Vitamin C regulates skeletal muscle post-injury regeneration by promoting myoblast proliferation through its direct interaction with the Pax7 protein.

Previous studies have shown that vitamin C (VC), an essential vitamin for the human body, can promote the differentiation of muscle satellite cells (MuSCs) in vitro and play an important role in skeletal muscle post-injury regeneration. However, the molecular mechanism of VC regulating MuSC proliferation has not been elucidated. In this study, the role of VC in promoting MuSC proliferation and its molecular mechanism were explored using cell molecular biology and animal experiments. The results showed that VC accelerates the progress of skeletal muscle post-injury regeneration by promoting MuSC proliferation in vivo. VC can also promote skeletal muscle regeneration in the case of atrophy. Using the C2C12 myoblast murine cell line, we observed that VC also stimulated cell proliferation. In addition, after an in vitro study establishing the occurrence of a physical interaction between VC and Pax7, we observed that VC also upregulated the total and nuclear Pax7 protein levels. This mechanism increased the expression of Myf5 (Myogenic Factor 5), a Pax7 target gene. This study establishes a theoretical foundation for understanding the regulatory mechanisms underlying VC-mediated MuSC proliferation and skeletal muscle regeneration. Moreover, it develops the application of VC in animal muscle nutritional supplements and treatment of skeletal muscle-related diseases.

Podocan promoting skeletal muscle post-injury regeneration by inhibiting TGF-ß signaling pathway.

Podocan, the fifth member of Small Leucine-Rich Proteoglycan (SLRP) family of extracellular matrix components, is poorly known in muscle development. Previous studies have shown that Podocan promotes C2C12 differentiation in mice. In this study, we elucidated the effect of Podocan on skeletal muscle post-injury regeneration and its underlying mechanism. Injection of Podocan protein promoted the process of mice skeletal muscle post-injury regeneration. This effect seemed to be from the acceleration of muscle satellite cell differentiation in vivo. Meanwhile, Podocan promoted myogenic differentiation in vitro by binding with TGF-ß1 to inhibit the activity of the TGF-ß signaling pathway. These results indicated that Podocan had the potential roles to enhance skeletal muscle post-injury regeneration. Its mechanism is likely the regulation of the expression of p-Smad2 and p-Smad4 related to the TGF-ß signaling pathway by interacting with TGF-ß1.

Leu promotes C2C12 cell differentiation by regulating the GSK3ß/ß-catenin signaling pathway through facilitating the interaction between SESN2 and RPN2.

BACKGROUND: Leucine (Leu) is an essential amino acid that facilitates skeletal muscle satellite cell differentiation, yet its mechanism remains underexplored. Sestrin2 (SESN2) serves as a Leu sensor, binding directly to Leu, while ribophorin II (RPN2) acts as a signaling factor in multiple pathways. This study aimed to elucidate Leu's impact on mouse C2C12 cell differentiation and skeletal muscle injury repair by modulating RPN2 expression through SESN2, offering a theoretical foundation for clinical skeletal muscle injury prevention and treatment. RESULTS: Leu addition promoted C2C12 cell differentiation compared to the control, enhancing early differentiation via myogenic determinant (MYOD) up-regulation. Sequencing revealed SESN2 binding to and interacting with RPN2. RPN2 overexpression up-regulated MYOD, myogenin and myosin heavy chain 2, concurrently decreased p-GSK3ß and increased nuclear ß-catenin. Conversely, RPN2 knockdown yielded opposite results. Combining RPN2 knockdown with Leu rescued increased p-GSK3ß and decreased nuclear ß-catenin compared to Leu absence. Hematoxylin and eosin staining results showed that Leu addition accelerated mouse muscle damage repair, up-regulating Pax7, MYOD and RPN2 in the cytoplasm, and nuclear ß-catenin, confirming that the role of Leu in muscle injury repair was consistent with the results for C2C12 cells. CONCLUSION: Leu, bound with SESN2, up-regulated RPN2 expression, activated the GSK3ß/ß-catenin pathway, enhanced C2C12 differentiation and expedited skeletal muscle damage repair. © 2024 Society of Chemical Industry.

