E3 Ubiquitin Ligase Smurf1 Research Articles

TWEAK causes myotube atrophy through coordinated activation of ubiquitin‐proteasome system, autophagy, and caspases

Proinflammatory cytokine TWEAK has now emerged as a key mediator of skeletal muscle-wasting in many catabolic conditions. However, the mechanisms by which TWEAK induces muscle proteolysis remain poorly understood. Here, we have investigated the role of ubiquitin-proteasome system, autophagy, and caspases in TWEAK-induced muscle wasting. Addition of TWEAK to C2C12 myotubes stimulated the ubiquitination of myosin heavy chain (MyHC) and augmented the expression of E3 ubiquitin ligase MuRF1. Pretreatment of myotubes with proteasome inhibitors MG132 or lactacystin or knockdown of MuRF1 by RNAi blocked the TWEAK-induced degradation of MyHC and myotube atrophy. TWEAK increased the expression of several autophagy-related molecules. Moreover, the inhibitors of autophagy improved the levels of MyHC in TWEAK-treated myotubes. TWEAK also increased activity of caspases in C2C12 myotubes. Pan-caspase or caspase 3 inhibitory peptide inhibited the TWEAK-induced loss of MyHC and myotube diameter. Our study demonstrates that nuclear factor-kappa B (NF-κB) transcription factor is essential for TWEAK-induced expression of MuRF1 and Beclin1. Furthermore, our results suggest that caspases contribute, at least in part, to the activation of NF-κB in response to TWEAK treatment. Collectively, the present study provides novel insight into the mechanisms of action of TWEAK in skeletal muscle.

Rescue of tropomyosin deficiency inDrosophilaand human cancer cells by synaptopodin reveals a role of tropomyosin α in RhoA stabilization

Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMβ causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.

Ubiquitination in Rho Signaling

The Rho family small GTPases of the Ras superfamily play key roles in regulating diverse signaling pathways that control a myriad of fundamental cellular processes such as cytoskeletal dynamics, cell cycle progression, gene expression, cell polarity, migration and cell transformation. The Rho GTPases cycle between an active GTP-bound and an inactive GDP-bound form, which is controlled by many regulators including GEFs, GAPs and GDIs. Recent studies have revealed a new layer of regulation for Rho GTPases, indicating that several members of the Rho family of small GTPases including RhoA, Rac1, and RhoBTB, as well as the Ras family member Rap1B, are also regulated by the ubiquitin-proteasome pathway, which plays important roles in controlling cell polarity, migration, cell transformation and actin dynamics. Importantly, regulators for Rho GTP-GDP cycling such as RhoGDI and Rho-GEF ECT2 were also found to be modulated by the ubiquitin pathway. In this review, we focus on how ubiquitin signaling guides the fate and function of Rho GTPases and their regulators, especially how the E3 ubiquitin ligase Smurf1 regulates cell polarity and motility through targeting RhoA for ubiquitination and degradation.

The SCF-Fbxo40 Complex Induces IRS1 Ubiquitination in Skeletal Muscle, Limiting IGF1 Signaling

Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/IRS1/PI3K/Akt pathway. However the effect of IGF1 in differentiated muscle is limited by IRS1 ubiquitination and proteasome-mediated breakdown. In skeletal muscle, IGF1R activation sensitizes IRS1 to degradation, and a screen for the responsible E3 ligase identified Fbxo40 as mediating this rapid turnover of IRS1, since IRS1 loss can be rescued by knockdown of Fbxo40. In biochemical assays, an SCF E3 ligase complex containing Fbxo40 directly ubiquitinates IRS1, and this activity is enhanced by increased tyrosine phosphorylation of IRS1. Fbxo40 is muscle specific in expression and is upregulated during differentiation. Knockdown of Fbxo40 induces dramatic hypertrophy of myofibers. Mice null for Fbxo40 have increased levels of IRS1 and demonstrate enhanced body and muscle size during the growth phase associated with elevated IGF1 levels. These findings establish an important means of restraining IGF1's effects on skeletal muscle.

