Genomic Profiling of Messenger RNAs and MicroRNAs
Reveals Potential Mechanisms of TWEAK-Induced
Skeletal Muscle Wasting in Mice
Siva K. Panguluri1., Shephali Bhatnagar1., Akhilesh Kumar1, John J. McCarthy2, Apurva K. Srivastava3,
Nigel G. Cooper1, Robert F. Lundy1, Ashok Kumar1*
1Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky, United States of America, 2Department of
Physiology, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America, 3Laboratory of Human Toxicology and Pharmacology, Applied &
Developmental Research Directorate SAIC-Frederick, National Cancer Institute, Frederick, Maryland, United States of America
Background: Skeletal muscle wasting is a devastating complication of several physiological and pathophysiological
conditions. Inflammatory cytokines play an important role in the loss of skeletal muscle mass in various chronic diseases. We
have recently reported that proinflammatory cytokine TWEAK is a major muscle-wasting cytokine. Emerging evidence
suggests that gene expression is regulated not only at transcriptional level but also at post-transcriptional level through the
expression of specific non-coding microRNAs (miRs) which can affect the stability and/or translation of target mRNA.
However, the role of miRs in skeletal muscle wasting is unknown.
Methodology/Principal Findings: To understand the mechanism of action of TWEAK in skeletal muscle, we performed
mRNA and miRs expression profile of control and TWEAK-treated myotubes. TWEAK increased the expression of a number
of genes involved in inflammatory response and fibrosis and reduced the expression of few cytoskeletal gene (e.g. Myh4,
Ankrd2, and TCap) and metabolic enzymes (e.g. Pgam2). Low density miR array demonstrated that TWEAK inhibits the
expression of several miRs including muscle-specific miR-1-1, miR-1-2, miR-133a, miR-133b and miR-206. The expression of a
few miRs including miR-146a and miR-455 was found to be significantly increased in response to TWEAK treatment.
Ingenuity pathway analysis showed that several genes affected by TWEAK are known/putative targets of miRs. Our cDNA
microarray data are consistent with miRs profiling. The levels of specific mRNAs and miRs were also found to be similarly
regulated in atrophying skeletal muscle of transgenic mice (Tg) mice expressing TWEAK.
Conclusions/Significance: Our results suggest that TWEAK affects the expression of several genes and microRNAs involved
in inflammatory response, fibrosis, extracellular matrix remodeling, and proteolytic degradation which might be responsible
for TWEAK-induced skeletal muscle loss.
Citation: Panguluri SK, Bhatnagar S, Kumar A, McCarthy JJ, Srivastava AK, et al. (2010) Genomic Profiling of Messenger RNAs and MicroRNAs Reveals Potential
Mechanisms of TWEAK-Induced Skeletal Muscle Wasting in Mice. PLoS ONE 5(1): e8760. doi:10.1371/journal.pone.0008760
Editor: Gisela Nogales-Gadea, University Hospital Vall d’Hebron, Spain
Received October 27, 2009; Accepted December 24, 2009; Published January 19, 2010
Copyright: ? 2010 Panguluri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grant RO1 AG129623 to AK. Support from NCRR award P20RR16481 is also gratefully
acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Skeletal muscle wasting or atrophy is a major cause of human
morbidity [1,2,3]. Proinflammatory cytokines are the key
mediators of muscle-wasting in various chronic conditions [4,5].
Besides directly inducing the degradation of selective muscle
proteins [6,7], elevated levels of inflammatory cytokines cause
extracellular matrix abnormalities  and prevents the regener-
ation of skeletal muscle fibers by inhibiting the differentiation of
muscle progenitor cells into myofibers [9,10]. Accumulating
evidence suggests that bulk of the muscle protein degradation in
atrophying skeletal muscle occurs through the activation of
ubiquitin-proteasome system [4,11,12]. In addition, it has been
also found that muscle-wasting conditions involve the activation of
nuclear-factor-kappa B (NF-kB), a proinflammatory transcription
factor, which regulates the expression of large number of genes
including the components of ubiquitin-proteasome system [1,13].