Vitamin A Promotes the Repair of Mice Skeletal Muscle Injury through RARα.

Vitamin A (VitA) is an important fat-soluble vitamin which plays an important role in cell growth and individual development. However, the effect of VitA on the repair process of muscle injury and its molecular mechanism are still unclear. In this study, VitA and RA were first added to the culture medium of differentiated cells. We then detected cell differentiation marker proteins and myotube fusion. Moreover, the effects of VitA on RARα expression and nuclear translocation were further examined. The results showed that VitA significantly promoted the differentiation of C2C12, and the expression of RARα was significantly increased. Furthermore, VitA was injected into skeletal muscle injury in mice. HE staining and Western Blot results showed that VitA could significantly accelerate the repair of skeletal muscle injury and VitA increase the expression of RARα in mice. This study provides a theoretical basis for elucidating the regulation mechanism of VitA-mediated muscle development and the development of therapeutic drugs for muscle diseases in animals.

Cyanocobalamin promotes muscle development through the TGF-ß signaling pathway.

Cyanocobalamin (CNCbl, the compound name of Vitamin B12) is the only mineral vitamin that is essential for growth and development and cannot be produced by animals. Some studies have found that CNCbl can promote the proliferation and migration of C2C12 cells, but the mechanism by which it affects muscle development is still unknown. In this study, we elucidated the effect of CNCbl on muscle development and studied its underlying mechanism. CNCbl could promote the differentiation of C2C12 cells and upregulate Acvr1, p-Smad2 and p-Smad3 in the TGF-ß signaling pathway in vitro. CD320 (the receptor in cell surface for binding with CNCbl transporter transcobalamin II) inhibition could reduce the uptake of CNCbl and significantly downregulate the expression of differentiation marker proteins MyoG and MYH2. Furthermore, the levels of p-Smad2 and p-Smad3 were also reduced with the inhibition of CD320, even though CNCbl was added to the C2C12 culture medium. In addition, the injection of CNCbl could accelerate the process of mouse muscle injury repair, enlarge the diameter of newly formed myofibers and upregulate the expression of MYH2, PAX7, CD320, Acvr1, p-Smad2 and p-Smad3 in vivo. These results suggest that CNCbl can promote muscle development and may play its role by regulating the expression of Acvr1, p-Smad2 and p-Smad3 related to the TGF-ß signaling pathway.

Two-Dimensional Ultrasound and Triplane Tissue Doppler Ultrasound of Patients with Severe Preeclampsia.

This study was to investigate the cardiac function characteristics under two-dimensional ultrasound and triplane tissue Doppler imaging (TDI) of patients with severe preeclampsia (SPE). 28 SPE patients with singleton pregnancy from January 2018 to December 2020 were included in the SPE group. 25 healthy nonpregnant women of reproductive age were taken as the control group (Ctrl group), and 26 normal pregnant women with singleton pregnancy were selected as the normal group (Norm group); all the research objects underwent ultrasonography. The morphological and functional indexes of left and right ventricles were compared among the cases in different groups. The results showed that the left ventricular end-diastolic period diameter (LVEDd), left ventricular relative wall thickness (LV-RWT), left ventricular mass index (LVMi), left anterior descending (LAd), left ventricular E/e and e/a values, right ventricular diameter (RV-D), right ventricular anterior wall thickness (RVAW), a value, right atrial septum (RA-S), pulmonary artery systolic pressure (PASP), left ventricular end-systolic period diameter (LVEds), interventricular septal thickness (IVSd), posterior wall thickness (PWd), end-diastolic period volume (EVD), end-systolic period volume (ESV), relative wall thickness (RWT), sphericity index (SpI), left atrium volume index (LAVi), and E/e value of patients in the SPE group were higher than those in the Ctrl group and the Norm group (P < 0.05). The mitral annular plane systolic excursion (MAPSE), s value, tricuspid annual plane systolic excursion (TAPSE), ratio of early diastolic blood flow velocity to late diastolic blood flow velocity (E/A), ratio of peak early diastolic velocity to peak late diastolic velocity (e/a), peak early diastolic velocity (e), and ejection fraction (EF) of the SPE group were lower than those of the Ctrl group and the Norm group (P < 0.05). The ratio of mitral valve early diastolic blood flow velocity to peak early diastolic velocity (E/e) of the Norm group was higher than that of the Ctrl group (P < 0.05). In two-dimensional ultrasound of the SPE group, the maximum difference in time from the start to the peak of systole (Ts) of the right ventricle between the basal and middle segments of the lateral wall and that of interventricular septum (RV-Ts-max) was 31.56 ± 0.39%. The maximum difference in time to peak of early diastole (Te) under the same condition (RV-Te-max) was 47.16 ± 0.19%. Left ventricular LV-Ts-max and LV-Te-max were 9.83 ± 0.80% and 8.37 ± 0.68%, respectively, in triplane TDI, which were considerably higher than those in the Ctrl and Norm groups (P < 0.05). It suggested that two-dimensional ultrasound and triplane TDI could reflect the ventricular morphology as well as diastolic and systolic function injury in patients, which offered a reference basis for the diagnosis of SPE.

