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TWEAK in Inclusion-Body Myositis MusclePossible Pathogenic Role of a Cytokine Inhibiting Myogenesis

Roberta Morosetti,* Carla Gliubizzi,*

Cristina Sancricca,† Aldobrando Broccolini,*

Teresa Gidaro,* Matteo Lucchini,* and

Massimiliano Mirabella*

From the Department of Neurosciences,* Institute of Neurology,

Università Cattolica, Rome; and the Don Gnocchi Onlus 

Foundation,† Milan, Italy 

 Tumor necrosis factor-like weak inducer of apoptosis(TWEAK) and its receptor Fn14 exert pleiotropic ef-fects, including regulation of myogenesis. Sporadicinclusion-body myositis (IBM) is the most common muscle disease of the elderly population and leads tosevere disability. IBM mesoangioblasts, different from 

mesoangioblasts in other inflammatory myopathies,display a myogenic differentiation defect. The objec-tive of the present study was to investigate TWEAK-Fn14 expression in IBM and other inflammatory my-opathies and explore whether TWEAK modulation affects myogenesis in IBM mesoangioblasts. TWEAK,Fn14, and NF- B expression was assessed by immu-nohistochemistry and Western blot in cell samplesfrom both muscle biopsies and primary cultures. Me-soangioblasts isolated from samples of IBM, dermat-omyositis, polymyositis, and control muscles weretreated with recombinant human TWEAK, Fn14-Fcchimera, and anti-TWEAK antibody. TWEAK-RNA in-

terference was performed in IBM and dermatomyosi-tis 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 werefound in differentiation medium from IBM mesoan-gioblasts. Moreover, TWEAK inhibited myogenic dif-ferentiation of mesoangioblasts. Consistent with thisevidence, 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 mesoangio- blast differentiation. Dysregulation of the TWEAK-Fn14axis in IBM muscle may induce progressive muscle at-rophy and reduce activation and differentiation of mus-

cle precursor cells. (Am J Pathol 2012, 180:xxx; DOI:

 10.1016/j.ajpath.2011.12.027)

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 activi-

ties, including proliferation, differentiation, migration, an-

giogenesis, apoptosis, and inflammation.1,2 TWEAK is

initially expressed as a type II transmembrane protein

that is proteolytically processed into a soluble biologically

active cytokine.2 In addition to the cleaved soluble mol-

ecule, also the membrane-anchored TWEAK isoform has

been shown to be functionally active in a juxtacrine man-

ner by binding to the fibroblast growth factor-inducible

gene 14 receptor [Fn14 or TWEAK receptor (TWEAKR)]on neighboring cells.3–5 Fn14 is a highly inducible small

cell-surface receptor linked to several intracellular signal-

ing pathways, including the nuclear factor-kappa B

(NFB) pathway, and it is widely expressed on a variety

of tissues, including the cardiac and skeletal mus-

cle.1,3,6,7 Fn14 is highly up-regulated in the context of

tissue injury, regeneration,8–10 and inflammatory re-

sponses.10,11 In fact, the TWEAK-Fn14 axis plays an im-

portant role in tissue repair following acute injury,12 and it

has been implicated in the pathogenesis of several

chronic inflammatory diseases and ischemic stroke.2,12

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

Supported by grants from The Myositis Association (M.M.), and the

Fondazione Don Gnocchi Onlus (M.M. and R.M.) and Università Cattolica

Linea D1 (M.M.), and a fellowship from Association Française contre les

Myopathies (T.G.).

Accepted for publication December 13, 2011.

Address reprint requests to Massimiliano Mirabella, M.D., Ph.D. or

Roberta Morosetti, M.D., Department of Neurosciences, Institute of Neu-

rology, Università Cattolica, Largo A. Gemelli 8, Rome, 00168 Italy. E-mail:

mirabella@rm.unicatt.it or rmorosetti@rm.unicatt.it.

The American Journal of Pathology, Vol. 180, No. 4, April 2012 

Copyright © 2012 American Society for Investigative Pathology.

Published by Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ajpath.2011.12.027 

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Cell Treatments

To study the effects of TWEAK on mesoangioblasts dif-

ferentiation 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 g/mL) (both from

R&D Systems, Minneapolis, MN).8,26 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 g/mL, as previously de-

scribed.13,27 Treatments were performed all over the dif-

ferentiation 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., Eu-

gene, 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 di-

rected 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 man-ufacturer’s instructions.