Specific inhibition of NF-kB activity has been found to attenuate
loss of skeletal muscle mass in response to various catabolic stimuli
including proinflammatory cytokines, tumor load, denervation,
and unloading [13,14,15,16].
TNF-like weak inducer of apoptosis (TWEAK) is an important
inflammation-related cytokine belonging to TNF super family
ligands [17,18]. The actions of TWEAK in target cells are
mediated through its binding to Fn14, a type I transmembrane
receptor, belonging to the TNF receptor super family [17,19].
Recently, we have reported that treatment of myotubes with
TWEAK leads to the degradation of select muscle proteins, which
PLoS ONE | www.plosone.org1January 2010 | Volume 5 | Issue 1 | e8760
in turn leads to atrophy, thus signifying TWEAK as a major
muscle-wasting cytokine . In fact, we have found that at
equimolar concentrations, TWEAK is more potent than its
structural homologue and well known muscle-wasting cytokine
TNF-a to induce the degradation of myosin heavy chain (MyHC)
in cultured myotubes . Chronic administration of soluble
TWEAK protein or transgenic overexpression of TWEAK in mice
also causes significant muscle-wasting . TWEAK also inhibits
the differentiation of myoblasts into multinucleated myotubes and
induces the degradation of myogenic regulatory factors (MRFs)
such as MyoD [21,22]. Our recent studies have further suggested
that the expression of TWEAK receptor Fn14 is increased in
skeletal muscle in disuse conditions (e.g. immobilization, unload-
ing, and denervation) and TWEAK is the major mediator of
skeletal muscle loss in response to denervation (Mittal et al.,
unpublished observations). However, the underpinning mecha-
nisms by which TWEAK induces skeletal muscle loss remain
Previous examinations of genome-wide gene expressions in
skeletal muscle has helped in identifying several known and novel
genes which mediate the loss of skeletal muscle mass in disuse
conditions such as unloading, sarcopenia, starvation, and dener-
vation [23,24,25,26,27]. However, the effects of proinflammatory
cytokines such as TWEAK on the gene expression and
intracellular pathways related to the acquisition and maintenance
of skeletal muscle mass remain unknown. MicroRNAs (miRNAs
or miRs), a new class of non-translating RNAs, plays critical role as
molecular switches for complex and extensive regulatory web
involving thousands of genes [28,29]. MicroRNAs are small 18 to
22 nucleotide long RNA molecules, which negatively regulate
expression of target genes by binding to specific sequences in
39UTR where partial complementarities inhibit their translation
and perfect complementarily induces degradation of mRNA
[28,29]. With the advent of these tiny regulatory RNAs, the
complexity of understanding the regulatory mechanisms of many
important pathways has been resolved [28,29,30]. miRs have been
shown to regulate a range of biological processes including
tumorigenesis, development of the limb, lung and hematopoietic
systems, and adipogenesis . Furthermore, a few skeletal muscle
specific miRs (e.g. miR-1, miR-133, and miR-206) have been
characterized as modulators of myogenic cells proliferation and
differentiation [31,32,33] and there is increasing evidence about
the involvement of miRs in skeletal muscle disorders such as
muscular dystrophy [34,35,36]. However, the role of miRs and
their potential gene targets in atrophying skeletal muscle remain
completely unknown. Identification and understanding the
mechanisms of actions of miRs that are differentially regulated
in atrophying skeletal muscle may provide novel molecular targets
towards therapeutic approaches in muscle-wasting.
In this study, using cDNA microarray, low density microRNA
array, TaqMan PCR assays, and bioinformatics tools, we have
investigated the potential mechanisms by which TWEAK
regulates skeletal muscle mass. Our results suggest that TWEAK
modulates the expression of selective muscle genes and miRs in
cultured myotubes and in skeletal muscle-specific TWEAK-Tg
mice. Furthermore, bioinformatics analyses of differentially
regulated genes and miRs have shown that TWEAK affects
diverse cellular responses such as proliferation, musculature
development, inflammation, and adipocyte formation.
