UBE4B regulates p27 expression in A549 NSCLC cells through regulating the interaction of HuR and the p27 5' UTR.

Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.

Melatonin and Exercise Counteract Sarcopenic Obesity through Preservation of Satellite Cell Function.

Sarcopenic obesity (SO) is characterized by atrophic skeletal muscle impairment (sarcopenia) and obesity, which is associated with adverse outcomes of morbidity and mortality in elderly people. We investigated the effects of melatonin and exercise training on SO in 32-week-old senescence-accelerated mouse-prone-8 (SAMP8) mice fed a normal diet or a high-fat diet for 16 weeks. Melatonin, exercise, or melatonin and exercise for 8 weeks displayed reductions in the SO-induced impairment of skeletal muscle function and atrophy. Specifically, a decrease in mitochondrial calcium retention capacity in skeletal muscles observed in the HFD-con group was attenuated in melatonin and/or exercise intervention groups. More importantly, HFD-con mice displayed a lower number of Pax7+ satellite cells (SCs) and higher expression of p16ink than P8ND mice, which were attenuated by melatonin and/or exercise interventions. The cellular senescence in SC-derived primary myoblasts from HFD-con mice was significantly attenuated in myoblasts from the melatonin and/or exercise groups, which was reproduced in a senescence model of H2O2-treated C2C12 myoblasts. Our results suggest that melatonin and exercise training attenuate SO-induced skeletal muscle dysfunction, at least in part, through preserving the SC pool by inhibiting cellular senescence and attenuating mitochondrial dysfunction.

Skeletal Muscle-Specific Bis Depletion Leads to Muscle Dysfunction and Early Death Accompanied by Impairment in Protein Quality Control.

Bcl-2-interacting cell death suppressor (BIS), also called BAG3, plays a role in physiological functions such as anti-apoptosis, cell proliferation, autophagy, and senescence. Whole-body Bis-knockout (KO) mice exhibit early lethality accompanied by abnormalities in cardiac and skeletal muscles, suggesting the critical role of BIS in these muscles. In this study, we generated skeletal muscle-specific Bis-knockout (Bis-SMKO) mice for the first time. Bis-SMKO mice exhibit growth retardation, kyphosis, a lack of peripheral fat, and respiratory failure, ultimately leading to early death. Regenerating fibers and increased intensity in cleaved PARP1 immunostaining were observed in the diaphragm of Bis-SMKO mice, indicating considerable muscle degeneration. Through electron microscopy analysis, we observed myofibrillar disruption, degenerated mitochondria, and autophagic vacuoles in the Bis-SMKO diaphragm. Specifically, autophagy was impaired, and heat shock proteins (HSPs), such as HSPB5 and HSP70, and z-disk proteins, including filamin C and desmin, accumulated in Bis-SMKO skeletal muscles. We also found metabolic impairments, including decreased ATP levels and lactate dehydrogenase (LDH) and creatine kinase (CK) activities in the diaphragm of Bis-SMKO mice. Our findings highlight that BIS is critical for protein homeostasis and energy metabolism in skeletal muscles, suggesting that Bis-SMKO mice could be used as a therapeutic strategy for myopathies and to elucidate the molecular function of BIS in skeletal muscle physiology.

Prominin-1-Radixin axis controls hepatic gluconeogenesis by regulating PKA activity.

Prominin-1 (Prom1) is a major cell surface marker of cancer stem cells, but its physiological functions in the liver have not been elucidated. We analyzed the levels of mRNA transcripts in serum-starved primary WT (Prom1+/+ ) and KO (Prom1-/- ) mouse hepatocytes using RNA sequencing (RNA-seq) data, and found that CREB target genes were downregulated. This initial observation led us to determine that Prom1 deficiency inhibited cAMP response element-binding protein (CREB) activation and gluconeogenesis, but not cyclic AMP (cAMP) accumulation, in glucagon-, epinephrine-, or forskolin-treated liver tissues and primary hepatocytes, and mitigated glucagon-induced hyperglycemia. Because Prom1 interacted with radixin, Prom1 deficiency prevented radixin from localizing to the plasma membrane. Moreover, systemic adenoviral knockdown of radixin inhibited CREB activation and gluconeogenesis in glucagon-treated liver tissues and primary hepatocytes, and mitigated glucagon-elicited hyperglycemia. Based on these results, we conclude that Prom1 regulates hepatic PKA signaling via radixin functioning as an A kinase-anchored protein (AKAP).