Twenty-four hours after transfection, cells were shifted

to myoblast-conditioned medium for 4 days and differen-

tiation medium for 7 days to induce skeletal muscle dif-

ferentiation. At different time points, cells were fixed for

immunocytochemistry or harvested for Western blot anal-

ysis. 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 exper-

imental 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-m-thick unfixed cryostat mus-

cle sections and on cells cultured on gelatin-coated op-

tical quality plastic -Dishes (ibidi, Munich, Germany)

and fixed with 4% paraformaldehyde in 0.1 M phosphate

buffer for 15 minutes.

The following primary antibodies were used: monoclo-

nal anti-skeletal Myosin (Sigma, St. Louis, MN), poly-

clonal 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), monoclo-

nal anti-CD68 (Covance, Princeton, New Jersey), poly-

clonal anti-MyoD (C-20, Santa Cruz Biotechnology, SantaCruz, CA).

 Table 1. Patients Included in the Study and Muscle of Origin

Patient Age (years) Sex Muscle Patient Age (years) Sex Muscle

IBM

1 66 M Q 9 64 M Q2 76 M D 10 76 M Q3 84 M D 11 61 F D4 79 F Q 12 60 F D5 62 F Q 13 62 M D6 72 M D 14 43 M D7 56 M D 15 79 M D8 78 F Q 16 66 M D

DM

1 32 M D 6 74 M D2 15 M Q 7 52 M D3 37 F D 8 53 F D4 54 F D 9 78 F Q5 44 M Q 10 71 M D

PM

1 77 F Q 6 71 M D2 56 F D 7 72 M D3 74 F D 8 73 F D4 22 M D 9 84 M D

5 59 F QCT

1 43 F D 5 80 F Q2 27 M D 6 78 F Q3 51 F D 7 83 M D4 80 M Q 8 67 M Q

CT, control; D, deltoid; DM, dermatomyositis; F, female; M, male; IBM, inclusion-body myositis; PM, polymyositis; Q, quadriceps.

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In IBM muscle biopsies, a very strong Fn14 immuno-

reactivity 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 mor-

phologically abnormal vacuolated and nonvacuolated fi-

bers and hypotrophic fibers) showed strong Fn14 mem-

brane 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. Like-

wise in PM, where a large number of MHCd-positive

regenerating fibers was seen, only rare abnormal fibers(probably damaged or degenerating) exhibited a mem-

brane Fn14 immunostaining (Figure 2B). In all control

muscles, staining for TWEAK and Fn14 were both negli-

gible (not shown). Omission of the primary antibody or

use of a nonrelevant primary antibody completely abol-

ished specific staining for TWEAK and Fn14.

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 mesoangio-

blasts samples expressed the TWEAK receptor, but IBM

mesoangioblasts showed a much higher expression of

Fn14 compared to PM, DM, and normal controls (Figure3, A and B).

Effects of TWEAK Treatment and TWEAK 

Neutralization on Differentiating

Mesoangioblasts

We previously demonstrated that IBM mesoangio-

blasts exhibit a differentiation block down skeletal mus-

cle, whereas DM (as well as PM and normal mesoan-

gioblasts) 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.9 The inhibitory effect of

TWEAK/TNFSF12 on myotubes formation was specific

since upon co-treatment with the anti-TWEAK/TNFSF12

neutralizing antibody (10g/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 ob-served in IBM mesoangioblasts. Then we explored the

Figure 1. Expression 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 withanti-CD68 antibody) and also detected along the sarcolemma of muscle fibers surrounded or partially invaded by monocytes (arrowhead ). In IBM muscle, a largenumber of fibers are TWEAK-positive, regardless the presence of nearby inflammatory cells. In DM, only muscle fibers invaded or surrounded by monocytes anddegenerating fibers next to inflammatory infiltrates show increased membrane TWEAK immunoreactivity. Scale bars, 80 m in upper and lower rows, and 20m 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 cytoplasmicimmunoreactivity 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 m. 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. Arepresentative experiment out of three is shown.