We have previously shown that treatment of C2C12 myotubes
with TWEAK augments the expression of muscle-specific E3
ubiquitin ligases atrogin and MuRF1 and augments the ubiqui-
tination of select muscle proteins within 12–24h of treatment .
In this study, we have performed mRNA and miRNA profiling
after 18h of TWEAK treatment to detect the expression of both
early and late responsive genes. To validate the effects of TWEAK
on expression of various genes and miRs in vivo, we have also
employed TWEAK-Tg mice. We have previously reported that
transgenic mice expressing very high levels (.14 fold) of TWEAK
in skeletal muscle died at perinatal/neonatal age . However,
the mice which expressed relatively low levels of TWEAK (4–5
folds higher than littermate controls) survived and developed into
adulthood. Our recent analysis of skeletal muscle revealed that
TWEAK-Tg mice show significant muscle fiber atrophy at the age
of 4–6 months (Mittal et al (2009), unpublished observation).
Therefore, we used skeletal muscle from 6-months old TWEAK-
Tg and their littermate control mice.
Microarray Analysis of Global Gene Expression in TWEAK-
Treated C2C12 Myotubes
C2C12 myotubes were treated with TWEAK (10ng/ml) and
the mRNA level of different genes was monitored by cDNA
microarray technique. The microarray gene expression profile
appeared normally distributed for TWEAK-treated samples
(Figure 1A) indicating that our analysis of differentially expressed
gene is not biased due to skewed distribution of certain genes. Out
of approximately 25,000 genes present on our microarray chips,
TWEAK significantly (p,0.05) affected the expression of a total of
6,938 genes (2,841 up regulated and 4,097 down regulated). Top
50 up-regulated and top 50 down-regulated known genes in
TWEAK-treated myotubes are presented in Table S1. Further
analysis of differentially regulated genes showed that about 67
genes were up-regulated and 26 down-regulated with fold values
$1.5 and p-value of #0.05. Only 12 up-regulated genes (e.g.
Nfkbia, Taf2 and Slc2a6 etc.) were with fold values $2. We
further observed that 13 out of 26 significantly down-regulated
genes were less than 2-fold and the functions of 10 genes with fold
value more than two is not yet known (Table. 1). Row and
normalized data of this microarray experiment has been submitted
to ArrayExpress database (http://www.ebi.ac.uk/microarray-as/
ae/) with accession number E-MEXP-2432.
Independent QRT-PCR assays were performed for the genes
which showed high fold change and/or have a direct or indirect
relation with skeletal muscle wasting. As shown in Figure 1B, the
expression of Nfkbia, Nfkb2, Psmb10, cyclin D1, Map3k14, and
Mmp9 was found to be significantly increased in TWEAK-treated
samples in QRT-PCR assays. Similarly, the reduced expression of
Notch1, Pgam2, Ankrd2, TCap, MyHC4, MMP-2 and TIMP2 in
TWEAK-treated samples was confirmed by independent QRT-
PCR assays (Figure 1C) suggesting direct correlation between
microarray and QRT-PCR analysis for almost all the genes tested.
Consistent with their mRNA levels, the protein levels of NF-kB2,
and MMP-9 were also increased whereas the levels of Notch1 and
MMP-2 were reduced in TWEAK-treated myotubes determined
by Western Blot (Figure 1D). QRT-PCR analysis further showed
that the expression levels of Nfkbia, Nfkb2, and Map3k14 were
significantly up-regulated in skeletal muscle of TWEAK-Tg mice
(Figure 2A). However, in contrast to TWEAK-treated myotubes,
the expression of Psmb1 was found to be significantly reduced in
skeletal muscle of TWEAK-Tg mice compared to littermate
control mice (Figure 2A). Although the exact reasons for this
anomalous regulation of Psmb1 in cultured myotubes and
TWEAK-Tg mice is not yet clear, it is possible that continued
presence of TWEAK in skeletal muscle of transgenic animal may
lead to its reduced expression due to compensatory (negative feed-
Effects of TWEAK on Muscles
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back) mechanisms. On the other hand, the reduced mRNA levels
of Notch1, phosphoglycerate mutase 2 (PGAM2), ankyrin repeat
domain 2 (Ankrd2), and TCap in skeletal muscle of TWEAK-Tg
mice (Figure 2B) was consistent with the data obtained in
TWEAK-treated C2C12 myotubes. Consistent with cell culture
data, the protein level of NF-kB2 was increased whereas the levels
of Notch1 and TIMP-2 were diminished in gastrocnemius muscle
of TWEAK-Tg mice compared to control mice (Figure 2C).