Vitamin C Promotes Muscle Development Mediated by the Interaction of CSRP3 with MyoD and MyoG.

Previous studies have reported that vitamin C (VC), an essential nutrient, exerts beneficial effects on muscle health. However, the molecular mechanism involved in the VC-mediated regulation of muscle development is still unclear. The roles of VC in muscle development and the underlying molecular mechanisms were examined using cell and molecular biology, transcriptomics, proteomics, and animal experiments in this study. VC upregulated the expression of sodium-dependent vitamin C transporter 2 (SVCT2) and cysteine rich protein 3 (CSRP3). Additionally, VC promoted the differentiation of C2C12 cells and the repair of mouse muscle injury by upregulating the nuclear translocation of CSRP3, which subsequently interacted with MyoD and MyoG. This study provided a theoretical basis for elucidating the mechanism underlying the VC-mediated regulation of muscle development, as well as for developing animal nutritional supplements and therapeutic drugs for muscle diseases.

Establishment of a bovine rumen epithelial cell line.

Rumen epithelium plays an essential role in absorption, transport, and metabolism of short-chain fatty acids, the main products of rumen fermentation, and in preventing microbes and other potentially harmful rumen contents from entering the systemic circulation. The objective of this study was to generate an immortal rumen epithelial cell line that can be used as a convenient model of rumen epithelial cells in vitro. We isolated primary rumen epithelial cells from a steer through trypsin digestion and transduced them with lentiviruses expressing the Simian Virus (SV) 40 T antigen. We cloned the transduced cells by limiting dilution. Western blotting analysis confirmed the expression of the SV40 T antigen in two single-cell clones. Cells from one clone, named bovine rumen epithelial clone 1 (BREC1), displayed a flat and squamous morphology in culture. RNA sequencing revealed that BREC1 cells expressed many markers of epithelial cells, including keratins, the epidermal growth factor receptor, and the short-chain fatty acid transporters monocarboxylic acid transporter (MCT) 1 (MCT-1) and MCT-4. RNA sequencing revealed that BREC1 cells expressed key enzymes such as 3-hydroxymethyl-3-methylglutaryl-CoA lyase and 3-hydroxy-3-methylglutaryl-CoA synthase 1 involved in ketogenesis, a unique function of rumen epithelial cells. RNA sequencing also revealed the expression of genes encoding tight junctions, desmosomes, anchoring junctions, and polarized plasma membranes, structures typical of epithelial cells, in BREC1 cells. Cell proliferation assays indicated that BREC1 cells were similar to primary rumen epithelial cells in response to insulin-like growth factor 1, insulin, and butyrate. In conclusion, BREC1 is not only a convenient but an appropriate model for studying the factors and mechanisms that control proliferation, apoptosis, differentiation, nutrient transport, metabolism, and barrier function in rumen epithelium.

MYOC Promotes the Differentiation of C2C12 Cells by Regulation of the TGF-ß Signaling Pathways via CAV1.

