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TWEAK in Inclusion-Body Myositis Muscle

Possible Pathogenic Role of a Cytokine Inhibiting Myogenesis
Published:February 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.027
      Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 exert pleiotropic effects, including regulation of myogenesis. Sporadic inclusion-body myositis (IBM) is the most common muscle disease of the elderly population and leads to severe disability. IBM mesoangioblasts, different from mesoangioblasts in other inflammatory myopathies, display a myogenic differentiation defect. The objective of the present study was to investigate TWEAK-Fn14 expression in IBM and other inflammatory myopathies and explore whether TWEAK modulation affects myogenesis in IBM mesoangioblasts. TWEAK, Fn14, and NF-κB expression was assessed by immunohistochemistry and Western blot in cell samples from both muscle biopsies and primary cultures. Mesoangioblasts isolated from samples of IBM, dermatomyositis, polymyositis, and control muscles were treated with recombinant human TWEAK, Fn14-Fc chimera, and anti-TWEAK antibody. TWEAK-RNA interference was performed in IBM and dermatomyositis mesoangioblasts. TWEAK levels in culture media were determined by enzyme-linked immunosorbent assay. In IBM muscle, we found increased TWEAK-Fn14 expression. Increased levels of TWEAK were found in differentiation medium from IBM mesoangioblasts. Moreover, TWEAK inhibited myogenic differentiation of mesoangioblasts. Consistent with this evidence, TWEAK inhibition by Fn14-Fc chimera or short interfering RNA induced myogenic differentiation of IBM mesoangioblasts. We provide evidence that TWEAK is a negative regulator of human mesoangioblast differentiation. Dysregulation of the TWEAK-Fn14 axis in IBM muscle may induce progressive muscle atrophy and reduce activation and differentiation of muscle precursor cells.
      Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional proinflammatory cytokine belonging to the tumor necrosis factor (TNF) superfamily that exerts pleiotropic effects on several cellular activities, including proliferation, differentiation, migration, angiogenesis, apoptosis, and inflammation.
      • Chicheportiche Y.
      • Bourdon P.R.
      • Xu H.
      • Hsu Y.M.
      • Scott H.
      • Hession C.
      • Garcia I.
      • Browning J.L.
      TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.
      • Winkles J.A.
      The TWEAK-Fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting.
      TWEAK is initially expressed as a type II transmembrane protein that is proteolytically processed into a soluble biologically active cytokine.
      • Winkles J.A.
      The TWEAK-Fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting.
      In addition to the cleaved soluble molecule, also the membrane-anchored TWEAK isoform has been shown to be functionally active in a juxtacrine manner by binding to the fibroblast growth factor-inducible gene 14 receptor [Fn14 or TWEAK receptor (TWEAKR)] on neighboring cells.
      • Marsters S.A.
      • Sheridan J.P.
      • Pitti R.M.
      • Brush J.
      • Goddard A.
      • Ashkenazi A.
      Identification of a ligand for the death-domain-containing receptor Apo3.
      • Wiley S.R.
      • Winkles J.A.
      TWEAK, a member of the TNF superfamily, is a multifunctional cytokine that binds the TWEAKR/Fn14 receptor.
      • Brown S.A.
      • Ghosh A.
      • Winkles J.A.
      Full-length, membrane-anchored TWEAK can function as a juxtacrine signalling molecule and activate the NF-kappaB pathway.
      Fn14 is a highly inducible small cell-surface receptor linked to several intracellular signaling pathways, including the nuclear factor-kappa B (NFκB) pathway, and it is widely expressed on a variety of tissues, including the cardiac and skeletal muscle.
      • Chicheportiche Y.
      • Bourdon P.R.
      • Xu H.
      • Hsu Y.M.
      • Scott H.
      • Hession C.
      • Garcia I.
      • Browning J.L.
      TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.
      • Marsters S.A.
      • Sheridan J.P.
      • Pitti R.M.
      • Brush J.
      • Goddard A.
      • Ashkenazi A.
      Identification of a ligand for the death-domain-containing receptor Apo3.
      • Meighan-Mantha R.L.
      • Hsu D.K.
      • Guo Y.
      • Brown S.A.
      • Feng S.L.
      • Peifley K.A.
      • Alberts G.F.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Richards C.M.
      • Winkles J.A.
      The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.
      • Feng S.L.
      • Guo Y.
      • Factor V.M.
      • Thorgeirsson S.S.
      • Bell D.W.
      • Testa J.R.
      • Peifley K.A.
      • Winkles J.A.
      The Fn14 immediate-early response gene is induced during liver regeneration and highly expressed in both human and murine hepatocellular carcinomas.
      Fn14 is highly up-regulated in the context of tissue injury, regeneration,
      • Dogra C.
      • Changotra H.
      • Mohan S.
      • Kumar A.
      Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
      • Dogra C.
      • Changotra H.
      • Wedhas N.
      • Qin X.
      • Wergedal J.E.
      • Kumar A.
      TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine.
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      and inflammatory responses.
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      • Mittal A.
      • Bhatnagar S.
      • Kumar A.
      • Lach-Trifilieff E.
      • Wauters S.
      • Li H.
      • Makonchuk D.Y.
      • Glass D.J.
      • Kumar A.
      The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice.
      In fact, the TWEAK-Fn14 axis plays an important role in tissue repair following acute injury,
      • Burkly L.C.
      • Michaelson J.S.
      • Hahm K.
      • Jakubowski A.
      • Zheng T.S.
      TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease.
      and it has been implicated in the pathogenesis of several chronic inflammatory diseases and ischemic stroke.
      • Winkles J.A.
      The TWEAK-Fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting.
      • Burkly L.C.
      • Michaelson J.S.
      • Hahm K.
      • Jakubowski A.
      • Zheng T.S.
      TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease.
      Several cytokines produced after damage by muscle fibers and invading inflammatory cells, in addition to their role in mediating inflammatory responses, may take part in skeletal muscle repair and regeneration. In particular, it has been shown that soluble TWEAK protein augments the degradation of specific muscle proteins, such as MyoD and myosin heavy chain fast type in cultured muscle, and promotes myoblasts proliferation inhibiting their differentiation into myotubes.
      • Dogra C.
      • Changotra H.
      • Mohan S.
      • Kumar A.
      Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
      • Girgenrath M.
      • Weng S.
      • Kostek C.A.
      • Browning B.
      • Wang M.
      • Brown S.A.
      • Winkles J.A.
      • Michaelson J.S.
      • Allaire N.
      • Schneider P.
      • Scott M.L.
      • Hsu Y.M.
      • Yagita H.
      • Flavell R.A.
      • Miller J.B.
      • Burkly L.C.
      • Zheng T.S.
      TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.
      Thus, increase of TWEAK-Fn14 in the skeletal muscle appears sufficient to inhibit the regeneration and growth of skeletal muscle in vivo, identifying TWEAK as a novel regulator of adult regenerative myogenesis.
      • Dogra C.
      • Changotra H.
      • Wedhas N.
      • Qin X.
      • Wergedal J.E.
      • Kumar A.
      TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine.
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      • Girgenrath M.
      • Weng S.
      • Kostek C.A.
      • Browning B.
      • Wang M.
      • Brown S.A.
      • Winkles J.A.
      • Michaelson J.S.
      • Allaire N.
      • Schneider P.
      • Scott M.L.
      • Hsu Y.M.
      • Yagita H.
      • Flavell R.A.
      • Miller J.B.
      • Burkly L.C.
      • Zheng T.S.
      TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.
      Furthermore, as previously demonstrated,
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      • Mittal A.
      • Bhatnagar S.
      • Kumar A.
      • Lach-Trifilieff E.
      • Wauters S.
      • Li H.
      • Makonchuk D.Y.
      • Glass D.J.
      • Kumar A.
      The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice.
      elevated levels of TWEAK cause muscle atrophy in vivo and the TWEAK-Fn14 system have been shown to mediate muscle loss in disuse conditions in transgenic mice overexpressing the cytokine, by inducing degradation of muscle proteins through the activation of the ubiquitin-proteasome and NFκB pathways. Conversely, genetic ablation of TWEAK in mice has been proven to significantly ameliorate skeletal muscle regeneration after cardiotoxin-mediated injury, whereas overexpression of TWEAK inhibits myofibers regeneration and increases interstitial fibrosis.
      • Mittal A.
      • Bhatnagar S.
      • Kumar A.
      • Paul P.K.
      • Kuang S.
      • Kumar A.
      Genetic ablation of TWEAK augments regeneration and post-injury growth of skeletal muscle in mice.
      This mounting evidence supports the hypothesis that blocking TWEAK activity might help to improve skeletal muscle regeneration in chronic degenerative muscle disorders in which impairment of regeneration plays a pivotal role in muscle wasting.
      Sporadic inclusion-body myositis (IBM), the most common muscle disease of the elderly population, has a progressive course leading to pronounced muscle weakness and wasting that result in severe disability.
      • Engel W.K.
      • Askanas V.
      Inclusion-body myositis: clinical, diagnostic, and pathologic aspects.
      • Needham M.
      • Mastaglia F.L.
      Inclusion body myositis: current pathogenetic concepts and diagnostic and therapeutic approaches.
      The cause of the disease is unknown, its pathogenesis is complex and multifactorial, and there is no enduring treatment. Muscle biopsies of IBM patients show the coexistence of two pathological processes (vacuolar degeneration and atrophy of muscle fibers and mononuclear cell inflammation),
      • Askanas V.
      • Engel W.K.
      Sporadic inclusion-body myositis: conformational multifactorial ageing-related degenerative muscle disease associated with proteasomal and lysosomal inhibition, endoplasmic reticulum stress, and accumulation of amyloid-β42 oligomers and phosphorylated tau.
      • Dalakas M.C.
      Sporadic inclusion body myositis – diagnosis, pathogenesis and therapeutic strategies.
      • Askanas V.
      • Engel W.K.
      • Nogalska A.
      Inclusion body myositis: a degenerative muscle disease associated with intra-muscle fiber multi-protein aggregates, proteasome inhibition, endoplasmic reticulum stress and decreased lysosomal degradation.
      • Dalakas M.C.
      Immunotherapy of myositis: issues, concerns and future prospects.
      but how each one relates to the pathogenesis remains unsettled.
      Thus, a distinctive feature of IBM, different from other inflammatory myopathies, is the concomitant accumulation of multi-protein aggregates containing, among others, amyloid-β 42, phosphorylated tau, ubiquitin, and a number of oxidative or cell stress-related proteins associated with a chronic inflammatory response characterized by overexpression of proinflammatory mediators and major histocompatibility class I (MHC-I) antigen (reviewed in Askanas and Engel
      • Askanas V.
      • Engel W.K.
      Sporadic inclusion-body myositis: conformational multifactorial ageing-related degenerative muscle disease associated with proteasomal and lysosomal inhibition, endoplasmic reticulum stress, and accumulation of amyloid-β42 oligomers and phosphorylated tau.
      , Dalakas
      • Dalakas M.C.
      Sporadic inclusion body myositis – diagnosis, pathogenesis and therapeutic strategies.
      , Askanas and colleagues
      • Askanas V.
      • Engel W.K.
      • Nogalska A.
      Inclusion body myositis: a degenerative muscle disease associated with intra-muscle fiber multi-protein aggregates, proteasome inhibition, endoplasmic reticulum stress and decreased lysosomal degradation.
      , and Dalakas
      • Dalakas M.C.
      Immunotherapy of myositis: issues, concerns and future prospects.
      ). Furthermore, although usually restricted to certain muscle groups, muscle atrophy is a prominent early aspect, even in those patients with minimal clinical weakness. We hypothesized that a dual effect of persistent TWEAK stimulation related to chronic lymphomonocytic inflammation could be operative in inhibiting muscle regeneration and promoting atrophy in IBM muscle.
      We have previously shown that mesoangioblasts (pericyte-derived stem cells) from IBM muscles display a defect of differentiation down skeletal muscle, although they are able to differentiate into smooth muscle or osteoblasts, both in vitro and in vivo, in contrast to mesoangioblasts from other inflammatory myopathies and normal muscle.
      • Morosetti R.
      • Mirabella M.
      • Gliubizzi C.
      • Broccolini A.
      • De Angelis L.
      • Tagliafico E.
      • Sampaolesi M.
      • Gidaro T.
      • Papacci M.
      • Roncaglia E.
      • Rutella S.
      • Ferrari S.
      • Tonali P.A.
      • Ricci E.
      • Cossu G.
      MyoD expression restores defective myogenic differentiation of human mesangioblasts from inclusion-body myositis muscle.
      The capacity of these cells to differentiate into skeletal muscle can be restored by MyoD expression or by silencing a myogenic inhibitory helix–loop–helix factor, B3 gene. These data have led to the hypothesis that putative factors, such as a cytokine, expressed in the milieu of IBM muscle may inhibit mesoangioblasts activation and subsequent skeletal muscle differentiation, thus contributing to defective muscle regeneration and progressive atrophy. It is known that TNF-α inhibits differentiation of cultured skeletal muscle by down-regulation of MyoD after activation of NFκB, providing a further possible link between inflammation and muscle atrophy in IBM.
      • Coletti D.
      • Yang E.
      • Marazzi G.
      • Sassoon D.
      TNFα inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways.
      It has been previously shown, moreover, that NFκB is accumulated in IBM muscle fibers
      • Yang C.C.
      • Askanas V.
      • Engel W.K.
      • Alvarez R.B.
      Immunolocalization of transcription factor NF-kappa B in inclusion-body myositis muscle and at normal human neuromuscular junctions.
      and is also activated in IBM in association with increased myostatin, another important protein leading to muscle fiber atrophy, suggesting a role for NFκB in IBM pathogenesis.
      • Nogalska A.
      • Wojcik S.
      • Engel W.K.
      • McFerrin J.
      • Askanas V.
      Endoplasmic reticulum stress induces myostatin precursor protein and NF-kappaB in cultured human muscle fibers: relevance to inclusion body myositis.
      So far, the expression of TWEAK in human diseased muscles has not been investigated. Although all progenitor cells of mesenchymal lineage express the TWEAK receptor Fn14,
      • Girgenrath M.
      • Weng S.
      • Kostek C.A.
      • Browning B.
      • Wang M.
      • Brown S.A.
      • Winkles J.A.
      • Michaelson J.S.
      • Allaire N.
      • Schneider P.
      • Scott M.L.
      • Hsu Y.M.
      • Yagita H.
      • Flavell R.A.
      • Miller J.B.
      • Burkly L.C.
      • Zheng T.S.
      TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.
      the possible effect of TWEAK on mesoangioblasts differentiation is not known.
      Our study was aimed to investigate TWEAK-Fn14 expression in human muscle biopsies from patients affected by IBM and other inflammatory myopathies, and to explore whether the modulation of TWEAK activity would affect skeletal muscle differentiation of mesoangioblasts isolated from IBM muscle.