Muscle actin is polyubiquitinylated in vitro and in vivo and targeted for breakdown by the E3 ligase MuRF1

Muscle atrophy prevails in numerous diseases (cancer cachexia, renal failure, infections, etc.), mainly results from elevated proteolysis, and is accelerated by bed rest. This largely contributes to increased health costs. Devising new strategies to prevent muscle wasting is a major clinical challenge. The ubiquitin proteasome system (UPS) degrades myofibrillar proteins, but the precise mechanisms responsible for actin breakdown are surprisingly poorly characterized. We report that chimeric flag-actin was destabilized and polyubiquitinylated in stably transfected C2C12 myotubes treated with the catabolic agent dexamethasone (1 μM) and that only proteasome inhibitors blocked its breakdown. Actin polyubiquitinylation was also detected in wild-type C2C12 myotubes and human muscle biopsies from control participants and patients with cancer. The muscle-specific E3 ubiquitin ligase MuRF1 is up-regulated in catabolic conditions and polyubiquitinylates components of the thick filament. We also demonstrate that recombinant GST-MuRF1 physically interacted and polyubiquitinylated actin in vitro and that MuRF1 is a critical component for actin breakdown, since MuRF1 siRNA stabilized flag-actin. These data identify unambiguously the abundant contractile protein actin as a target of the UPS in skeletal muscle both in vitro and in vivo, further supporting the need for new strategies blocking specifically the activation of this pathway in muscle wasting conditions.

Abstract 1982: TGF-beta induced SIK cooperates with Smurf2 to negatively regulate type I receptor kinase signaling

Abstract Signal transduction pathways, like transforming growth factor beta (TGFβ), regulate many physiological processes and require control mechanisms to safeguard proper and timely action. Dysregulation of TGFβ signaling is established as a major player in cancer progression. We have recently described how negative regulation of TGFβ signaling is controlled by the serine/threonine kinase salt-inducible kinase (SIK). SIK is induced by TGFβ and forms protein complexes with the TGFβ type I receptor and with the inhibitory Smad7, which leads to type I receptor downregulation. We now investigate this mechanism further and demonstrate similarities between TGFβ/Smad-mediated immediate-early induction of SIK and Smad7. We also relate another protein of the negative feedback loop of TGFβ signaling, the E3 ubiquitin ligase Smurf2, to this mechanism of regulation. We provide evidence that Smurf2 cooperates with SIK to downregulate the type I receptor. Both the kinase activity of SIK and the ubiquitin ligase activity of Smurf2 are important for proper receptor turnover. However, Smurf2 does not directly bind to SIK, but participates in the complex via the PY-motif of Smad7. We thus establish a central role for Smad7 during SIK-mediated receptor downregulation. Finally, we explore the role of SIK in a cancer context by investigating its expression profile in human prostate cancer tissues. In conclusion, we have uncovered a phosphorylation/ubiquitination-dependent mechanism for TGFβ receptor downregulation that could play important roles during cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1982. doi:10.1158/1538-7445.AM2011-1982

Crosstalk between Glucocorticoid Receptor and Nutritional Sensor mTOR in Skeletal Muscle

Maintenance of skeletal muscle mass relies on the dynamic balance between anabolic and catabolic processes and is important for motility, systemic energy homeostasis, and viability. We identified direct target genes of the glucocorticoid receptor (GR) in skeletal muscle, i.e., REDD1 and KLF15. As well as REDD1, KLF15 inhibits mTOR activity, but via a distinct mechanism involving BCAT2 gene activation. Moreover, KLF15 upregulates the expression of the E3 ubiquitin ligases atrogin-1 and MuRF1 genes and negatively modulates myofiber size. Thus, GR is a liaison involving a variety of downstream molecular cascades toward muscle atrophy. Notably, mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids. This mutually exclusive crosstalk between GR and mTOR, a highly coordinated interaction between the catabolic hormone signal and the anabolic machinery, may be a rational mechanism for fine-tuning of muscle volume and a potential therapeutic target for muscle wasting.

Phosphorylation of E3 Ligase Smurf1 Switches Its Substrate Preference in Support of Axon Development

Ubiquitin E3 ligases serve for ubiquitination of specific substrates, and its ligase efficacy is regulated by interacting proteins or substrate modifications. Whether and how the ligases themselves are modified by cellular signaling is unclear. Here we report that protein kinase A (PKA)-dependent phosphorylation of Smad Ubiquitin Regulatory Factor 1 (Smurf1) can switch its substrate preference between two proteins of opposing actions on axon development. Extracellular factors that promote axon formation elevated Smurf1 phosphorylation at a PKA site Thr³⁰⁶, and preventing this phosphorylation reduced axon formation in cultured hippocampal neurons and impaired polarization of cortical neurons in vivo. Thr³⁰⁶-phosphorylation changed the relative affinities of Smurf1 for its substrates, leading to reduced degradation of polarity protein Par6 and increased degradation of growth-inhibiting RhoA. Thus, PKA-dependent phosphorylation of the E3 ligase could switch its substrate preference, contributing to selective protein degradation required for localized cellular function.