Myocilin (MYOC) is a glycoprotein encoded by a gene associated with glaucoma pathology. In addition to the eyes, it also expresses at high transcription levels in the heart and skeletal muscle. MYOC affects the formation of the murine gastrocnemius muscle and is associated with the differentiation of mouse osteoblasts, but its role in the differentiation of C2C12 cells has not yet been reported. Here, MYOC expression was found to increase gradually during the differentiation of C2C12 cells. Overexpression of MYOC resulted in enhanced differentiation of C2C12 cells while its inhibition caused reduced differentiation. Furthermore, immunoprecipitation indicated that MYOC binds to Caveolin-1 (CAV1), a protein that influences the TGF-ß pathway. Laser confocal microscopy also revealed the common sites of action of the two during the differentiation of C2C12 cells. Additionally, CAV1 was upregulated significantly as C2C12 cells differentiated, with CAV1 able to influence the differentiation of the cells. Furthermore, the Western blotting analysis demonstrated that the expression of MYOC affected the TGF-ß pathway. Finally, MYOC was overexpressed while CAV1 was inhibited. The results indicate that reduced CAV1 expression blocked the promotion of C2C12 cell differentiation by MYOC. In conclusion, the results demonstrated that MYOC regulates TGF-ß by influencing CAV1 to promote the differentiation of C2C12 cells.

Fibronectin type III domain-containing 4 promotes the migration and differentiation of bovine skeletal muscle-derived satellite cells via focal adhesion kinase.

FNDC4 is an anti-inflammatory factor that alters the activation state of macrophages; it is used to treat colitis in mice. However, its role in muscle formation and mechanism of function remains unknown. We found that FNDC4 promotes the bovine MDSCs migration and differentiation. Furthermore, we reported that it interacts with integrin ß1 (ITGß1). FAK, mediated by ITGß1, regulates cell migration. Our results found FNDC4 to influence the expression of p-FAK, p-paxillin, and vinculin. Then, overexpressed or added FNDC4 protein could not influence migration and differentiation any more when the activated form of FAK was reduced. Therefore, we concluded that FNDC4 promotes the differentiation and migration of bovine MDSCs via the FAK, mediated by the ITGß1 receptor.

SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway.

As an extracellular matrix protein, secreted protein acidic and rich in cysteine (SPARC)-like 1 (SPARCL1) is involved in various cell functions. It was previously implicated in bovine skeletal muscle-derived satellite cell (MDSC) differentiation; however, the underlying mechanism remains unknown. In this study, immunoprecipitation and mass spectrometry revealed that integrin ß1 (ITGB1) combines with SPARCL1. Further, co-immunoprecipitation demonstrated that SPARCL1 interacts with ITGB1. Cell scratch assays explored the influence of SPARCL1 on MDSC migration through ITGB1. In addition, desmin staining for myotube fusion rate and MyoD protein expression results showed that SPARCL1 promotes MDSC early differentiation through ITGB1. Furthermore, Western blotting results demonstrated that SPARCL1 regulates the expression of p-FAK, p-paxillin, vinculin, Cdc42, and Arp2/3 through ITGB1. These findings indicate that SPARCL1 may influence bovine MDSC migration and differentiation through an ITGB1-mediated cell signaling pathway. Herein, we elucidated the mechanism through which SPARCL1 affects MDSC differentiation. Our results provide insight into the molecular mechanism of muscle development and may in the future facilitate skeletal muscle regeneration and treatment.

WISP1 promotes bovine MDSC differentiation via recruitment of ANXA1 for the regulation of the TGF-ß signalling pathway.