      Materials and Methods

      Patients

      Diagnostic muscle biopsies were performed after informed consent at the Neurology Department of the Catholic University. We used fresh and fresh-frozen muscles from 8 normal controls (27 to 83 years; average, 63.625 ± 20.9) and 30 patients with inflammatory myopathies: 16 sporadic IBM (43 to 84 years; average, 67.7 ± 10.7), 10 dermatomyositis (DM) (15 to 78 years; average, 51 ± 19.9), and 9 polymyositis (PM) (22 to 84 years; average, 65.3 ± 18.4) (Table 1). Diagnosis was based on clinical evaluation and laboratory studies. None of the patients received steroids or immunosuppressive therapy before biopsy. This research was approved by the Ethical Committee of our institution.
      Table 1Patients Included in the Study and Muscle of Origin
      PatientAge (years)SexMusclePatientAge (years)SexMuscle
      IBM
      166MQ964MQ
      276MD1076MQ
      384MD1161FD
      479FQ1260FD
      562FQ1362MD
      672MD1443MD
      756MD1579MD
      878FQ1666MD
      DM
      132MD674MD
      215MQ752MD
      337FD853FD
      454FD978FQ
      544MQ1071MD
      PM
      177FQ671MD
      256FD772MD
      374FD873FD
      422MD984MD
      559FQ
      CT
      143FD580FQ
      227MD678FQ
      351FD783MD
      480MQ867MQ
      CT, control; D, deltoid; DM, dermatomyositis; F, female; M, male; IBM, inclusion-body myositis; PM, polymyositis; Q, quadriceps.