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effect of recombinant human Fn14-Fc chimera (1g/mL) on

IBM mesoangioblasts. The addition of Fn14-Fc protein con-

sistently led to the formation of spare multinucleated myo-

tubes, indicating the ability of Fn14-Fc chimera to partially

revert the inhibitory effects of TWEAK (Figure 4B).

 siRNA Inhibition of TWEAK Expression Restores

Myogenic Differentiation in IBM

Mesoangioblasts

To further investigate the possible inhibitory role ofTWEAK on the differentiation process of IBM mesoangio-

blasts, we examined the effect of siRNA-mediated sup-

pression 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 me-

dium, siRNA-transfected IBM mesoangioblasts were able

to fuse into multinucleated myosin-positive myotubes (fu-

sion index, 0.55 0.21), whereas control IBM mesoan-gioblasts displayed a differentiation defect with only

spare, mainly bi-nucleated, myosin-positive myotubes.

As expected, we observed a dramatic increase of myo-

tubes formation in DM siRNA-treated mesoangioblasts

(Figure 5B).

Different studies have shown that TWEAK has the ca-

pability to activate the NFB pathway, thus promoting cell

proliferation and inhibition of skeletal muscle differentia-

tion, in part through MyoD inactivation.8,10 Recently, it

has been demonstrated that genetic ablation or overex-

pression of TWEAK leads to suppression or activation of

NFB in regenerating myofibers, respectively.14 Interest-

ingly, siRNA-mediated suppression of TWEAK dimin-ished NFB 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 capabil-

ity to differentiate into multinucleated myotubes (Figure

5C). Moreover TWEAK siRNA induced Akt phosphoryla-

tion (Figure 5D) and enhanced MyoD levels in differenti-

ating 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 col-

lected from human mesoangioblasts during skeletal mus-

cle differentiation. In particular, supernatants were col-

Figure 2. Expression of Fn14 in muscle biopsy of inclusion-body myositis(IBM), dermatomyositis (DM) and polymyositis (PM) patients. A: In IBMmuscle, very strong Fn14 immunoreactivity is present as sarcolemmal stain-ing in muscle fibers partially invaded or surrounded by infiltrating monocytes(identified by double staining with anti-CD68 antibody). In PM, in addition tomonocytes, only muscle fibers undergoing invasion by macrophages show

 weak Fn14 immunoreactivity. Scales bar, 80 m in the top row, 20 m in themiddle row, and 40 m 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 intenseFn14 membrane staining, also independent from the presence of inflamma-tory cells. The rare regenerating fibers (identified by double staining withanti-MHCd antibody) usually do not have Fn14 expression (arrows) exceptfor one isolated morphologically abnormal fiber (arrowhead ). Likewise inPM, where a large quantity of Myosin heavy chain developmental type(MHCd)-positive regenerating fibers is seen, only rare abnormal fibers (prob-

ably damaged or degenerating fibers) exhibit a positive membrane Fn14immunostaining (arrows). Scale bar 80 m.

Figure 3. Expression of Fn14 in mesoangioblasts from inclusion-body my-ositis (IBM), dermatomyositis (DM) and polymyositis (PM), and controls.Mesoangioblasts from IBM muscles express a higher level of Fn14 thanmesoangioblasts isolated from DM, PM, and normal control muscles. -actinserved as a loading control. A: A representative Western blot out of three isshown. B: Densitometric analysis from three different Western blots per-formed for 8 IBM samples, 2 DM, 1 PM, and 1 normal control. Ratios werecalculated between Fn14 and -actin values. Data represent the mean SD.

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lected from mesoangioblasts of IBM, DM, PM patients,

and normal controls after 7 days in serum-free differenti-

ation 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 con-

ducted on supernatants from age-matched mesoangio-

blast cultures (4 IBM, 3 DM, 3 PM, and 4 controls), thus

Figure 4. Effects 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, nucleiare labeled in blue by Hoechst staining). A: Dermatomyositis (DM) mesoangioblasts treated with TWEAK (100 ng/mL) show a significant decrease of myotubeformation compared to untreated DM cells. Specificity of the negative modulatory effect of TWEAK treatment on DM mesoangioblasts is demonstrated by therestoration of normal myogenic differentiation on a combination treatment of TWEAK (100 ng/mL) and anti-TWEAK neutralizing antibody (10 g/mL). B:Treatment with recombinant human Fn14-Fc chimera (1 g/mL) induces significant skeletal muscle differentiation of inclusion-body myositis (IBM) mesoangio-blasts. Scale bar 80 m. Each experiment was performed in duplicate at least 2 times for each patient.