Factors and Pathways Modulating Endothelial Cell Senescence in Vascular Aging.

Aging causes a progressive decline in the structure and function of organs. With advancing age, an accumulation of senescent endothelial cells (ECs) contributes to the risk of developing vascular dysfunction and cardiovascular diseases, including hypertension, diabetes, atherosclerosis, and neurodegeneration. Senescent ECs undergo phenotypic changes that alter the pattern of expressed proteins, as well as their morphologies and functions, and have been linked to vascular impairments, such as aortic stiffness, enhanced inflammation, and dysregulated vascular tone. Numerous molecules and pathways, including sirtuins, Klotho, RAAS, IGFBP, NRF2, and mTOR, have been implicated in promoting EC senescence. This review summarizes the molecular players and signaling pathways driving EC senescence and identifies targets with possible therapeutic value in age-related vascular diseases.

Sulfated syndecan 1 is critical to preventing cellular senescence by modulating fibroblast growth factor receptor endocytosis.

Cellular senescence can be triggered by various intrinsic and extrinsic stimuli. We previously reported that silencing of 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) induces cellular senescence through augmented fibroblast growth factor receptor 1 (FGFR1) signaling. However, the exact molecular mechanism connecting heparan sulfation and cellular senescence remains unclear. Here, we investigated the potential involvement of heparan sulfate proteoglycans (HSPGs) in augmented FGFR1 signaling and cellular senescence. Depletion of several types of HSPGs revealed that cells depleted of syndecan 1 (SDC1) exhibited typical senescence phenotypes, and those depleted of PAPSS2-, SDC1-, or heparan sulfate 2-O sulfotransferase 1 (HS2ST1) showed decreased FGFR1 internalization along with hyperresponsiveness to and prolonged activation of fibroblast growth factor 2 (FGF2)-stimulated FGFR1- v-akt murine thymoma viral oncogene homolog (AKT) signaling. Clathrin- and caveolin-mediated FGFR1 endocytosis contributed to cellular senescence through the FGFR1-AKT-p53-p21 signaling pathway. Dynasore treatment triggered senescence phenotypes, augmented FGFR1-AKT-p53-p21 signaling, and decreased SDC1 expression. Finally, the replicatively and prematurely senescent cells were characterized by decreases of SDC1 expression and FGFR1 internalization, and an increase in FGFR1-AKT-p53-p21 signaling. Together, our results demonstrate that properly sulfated SDC1 plays a critical role in preventing cellular senescence through the regulation of FGFR1 endocytosis.

Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo.

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.

Transglutaminase 2-Mediated p53 Depletion Promotes Angiogenesis by Increasing HIF-1α-p300 Binding in Renal Cell Carcinoma.

Angiogenesis and the expression of vascular endothelial growth factor (VEGF) are increased in renal cell carcinoma (RCC). Transglutaminase 2 (TGase 2), which promotes angiogenesis in endothelial cells during wound healing, is upregulated in RCC. Tumor angiogenesis involves three domains: cancer cells, the extracellular matrix, and endothelial cells. TGase 2 stabilizes VEGF in the extracellular matrix and promotes VEGFR-2 nuclear translocation in endothelial cells. However, the role of TGase 2 in angiogenesis in the cancer cell domain remains unclear. Hypoxia-inducible factor (HIF)-1α-mediated VEGF production underlies the induction of angiogenesis in cancer cells. In this study, we show that p53 downregulated HIF-1α in RCC, and p53 overexpression decreased VEGF production. Increased TGase 2 promoted angiogenesis by inducing p53 degradation, leading to the activation of HIF-1α. The interaction of HIF-1α and p53 with the cofactor p300 is required for stable transcriptional activation. We found that TGase 2-mediated p53 depletion increased the availability of p300 for HIF-1α-p300 binding. A preclinical xenograft model suggested that TGase 2 inhibition can reverse angiogenesis in RCC.