      Cell Cultures

      Human mesoangioblasts were obtained from muscle biopsy fragments, including intramuscular vessels and surrounding mesenchymal tissue, as previously described,
      • Morosetti R.
      • Mirabella M.
      • Gliubizzi C.
      • Broccolini A.
      • De Angelis L.
      • Tagliafico E.
      • Sampaolesi M.
      • Gidaro T.
      • Papacci M.
      • Roncaglia E.
      • Rutella S.
      • Ferrari S.
      • Tonali P.A.
      • Ricci E.
      • Cossu G.
      MyoD expression restores defective myogenic differentiation of human mesangioblasts from inclusion-body myositis muscle.
      • Dellavalle A.
      • Sampaolesi M.
      • Tonlorenzi R.
      • Tagliafico E.
      • Sacchetti B.
      • Perani L.
      • Innocenzi A.
      • Galvez B.G.
      • Messina G.
      • Morosetti R.
      • Li S.
      • Belicchi M.
      • Peretti G.
      • Chamberlain J.S.
      • Wright W.E.
      • Torrente Y.
      • Ferrari S.
      • Bianco P.
      • Cossu G.
      Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.
      from 12 IBM, 5 DM, 3 PM, and 7 normal controls. Cells were maintained in culture for several passages. At every passage, cells were counted and viability was determined by trypan blue exclusion.
      For skeletal muscle differentiation, human mesoangioblasts were cultured in normal human myoblast-conditioned medium for 4 days and then exposed for 7 days to differentiation medium with 1% bovine serum albumin (without serum), as previously described.
      • Morosetti R.
      • Mirabella M.
      • Gliubizzi C.
      • Broccolini A.
      • De Angelis L.
      • Tagliafico E.
      • Sampaolesi M.
      • Gidaro T.
      • Papacci M.
      • Roncaglia E.
      • Rutella S.
      • Ferrari S.
      • Tonali P.A.
      • Ricci E.
      • Cossu G.
      MyoD expression restores defective myogenic differentiation of human mesangioblasts from inclusion-body myositis muscle.
      At each time point cells were fixed for immunocytochemistry or harvested for Western blot analysis. Each experiment was performed in duplicate at least three times.

      Cell Treatments

      To study the effects of TWEAK on mesoangioblasts differentiation down skeletal muscle, cells were treated with either recombinant human TWEAK (TWEAK/TNFSF12, 100 ng/mL) and/or recombinant human TWEAKR/TNFSFR12/Fc (Fn14-Fc) chimera (1 μg/mL) (both from R&D Systems, Minneapolis, MN).
      • Dogra C.
      • Changotra H.
      • Mohan S.
      • Kumar A.
      Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
      • Ando T.
      • Ichikawa J.
      • Wako M.
      • Hatsushika K.
      • Watanabe Y.
      • Sakuma M.
      • Tasaka K.
      • Ogawa H.
      • Hamada Y.
      • Yagita H.
      • Nakao A.
      TWEAK/Fn14 interaction regulates RANTES production BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells.
      The specificity of TWEAK effects was evaluated by treating DM and control mesoangioblasts with TWEAK/TNFSF12 (100 ng/mL) with or without the neutralizing anti-human TWEAK/TNFSF12 antibody (R&D Systems) 10 μg/mL, as previously described.
      • Girgenrath M.
      • Weng S.
      • Kostek C.A.
      • Browning B.
      • Wang M.
      • Brown S.A.
      • Winkles J.A.
      • Michaelson J.S.
      • Allaire N.
      • Schneider P.
      • Scott M.L.
      • Hsu Y.M.
      • Yagita H.
      • Flavell R.A.
      • Miller J.B.
      • Burkly L.C.
      • Zheng T.S.
      TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.
      • Perper S.J.
      • Browning B.
      • Burkly L.C.
      • Weng S.
      • Gao C.
      • Giza K.
      • Su L.
      • Tarilonte L.
      • Crowell T.
      • Rajman L.
      • Runkel L.
      • Scott M.
      • Atkins G.J.
      • Findlay D.M.
      • Zheng T.S.
      • Hess H.
      TWEAK is a novel arthritogenic mediator.
      Treatments were performed all over the differentiation process (for 4 days in conditioned medium and 7 days in differentiation medium), and medium was replenished every 2 days. Fusion index was expressed as number of myonuclei/number of total nuclei, visualized by Hoechst 33258 staining (Molecular Probes Inc., Eugene, OR).

      RNA Interference

      Silencing of TWEAK gene was achieved using the small-interfering RNA (siRNA) technique. Mesoangioblasts from 8 IBM, 2 DM, and 1 normal control were transfected with pre-designed chimera siRNA oligonucleotide directed against human TWEAK (TNFSF12A Pre-Design Chimera RNAi Duplex; Abnova, Heidelberg, Germany).
      Cells seeded on 30-mm/60-mm/100-mm dishes at 80% confluency were transfected with 250/500/1500 pmol of siRNA, using Lipofectamine 2000 as transfection reagent (Invitrogen, Carlsbad, CA) by following the manufacturer's instructions.
      Twenty-four hours after transfection, cells were shifted to myoblast-conditioned medium for 4 days and differentiation medium for 7 days to induce skeletal muscle differentiation. At different time points, cells were fixed for immunocytochemistry or harvested for Western blot analysis. To establish efficiency of transfection, RNA was extracted 48 hours after treatment. To determine any nonspecific effect caused by the transfection reagent or process, mock-transfected cells went through the experimental procedure without addition of siRNA (treated with transfection reagent only). Untreated controls were used as well throughout the duration of the experiment for immunocytochemistry, protein, and gene expression analyses. Each experiment was performed in duplicate at least 3 times.

      Immunochemistry

      This was performed on 8-μm-thick unfixed cryostat muscle sections and on cells cultured on gelatin-coated optical quality plastic μ-Dishes (ibidi, Munich, Germany) and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 15 minutes.
      The following primary antibodies were used: monoclonal anti-skeletal myosin (Sigma, St. Louis, MN), polyclonal anti-human TWEAKR/TNFRSF12, polyclonal anti-human TWEAK/TNFSF12 (both from R&D Systems), monoclonal anti-myosin heavy chain developmental type (MHCd) (Novocastra, Newcastle On Tyne, UK), monoclonal anti-CD68 (Covance, Princeton, New Jersey), polyclonal anti-MyoD (C-20, Santa Cruz Biotechnology, Santa Cruz, CA).
      Detection of immunocomplexes was performed using the appropriate Alexa Fluor 488 or 594-conjugated secondary antibodies (Invitrogen). Hoechst 33258 (Molecular Probes Inc., Eugene, OR) staining was used to visualize cell nuclei. In all experiments, the omission of primary antibody or the incubation with an irrelevant primary antibody were used to verify the specificity of the immunoreaction.
      Samples were analyzed using a TCS SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).

      Western Blot Analysis

      Diagnostic muscle biopsies from 4 IBM, 4 DM, 4 PM, and 1 normal control were homogenized in lysis buffer for Western blot analysis. Mesoangioblasts were collected at different time points and homogenates were obtained according to standard protocols. Nuclear extracts were prepared according to standard protocols. An equal amount of total lysates or nuclear extracts (30 μg) from each experimental sample was separated by SDS-PAGE and blotted onto nitrocellulose membranes (Schleicher & Schuell, Relliehausen, Germany). Blots were incubated with one of the following antibodies: polyclonal anti-human TWEAKR/TNFRSF12, polyclonal anti-human TWEAK/TNFSF12 (R&D Systems), monoclonal anti-human MyoD1 (Clone 5.8A, DakoCytomation, Glostrup, Denmark), polyclonal anti-phosphorylated NF-κB p65 (pNFκB-p65, Ser536), polyclonal anti-NFκB p65, polyclonal anti-phosphorylated Akt (pAkt, Ser473), and polyclonal anti-Akt (all from Cell Signaling Technology, Beverly, MA). The equal loading of samples was confirmed by probing the membranes with the monoclonal anti-β-Actin antibody or polyclonal anti-Retinoic X Receptor α (RXRα), (both from Santa Cruz Biotechnology). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, blots were visualized using ECL (Amersham Bioscience, Health care, Piscataway, NJ) and quantified by densitometry using the TotalLab 2.01 software (Nonlinear Dynamics Ltd, Newcastle on Tyne, UK).

      RT-PCR

      Two μg of total RNA was used as template for first-strand cDNA synthesis according to standard methods. Briefly, cDNAs were amplified for TWEAK and β-actin using the following primers: TWEAK, forward: 5′-CCCTGCGCTGCCTGGAGGAA-3′, reverse: 5′-AGACCAGGGCCCCTCAGTGA-3; β-actin, forward: 5′-AAGAGAGGCATCCTCACCCT-3′, reverse: 5′-TACATGGCTGGGGTGTTGAA-3′. PCR products were electrophoresed on 2% agarose gels.

      Determination of TWEAK Levels by Enzyme-Linked Immunosorbent Assay

      Measurement of TWEAK concentration on supernatants from differentiating mesoangioblasts was performed using a commercial human TWEAK instant enzyme-linked immunosorbent assay kit, according to the manufacturer's suggested protocol (Bender Medsystems, Vienna, Austria). All assays were performed in triplicate.
      Supernatants from differentiating mesoangioblasts from 6 IBM, 4 DM, 3 PM patients, and 7 normal controls were collected after 7 days. The medium was centrifuged at 12,000 × g to remove cells, debris, and proteins were concentrated using Ultrafree 0.5 columns (Millipore Corporation, Billerica, MA). Fifty μL of each sample was loaded in wells of a microwell plate pre-coated with a monoclonal antibody against TWEAK and incubated for 3 hours at room temperature. Absorbance of each reaction was read on a spectrophotometer using 450 nm as the primary wavelength.