Figure 5. Induction of myogenic differentiation of inclusion-body myositis (IBM) and dermatomyositis (DM) mesoangioblasts by tumor necrosis factor-like weakinducer of apoptosis (TWEAK) small-interfering RNA (siRNA). A pre-designed chimera siRNA oligonucleotide directed against human TWEAK was transfected intoIBM and DM mesoangioblasts. A: Efficiency of transfection. Forty-eight hours after transfection, cells were harvested for RNA extraction and reverse transcription-polymerase chain reaction analysis. -actin served as a control. B: After 24 hours, siRNA-transfected cells were exposed to myoblast-conditioned medium for 4days and subsequently shifted to differentiation medium. After 7 days of culture, cells were stained with anti-myosin antibody (red) or harvested for Western blotanalysis. Representative cultures from three different IBM patients and 1 DM patient are shown. siRNA-transfected IBM mesoangioblasts were able to fuse intomultinucleated myosin-positive myotubes, whereas control IBM mesoangioblasts displayed a differentiation defect with only spare and mainly bi-nucleatedmyosin-positive cells. A dramatic increase of myotubes formation was also observed in DM siRNA-treated mesoangioblasts. Scale bar 100 m. C: Representative

 Western blot showing decrease of NFB phospho-p65 (Ser536) subunit (pNFB-p65) in total lysates of siRNA-transfected IBM and DM mesoangioblasts (total). Western blot on nuclear extracts shows marked reduction of pNFB-p65 and, to a lesser extent, of total NFB levels after siRNA (nuclear). An anti-Retinoic X receptor (RXR ) antibody was used as loading control for nuclear fraction. D: Representative Western blots showing induction of MyoD expression and Aktphosphorylation (pAkt) in siRNA-transfected IBM mesoangioblasts. -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 m.

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confirming that such increased TWEAK secretion wasindeed 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 su-

pernatants 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.

Discussion 

Various cytokines secreted by inflammatory cells directly

influence the properties of progenitor cells that reside in

tissues undergoing repair and regeneration.28 Likewise,

soluble factors secreted by macrophages or muscle fi-

bers themselves, and delivered into the muscle microen-

vironment, can also drive the proliferation of satellite cells

during muscle regeneration.29 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, persistentTWEAK-Fn14 axis signaling is believed to play a role in

human diseases caused by an excessive or abnormal

inflammatory response (eg, systemic lupus erythemato-

sus, rheumatoid arthritis, multiple sclerosis).27,30–32 As a

matter of fact, TWEAK-Fn14 inhibition is efficacious in

experimental autoimmune encephalitis models.33 When

persistently activated in chronic inflammatory disorders,

TWEAK has been demonstrated to promote abnormal

hyperplasia and angiogenesis, and proliferation of pro-

genitor cells with inhibition of their differentiation.12

Several studies8,10,13 have previously shown that the

interplay between TWEAK and its receptor Fn14 is crucial

for the proliferation and differentiation of murine myo-blasts. In fact, TWEAK is able to potently inhibit myotube

formation and the expression of muscle-specific tran-scription factors in C2C12 cells and in primary murine

myoblasts.8

In a previous study,21 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 iso-

lated from IBM, DM, and PM patients. Both TWEAK and

Fn14 were increased in IBM skeletal muscle. Moreover,

TWEAK was able to inhibit skeletal muscle differentiationof human mesoangioblasts. In fact, our in vitro experi-

ments demonstrate that TWEAK/TNFSF12 treatment led

to significant reduction of skeletal muscle differentiation

(fusion index, mean myotube diameter) of DM mesoan-

gioblasts. 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 de-

tectable after TWEAK/TNFSF12 treatment. As expected

in progenitor cells of mesenchymal lineage, all mesoan-

gioblasts expressed the TWEAK receptor; however IBM

mesoangioblasts showed a much higher expression of

Fn14 compared to PM, DM, and normal controls. Giventhat IBM mesoangioblasts are greatly defective in skele-

tal 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 effi-

ciency, thus suggesting a possible role of TWEAK-Fn14

activation in the differentiation block of IBM mesoangio-

blasts. 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 me-

soangioblasts were able to fuse into multinucleated my-

osin-positive myotubes, whereas control IBM mesoangio-blasts produced only spare myosin-positive cells. As

Figure 6. Quantification by enzyme-linked immunosorbent assay of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) concentration in mediumfrom 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) (3patients), 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 weredetected in medium collected from siRNA-treated mesoangioblasts ( *P  0.05). Mean values from three wells of one representative experiment out of twoindependent ones are shown.