Transcription alterations of members of the ubiquitin–proteasome network in prostate carcinoma

The purpose of this work was to investigate the role of the ubiquitin-proteasome network (UPN) in prostate cancer (PCA) and to elicit potential markers for this disease. The UPN represents a key factor in the maintenance of cellular homoeostasis as a result of its fundamental function in the regulation of intracellular protein degradation. Members of this network have a role in the biology of haematological and solid tumours. Tumour cells and normal epithelial cells from 22 prostatectomy specimens were isolated by laser microdissection. Prostate biopsy samples from healthy individuals served for technical calibration and as controls. Transcript levels of eight selected genes with E3 ubiquitin ligase activity (labelling target proteins for proteasome degradation) and two genes belonging to the proteasome-multienzyme complex itself were analysed by quantitative real-time RT-PCR. The proteasome genes PSMC4 and PSMB5 and the E3 ubiquitin ligase NEDD4L were significantly and coherently upregulated in PCA cells compared with the corresponding adjacent normal prostate tissue. Transcription of the E3 ubiquitin ligase SMURF2 was significantly higher in organ-confined tumours (pT2) compared with non-organ-confined cancers (pT3). The results indicate a role for PSMC4 and PSMB5 and the E3 ubiquitin ligase NEDD4L in prostate tumourigenesis, whereas SMURF2 downregulation could be associated with clinical progression. NEDD4L and SMURF2 both target transforming growth factor (TGF)-β for degradation. This reflects the pleiotropic role of the TGF-β signalling pathway acting as a tumour suppressor in normal and pre-cancerous cells, but having oncogenic properties in progressing cancer. Further studies have to elucidate whether these alterations could represent clinically relevant PCA-diagnostic and progression markers.

Myogenin and Class II HDACs Control Neurogenic Muscle Atrophy by Inducing E3 Ubiquitin Ligases

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.

Smurf control in bone cells

The homologous to the E6-associated protein carboxyl terminus (HECT) domain E3 ubiquitin ligase Smurf1 is the first E3 ligase to be implicated in regulating bone cell function. The involvement of Smurf1 in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review highlights recent works exploring Smurf-regulated biological processes in bone cells and highlights recent discoveries surrounding the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Moreover, we discuss the relevance of targeting the HECT E3s through the development of small-molecule inhibitors as an anticancer therapeutic strategy.

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

Skeletal muscle atrophy occurs in a variety of clinical settings, including cachexia, disuse, and denervation. Inflammatory cytokines have been shown to be mediators of cancer cachexia; however, the role of cytokines in denervation- and immobilization-induced skeletal muscle loss remains unknown. In this study, we demonstrate that a single cytokine, TNF-like weak inducer of apoptosis (TWEAK), mediates skeletal muscle atrophy that occurs under denervation conditions. Transgenic expression of TWEAK induces atrophy, fibrosis, fiber-type switching, and the degradation of muscle proteins. Importantly, genetic ablation of TWEAK decreases the loss of muscle proteins and spared fiber cross-sectional area, muscle mass, and strength after denervation. Expression of the TWEAK receptor Fn14 (fibroblast growth factor-inducible receptor 14) and not the cytokine is significantly increased in muscle upon denervation, demonstrating an unexpected inside-out signaling pathway; the receptor up-regulation allows for TWEAK activation of nuclear factor kappaB, causing an increase in the expression of the E3 ubiquitin ligase MuRF1. This study reveals a novel mediator of skeletal muscle atrophy and indicates that the TWEAK-Fn14 system is an important target for preventing skeletal muscle wasting.

Abstract 5662: Reductions in Atrophic E3 Ubiquitin Ligase Expression in Pulmonary Arteries Correlate With the Development of Pulmonary Hypertension

Vascular remodeling in pulmonary hypertensive syndromes is driven by increased activity of nuclear factor of activated T-cells (NFAT) in smooth muscle cells. Although much is known about the regulation of NFAT activity by calcineurin, very little is known about vascular smooth muscle atrophy F-box ubiquitin E3 ligase expression, which targets calcineurin for proteosomal degradation and inhibits pathological hypertrophy. Therefore, we hypothesized the ubiquitin E3 ligases muscle atrophy F-box (MAFbx) and muscle ring finger (MuRF-1) expression would decline during the development of pulmonary hypertension. To investigate this hypothesis, rats were given a single injection of MCT (50 mg/kg i.p.) and indices of pulmonary hypertension (RVSP and RV/LVS) were measured at 0, 7, 14, 28 and 42 days post MCT injection (n=7–10/group). Left and right pulmonary arteries were harvested from each rat at the conclusion of these measures and mRNA expression was assessed by real-time multiplex PCR analysis for MAFbx and MuRF-1. Consistent with our hypothesis, pulmonary vascular smooth muscle MAFbx and MuRF-1 expression were attenuated from 14d to 42d time points compared to 0d when normalized to b-actin mRNA expression, which correlated negatively with RVSP and right ventricular hypertrophy. Interestingly, while treatment with sildenafil from days 28 – 42 reduced RVSP and RV/LVS, MAFbx expression was further compromised. These results suggest that the E3 ubiquitin ligases MAFbx and MuRF-1 may play a role in the development of MCT-induced pulmonary hypertension.

Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-beta (TGF-beta) promotes cancer progression and fibrosis. Here we show that expression of the TGF-beta receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-beta and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-beta signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. We find that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle alpha-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. We therefore examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation.

Numb regulates cell–cell adhesion and polarity in response to tyrosine kinase signalling

Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell–cell junction. Moreover, Numb engages the polarity complex Par3–aPKC–Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC–Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and β-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell–cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell–cell adhesion and a sensor of HGF signalling or Src activity during EMT.

Talin phosphorylation by Cdk5 regulates Smurf1-mediated talin head ubiquitylation and cell migration

Cell migration is a dynamic process that requires temporal and spatial regulation of integrin activation and focal adhesion assembly-disassembly1. Talin, an actin and β integrin tail-binding protein, is essential for integrin activation and focal adhesion formation2,3. Calpain-mediated cleavage of talin plays a key role in focal adhesion turnover3; however, the talin head (TH) domain, one of the two cleavage products, stimulates integrin activation, localizes to focal adhesions, and maintains cell edge protrusions2,4,5, suggesting that additional steps, downstream of talin proteolysis, are required for focal adhesion disassembly. Here we show that TH binds Smurf1, an E3 ubiquitin ligase involved in cell polarity and migration6,7, more tightly than full length talin and that this interaction leads to TH ubiquitination and degradation. TH was a substrate for Cdk5, a regulator of cell migration and cancer metastasis8–11. Cdk5 phosphorylated TH at Ser425, inhibiting its binding to Smurf1, thus preventing TH ubiquitination and degradation. Expression of talS425A, which resists Cdk5 phosphorylation thereby increasing its susceptibility to Smurf1-mediated ubiqitination, resulted in extensive focal adhesion turnover and inhibited cell migration. Thus, TH produced by calpain cleavage of talin, is degraded via Smurf1-mediated ubiquitination; moreover, phosphorylation by Cdk5 regulates Smurf1 binding to TH and, in this way, controls TH turnover and adhesion stability and, ultimately, cell migration.

Ghrelin inhibits skeletal muscle protein breakdown in rats with thermal injury through normalizing elevated expression of E3 ubiquitin ligases MuRF1 and MAFbx

We previously determined that ghrelin synthesis was downregulated after burn injury and that exogenous ghrelin retained its ability both to stimulate food intake and to restore plasma growth hormone levels in burned rats. These observations and the finding that anabolic hormones can attenuate skeletal muscle catabolism led us to investigate whether ghrelin could attenuate burn-induced skeletal muscle protein breakdown in rats. These studies were performed in young rats (50-60 g) 24 h after approximately 30% total body surface area burn injury. Burn injury increased total and myofibrillar protein breakdown in extensor digitorum longus (EDL) muscles assessed by in vitro tyrosine and 3-methyl-histidine release, respectively. Continuous 24-h administration of ghrelin (0.2 mg.kg(-1).h(-1)) significantly inhibited both total and myofibrillar protein breakdown in burned rats. Ghrelin significantly attenuated burn-induced changes in mRNA expression of IGFBP-1 and IGFBP-3 in liver. In EDL, ghrelin attenuated the increases in mRNA expression of the binding proteins, but had no significant effect on reduced expression of IGF-I. Ghrelin markedly reduced the elevated mRNA expression of TNF-alpha and IL-6 in EDL muscle that occurred after burn. Moreover, ghrelin normalized plasma glucocorticoid levels, which were elevated after burn. Expression of the muscle-specific ubiquitin-ligating enzyme (E3) ubiquitin ligases MuRF1 and MAFbx were markedly elevated in both EDL and gastrocnemius and were normalized by ghrelin. These results suggest that ghrelin is a powerful anticatabolic compound that reduces skeletal muscle protein breakdown through attenuating multiple burn-induced abnormalities.