      Statistical Analysis

      Data are reported as means ± SD. Statistical analysis was performed in all experiments by analysis of variance for repeated measures and comparisons between groups were assessed by Student's t-test. Adjustment for multiple comparisons was made, when necessary, by the use of the Bonferroni correction method. Statistical significance was set at P ≤ 0.05.

      Results

      IBM Muscle Biopsies and IBM Mesoangioblasts Display Increased Levels of TWEAK and Fn14

      We analyzed the expression of TWEAK and Fn14 in muscle biopsies of 14 patients affected by IBM, 6 DM, 6 PM, and 3 normal controls by immunohistochemistry and Western blot. We observed a strong expression of both TWEAK and its receptor Fn14 in infiltrating monocytes in all muscle biopsies that contained them. In IBM muscle biopsies strong TWEAK immunoreactivity was also observed around the sarcolemma of muscle fibers partially invaded or surrounded by monocytes, in the form of membrane-bound protein, and as diffuse staining in the endomysium close to degenerating or apparently normal fibers bounded by infiltrating cells, probably corresponding to the secreted soluble protein (Figure 1A). In IBM muscle biopsies, membrane staining was also visible in a large number of fibers, apparently regardless of the presence of nearby inflammatory cells. In DM and PM, only muscle fibers invaded or surrounded by monocytes and degenerating fibers next to inflammatory infiltrates showed increased membrane TWEAK immunoreactivity. In IBM biopsies, the majority of muscle fibers showing positive TWEAK immunosignal on the membrane were not regenerating fibers with only rare regenerating fibers being weakly positive (Figure 1B). In PM, TWEAK immunoreactivity was observed only in muscle fibers undergoing phagocytosis, whereas regenerating fibers were negative. Western blot analysis on whole muscle homogenates showed increased TWEAK expression in IBM muscle biopsy samples compared to DM, PM, and normal controls (Figure 1C).
      Figure thumbnail gr1
      Figure 1Expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in muscle biopsies of inclusion-body myositis (IBM), dermatomyositis (DM) and polymyositis (PM) patients. A: In IBM muscle biopsies a strong TWEAK immunoreactivity is observed in monocytes (identified by double staining with anti-CD68 antibody) and also detected along the sarcolemma of muscle fibers surrounded or partially invaded by monocytes (arrowhead). In IBM muscle, a large number of fibers are TWEAK-positive, regardless the presence of nearby inflammatory cells. In DM, only muscle fibers invaded or surrounded by monocytes and degenerating fibers next to inflammatory infiltrates show increased membrane TWEAK immunoreactivity. Scale bars, 80 μm in upper and lower rows, and 20 μm in the middle row. B: In IBM biopsies, the majority of muscle fibers showing positive TWEAK immunosignal on the membrane are not regenerating fibers (identified by double staining with anti-Myosin heavy chain developmental type (MHCd) antibody). Rare regenerating fibers show a faint cytoplasmic immunoreactivity in IBM muscle (arrowheads). In PM, TWEAK immunoreactivity is observed only in muscle fibers undergoing phagocytosis (arrowhead), whereas regenerating fibers are negative (arrow). Scale bar = 80 μm. Hoechst staining identifies cell nuclei. C: Western blot analysis of TWEAK expression in whole muscle homogenates shows an increased protein level in IBM muscle biopsy samples compared to PM, DM, and normal control (CT) muscles. A representative experiment out of three is shown.
      In IBM muscle biopsies, a very strong Fn14 immunoreactivity was present along the sarcolemma in muscle fibers partially invaded or surrounded by infiltrating monocytes and in hypotrophic and apparently normal fibers close to inflammatory infiltrates (Figure 2A). The vast majority of nonregenerating fibers (including morphologically abnormal vacuolated and nonvacuolated fibers and hypotrophic fibers) showed strong Fn14 membrane staining also independently from the presence of inflammatory cells. The rare regenerating fibers observed in IBM muscle usually did not show Fn14 expression, except for isolated morphologically abnormal ones. Likewise in PM, where a large number of MHCd-positive regenerating fibers was seen, only rare abnormal fibers (probably damaged or degenerating) exhibited a membrane Fn14 immunostaining (Figure 2B). In all control muscles, staining for TWEAK and Fn14 were both negligible (not shown). Omission of the primary antibody or use of a nonrelevant primary antibody completely abolished specific staining for TWEAK and Fn14.
      Figure thumbnail gr2
      Figure 2Expression of Fn14 in muscle biopsy of inclusion-body myositis (IBM), dermatomyositis (DM) and polymyositis (PM) patients. A: In IBM muscle, very strong Fn14 immunoreactivity is present as sarcolemmal staining in muscle fibers partially invaded or surrounded by infiltrating monocytes (identified by double staining with anti-CD68 antibody). In PM, in addition to monocytes, only muscle fibers undergoing invasion by macrophages show weak Fn14 immunoreactivity. Scales bar, 80 μm in the top row, 20 μm in the middle row, and 40 μm in the bottom row. B: In IBM muscle biopsies, the vast majority of nonregenerating fibers (including morphologically abnormal vacuolated and nonvacuolated fibers and hypotrophic fibers) show intense Fn14 membrane staining, also independent from the presence of inflammatory cells. The rare regenerating fibers (identified by double staining with anti-MHCd antibody) usually do not have Fn14 expression (arrows) except for one isolated morphologically abnormal fiber (arrowhead). Likewise in PM, where a large quantity of Myosin heavy chain developmental type (MHCd)-positive regenerating fibers is seen, only rare abnormal fibers (probably damaged or degenerating fibers) exhibit a positive membrane Fn14 immunostaining (arrows). Scale bar = 80 μm.
      In addition, Fn14 expression was also investigated by Western blot analysis on proliferating mesoangioblasts obtained from 8 IBM primary cultures. As expected in progenitor cells of mesenchymal lineage, all mesoangioblasts samples expressed the TWEAK receptor, but IBM mesoangioblasts showed a much higher expression of Fn14 compared to PM, DM, and normal controls (Figure 3, A and B).
      Figure thumbnail gr3
      Figure 3Expression of Fn14 in mesoangioblasts from inclusion-body myositis (IBM), dermatomyositis (DM) and polymyositis (PM), and controls. Mesoangioblasts from IBM muscles express a higher level of Fn14 than mesoangioblasts isolated from DM, PM, and normal control muscles. β-actin served as a loading control. A: A representative Western blot out of three is shown. B: Densitometric analysis from three different Western blots performed for 8 IBM samples, 2 DM, 1 PM, and 1 normal control. Ratios were calculated between Fn14 and β-actin values. Data represent the mean ± SD.

      Effects of TWEAK Treatment and TWEAK Neutralization on Differentiating Mesoangioblasts

      We previously demonstrated that IBM mesoangioblasts exhibit a differentiation block down skeletal muscle, whereas DM (as well as PM and normal mesoangioblasts) differentiate into skeletal muscle with a very high efficiency (fusion index, 0.6 to 0.7). First, to assess a possible inhibitory role of TWEAK on mesoangioblasts differentiation, we investigated the direct effects of TWEAK on DM mesoangioblasts. The exposure of DM mesoangioblasts to recombinant human TWEAK/TNFSF12 (100 ng/mL) during myogenic differentiation resulted in a significant decrease of myotubes formation (fusion index, 0.3 to 0.4), compared to untreated DM cells (Figure 4A). In addition, TWEAK/TNFSF12-treated cultures displayed thinner myotubes compared to untreated ones, similarly to what was previously reported for TWEAK/TNFSF12-treated C2C12 myotubes.
      • Dogra C.
      • Changotra H.
      • Wedhas N.
      • Qin X.
      • Wergedal J.E.
      • Kumar A.
      TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine.
      The inhibitory effect of TWEAK/TNFSF12 on myotubes formation was specific since upon co-treatment with the anti-TWEAK/TNFSF12 neutralizing antibody (10 μg/mL) the differentiation rate of DM mesoangioblasts was comparable to differentiation rate of untreated cells (fusion index, 0.6 to 0.7) (Figure 4A). No effect of TWEAK/TNFSF12 treatment was observed in IBM mesoangioblasts. Then we explored the effect of recombinant human Fn14-Fc chimera (1 μg/mL) on IBM mesoangioblasts. The addition of Fn14-Fc protein consistently led to the formation of spare multinucleated myotubes, indicating the ability of Fn14-Fc chimera to partially revert the inhibitory effects of TWEAK (Figure 4B).
      Figure thumbnail gr4
      Figure 4Effects of recombinant human tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/TNFSF12 (TWEAK), anti-TWEAK/tumor necrosis factor (TNF)SF12 (anti-TWEAK) antibody, and Fn14-Fc chimera on differentiation of mesoangioblasts into skeletal muscle. Immunofluorescence for myosin (red, nuclei are labeled in blue by Hoechst staining). A: Dermatomyositis (DM) mesoangioblasts treated with TWEAK (100 ng/mL) show a significant decrease of myotube formation compared to untreated DM cells. Specificity of the negative modulatory effect of TWEAK treatment on DM mesoangioblasts is demonstrated by the restoration of normal myogenic differentiation on a combination treatment of TWEAK (100 ng/mL) and anti-TWEAK neutralizing antibody (10 μg/mL). B: Treatment with recombinant human Fn14-Fc chimera (1 μg/mL) induces significant skeletal muscle differentiation of inclusion-body myositis (IBM) mesoangioblasts. Scale bar = 80 μm. Each experiment was performed in duplicate at least 2 times for each patient.