Collectively, these data indicate that our microarray analysis
represents the set of the genes that are differentially regulated in
response to TWEAK.
TWEAK Regulates the Activity of Toxic Pathways in
To understand the effects of TWEAK on various canonical
pathways, we used Ingenuity Pathway Analysis (IPA) software. We
first used a set of differentially regulated genes with fold values
$1.5 and p-value of #0.05 in microarray analysis as an input in
IPA software. However, this set of genes was not sufficient to
generate pathways affected by TWEAK. We then reduced the
stringency and used the set of genes with fold change (both up- and
down-regulated genes) values $1.2 and p-value of #0.05 in the
microarray experiment. We found that TWEAK affects the
expression of genes that are involved in distinct molecular
pathways. The major pathways affected by TWEAK in myotubes
were those that regulate hepatic fibrosis, oxidative stress, NF-kB,
mitochondrial dysfunction, TGF-b, and anti-apoptotic response
(Table 2). Interestingly, our bioinformatics analysis of pathways
using differentially regulated gene is consistent with the experi-
mental evidence that skeletal muscle-wasting and other muscular
disorders such as muscular dystrophy involves the activation of
some/all of these molecular pathways [1,2,4,37,38,39]. These data
Figure 1. Differential expression of genes by TWEAK in C2C12 myotubes. A). Distribution curve of differentially expressed genes in
response to TWEAK treatment detected by cDNA microarray analysis. The normalized fold changes were plotted on y-axis on logarithmic scale. B &
C). C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h followed by isolation of total RNA and QRT-PCR. Untreated cells under similar
conditions were taken as control. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean 6 SD (n=3). The
numbers above the bar represents the fold changes with TWEAK treatment against control, and ‘*’ represents the statistical significance (p-value #
0.01). Data presented here show that mRNA levels of Nfkbia, Nfkb2, cyclinD1, Map3k14, and Mmp9 was significantly increased whereas the levels of
Notch1, Pgam2, Ankrd2, TCap, Mhc4, Mmp2, and Timp2 are reduced in TWEAK-treated C2C12 cells. The relative expression values from the QRT-PCR
analysis were plotted for each gene are mean 6 SD (n=3). The numbers above the bar represents the fold changes with TWEAK treatment against
control, and ‘*’ represents the statistical significance (p-value #0.01). D). Differential expression of NF-kB2, MMP-9, Notch1, and MMP-2. C2C12
myotubes were treated with 10 ng/ml of TWEAK for 18h following isolation of total protein for Western blotting. All the samples were quantified and
equal amounts of proteins were loaded on 10% SDS-PAGE gel. Representative immunoblots from three independent experiments (n=3) presented
here showed that TWEAK treatment increases the protein levels of NF-kB2 and MMP-9 and reduces the levels of Notch1 and MMP-2.
Effects of TWEAK on Muscles
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Table 1. List of differentially expressed genes in TWEAK-treated C2C12 cells by cDNA microarray with p-values #0.05 and fold
Gene Namep-valueFold Gene Description
21.74825ankyrin repeat domain 2 (stretch responsive muscle)
21.66113 phosphoglycerate mutase 2
21.63934Bacillus subtillis sporulation protein (spoOB), GTP-binding protein (obg), phenylalanine
biosynthesis associated protein (pheB), and monofunctional prephenate dehydratase
(pheA) genes, complete cds.