Abstract 3789: Induction of Murf-1 is Required for Tnf-alpha Induced Loss of Skeletal Muscle Function

Background: Inflammatory cytokines like tumor necrosis factor alpha (TNF-α) are known to impair skeletal muscle (SM) function. Furthermore, TNF-α induces the expression of atrogin-like muscle specific ubiquitin E3-ligases, presumed to mediate muscle atrophy. The relative contributions of respective ubiquitin ligases, like MuRF1 for the TNF-α induced reduction in muscle function are not known. Methods: TNF-α or saline was injected either into C57Bl6 or MuRF1 −/− mice. After 16 –24h the expression of MuRF1 in the SM was quantified by qRT-PCR and western blot. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, 2D-gel electrophoresis was performed. Results: WT animals injected with TNF-α had higher MuRF1 mRNA (saline: 56.6±12.1 vs. TNF-α: 133.6±30.3 arb. Units; p<0.05) and protein expression (saline: 0.38±0.11 vs. TNF-α: 1.07±0.25 arb. Units; p<0.05) as compared to saline injected littermates. However, TNF- α was unable to induce MurRF1 expression in MuRF1 −/− mice. Furthermore, TNF- α reduced force development at 150Hz by 25% in C57Bl6 animals (saline: 2412±120 vs. TNF-α: 1799±114 g/cm2; p<0.05), but not in MuRF1 −/− mice (saline: 2424±198 vs. TNF-α: 2431±180 g/cm2; p=NS). The proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T (TNNT3) in WT animals treated with TNF- α as compared to MuRF1 −/− mice receiving TNF-α . In addition, TNF-α injection into C57Bl6 animals resulted in a down-regulation of eEF1γ ( WT: 0.60±0.02 vs. WT+TNF-α: 0.39±0.05 arb. Units; p<0.05). This reduction was not seen in MuRF1 −/− mice receiving TNF- α (KO: 0.59±0.03 vs. KO+TNF-α: 0.68±0.01 arb. Units; p<0.05) Conclusion: The results of this study demonstrate for the first time, that the TNF- α induced reduction in SM force development depends on the induction of the atrophy related E3-ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-α-MuRF1-mediated down-regulation of TNNT3 and the elongation factor eEF1γ.

Induction of MuRF1 Is Essential for TNF-α-Induced Loss of Muscle Function in Mice

BackgroundHumoral circulating inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) can impair skeletal muscle contractility. Furthermore, TNF-α expression correlates with elevated levels of atrogin-like muscle-specific ubiquitin E3 ligases, which are presumed to mediate muscle protein breakdown and atrophy. However, the casual relationships between MuRF1 and TNF-α and their relative contributions to muscle function impairment are not known. MethodsTNF-α or saline was injected into either C57Bl6 or MuRF1−/− mice. After 16–24 h, the expression of MuRF1 in skeletal muscle was quantified by quantitative reverse transcription–PCR and Western blot analysis. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, two-dimensional gel electrophoresis was performed. ResultsWild-type animals injected with TNF-α had higher MuRF1 mRNA expression (saline versus TNF-α: 56.6±12.1 versus 133.6±30.3 arbitrary units; p<0.05) and protein expression (saline versus TNF-α: 0.38±0.11 versus 1.07±0.25 arbitrary units; p<0.05) as compared to saline-injected littermates. Furthermore, TNF-α reduced force development at 150 Hz by 25% in C57Bl6 animals (saline versus TNF-α: 2412±120 versus 1799±114 g/cm2; p<0.05), but not in MuRF1−/− mice (saline versus TNF-α: 2424±198 versus 2431±180 g/cm2; p=NS). Proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T in wild-type animals treated with TNF-α as compared to MuRF1−/− mice that received TNF-α. ConclusionThe results of this study demonstrate for the first time that TNF-α-induced reduction in skeletal muscle force development depends on the induction of the atrophy-related E3 ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-α/MuRF1-mediated down-regulation of fast skeletal muscle troponin T.

Smurf2 is a TRAF2 binding protein that triggers TNF-R2 ubiquitination and TNF-R2-induced JNK activation

TRAF2 plays a central role in TNF-induced signalling to NF-κB and JNK/p38 MAPK. To better understand the molecular mechanisms that mediate this dual function of TRAF2, we performed a yeast two-hybrid screening for TRAF2 interacting proteins using the Sos recruitment system. This resulted in the identification of the E3 ubiquitin ligase Smurf2 as a TRAF2 binding protein. TRAF2 overexpression was shown to trigger Smurf2 ubiquitination and the formation of a TNF-R2/Smurf2 complex. Smurf2 on its turn promoted TNF-R2 ubiquitination and the relocalization of TNF-R2 as well as TRAF2 to a detergent-insoluble cell fraction. This was associated with enhanced TNF-R2-induced JNK activation, whereas TNF-R2-induced NF-κB activation remained unaffected. These results suggest an important role for Smurf2 binding to TRAF2 in determining specific signalling outputs of TNF-R2.