      siRNA Inhibition of TWEAK Expression Restores Myogenic Differentiation in IBM Mesoangioblasts

      To further investigate the possible inhibitory role of TWEAK on the differentiation process of IBM mesoangioblasts, we examined the effect of siRNA-mediated suppression of TWEAK gene expression in cells from 4 IBM and 1 DM patients (Figure 5A). Interestingly, after 4 days in conditioned medium and 7 days in differentiation medium, siRNA-transfected IBM mesoangioblasts were able to fuse into multinucleated myosin-positive myotubes (fusion index, 0.55 ± 0.21), whereas control IBM mesoangioblasts displayed a differentiation defect with only spare, mainly bi-nucleated, myosin-positive myotubes. As expected, we observed a dramatic increase of myotubes formation in DM siRNA-treated mesoangioblasts (Figure 5B).
      Figure thumbnail gr5
      Figure 5Induction of myogenic differentiation of inclusion-body myositis (IBM) and dermatomyositis (DM) mesoangioblasts by tumor necrosis factor-like weak inducer of apoptosis (TWEAK) small-interfering RNA (siRNA). A pre-designed chimera siRNA oligonucleotide directed against human TWEAK was transfected into IBM and DM mesoangioblasts. A: Efficiency of transfection. Forty-eight hours after transfection, cells were harvested for RNA extraction and RT-PCR analysis. β-actin served as a control. B: After 24 hours, siRNA-transfected cells were exposed to myoblast-conditioned medium for 4 days and subsequently shifted to differentiation medium. After 7 days of culture, cells were stained with anti-myosin antibody (red) or harvested for Western blot analysis. Representative cultures from three different IBM patients and 1 DM patient are shown. siRNA-transfected IBM mesoangioblasts were able to fuse into multinucleated myosin-positive myotubes, whereas control IBM mesoangioblasts displayed a differentiation defect with only spare and mainly bi-nucleated myosin-positive cells. A dramatic increase of myotubes formation was also observed in DM siRNA-treated mesoangioblasts. Scale bar = 100 μm. C: Representative Western blot showing decrease of NFκB phospho-p65 (Ser536) subunit (pNFκB-p65) in total lysates of siRNA-transfected IBM and DM mesoangioblasts (total). Western blot on nuclear extracts shows marked reduction of pNFκB-p65 and, to a lesser extent, of total NFκB levels after siRNA (nuclear). An anti-Retinoic X receptor α (RXRα) antibody was used as loading control for nuclear fraction. D: Representative Western blots showing induction of MyoD expression and Akt phosphorylation (pAkt) in siRNA-transfected IBM mesoangioblasts. β-actin served as a loading control. E: Immunocytochemistry showing nuclear staining (arrows) for MyoD in wild-type and siRNA-transfected IBM mesoangioblasts. Scale bar = 10 μm.
      Different studies have shown that TWEAK has the capability to activate the NFκB pathway, thus promoting cell proliferation and inhibition of skeletal muscle differentiation, in part through MyoD inactivation.
      • Dogra C.
      • Changotra H.
      • Mohan S.
      • Kumar A.
      Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      Recently, it has been demonstrated that genetic ablation or overexpression of TWEAK leads to suppression or activation of NFκB in regenerating myofibers, respectively.
      • Mittal A.
      • Bhatnagar S.
      • Kumar A.
      • Paul P.K.
      • Kuang S.
      • Kumar A.
      Genetic ablation of TWEAK augments regeneration and post-injury growth of skeletal muscle in mice.
      Interestingly, siRNA-mediated suppression of TWEAK diminished NFκB signaling and nuclear translocation through decreased phosphorylation of the p65 subunit at Ser536, as evidenced in total lysates and nuclear extracts of IBM mesoangioblasts, which displayed an increased capability to differentiate into multinucleated myotubes (Figure 5C). Moreover TWEAK siRNA induced Akt phosphorylation (Figure 5D) and enhanced MyoD levels in differentiating IBM mesoangioblasts, as demonstrated by Western blot and immunocytochemistry (Figure 5, D and E).

      TWEAK Levels in Culture Medium from Differentiating Mesoangioblasts

      TWEAK concentration was determined in medium collected from human mesoangioblasts during skeletal muscle differentiation. In particular, supernatants were collected from mesoangioblasts of IBM, DM, PM patients, and normal controls after 7 days in serum-free differentiation medium. All mesoangioblast cultures released the cytokine in the medium with significantly higher levels of TWEAK detected in the medium obtained from IBM cells. A similar result was obtained when the analysis was conducted on supernatants from age-matched mesoangioblast cultures (4 IBM, 3 DM, 3 PM, and 4 controls), thus confirming that such increased TWEAK secretion was indeed disease-related rather than simply reflecting the age of patients from whom cells were obtained (Figure 6, A and B) (P ≤ 0.01). To verify whether siRNA would affect TWEAK production and release by mesoangioblasts, we determined the concentration of the cytokine in the supernatants of IBM cells previously treated with TWEAK siRNA. As expected, the level of TWEAK in the medium of treated mesoangioblasts was significantly lower than that from untreated ones (Figure 6C), and this was associated with an induction of skeletal muscle differentiation. Our data indicate that mesoangioblasts from IBM muscle, besides expressing higher levels of Fn14, also release more cytokine than DM and controls.
      Figure thumbnail gr6
      Figure 6Quantification by enzyme-linked immunosorbent assay of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) concentration in medium from human mesoangioblasts during skeletal muscle differentiation. A: Medium from differentiating mesoangioblasts from four inclusion-body myositis (IBM) shows significantly higher levels of TWEAK when compared to medium collected from age-matched dermatomyositis (DM) (3 patients), polymyositis (PM) (3 patients), and control (CT, 4 subjects) mesoangioblasts (*P ≤ 0.01). B: Medium from differentiating mesoangioblasts from 6 IBM patients displays significantly higher levels of TWEAK compared to medium collected from 3 DM mesoangioblasts (*P ≤ 0.05) and from 3 control mesoangioblasts (*P ≤ 0.01). C: Effect of TWEAK small-interfering RNA (siRNA) on TWEAK levels in differentiating medium from IBM mesoangioblasts. Significant lower levels of the cytokine were detected in medium collected from siRNA-treated mesoangioblasts (*P ≤ 0.05). Mean values from three wells of one representative experiment out of two independent ones are shown.