21.63666myosin, heavy polypeptide 4, skeletal muscle
21.61812 proline arginine-rich end leucine-rich repeat
21.60772 RIKEN cDNA 1110059G02 gene
21.5949 Notch gene homolog 1 (Drosophila)
21.5949 RIKEN cDNA 4732473B16 gene
21.55039inhibitor of DNA binding 3
21.53846DEP domain containing 6
21.51745olfactory receptor 297
21.51515nebulin-related anchoring protein
Isyna10.03141.501myo-inositol 1-phosphate synthase A1
Fkhl180.001991.502forkhead-like 18 (Drosophila)
Map3k140.002521.503mitogen-activated protein kinase kinase kinase 14
1110020C13Rik0.009671.503RIKEN cDNA 1110020C13 gene
Mmp90.001721.509matrix metalloproteinase 9
C030034P18Rik0.01271.525RIKEN cDNA C030034P18 gene
V1rd210.000812 1.526 vomeronasal 1 receptor, D21
2310031L18Rik 0.01931.53RIKEN cDNA 2310031L18 gene
Cd200r40.001191.531Cd200 receptor 4
Psmb100.0004341.536proteasome (prosome, macropain) subunit, beta type 10
Olfr13920.01311.544olfactory receptor 1392
Nmyc10.001811.545neuroblastoma myc-related oncogene 1
Zfp9 0.00695 1.553zinc finger protein 9
AF3101340.002891.568Mus musculus krev interaction trapped 1 mRNA, complete cds.
C730014E05Rik 0.04671.569RIKEN cDNA C730014E05 gene
Olfr1860.0002171.577olfactory receptor 186
Effects of TWEAK on Muscles
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Gene Namep-value FoldGene Description
Krt1-140.000641 1.579 keratin complex 1, acidic, gene 14
Cstb0.000164 1.581cystatin B
9430078K10Rik 0.0288 1.582RIKEN cDNA 9430078K10 gene
V1rd18 4.72E-051.602 vomeronasal 1 receptor, D18
Defb1 0.002351.602defensin beta 1
Hsd17b90.00192 1.605 hydroxysteroid (17-beta) dehydrogenase 9
AI1823711.01E-05 1.606 expressed sequence AI182371
B230208H210.00961 1.606 hypothetical protein B230208H21
BC0510760.000235 1.607cDNA sequence BC051076
1200014M14Rik0.00663 1.607RIKEN cDNA 1200014M14 gene
Gzmb0.000451 1.608 granzyme B
Slc9a30.00148 1.609 solute carrier family 9 (sodium/hydrogen exchanger), member 3
4933433J03Rik0.00449 1.634 RIKEN cDNA 4933433J03 gene
mKIAA1696 0.002061.638 Mus musculus mRNA for mKIAA1696 protein.
Adam2 0.00157 1.644a disintegrin and metalloprotease domain 2
Krtap16-2 9.19E-051.673 keratin associated protein 16-2
H2-K1 0.004291.681 histocompatibility 2, K1, K region
Mt2 0.0001121.701 metallothionein 2
C3 0.000787 1.719complement component 3
5730530J16Rik 0.0001731.737 RIKEN cDNA 5730530J16 gene
X66118 0.0006951.742M.musculus mRNA for glutamate receptor subunit GluR5-2c.
Nfkb20.000273 1.762 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, p49/p100
4930432K09Rik 0.0009191.792 RIKEN cDNA 4930432K09 gene
Polr3k 0.02221.798 polymerase (RNA) III (DNA directed) polypeptide K
4930580F03Rik0.000366 1.821 RIKEN cDNA 4930580F03 gene
2300002C06Rik6.83E-051.859RIKEN cDNA 2300002C06 gene
mCD037925 0.0191 1.865
Dlgap20.03791.865discs, large (Drosophila) homolog-associated protein 2
Slc2a63.20E-05 2.142 solute carrier family 2 (facilitated glucose transporter), member 6
E230016D10 0.005752.183 hypothetical protein E230016D10
mCD037850 0.000558 2.204
Nfkbia2.16E-05 2.54 nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha
Taf20.000652.761 TAF2 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 150kDa
Table 1. Cont.