Anti-cancer effect of doxorubicin is mediated by downregulation of HMG-Co A reductase via inhibition of EGFR/Src pathway.

Doxorubicin is a widely used DNA damage-inducing anti-cancer drug. However, its use is limited by its dose-dependent side effects, such as cardiac toxicity. Cholesterol-lowering statin drugs increase the efficacy of some anti-cancer drugs. Cholesterol is important for cell growth and a critical component of lipid rafts, which are plasma membrane microdomains important for cell signaling. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-CR) is a critical enzyme in cholesterol synthesis. Here, we show that doxorubicin downregulated HMG-CR protein levels and thus reduced levels of cholesterol and lipid rafts. Cholesterol addition attenuated doxorubicin-induced cell death, and cholesterol depletion enhanced it. Reduction of HMG-CR activity by simvastatin, a statin that acts as an HMG-CR inhibitor, or by siRNA-mediated HMG-CR knockdown enhanced doxorubicin cytotoxicity. Doxorubicin-induced HMG-CR downregulation was associated with inactivation of the EGFR-Src pathway. Furthermore, a high-cholesterol-diet attenuated the anti-cancer activity of doxorubicin in a tumor xenograft mouse model. In a multivulva model of Caenorhabditis elegans expressing an active-EGFR mutant, doxorubicin decreased hyperplasia more efficiently in the absence than in the presence of cholesterol. These data indicate that EGFR/Src/HMG-CR is a new pathway mediating doxorubicin-induced cell death and that cholesterol control could be combined with doxorubicin treatment to enhance efficacy and thus reduce side effects.

WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA.

Quinacrine-Mediated Inhibition of Nrf2 Reverses Hypoxia-Induced 5-Fluorouracil Resistance in Colorectal Cancer.

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for the systemic treatment of colorectal cancer (CRC), but its effectiveness against CRC is limited by increased 5-FU resistance caused by the hypoxic tumor microenvironment. The purpose of our study was to assess the feasibility of using quinacrine (QC) to increase the efficacy of 5-FU against CRC cells under hypoxic conditions. QC reversed the resistance to 5-FU induced by hypoxia in CRC cell lines, as determined using ATP-Glo cell viability assays and clonogenic survival assays. Treatment of cells with 5-FU under hypoxic conditions had no effect on the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a regulator of cellular resistance to oxidative stress, whereas treatment with QC alone or in combination with 5-FU reduced Nrf2 expression in all CRC cell lines tested. Overexpression of Nrf2 effectively prevented the increase in the number of DNA double-strand breaks induced by QC alone or in combination with 5-FU. siRNA-mediated c-Jun N-terminal kinase-1 (JNK1) knockdown inhibited the QC-mediated Nrf2 degradation in CRC cells under hypoxic conditions. The treatment of CRC xenografts in mice with the combination of QC and 5-FU was more effective in suppressing tumor growth than QC or 5-FU alone. QC increases the susceptibility of CRC cells to 5-FU under hypoxic conditions by enhancing JNK1-dependent Nrf2 degradation.

Allosteric inhibition site of transglutaminase 2 is unveiled in the N terminus.

Previously we have demonstrated transglutaminase 2 (TGase 2) inhibition abrogated renal cell carcinoma (RCC) using GK921 (3-(phenylethynyl)-2-(2-(pyridin-2-yl)ethoxy)pyrido[3,2-b]pyrazine), although the mechanism of TGase 2 inhibition remains unsolved. Recently, we found that the increase of TGase 2 expression is required for p53 depletion in RCC by transporting the TGase 2 (1-139 a.a)-p53 complex to the autophagosome, through TGase 2 (472-687 a.a) binding p62. In this study, mass analysis revealed that GK921 bound to the N terminus of TGase 2 (81-116 a.a), which stabilized p53 by blocking TGase 2 binding. This suggests that RCC survival can be stopped by p53-induced cell death through blocking the p53-TGase 2 complex formation using GK921. Although GK921 does not bind to the active site of TGase 2, GK921 binding to the N terminus of TGase 2 also inactivated TGase 2 activity through acceleration of non-covalent self-polymerization of TGase 2 via conformational change. This suggests that TGase 2 has an allosteric binding site (81-116 a.a) which changes the conformation of TGase 2 enough to accelerate inactivation through self-polymer formation.