      Discussion

      Various cytokines secreted by inflammatory cells directly influence the properties of progenitor cells that reside in tissues undergoing repair and regeneration.
      • Duffield J.S.
      The inflammatory macrophage: a story of Jekyll and Hyde.
      Likewise, soluble factors secreted by macrophages or muscle fibers themselves, and delivered into the muscle microenvironment, can also drive the proliferation of satellite cells during muscle regeneration.
      • Merly F.
      • Lescaudron L.
      • Rouaud T.
      • Crossin F.
      • Gardahaut M.F.
      Macrophages enhance muscle satellite cell proliferation and delay their differentiation.
      Cell culture studies have shown that inflammatory cytokines can modulate both proliferation and differentiation of myogenic cells. TWEAK is a proinflammatory cytokine and, as such, persistent TWEAK-Fn14 axis signaling is believed to play a role in human diseases caused by an excessive or abnormal inflammatory response (eg, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis).
      • Perper S.J.
      • Browning B.
      • Burkly L.C.
      • Weng S.
      • Gao C.
      • Giza K.
      • Su L.
      • Tarilonte L.
      • Crowell T.
      • Rajman L.
      • Runkel L.
      • Scott M.
      • Atkins G.J.
      • Findlay D.M.
      • Zheng T.S.
      • Hess H.
      TWEAK is a novel arthritogenic mediator.
      • Kumar M.
      • Makonchuk D.Y.
      • Li H.
      • Mittal A.
      • Kumar A.
      TNF-like weak inducer of apoptosis (TWEAK) activates proinflammatory signaling pathways and gene expression through the activation of TGF-beta-activated kinase 1.
      • Liu Z.C.
      • Zhou Q.L.
      • Li X.Z.
      • Yang J.H.
      • Ao X.
      • Veeraragoo P.
      • Zuo X.X.
      Elevation of human tumor necrosis factor-like weak inducer of apoptosis in peripheral blood mononuclear cells is correlated with disease activity and lupus nephritis in patients with systemic lupus erythematosus.
      • Desplat-Jégo S.
      • Feuillet L.
      • Creidy R.
      • Malikova I.
      • Rance R.
      • Khrestchatisky M.
      • Hahm K.
      • Burkly L.C.
      • Pelletier J.
      • Boucraut J.
      TWEAK is expressed at the cell surface of monocytes during multiple sclerosis.
      As a matter of fact, TWEAK-Fn14 inhibition is efficacious in experimental autoimmune encephalitis models.
      • Mueller A.M.
      • Pedré X.
      • Kleiter I.
      • Hornberg M.
      • Steinbrecher A.
      • Giegerich G.
      Targeting fibroblast growth factor-inducible-14 signaling protects from chronic relapsing experimental autoimmune encephalomyelitis.
      When persistently activated in chronic inflammatory disorders, TWEAK has been demonstrated to promote abnormal hyperplasia and angiogenesis, and proliferation of progenitor cells with inhibition of their differentiation.
      • Burkly L.C.
      • Michaelson J.S.
      • Hahm K.
      • Jakubowski A.
      • Zheng T.S.
      TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease.
      Several studies
      • Dogra C.
      • Changotra H.
      • Mohan S.
      • Kumar A.
      Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      • Girgenrath M.
      • Weng S.
      • Kostek C.A.
      • Browning B.
      • Wang M.
      • Brown S.A.
      • Winkles J.A.
      • Michaelson J.S.
      • Allaire N.
      • Schneider P.
      • Scott M.L.
      • Hsu Y.M.
      • Yagita H.
      • Flavell R.A.
      • Miller J.B.
      • Burkly L.C.
      • Zheng T.S.
      TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.
      have previously shown that the interplay between TWEAK and its receptor Fn14 is crucial for the proliferation and differentiation of murine myoblasts. In fact, TWEAK is able to potently inhibit myotube formation and the expression of muscle-specific transcription factors in C2C12 cells and in primary murine myoblasts.
      • Dogra C.
      • Changotra H.
      • Mohan S.
      • Kumar A.
      Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
      In a previous study,
      • Morosetti R.
      • Mirabella M.
      • Gliubizzi C.
      • Broccolini A.
      • De Angelis L.
      • Tagliafico E.
      • Sampaolesi M.
      • Gidaro T.
      • Papacci M.
      • Roncaglia E.
      • Rutella S.
      • Ferrari S.
      • Tonali P.A.
      • Ricci E.
      • Cossu G.
      MyoD expression restores defective myogenic differentiation of human mesangioblasts from inclusion-body myositis muscle.
      we have demonstrated that DM, PM, and normal control mesoangioblasts are able to fully differentiate into skeletal muscle with high efficiency in vitro and in vivo, whereas IBM cells, despite a comparable proliferating activity, display a differentiation block that can be overcome by MyoD ectopic expression. Here we have investigated TWEAK and Fn14 expression in human muscle biopsies and in cultured mesoangioblasts isolated from IBM, DM, and PM patients. Both TWEAK and Fn14 were increased in IBM skeletal muscle. Moreover, TWEAK was able to inhibit skeletal muscle differentiation of human mesoangioblasts. In fact, our in vitro experiments demonstrate that TWEAK/TNFSF12 treatment led to significant reduction of skeletal muscle differentiation (fusion index, mean myotube diameter) of DM mesoangioblasts. This effect was specific because it could be reversed on co-treatment with the anti-TWEAK/TNFSF12 neutralizing antibody. No further reduction of skeletal muscle differentiation of IBM mesoangioblasts was detectable after TWEAK/TNFSF12 treatment. As expected in progenitor cells of mesenchymal lineage, all mesoangioblasts expressed the TWEAK receptor; however IBM mesoangioblasts showed a much higher expression of Fn14 compared to PM, DM, and normal controls. Given that IBM mesoangioblasts are greatly defective in skeletal muscle differentiation, we asked whether modulation of TWEAK-Fn14 axis would affect their myogenic ability. First, we investigated the effect of Fn14-Fc chimera on IBM mesoangioblasts, and interestingly we observed an induction of myotubes formation, although with low efficiency, thus suggesting a possible role of TWEAK-Fn14 activation in the differentiation block of IBM mesoangioblasts. To further study this pathway, we examined the effect of siRNA-mediated suppression of TWEAK in cells from IBM and DM patients. siRNA-transfected IBM mesoangioblasts were able to fuse into multinucleated myosin-positive myotubes, whereas control IBM mesoangioblasts produced only spare myosin-positive cells. As expected, we also observed a dramatic increase of myotubes formation in siRNA-treated DM mesoangioblasts cultures. Moreover, by analyzing TWEAK levels in serum-free differentiation medium collected from IBM, DM, and control mesoangioblasts, we found significantly higher levels of TWEAK protein in the supernatants of IBM cells. This underlines the ability of mesoangioblasts to secrete the cytokine and further support the hypothesis of a negative role of TWEAK on skeletal muscle differentiation of human mesoangioblasts, similar to that described in murine myoblasts.
      • Dogra C.
      • Changotra H.
      • Mohan S.
      • Kumar A.
      Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
      • Dogra C.
      • Changotra H.
      • Wedhas N.
      • Qin X.
      • Wergedal J.E.
      • Kumar A.
      TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine.
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      TWEAK is constitutively expressed in adult murine skeletal muscle, although it is known that its receptor is not significantly expressed. Catabolic conditions, however, can up-regulate the expression of Fn14 in muscle, and the subsequent interaction with TWEAK eventually leads to the activation of ubiquitin-proteasome system resulting in atrophy.
      • Mittal A.
      • Bhatnagar S.
      • Kumar A.
      • Lach-Trifilieff E.
      • Wauters S.
      • Li H.
      • Makonchuk D.Y.
      • Glass D.J.
      • Kumar A.
      The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice.
