB and MyoG knockdown and MyoG overexpression. siRNAs against MyoD, PAX7, CREB and MyoG were designed as described previously [43][44][45][46] and synthesized with the control siRNA (GenePharma Co., Ltd., Shanghai, China). The NC siRNA served as a negative control. ...
The SIX1 gene belongs to the family of six homeodomain transcription factors (TFs), that regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and mediate skeletal muscle growth and regeneration. Previous studies have demonstrated that SIX1 is positively correlated with body measurement traits (BMTs). However, the transcriptional regulation of SIX1 remains unclear. In the present study, we determined that bovine SIX1 was highly expressed in the longissimus thoracis. To elucidate the molecular mechanisms involved in bovine SIX1 regulation, 2-kb of the 5′ regulatory region were obtained. Sequence analysis identified neither a consensus TATA box nor a CCAAT box in the 5′ flanking region of bovine SIX1. However, a CpG island was predicted in the region −235 to +658 relative to the transcriptional start site (TSS). An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with serial deletion constructs of the 5′ flanking region, site-directed mutation and siRNA interference demonstrated that MyoD, PAX7 and CREB binding occur in region −689/−40 and play important roles in bovine SIX1 transcription. In addition, MyoG drives SIX1 transcription indirectly via the MEF3 motif. Taken together these interactions suggest a key functional role for SIX1 in mediating skeletal muscle growth in cattle.
... TRIM27 contains an N-terminal RING motif that binds to E2 ubiquitin-conjugating enzymes followed by a zinc-containing B-box motif, coiled-coil multimerization motif, and a C-terminal PRY-SPRY protein-protein interaction motif. TRIM27 has been implicated in a number of cellular and disease processes, including PTEN, AKT, and NF-kB signaling, transcription, T-cell activation, apoptosis, innate immunity, spermatogenesis, muscle atrophy, cancer, and autism [13,[38][39][40][41][42][43][44][45]. MAGEL2 binds only a fraction of the total cellular pool of TRIM27 and it remains to be determined which of the known TRIM27 activities are regulated by MAGEL2 [25]. ...
... The original seven infants identified with USP7 haploinsufficiency manifested several symptoms of PWS and SHFYNG and a high prevalence of ASD, similar to that seen in SHFYNG (Table 1). However, children with USP7 mutation often present with seizures, which is less common in those with PWS and SHFYNG [87,92], suggesting that MAGEL2-independent cellular functions of USP7 and possibly TRIM27 contribute to the phenotypes seen in patients with USP7 haploinsufficiency [13,[38][39][40][41][42][43][44][45]78,79,81]. A number of additional cases have been identified that await further characterization and will help to improve understanding of the molecular mechanisms underlying the observed phenotypes. ...
Melanoma antigen L2 (MAGEL2 or MAGE-L2) is a member of the MAGE family of ubiquitin ligase regulators. It is maternally imprinted and often paternally deleted or mutated in the related neurodevelopmental syndromes, Prader-Willi Syndrome (PWS) and Schaaf- Yang Syndrome (SHFYNG). MAGEL2 is highly expressed in the hypothalamus and plays an important role in a fundamental cellular process that recycles membrane proteins from endosomes through the retromer sorting pathway. MAGEL2 is part of a multi-subunit protein complex consisting of MAGEL2, the TRIM27 E3 ubiquitin ligase, and the USP7 deubiquitinating enzyme. The MAGEL2-USP7-TRIM27 (or MUST) complex facilitates the retromer recycling pathway through ubiquitination and activation of the WASH actin nucleation promoting factor. This review provides an overview of the MAGE protein family of ubiquitin ligases regulators and details the molecular and cellular role of MAGEL2 in ubiquitination, actin regulation and endosomal sorting processes, as well as MAGEL2 implications in PWS and SHFYNG disorders. The physiological functions of MAGEL2, elucidated through the study of Magel2 knockout mouse models, are also discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
... MyoD, a key myogenic differentiation factor, contributes to muscle plasticity and regeneration (Joung et al., 2014) and regulates skeletal myogenesis (Han et al., 2011). The luciferase activity analysis as well as qPCR and Western blotting results showed that MyoD positively regulated FGF21 expression by binding to the FGF21 promoter region. ...
Fibroblast growth factor 21(FGF21) is a pivotal regulator of energy metabolism, which is currently being assessed as a potential drug target for the treatment of insulin-resistant conditions. However, the cellular mechanisms by which FGF21 affects myogenesis remain unclear. In this study, we explored the function of FGF21 in myogenesis both in vitro and in vivo. Our experiments showed for the first time that FGF21 promotes myoblast differentiation and serves as a switch of molecular transformation from anaerobic myofibers to aerobic myofibers via the FGF21-SIRT1-AMPK-PGC1α axis. Furthermore, we employed the Dual-Luciferase Reporter Assay System and Electrophoretic Mobility Shift Assay (EMSA) and demonstrated that MYOD, a major myogenic transcription factor, binds directly to the promoter region of Fgf21, leading to the activation of Fgf21 expression in mouse C2C12 myoblasts. Our study revealed a novel mechanism of myogenesis and muscle fiber transformation and indicated that FGF21 serves as a vital regulator of muscle development and important contributor to the pathogenesis of myopathy. This article is protected by copyright. All rights reserved.
... Stuelsatz et al. [19] reported that calpain 3 up-regulation negatively affects the differentiation process of C2C12 myoblast cells, and promotes generation of reserve cells in C2C12 myoblasts. Denervation injury reduced the MyoD protein amount [20]. Citing the views of related literature, our results showed an increasing level of CAPN3 expression during muscle degeneration phase, may inhibit myogenic differentiation; in this scenario, the down-regulation of CAPN3 during re-innervation could stimulate myogenic differentiation. ...
Calpain 3 (CAPN3), also known as p94, is a skeletal muscle-specific member of the calpain family that is involved in muscular dystrophy; however, the roles of CAPN3 in muscular atrophy and regeneration are yet to be understood. In the present study, we attempted to explain the effect of CAPN3 in muscle atrophy by evaluating CAPN3 expression in rat gastrocnemius muscle following reversible sciatic nerve injury. After nerve injury, the wet weight ratio and cross sectional area (CSA) of gastrocnemius muscle were decreased gradually from 1-14 days and then recovery from 14-28 days. The active form of CAPN3 (~62 kDa) protein decreased slightly on day 3 and then increased from day 7 to 14 before a decrease from day 14 to 28. The result of linear correlation analysis showed that expression of the active CAPN3 protein level was negatively correlated with muscle wet weight ratio. CAPN3 knockdown by short interfering RNA (siRNA) injection improved muscle recovery on days 7 and 14 after injury as compared to that observed with control siRNA treatment. Depletion of CAPN3 gene expression could promote myoblast differentiation in L6 cells. Based on these findings, we conclude that the expression pattern of the active CAPN3 protein is linked to muscle atrophy and regeneration following denervation: its upregulation during early stages may promote satellite cell renewal by inhibiting differentiation, whereas in later stages, CAPN3 expression may be downregulated to stimulate myogenic differentiation and enhance recovery. These results provide a novel mechanistic insight into the role of CAPN3 protein in muscle regeneration after peripheral nerve injury.