Skeletal muscle is one of the most important tissues of the human body necessary for sporting activities. The differentiation of muscle-derived satellite cells (MDSCs) plays an important role in the development and regeneration of skeletal muscles. Similarly, the Wnt/ß-catenin signalling pathway plays an important role in the process of muscle differentiation. Wnt1-inducible signalling pathway protein-1 (WISP1), a downstream protein of the Wnt/ß-catenin signalling pathway and a member of the CCN family that also plays an important role in the differentiation process, and its expression increase during the differentiation of bovine MDSCs. However, its role in MDSC differentiation is poorly understood. Therefore, we investigated the mechanisms regulating this process via Western blot and immunofluorescence staining. Immunoprecipitation and mass spectrometry detected annexin A1 (ANXA1), a protein that interacts with WISP1. To determine whether WISP1 influences TGF-ß signalling and differentiation independently of ANXA1, the latter was knocked down, while WISP1 was activated. WISP1 expression increased significantly during bovine MDSC differentiation. However, WISP1 did not affect the TGF-ß signalling pathway protein marker when ANXA1 was inhibited. Taken together, WISP1 regulates the TGF-ß signalling pathway through ANXA1 recruitment, thereby promoting bovine MDSC differentiation, suggesting the Wnt/ß-catenin signalling pathway as another target to promote cell differentiation.

Fibronectin type III domain containing four promotes differentiation of C2C12 through the Wnt/ß-catenin signaling pathway.

Fibronectin type III domain containing 4 (FNDC4) belongs to the fibronectin type III domain containing protein family. FNDC5, which is highly homologous to FNDC4, can promote the differentiation of cardiac cells. We aimed to investigate the role of FNDC4 in the differentiation of C2C12 mouse skeletal muscle cells. Western blotting and immunofluorescence analysis showed that FNDC4 gradually increased with the differentiation of C2C12. Muscle injury repair experiments indicated that FNDC4 may promote the repair of injured muscles. When FNDC4 was either overexpressed or knocked down, the expression of desmin and myogenin myogenic marker molecules followed that of FNDC4, suggesting that FNDC4 can influence the differentiation of C2C12. In addition, immunoprecipitation results showed that FNDC4 can interact with the Wnt/ß-catenin signaling pathway receptor low-density lipoprotein receptor-related protein 6 (LRP6), and that ß-catenin levels in the nucleus decreased after knocking down FNDC4. Exogenous addition of FNDC4 protein could not restore the blocking of differentiation due to inhibition of both Wnt/ß-catenin signal transduction and LRP6 activity via the ß-catenin inhibitor XAV-939. Overall, our findings indicate that FDNC4 can influence the differentiation of C2C12 by activating Wnt/ß-catenin signal transduction.

TCP11L2 promotes bovine skeletal muscle-derived satellite cell migration and differentiation via FMNL2.

T-complex 11 like 2 (TCP11L2) is a protein containing a serine-rich region in its N-terminal region. However, the function of TCP11L2 is unclear. Here, we showed that TCP11L2 expression gradually increased during muscle-derived satellite cell (MDSC) differentiation in vitro, reaching a peak on Day 3, which is the migration and fusion stage of MDSCs. Using CRISPR/dCas9 gene-editing technology to elevate or repress the expression of TCP11L2, we also showed that TCP11L2 promoted MDSC differentiation. Moreover, wound-healing assays showed that TCP11L2 promoted the migration of MDSCs during differentiation. Additionally, immunofluorescence analyses showed that TCP11L2 was mainly distributed around the microfilament and microtubules. Furthermore, the expression of TCP11L2 affected the expression of actin-related protein 2/3 (ARP2/3) complex. Co-immunoprecipitation assays and immunofluorescence analysis showed that TCP11L2 interacted with formin-like 2 (FMNL2). This protein promoted migration of bovine MDSCs by affecting the expression of ARP2/3. Finally, the activities of TCP11L2 during MDSC differentiation and migration were blocked when FMNL2 was inhibited. Taken together, our data established that TCP11L2 interacted with FMNL2 to promote MDSC migration and differentiation.

SPARCL1 promotes C2C12 cell differentiation via BMP7-mediated BMP/TGF-ß cell signaling pathway.