      Our study shows that TWEAK protein expression was increased in muscle homogenates of IBM compared to DM and PM and by using immunohistochemistry, this cytokine was detectable in a large number of non-necrotic fibers and in fibers close to or invaded by monocytes, unlike PM and DM, where its expression was restricted to this latter subpopulation of fibers. This may theoretically reflect both a quantitative difference in the composition of the inflammatory infiltrate with a higher percentage of monocytes in IBM, or more probably a disease-specific feature related to chronicity of both inflammation and muscle fiber degeneration in IBM compared to the acute–subacute inflammatory response seen in PM/DM. It is well-known that proinflammatory cytokines (such as TNFα, interleukin-1β, and interleukin-6) produced by both the immune system and skeletal muscle, stimulate proliferation of progenitor cells and inhibit terminal differentiation of myoblasts into mature fibers.
      • Langen R.C.
      • Schols A.M.
      • Kelders M.C.
      • Wouters E.F.
      • Janssen-Heininger Y.M.
      Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappa B.
      • Langen R.C.
      • Van Der Velden J.L.
      • Schols A.M.
      • Kelders M.C.
      • Wouters E.F.
      • Janssen-Heininger Y.M.
      Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization.
      Similarly, a transient rapid TWEAK up-regulation after acute inflammatory injury may be instrumental in actively promoting expansion of progenitor cells, followed by a decline of its level coincident with extensive differentiation of myofibers. On the contrary, in IBM a persistent TWEAK stimulation during chronic inflammation may negatively influence the activation of pro-myogenic pathways exacerbating the inadequate regeneration of myofibers typical of the disease.
      The widespread expression of Fn14 observed by immunohistochemistry in IBM nonregenerating muscle fibers is in agreement with the fact that Fn14 is highly regulated in vivo, and its induction has been reported in various rodent models of tissue injury. Fn14 has been demonstrated to function during muscle regeneration, also independently from TWEAK, and its knockdown by siRNA reduces the expression of myogenic regulatory factors and myogenic differentiation of murine myoblasts.
      • Dogra C.
      • Hall S.L.
      • Wedhas N.
      • Linkhart T.A.
      • Kumar A.
      Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes Evidence for TWEAK-independent functions of Fn14 during myogenesis.
      Therefore, Fn14 expression in IBM muscle could also reflect a positive attempt of myofibers to activate pro-myogenic signaling pathways, because multiple potential repairing mechanisms normally involved in muscle development are usually activated in abnormal muscle fibers of IBM.
      • Broccolini A.
      • Gidaro T.
      • Morosetti R.
      • Gliubizzi C.
      • Servidei T.
      • Pescatori M.
      • Tonali P.A.
      • Ricci E.
      • Mirabella M.
      Neprilysin participates in skeletal muscle regeneration and is accumulated in abnormal muscle fibres of inclusion body myositis.
      • Broccolini A.
      • Ricci E.
      • Pescatori M.
      • Papacci M.
      • Gliubizzi C.
      • D’Amico A.
      • Servidei S.
      • Tonali P.A.
      • Mirabella M.
      Insulin-like growth factor I in inclusion-body myositis and human muscle cultures.
      Previous studies have shown the capability of TWEAK to activate the NFκB pathway, thus inhibiting skeletal muscle differentiation. In addition, it has been described that overexpression or genetic ablation of TWEAK in mice provokes activation or suppression of NFκB in regenerating myofibers, respectively.
      • Mittal A.
      • Bhatnagar S.
      • Kumar A.
      • Paul P.K.
      • Kuang S.
      • Kumar A.
      Genetic ablation of TWEAK augments regeneration and post-injury growth of skeletal muscle in mice.
      • Li H.
      • Mittal A.
      • Paul P.K.
      • Kumar M.
      • Srivastava D.S.
      • Tyagi S.C.
      • Kumar A.
      Tumor necrosis factor-related weak inducer of apoptosis augments matrix metalloproteinase 9 (MMP-9) production in skeletal muscle through the activation of nuclear factor-kappaB-inducing kinase and p38 mitogen-activated protein kinase: a potential role of MMP-9 in myopathy.
      Of note, we observed a reduced activation of NFκB in siRNA-transfected IBM mesoangioblasts, which displayed an increased capability to differentiate into multinucleated myotubes associated with increased levels of pro-myogenic factors, such as MyoD and phosphorylated Akt. Moreover, the binding of TWEAK to Fn14 has been shown to induce the expression of several known NFκB-target genes and of Fn14 itself.
      • Tran N.L.
      • McDonough W.S.
      • Savitch B.A.
      • Fortin S.P.
      • Winkles J.A.
      • Symons M.
      • Nakada M.
      • Cunliffe H.E.
      • Hostetter G.
      • Hoelzinger D.B.
      • Rennert J.L.
      • Michaelson J.S.
      • Burkly L.C.
      • Lipinski C.A.
      • Loftus J.C.
      • Mariani L.
      • Berens M.E.
      Increased fibroblast growth factor-inducible 14 expression levels promote glioma cell invasion via Rac1 and nuclear factor-kappa B and correlate with poor patient outcome.
      In IBM muscle, a more widespread expression of Fn14 may be related to the chronic injury of muscle fibers with locally increased TWEAK. It has been hypothesized that (depending on TWEAK expression levels in the tissue) an increase of Fn14 expression after tissue injury would trigger TWEAK-dependent (basal TWEAK levels high) or TWEAK-independent (TWEAK levels low) Fn14 signaling that can also contribute to disease pathology.
      • Winkles J.A.
      The TWEAK-Fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting.
      Whatever the mechanism of Fn14 activation, however, it remains to be determined whether Fn14 signaling also plays a pathogenic role in IBM independently from TWEAK.
      Dysregulation of TWEAK-Fn14 axis may play a twofold role in the pathogenesis of IBM. In fact, by acting as a powerful muscle-wasting cytokine, TWEAK may induce progressive muscle fiber atrophy, and simultaneously as a negative regulator of regenerative myogenesis, it might reduce the activation and differentiation of muscle precursor cells in vivo. TWEAK could represent an important linkage molecule connecting the persisting inflammation with the defective regeneration and the progressive muscle atrophy, possibly in conjunction with other putative cytokines or growth factors released in the muscle milieu of IBM with similar or synergistic activity.
      More studies are required to quantitatively assess how much the activation of this ligand-receptor dyad specifically contributes to the pathogenic mechanism of IBM and to understand how they act in concert with other pathological stressors, such as accumulation of multi-protein aggregates, proteasome inhibition, and oxidative and endoplasmic reticulum stress.
      IBM represents, to our knowledge, the first human myopathy showing an increase of TWEAK-Fn14 axis in skeletal muscle. This may open the way to selective targeting of TWEAK-Fn14 to concurrently suppress degenerative and inflammatory components of IBM and counteract muscle atrophy, a strategy that may prove to be therapeutically rewarding.
      The results of the present study provide the first evidence that TWEAK is a negative regulator of mesoangioblasts differentiation, and furthermore suggest that TWEAK-Fn14 interaction may regulate mesoangioblasts functions. Blocking TWEAK-Fn14 activity, therefore, may restore mesoangioblasts myogenic capacity in the IBM diseased muscle. In general terms, because mesoangioblasts isolated from IBM muscle fail to normally differentiate into skeletal muscle, to use autologous mesoangioblasts in a possible muscle regenerative cell therapy, it is indispensable to stimulate these cells in vitro to enhance their defective myogenic differentiation. It would be even more important to successfully activate endogenous mesoangioblasts located in IBM muscle niche in vivo, thus inducing them to make new regenerating fibers. To this end, it is of utmost importance the identification of factors, such as TWEAK, produced by muscle and inflammatory cells and released in the surrounding milieu, that are able to regulate the differentiation ability of IBM mesoangioblasts. Modulation of such target molecules, that are selectively dysregulated in IBM muscle, appear to be a more handy approach to enhance muscle regeneration compared to transplantation techniques.