Effects of TWEAK on Muscles
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suggest that TWEAK may utilize many common pathways that
are also activated by other catabolic stimuli to cause the loss of
skeletal muscle mass and accumulation of fibrotic tissues (Table 2).
Identification of Differentially Expressed MicroRNAs
(miRs) in TWEAK-Treated Myotubes
It is estimated that among several thousand human genes, up to
one-third of the mRNA, are potential targets for regulation by
miRNAs encoded in the genome . To understand the
TWEAK-induced regulatory mechanisms that occurs at post-
transcriptional level and involves miRs interaction with a target
site in the mRNA, we investigated the effect of TWEAK on the
expression of various miRs using low density miR array. Out of
nearly 760 miRNAs present in our array experiment, about 150
miRs were differentially regulated by TWEAK with p-value#0.05
and $2-fold change. Some of the important miRs with known/
putative targets and differentially regulated by TWEAK are
presented in Figure 3. Our results showed that TWEAK reduced
the expression of muscle-specific miR-1, miR-133a, miR-133b and
miR-206 in addition to several other miRs including miR-27,
miR-23, miR-93, miR-199, miR-107, and miR-192 (Figure 3A).
Moreover, TWEAK also significantly increased the expression of
miR-715, miR- 146a, miR-455, miR-322, mir-98, and miR-470 in
TWEAK-treated C2C12 myotubes (Figure 3B).
We next investigated whether the expression of some of the
miRs found to be altered in response to TWEAK treatment in our
array experiment can be validated by independent TaqMan
QRT-PCR assays. We studied the expression of miR-1-1, miR-1-
2, miR-133a, miR-133b, miR-206, miR-146a, and miR-455. The
TaqMan QRT-PCR analysis showed directional correspondence
with our low density miRNA-array (Figure 4A). Since our array
experiment and QRT-PCR assays were designed to measure the
levels of only mature miRs, we also investigated whether the
TWEAK regulates the differential expression of these miRNAs at
transcriptional level or at post-transcriptional level by measuring
the expression levels of their processing enzymes using QRT-PCR
assays. Processing of pre-miRs into mature miRs involves a series
of reactions that involves regulatory enzymes such as Dicer,
Dorsha, and Exportin-5. The altered expression of these enzymes
can affect the levels of mature miRs . As shown in Figure 4B,
treatment of myotubes with TWEAK for 18h did not affect the
transcript levels of Dicer, Dorsha, or Exportin-5 indicating that
Figure 2. Differential expression of genes in skeletal muscle of TWEAK-Tg mice. Gastrocnemius muscle of 6 months old TWEAK-Tg mice
and littermate control mice were used for total RNA isolation and QRT-PCR analysis. The relative expression values from the QRT-PCR analysis were
plotted for each gene are mean 6 SD (n=3). The numbers above the bar represents the fold changes in TWEAK-Tg against littermate control mice,
and ‘*’ represents the statistical significance (p-value #0.01). A) The levels of Nfkbia, Nfkb2, and Map3k14 were increased whereas the level of
Psmb10 was found to be reduced in TWEAK-Tg mice compared to littermate control mice (n=3 in each group). B). QRT-PCR analysis showed that the
levels of Notch1, Pgam2, Ankrd1, and TCap were reduced in TWEAK-Tg mice compared to control mice (n=3 in each group). C). Western blot
analysis of NF-kB2, Notch1 and TIMP2 protein expression profiles in TWEAK-Tg compared to control mice. The gel pictures presented here from two
independent experiments (n=4) showed that protein levels of Notch1, and TIMP-2 were significantly reduced whereas NF-kB2 protein levels were
increased in gastrocnemius muscle of TWEAK-Tg compared to littermate control.
Effects of TWEAK on Muscles
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TWEAK may be affecting the expression of miRs but not their
processing from pre-miRs to mature miRs.