The extracellular matrix (ECM) is known to regulate tissue development and cell morphology, movement, and differentiation. SPARCL1 is an ECM protein, but its role in mouse cell differentiation has not been widely investigated. The results of western blotting and immunofluorescence showed that SPARCL1 is associated with the repair of muscle damage in mice and that SPARCL1 binds to bone morphogenetic protein 7 (BMP7) by regulating BMP/transforming growth factor (TGF)-ß cell signaling. This pathway promotes the differentiation of C2C12 cells. Using CRISPR/Cas9 technology, we also showed that SPARCL1 activates BMP/TGF-ß to promote the differentiation of C2C12 cells. BMP7 molecules were found to interact with SPARCL1 by immunoprecipitation analysis. Western blotting and immunofluorescence were performed to verify the effect of BMP7 on C2C12 cell differentiation. Furthermore, SPARCL1 was shown to influence the expression of BMP7 and activity of the BMP/TGF-ß signaling pathway. Finally, SPARCL1 activation was accompanied by BMP7 inhibition in C2C12 cells, which confirmed that SPARCL1 affects BMP7 expression and can promote C2C12 cell differentiation through the BMP/TGF-ß pathway. The ECM is essential for muscle regeneration and damage repair. This study intends to improve the understanding of the molecular mechanisms of muscle development and provide new treatment ideas for muscle injury diseases.

Podocan Promotes Differentiation of Bovine Skeletal Muscle Satellite Cells by Regulating the Wnt4-ß-Catenin Signaling Pathway.

BACKGROUND: Small leucine-rich repeat proteins (SLRPs) are highly effective and selective modulators of cell proliferation and differentiation. Podocan is a newly discovered member of the SLRP family. Its potential roles in the differentiation of bovine muscle-derived satellite cells (MDSCs) and its underlying functional mechanism remain unclear. Our study aimed to characterize the function of the podocan gene in the differentiation of bovine MDSCs and to clarify the molecular mechanism by which podocan functions in order to contribute to a better understanding of the molecular mechanism by which extracellular matrix promotes bovine MDSC differentiation and provide a theoretical basis for the improvement of beef quality. METHODS: Bovine MDSCs were transfected with vectors to overexpress or inhibit podocan, and podocan protein was added to differentiation culture medium. qRT-PCR, western blotting, and immunofluorescence were performed to investigate the effects of podocan on MDSC differentiation. Confocal microscopy and western blotting were used to assess the nuclear translocation and expression of ß-catenin. An inhibitor and activator of ß-catenin were used to assess the effects of the Wnt/ß-catenin signaling pathway on MDSC differentiation. We inhibited ß-catenin while overexpressing podocan in MDSCs. Then, we performed mass spectrometry to identify which proteins interact with podocan to regulate the Wnt/ß-catenin signaling pathway. Finally, we confirmed the relationship between podocan and Wnt4 by co-immunoprecipitation and western blotting. RESULTS: Podocan protein expression increased significantly during bovine MDSC differentiation. Differentiation of bovine MDSC was promoted and suppressed by podocan overexpression or inhibition, respectively. Podocan was also shown to modulate the Wnt/ß-catenin signaling pathway. Treatment of bovine MDSCs with ß-catenin inhibitor and activator showed that the Wnt/ß-catenin pathway is involved in bovine MDSC differentiation. Furthermore, the effect of podocan on bovine MDSC differentiation was suppressed when this pathway was inhibited. We also found that podocan interacts with Wnt4. When Wnt4 was inhibited, podocan-induced promotion of bovine MDSC differentiation was attenuated through Wnt/ß-catenin signaling. CONCLUSION: Podocan regulates Wnt/ß-catenin through Wnt4 to promote bovine MDSC differentiation.

Platelet endothelial aggregation receptor-1 regulates bovine muscle satellite cell migration and differentiation via integrin beta-1 and focal adhesion kinase.

PEAR1 is highly expressed at bovine MDSC differentiation. However, its biological function remains unclear. Western blotting results showed that PEAR1 increased between day 0 and day 2 of cell differentiation and decreased from day 3. Moreover, scratch test showed that wound healing rate increased after PEAR1 overexpression and decreased upon its suppression. Meanwhile, we found that, upon PEAR1 induction, both the expression of the focal adhesion-associated and MyoG, and the myotube fusion rate increased. However, when PEAR1 was suppressed, opposite results were obtained. Immunoprecipitation revealed an association between PEAR1 and ITGB1. Notably, inhibition of FAK and ITGB1 repressed cell differentiation. In conclusion, our study indicated that PEAR1 is involved in the regulation of bovine MDSC migration and differentiation.