      Acknowledgments

      We thank Manuela Papacci for helpful assistance. We also thank all of our patients without whom these studies would not have been possible and to whose ultimate benefit our efforts are directed.

      References

        • Chicheportiche Y.
        • Bourdon P.R.
        • Xu H.
        • Hsu Y.M.
        • Scott H.
        • Hession C.
        • Garcia I.
        • Browning J.L.
        TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.
        J Biol Chem. 1997; 272: 32401-32410
        • Winkles J.A.
        The TWEAK-Fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting.
        Nat Rev Drug Discov. 2008; 7: 411-425
        • Marsters S.A.
        • Sheridan J.P.
        • Pitti R.M.
        • Brush J.
        • Goddard A.
        • Ashkenazi A.
        Identification of a ligand for the death-domain-containing receptor Apo3.
        Curr Biol. 1998; 8: 525-528
        • Wiley S.R.
        • Winkles J.A.
        TWEAK, a member of the TNF superfamily, is a multifunctional cytokine that binds the TWEAKR/Fn14 receptor.
        Cytokine Growth Factor Rev. 2003; 14: 241-249
        • Brown S.A.
        • Ghosh A.
        • Winkles J.A.
        Full-length, membrane-anchored TWEAK can function as a juxtacrine signalling molecule and activate the NF-kappaB pathway.
        J Biol Chem. 2010; 285: 17432-17441
        • Meighan-Mantha R.L.
        • Hsu D.K.
        • Guo Y.
        • Brown S.A.
        • Feng S.L.
        • Peifley K.A.
        • Alberts G.F.
        • Copeland N.G.
        • Gilbert D.J.
        • Jenkins N.A.
        • Richards C.M.
        • Winkles J.A.
        The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.
        J Biol Chem. 1999; 274: 33166-33176
        • Feng S.L.
        • Guo Y.
        • Factor V.M.
        • Thorgeirsson S.S.
        • Bell D.W.
        • Testa J.R.
        • Peifley K.A.
        • Winkles J.A.
        The Fn14 immediate-early response gene is induced during liver regeneration and highly expressed in both human and murine hepatocellular carcinomas.
        Am J Pathol. 2000; 156: 1253-1261
        • Dogra C.
        • Changotra H.
        • Mohan S.
        • Kumar A.
        Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein.
        J Biol Chem. 2006; 281: 10327-10336
        • Dogra C.
        • Changotra H.
        • Wedhas N.
        • Qin X.
        • Wergedal J.E.
        • Kumar A.
        TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine.
        FASEB J. 2007; 21: 1857-1869
        • Dogra C.
        • Hall S.L.
        • Wedhas N.
        • Linkhart T.A.
        • Kumar A.
        Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.
        J Biol Chem. 2007; 282: 15000-15010
        • Mittal A.
        • Bhatnagar S.
        • Kumar A.
        • Lach-Trifilieff E.
        • Wauters S.
        • Li H.
        • Makonchuk D.Y.
        • Glass D.J.
        • Kumar A.
        The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice.
        J Cell Biol. 2010; 188: 833-849
        • Burkly L.C.
        • Michaelson J.S.
        • Hahm K.
        • Jakubowski A.
        • Zheng T.S.
        TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease.
        Cytokine. 2007; 40: 1-16
        • Girgenrath M.
        • Weng S.
        • Kostek C.A.
        • Browning B.
        • Wang M.
        • Brown S.A.
        • Winkles J.A.
        • Michaelson J.S.
        • Allaire N.
        • Schneider P.
        • Scott M.L.
        • Hsu Y.M.
        • Yagita H.
        • Flavell R.A.
        • Miller J.B.
        • Burkly L.C.
        • Zheng T.S.
        TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.
        EMBO J. 2006; 25: 5826-5839
        • Mittal A.
        • Bhatnagar S.
        • Kumar A.
        • Paul P.K.
        • Kuang S.
        • Kumar A.
        Genetic ablation of TWEAK augments regeneration and post-injury growth of skeletal muscle in mice.
        Am J Pathol. 2010; 177: 1732-1742
        • Engel W.K.
        • Askanas V.
        Inclusion-body myositis: clinical, diagnostic, and pathologic aspects.
        Neurology. 2006; 66: S20-S29
        • Needham M.
        • Mastaglia F.L.
        Inclusion body myositis: current pathogenetic concepts and diagnostic and therapeutic approaches.
        Lancet Neurol. 2007; 6: 620-631
        • Askanas V.
        • Engel W.K.
        Sporadic inclusion-body myositis: conformational multifactorial ageing-related degenerative muscle disease associated with proteasomal and lysosomal inhibition, endoplasmic reticulum stress, and accumulation of amyloid-β42 oligomers and phosphorylated tau.
        Presse Med. 2011; 40: e219-e235
        • Dalakas M.C.
        Sporadic inclusion body myositis – diagnosis, pathogenesis and therapeutic strategies.
        Nat Clin Pract Neurol. 2006; 2: 437-447
        • Askanas V.
        • Engel W.K.
        • Nogalska A.
        Inclusion body myositis: a degenerative muscle disease associated with intra-muscle fiber multi-protein aggregates, proteasome inhibition, endoplasmic reticulum stress and decreased lysosomal degradation.
        Brain Pathol. 2009; 19: 493-506
        • Dalakas M.C.
        Immunotherapy of myositis: issues, concerns and future prospects.
        Nat Rev Rheumatol. 2010; 6: 129-137
        • Morosetti R.
        • Mirabella M.
        • Gliubizzi C.
        • Broccolini A.
        • De Angelis L.
        • Tagliafico E.
        • Sampaolesi M.
        • Gidaro T.
        • Papacci M.
        • Roncaglia E.
        • Rutella S.
        • Ferrari S.
        • Tonali P.A.
        • Ricci E.
        • Cossu G.
        MyoD expression restores defective myogenic differentiation of human mesangioblasts from inclusion-body myositis muscle.
        Proc Natl Acad Sci USA. 2006; 103: 16995-17000
        • Coletti D.
        • Yang E.
        • Marazzi G.
        • Sassoon D.
        TNFα inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways.
        EMBO J. 2002; 21: 631-642
        • Yang C.C.
        • Askanas V.
        • Engel W.K.
        • Alvarez R.B.
        Immunolocalization of transcription factor NF-kappa B in inclusion-body myositis muscle and at normal human neuromuscular junctions.
        Neurosci Lett. 1998; 254: 77-80
        • Nogalska A.
        • Wojcik S.
        • Engel W.K.
        • McFerrin J.
        • Askanas V.
        Endoplasmic reticulum stress induces myostatin precursor protein and NF-kappaB in cultured human muscle fibers: relevance to inclusion body myositis.
        Exp Neurol. 2007; 204: 610-618
        • Dellavalle A.
        • Sampaolesi M.
        • Tonlorenzi R.
        • Tagliafico E.
        • Sacchetti B.
        • Perani L.
        • Innocenzi A.
        • Galvez B.G.
        • Messina G.
        • Morosetti R.
        • Li S.
        • Belicchi M.
        • Peretti G.
        • Chamberlain J.S.
        • Wright W.E.
        • Torrente Y.
        • Ferrari S.
        • Bianco P.
        • Cossu G.
        Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.
        Nat Cell Biol. 2007; 9: 255-267
        • Ando T.
        • Ichikawa J.
        • Wako M.
        • Hatsushika K.
        • Watanabe Y.
        • Sakuma M.
        • Tasaka K.
        • Ogawa H.
        • Hamada Y.
        • Yagita H.
        • Nakao A.
        TWEAK/Fn14 interaction regulates RANTES production.
        Arthritis Res Ther. 2006; 8: R146
        • Perper S.J.
        • Browning B.
        • Burkly L.C.
        • Weng S.
        • Gao C.
        • Giza K.
        • Su L.
        • Tarilonte L.
        • Crowell T.
        • Rajman L.
        • Runkel L.
        • Scott M.
        • Atkins G.J.
        • Findlay D.M.
        • Zheng T.S.
        • Hess H.
        TWEAK is a novel arthritogenic mediator.
        J Immunol. 2006; 177: 2610-2620
        • Duffield J.S.
        The inflammatory macrophage: a story of Jekyll and Hyde.
        Clin Sci (Lond). 2003; 104: 27-38
        • Merly F.
        • Lescaudron L.
        • Rouaud T.
        • Crossin F.
        • Gardahaut M.F.
        Macrophages enhance muscle satellite cell proliferation and delay their differentiation.
        Muscle Nerve. 1999; 22: 724-732
        • Kumar M.
        • Makonchuk D.Y.
        • Li H.
        • Mittal A.
        • Kumar A.
        TNF-like weak inducer of apoptosis (TWEAK) activates proinflammatory signaling pathways and gene expression through the activation of TGF-beta-activated kinase 1.
        J Immunol. 2009; 182: 2439-2448
        • Liu Z.C.
        • Zhou Q.L.
        • Li X.Z.
        • Yang J.H.
        • Ao X.
        • Veeraragoo P.
        • Zuo X.X.
        Elevation of human tumor necrosis factor-like weak inducer of apoptosis in peripheral blood mononuclear cells is correlated with disease activity and lupus nephritis in patients with systemic lupus erythematosus.
        Cytokine. 2011; 53: 295-300
        • Desplat-Jégo S.
        • Feuillet L.
        • Creidy R.
        • Malikova I.
        • Rance R.
        • Khrestchatisky M.
        • Hahm K.
        • Burkly L.C.
        • Pelletier J.
        • Boucraut J.
        TWEAK is expressed at the cell surface of monocytes during multiple sclerosis.
        J Leukoc Biol. 2009; 85: 132-135
        • Mueller A.M.
        • Pedré X.
        • Kleiter I.
        • Hornberg M.
        • Steinbrecher A.
        • Giegerich G.
        Targeting fibroblast growth factor-inducible-14 signaling protects from chronic relapsing experimental autoimmune encephalomyelitis.
        J Neuroimmunol. 2005; 159: 55-65
        • Langen R.C.
        • Schols A.M.
        • Kelders M.C.
        • Wouters E.F.
        • Janssen-Heininger Y.M.
        Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappa B.
        FASEB J. 2001; 15: 1169-1180
        • Langen R.C.
        • Van Der Velden J.L.
        • Schols A.M.
        • Kelders M.C.
        • Wouters E.F.
        • Janssen-Heininger Y.M.
        Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization.
        FASEB J. 2004; 18: 227-237
        • Broccolini A.
        • Gidaro T.
        • Morosetti R.
        • Gliubizzi C.
        • Servidei T.
        • Pescatori M.
        • Tonali P.A.
        • Ricci E.
        • Mirabella M.
        Neprilysin participates in skeletal muscle regeneration and is accumulated in abnormal muscle fibres of inclusion body myositis.
        J Neurochem. 2006; 96: 777-789
        • Broccolini A.
        • Ricci E.
        • Pescatori M.
        • Papacci M.
        • Gliubizzi C.
        • D’Amico A.
        • Servidei S.
        • Tonali P.A.
        • Mirabella M.
        Insulin-like growth factor I in inclusion-body myositis and human muscle cultures.
        J Neuropathol Exp Neurol. 2004; 63: 650-659
        • Li H.
        • Mittal A.
        • Paul P.K.
        • Kumar M.
        • Srivastava D.S.
        • Tyagi S.C.
        • Kumar A.
        Tumor necrosis factor-related weak inducer of apoptosis augments matrix metalloproteinase 9 (MMP-9) production in skeletal muscle through the activation of nuclear factor-kappaB-inducing kinase and p38 mitogen-activated protein kinase: a potential role of MMP-9 in myopathy.
        J Biol Chem. 2009; 284: 4439-4450
        • Tran N.L.
        • McDonough W.S.
        • Savitch B.A.
        • Fortin S.P.
        • Winkles J.A.
        • Symons M.
        • Nakada M.
        • Cunliffe H.E.
        • Hostetter G.
        • Hoelzinger D.B.
        • Rennert J.L.
        • Michaelson J.S.
        • Burkly L.C.
        • Lipinski C.A.
        • Loftus J.C.
        • Mariani L.
        • Berens M.E.
        Increased fibroblast growth factor-inducible 14 expression levels promote glioma cell invasion via Rac1 and nuclear factor-kappa B and correlate with poor patient outcome.
        Cancer Res. 2006; 66: 9535-9542