MG53-IRS-1 (Mitsugumin 53-Insulin Receptor Substrate-1) Interaction Disruptor Sensitizes Insulin Signaling in Skeletal Muscle.

Mitsugumin 53 (MG53) is an E3 ligase that interacts with and ubiquitinates insulin receptor substrate-1 (IRS-1) in skeletal muscle; thus, an MG53-IRS-1 interaction disruptor (MID), which potentially sensitizes insulin signaling with an elevated level of IRS-1 in skeletal muscle, is an excellent candidate for treating insulin resistance. To screen for an MID, we developed a bimolecular luminescence complementation system using an N-terminal luciferase fragment fused with IRS-1 and a C-terminal luciferase fragment fused with an MG53 C14A mutant that binds to IRS-1 but does not have E3 ligase activity. An MID, which was discovered using the bimolecular luminescence complementation system, disrupted the molecular association of MG53 with IRS-1, thus abolishing MG53-mediated IRS-1 ubiquitination and degradation. Thus, the MID sensitized insulin signaling and increased insulin-elicited glucose uptake with an elevated level of IRS-1 in C2C12 myotubes. These data indicate that this MID holds promise as a drug candidate for treating insulin resistance.

FOXO1 reduces tumorsphere formation capacity and has crosstalk with LGR5 signaling in gastric cancer cells.

Gastric cancer (GC) is a major of cause of cancer-related death and is characterized by its heterogeneity and molecular complexity. FOXO1 is a transcription factor that plays a key role in GC growth and metastasis. However, the implication of FOXO1 in GC cell stemness has been elusive. This study, for the first time, demonstrates that FOXO1 regulates GC cell stemness in association with LGR5. FOXO1 expression was significantly lower in GC tumorsphere cells than in adherent GC cells. FOXO1 silencing and overexpression promoted and inhibited the tumorsphere formation capacity of GC cells, respectively. Additionally, there was an inverse correlation between FOXO1 and GC stem cell marker LGR5 in human GC specimens. Further in vitro and in vivo experiments showed that negative crosstalk between these two molecules exists and that LGR5 silencing reversed the FOXO1 shRNA-induced tumorsphere formation even without FOXO1 restoration. Taken together, our results suggest that FOXO1 inhibits the self-renewal capacity of GC cells through interaction with LGR5. Thus, FOXO1/LGR5 signaling pathway may provide a novel targeted therapy for GC.

A novel role for flotillin-1 in H-Ras-regulated breast cancer aggressiveness.

Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p < 0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer.

Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment.

Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence.

Glutaminase 1 inhibition reduces thymidine synthesis in NSCLC.

We found that non-small cell lung cancer (NSCLC) is remarkably sensitive to the regulation of glutamine supply by testing the metabolic dependency of 11 cancer cell lines against regulation of glycolysis, autophagy, fatty acid synthesis, and glutamine supply. Glutamine is known as a key supplement of cancer cell growth that is converted to α-ketoglutarate for anabolic biogenesis via glutamate by glutaminase 1 (GLS1). GLS1 inhibition using 10 µM of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) showed about 50% cell growth arrest by SRB assay. By testing the synergistic effects of conventional therapeutics, BPTES combined with 5-fluorouracil (5-FU), an irreversible inhibitor of thymidylate synthase, significant effects were observed on cell growth arrest in NSCLC. We found that GLS1 inhibition using BPTES reduced metabolic intermediates including thymidine and carbamoyl phosphate. Reduction of thymidine and carbamoyl-phosphate synthesis by BPTES treatment exacerbated pyrimidine supply by combination with 5-FU, which induced cell death synergistically in NSCLC.

Identification of a mitochondrial defect gene signature reveals NUPR1 as a key regulator of liver cancer progression.