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expected, we also observed a dramatic increase of myo-

tubes 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 secretethe cytokine and further support the hypothesis of a neg-

ative role of TWEAK on skeletal muscle differentiation of

human mesoangioblasts, similar to that described in mu-

rine myoblasts.8–10

TWEAK is constitutively expressed in adult murine

skeletal muscle, although it is known that its receptor is

not significantly expressed. Catabolic conditions, how-

ever, 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.11 Our study shows that TWEAK pro-

tein expression was increased in muscle homogenates of

IBM compared to DM and PM and by using immunohis-tochemistry, this cytokine was detectable in a large num-

ber of non-necrotic fibers and in fibers close to or in-

vaded 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 re-

sponse seen in PM/DM. It is well-known that proinflam-

matory cytokines (such as TNF, interleukin-1, and in-

terleukin-6) produced by both the immune system and

skeletal muscle, stimulate proliferation of progenitor cells

and inhibit terminal differentiation of myoblasts into ma-

ture fibers.34,35 Similarly, a transient rapid TWEAK up-

regulation after acute inflammatory injury may be instru-

mental 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 in-

flammation may negatively influence the activation of pro-

myogenic pathways exacerbating the inadequate regen-

eration of myofibers typical of the disease.

The widespread expression of Fn14 observed by im-

munohistochemistry in IBM nonregenerating muscle fi-

bers is in agreement with the fact that Fn14 is highlyregulated 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 myo-

blasts.10 Therefore, Fn14 expression in IBM muscle could

also reflect a positive attempt of myofibers to activate

pro-myogenic signaling pathways, because multiple po-

tential repairing mechanisms normally involved in muscle

development are usually activated in abnormal muscle

fibers of IBM.36,37

Previous studies have shown the capability of TWEAKto activate the NFB 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 NFB in regener-

ating myofibers, respectively.14,38 Of note, we observed

a reduced activation of NFB in siRNA-transfected IBM

mesoangioblasts, which displayed an increased capabil-

ity to differentiate into multinucleated myotubes associ-ated 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 expres-

sion of several known NFB-target genes and of Fn14

itself.39 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 tis-

sue) 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.2

Whatever the mechanism of Fn14 activation, however, itremains 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 pre-

cursor cells in vivo. TWEAK could represent an important

linkage molecule connecting the persisting inflammation

with the defective regeneration and the progressive mus-

cle 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 specifi-

cally 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 my-

opathy showing an increase of TWEAK-Fn14 axis in skel-

etal muscle. This may open the way to selective targeting

of TWEAK-Fn14 to concurrently suppress degenerative

and inflammatory components of IBM and counteractmuscle atrophy, a strategy that may prove to be thera-

peutically rewarding.

The results of the present study provide the first evi-

dence that TWEAK is a negative regulator of mesoangio-

blasts 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 mesoangio-

blasts isolated from IBM muscle fail to normally differen-

tiate into skeletal muscle, to use autologous mesoangio-

blasts in a possible muscle regenerative cell therapy, it is

indispensable to stimulate these cells in vitro to enhancetheir defective myogenic differentiation. It would be even

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more important to successfully activate endogenous me-

soangioblasts 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 mesoangio-blasts. Modulation of such target molecules, that are se-

lectively 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 

1. Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C,

Garcia I, Browning JL: TWEAK, a new secreted ligand in the tumor

necrosis factor family that weakly induces apoptosis. J Biol Chem

1997, 272:32401–32410

2. Winkles JA: The TWEAK-Fn14 cytokine-receptor axis: discovery, bi-

ology and therapeutic targeting. Nat Rev Drug Discov 2008, 7:411–

425

3. Marsters SA, Sheridan JP, Pitti RM, Brush J, Goddard A, Ashkenazi A:

Identification of a ligand for the death-domain-containing receptor

Apo3. Curr Biol 1998, 8:525–528

4. Wiley SR, Winkles JA: TWEAK, a member of the TNF superfamily, is a

multifunctional cytokine that binds the TWEAKR/Fn14 receptor. Cyto-

kine Growth Factor Rev 2003, 14:241–2495. Brown SA, Ghosh A, Winkles JA: 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