WDR13 promotes the differentiation of bovine skeletal muscle-derived satellite cells by affecting PI3K/AKT signaling.

Muscle satellite cells are usually at rest, and when externally stimulated or regulated, they can be further differentiated by cell fusion to form new myotubes and muscle fibers. WD repeat domain 13 (WDR13) is highly conserved in vertebrates. Studies have shown that mice lacking the Wdr13 gene develop mild obesity, hyperinsulinemia, and increased islet ß cell proliferation. However, the role of WDR13 in bovine cells is unclear. Here, we investigated the effect of WDR13 on bovine skeletal muscle-derived satellite cells (MDSCs). We found that WDR13 was upregulated in bovine MDSCs using western blotting and immunofluorescence experiments. Moreover, activation and inhibition of WDR13 expression increased and decreased cell differentiation, respectively, suggesting that WDR13 promotes bovine MDSC differentiation. To further understand the mechanism of action of WDR13, we examined changes in the PI3K/AKT signaling pathway following WDR13 activation or inhibition. In addition, cells were treated with a phosphoinositide kinase 3 (PI3K) inhibitor, LY294004, to observe cell differentiation. The results showed that activation of WDR13 inhibited the PI3K/AKT signaling pathway and enhanced cell differentiation. These data suggest that WDR13 can promote the differentiation of bovine MDSCs by affecting the PI3K/AKT signaling pathway.

GRP94 promotes muscle differentiation by inhibiting the PI3K/AKT/mTOR signaling pathway.

The glucose-regulated endoplasmic reticulum chaperone protein 94 (GRP94) is required for many biological processes, such as secretion of immune factors and mesoderm induction. Here, we demonstrated that GRP94 promotes muscle differentiation in vitro and in vivo. Moreover, GRP94 inhibited the PI3K/AKT/mTOR signaling pathway. Using both in vitro and in vivo approaches, in myoblasts, we found that this inhibition resulted in reduced proliferation and increased differentiation. To further investigate the mechanism of GRP94-induced muscle differentiation, we used co-immunoprecipitation and proximity ligation assays and found that GRP94 interacted with PI3K-interacting protein 1 (Pik3ip1). The latter protein promoted muscle differentiation by inhibiting the PI3K/AKT/mTOR pathway. Furthermore, GRP94 was found to regulate Pik3ip1 expression. Finally, when Pik3ip1 expression was inhibited, GRP94-induced promotion of muscle differentiation was diminished. Taken together, our data demonstrated that GRP94 promoted muscle differentiation, mediated by Pik3ip1-dependent inhibition of the PI3K/AKT/mTOR signaling pathway.

Podocan affects C2C12 myogenic differentiation by enhancing Wnt/ß-catenin signaling.

Podocan, a small leucine-rich repeat protein, is a negative regulator of cell proliferation. In this study, we demonstrated that podocan is involved in the differentiation of C2C12 murine myoblasts. Podocan expression increased with the progression of C2C12 differentiation. As expect, siRNA-mediated podocan knockdown inhibited C2C12 differentiation, as indicated by inhibition of MYOG, MYH2, and desmin expression, as well as reductions in the differentiation and fusion indices. Overexpression of podocan using dCas9 technology promoted C2C12 cell differentiation. In addition, supplementation of culture medium with podocan influenced C2C12 differentiation. Podocan knockdown reduced Wnt/ß-catenin signaling, characterized by a reduction in the nuclear translocation of ß-catenin, whereas podocan overexpression had the opposite effect. Furthermore, treatment with XAV939, an inhibitor of Wnt/ß-catenin, reduced the podocan-mediated promotion of C2C12 differentiation. Induction of muscle injury in mice by bupivacaine administration suggested that podocan may play a role in muscle regeneration. In summary, our results suggest that podocan is required for normal C2C12 differentiation and that its role in myogenesis is mediated by the Wnt/ß-catenin pathway.