UNLABELLED: Many cancer cells require more glycolytic adenosine triphosphate production due to a mitochondrial respiratory defect. However, the roles of mitochondrial defects in cancer development and progression remain unclear. To address the role of transcriptomic regulation by mitochondrial defects in liver cancer cells, we performed gene expression profiling for three different cell models of mitochondrial defects: cells with chemical respiratory inhibition (rotenone, thenoyltrifluoroacetone, antimycin A, and oligomycin), cells with mitochondrial DNA depletion (Rho0), and liver cancer cells harboring mitochondrial defects (SNU354 and SNU423). By comparing gene expression in the three models, we identified 10 common mitochondrial defect-related genes that may be responsible for retrograde signaling from cancer cell mitochondria to the intracellular transcriptome. The concomitant expression of the 10 common mitochondrial defect genes is significantly associated with poor prognostic outcomes in liver cancers, suggesting their functional and clinical relevance. Among the common mitochondrial defect genes, we found that nuclear protein 1 (NUPR1) is one of the key transcription regulators. Knockdown of NUPR1 suppressed liver cancer cell invasion, which was mediated in a Ca(2+) signaling-dependent manner. In addition, by performing an NUPR1-centric network analysis and promoter binding assay, granulin was identified as a key downstream effector of NUPR1. We also report association of the NUPR1-granulin pathway with mitochondrial defect-derived glycolytic activation in human liver cancer. CONCLUSION: Mitochondrial respiratory defects and subsequent retrograde signaling, particularly the NUPR1-granulin pathway, play pivotal roles in liver cancer progression.

Mitsugumin 53 (MG53) ligase ubiquitinates focal adhesion kinase during skeletal myogenesis.

The striated muscle-specific mitsugumin 53 (MG53) is a novel E3 ligase that induces the ubiquitination of insulin receptor substrate 1 (IRS-1) during skeletal myogenesis, negatively regulating insulin-like growth factor and insulin signaling. Here we show that focal adhesion kinase (FAK) is the second target of MG53 during skeletal myogenesis. The FAK protein level gradually decreased, whereas its mRNA level was constant during myogenesis in C2C12 cells and MyoD-overexpressing mouse embryonic fibroblasts. The FAK protein was associated with the E2 enzyme UBE2H and the E3 enzyme MG53 in endogenous and exogenous immunoprecipitation experiments. FAK ubiquitination and degradation was induced by MG53 overexpression in myoblasts but abolished by MG53 or UBE2H knockdown in myotubes. Because RING-disrupted MG53 mutants (C14A and ΔR) did not induce FAK ubiquitination and degradation, the RING domain was determined to be required for MG53-induced FAK ubiquitination. Taken together, these data indicate that MG53 induces FAK ubiquitination with the aid of UBE2H during skeletal myogenesis.

Mitochondrial complex I deficiency enhances skeletal myogenesis but impairs insulin signaling through SIRT1 inactivation.

To address whether mitochondrial biogenesis is essential for skeletal myogenesis, C2C12 myogenesis was investigated after knockdown of NADH dehydrogenase (ubiquintone) flavoprotein 1 (NDUFV1), which is an oxidative phosphorylation complex I subunit that is the first subunit to accept electrons from NADH. The NDUFVI knockdown enhanced C2C12 myogenesis by decreasing the NAD(+)/NADH ratio and subsequently inactivating SIRT1 and SIRT1 activators (pyruvate, SRT1720, and resveratrol) abolished the NDUFV1 knockdown-induced myogenesis enhancement. However, the insulin-elicited activation of insulin receptor ß (IRß) and insulin receptor substrate-1 (IRS-1) was reduced with elevated levels of protein-tyrosine phosphatase 1B after NDUFV1 knockdown in C2C12 myotubes. The NDUFV1 knockdown-induced blockage of insulin signaling was released by protein-tyrosine phosphatase 1B knockdown in C2C12 myotubes, and we found that NDUFV1 or SIRT1 knockdown did not affect mitochondria biogenesis during C2C12 myogenesis. Based on these data, we can conclude that complex I dysfunction-induced SIRT1 inactivation leads to myogenesis enhancement but blocks insulin signaling without affecting mitochondria biogenesis.