6. Meighan-Mantha RL, Hsu DK, Guo Y, Brown SA, Feng SL, Peifley KA,

Alberts GF, Copeland NG, Gilbert DJ, Jenkins NA, Richards CM,

Winkles JA: The mitogen-inducible Fn14 gene encodes a type I

transmembrane protein that modulates fibroblast adhesion and mi-

gration. J Biol Chem 1999, 274:33166–33176

7. Feng SL, Guo Y, Factor VM, Thorgeirsson SS, Bell DW, Testa JR,

Peifley KA, Winkles JA: The Fn14 immediate-early response gene is

induced during liver regeneration and highly expressed in both hu-

man and murine hepatocellular carcinomas. Am J Pathol 2000, 156:

1253–1261

8. Dogra C, Changotra H, Mohan S, Kumar A: Tumor necrosis factor-like

weak inducer of apoptosis inhibits skeletal myogenesis through sus-

tained activation of nuclear factor-kappaB and degradation of MyoD

protein. J Biol Chem 2006, 281:10327–10336

9. Dogra C, Changotra H, Wedhas N, Qin X, Wergedal JE, Kumar A:

TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal

muscle-wasting cytokine. FASEB J 2007, 21:1857–1869

10. Dogra C, Hall SL, Wedhas N, Linkhart TA, 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 myogen-

esis. J Biol Chem 2007, 282:15000–15010

11. Mittal A, Bhatnagar S, Kumar A, Lach-Trifilieff E, Wauters S, Li H,

Makonchuk DY, Glass DJ, 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

12. Burkly LC, Michaelson JS, Hahm K, Jakubowski A, Zheng TS:

TWEAKing tissue remodeling by a multifunctional cytokine: role of

TWEAK/Fn14 pathway in health and disease. Cytokine 2007, 40:1–16

13. Girgenrath M, Weng S, Kostek CA, Browning B, Wang M, Brown SA,Winkles JA, Michaelson JS, Allaire N, Schneider P, Scott ML, Hsu YM,

Yagita H, Flavell RA, Miller JB, Burkly LC, Zheng TS: TWEAK, via its

receptor Fn14, is a novel regulator of mesenchymal progenitor cells

and skeletal muscle regeneration. EMBO J 2006, 25:5826–5839

14. Mittal A, Bhatnagar S, Kumar A, Paul PK, 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

15. Engel WK, Askanas V. Inclusion-body myositis: clinical, diagnostic,

and pathologic aspects. Neurology. 2006, 66(2 Suppl 1):S20–S2916. Needham M, Mastaglia FL: Inclusion body myositis: current patho-

genetic concepts and diagnostic and therapeutic approaches. Lan-

cet Neurol 2007, 6:620–631

17. Askanas V, Engel WK: Sporadic inclusion-body myositis: conforma-

tional multifactorial ageing-related degenerative muscle disease as-

sociated with proteasomal and lysosomal inhibition, endoplasmic

reticulum stress, and accumulation of amyloid-42 oligomers and

phosphorylated tau. Presse Med 2011, 40(4 Pt 2):e219–e235

18. Dalakas MC: Sporadic inclusion body myositis – diagnosis, patho-

genesis and therapeutic strategies. Nat Clin Pract Neurol 2006,

2:437–447

19. Askanas V, Engel WK, Nogalska A: Inclusion body myositis: a de-

generative 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

20. Dalakas MC: Immunotherapy of myositis: issues, concerns and future

prospects. Nat Rev Rheumatol 2010, 6:129–137

21. 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 PA, 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

22. Coletti D, Yang E, Marazzi G, Sassoon D: TNF inhibits skeletal

myogenesis through a PW1-dependent pathway by recruitment of

caspase pathways. EMBO J 2002, 21:631–642

23. Yang CC, Askanas V, Engel WK, Alvarez RB: Immunolocalization of

transcription factor NF-kappa B in inclusion-body myositis muscle

and at normal human neuromuscular junctions. Neurosci Lett 1998,

254(2):77–80

24. Nogalska A, Wojcik S, Engel WK, McFerrin J, Askanas V: Endoplas-

mic reticulum stress induces myostatin precursor protein and NF-

kappaB in cultured human muscle fibers: relevance to inclusion body

myositis. Exp Neurol 2007, 204(2):610 –618

25. Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B,

Perani L, Innocenzi A, Galvez BG, Messina G, Morosetti R, Li S,

Belicchi M, Peretti G, Chamberlain JS, Wright WE, Torrente Y, Ferrari

S, Bianco P, Cossu G: Pericytes of human skeletal muscle are myo-

genic precursors distinct from satellite cells. Nat Cell Biol 2007,

9:255–267

26. 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 differenti-

ation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Arthritis Res Ther 2006, 8:R146

27. Perper SJ, Browning B, Burkly LC, Weng S, Gao C, Giza K, Su L,

Tarilonte L Crowell T, Rajman L, Runkel L, Scott M, Atkins GJ, Findlay

DM, Zheng TS, Hess H: TWEAK is a novel arthritogenic mediator.

J Immunol 2006, 177:2610–2620

28. Duffield JS: The inflammatory macrophage: a story of Jekyll and

Hyde. Clin Sci (Lond) 2003, 104:27–38

29. Merly F, Lescaudron L, Rouaud T, Crossin F, Gardahaut MF: Macro-

phages enhance muscle satellite cell proliferation and delay their

differentiation. Muscle Nerve 1999, 22:724–732

30. Kumar M, Makonchuk DY, 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

31. Liu ZC, Zhou QL, Li XZ, Yang JH, Ao X, Veeraragoo P, Zuo XX:

Elevation of human tumor necrosis factor-like weak inducer of apop-

tosis in peripheral blood mononuclear cells is correlated with disease

activity and lupus nephritis in patients with systemic lupus erythem-

atosus. Cytokine 2011, 53:295–300

32. Desplat-Jégo S, Feuillet L, Creidy R, Malikova I, Rance R, Khrest-chatisky M, Hahm K, Burkly LC, Pelletier J, Boucraut J: TWEAK is

10 Morosetti et alAJP April 2012, Vol. 180, No. 4 

AQ: 14

8/3/2019 TWEAK Inhibits Myogenesis in Myositis AJP

http://slidepdf.com/reader/full/tweak-inhibits-myogenesis-in-myositis-ajp 11/11

expressed at the cell surface of monocytes during multiple sclerosis.

J Leukoc Biol 2009, 85:132–135

33. Mueller AM, 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

34. Langen RC, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger

YM: Inflammatory cytokines inhibit myogenic differentiation through ac-tivation of nuclear factor-kappa B. FASEB J 2001, 15:1169–1180

35. Langen RC, Van Der Velden JL, Schols AM, Kelders MC, Wouters EF,

Janssen-Heininger YM: Tumor necrosis factor-alpha inhibits myo-

genic differentiation through MyoD protein destabilization. FASEB J

2004, 18:227–237

36. Broccolini A, Gidaro T, Morosetti R, Gliubizzi C, Servidei T, Pescatori

M, Tonali PA, 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

37. Broccolini A, Ricci E, Pescatori M, Papacci M, Gliubizzi C, D’Amico A,

Servidei S, Tonali PA, Mirabella M: Insulin-like growth factor I in

inclusion-body myositis and human muscle cultures. J Neuropathol

Exp Neurol 2004, 63:650–659

38. Li H, Mittal A, Paul PK, Kumar M, Srivastava DS, Tyagi SC, 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 andp38 mitogen-activated protein kinase: a potential role of MMP-9 in

myopathy. J Biol Chem 2009, 284:4439–4450

39. Tran NL, McDonough WS, Savitch BA, Fortin SP, Winkles JA, Symons

M, Nakada M, Cunliffe HE, Hostetter G, Hoelzinger DB, Rennert JL,

Michaelson JS, Burkly LC, Lipinski CA, Loftus JC, Mariani L, Berens

ME: 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

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