Previous studies have suggested that myogenic transcription
factors (MRFs) such as MEF2c regulates the expression of various
muscle-specific miRs by binding in their promoter/enhancer
regions [35,42,43]. Indeed, consensus binding sites for MEF2 have
been identified in the enhancer and promoter region of miR-1 and
miR-133 [42,43]. By performing QRT-PCR, we validated that the
mRNA levels of MEF2C are significantly diminished in TWEAK-
treated myotubes (Figure 4C).
Although TWEAK was found to regulate the expression of
various miRNAs, it was not clear whether the altered levels of
miRs also affect the expression of their target genes in
myotubes. A recent study has suggested that NF-kB transcrip-
tion factor induces the expression of miR-146a, which is found
to be up-regulated in many muscle disorders [34,44]. To
identify the putative targets for this miR, we used miRDB
(http://mirdb.org/miRDB/), an online database for miR
target prediction and functional annotations in animals. This
database uses a bioinformatics tool called MirTarget2, which
was developed by analyzing thousands of genes down-regulated
by miRs . From this analysis, we found that miR-146a can
target TNF receptor-associated factor 6 (TRAF6) with target
score more than 95 (Table 3). The role of miR-146a in
regulation of TRAF6 levels have also been previously validated
by Taganov et al . Moreover, our Western blot experiments
confirmed that the protein levels of TRAF6 are reduced in
TWEAK-treated C2C12 myotubes (Figure 4D), which is
consistent with the increased expression of miR-146a. We also
investigated the in vivo effects of TWEAK on the expression of
various miRs in skeletal muscle. Similar to TWEAK-treated
myotubes, the levels of miR1-1 and mir-133b were found to be
reduced whereas the levels of miR-146a were increased in
skeletal muscle of TWEAK-Tg mice compared to their
littermate controls (Figure 5A). The level of miR-133b was
also somewhat reduced in TWEAK-Tg mice but it was not
significantly different from control mice (Figure 5A). Further-
more, we also found that the levels TRAF6 (a target for miR-
146a) were significantly diminished in skeletal muscle of
TWEAK-Tg mice compared to control mice (Figure 5B).
TWEAK Regulates Distinct Cellular Networks in C2C12
In order to understand the interaction between different genes,
we generated common networks using Ingenuity Pathway Analysis
(IPA) software. The dataset of differentially expressed genes by
TWEAK in C2C12 myotubes with selected stringency (p
value#0.05 and fold $1.5) was uploaded into the IPA software
tool. Networks of these genes were then algorithmically generated
based on their connectivity. The graphical representation of the
Table 2. List of top 20 toxicity pathways induced by TWEAK in C2C12 cells. Ingenuity pathway analysis was used to generate the
toxicity pathways involved by differentially expressed genes by TWEAK with p-values#0.05 and $1.2-fold. Negative logarithmic p-
values in the table are Fisher’s exact test p-value which determines the probability of the association between the genes in the
data set and the canonical pathway. Ratio was calculated by the genes in the data set involved in a particular toxicity pathway
divided by total number of genes involved in that pathway.
Ingenuity Toxicity Lists
2 2Log(P-value)Ratio Molecules
Hepatic Fibrosis 5.6400.200 ELN, IGFBP6, LEP, BGN, COL4A3, MMP2, IGFBP7, COL4A2, COL1A2, COL5A1,
CCL2, CSF1, TIMP1, TGFB3, SPARC, MMP9, AGT
Hepatic Stellate Cell Activation3.3600.229 RELA, CCL2, TIMP1, TGFB3, NFKB2, NFKB1, PDGFRB, AGT
RAR Activation2.2800.113 ADCY9, RELA, STAT5A, ADCY3, MAPK13, NFKB2, NFKB1, RXRG, AKT1, JUN, TAF4,
DUSP1, CRABP2, TGFB3, PRKCB
NFkB Signaling Pathway1.410 0.098 MAP3K14, RELA, AKT1, NFKBIA, BCL10, HDAC1, TNFAIP3, MAP3K8, TRAF5, NFKB2,
G1/S Transition of the Cell Cycle1.3700.125RB1, CCNE2, HDAC1, TGFB3, CCND1, SKP1
Oxidative Stress Response Mediated by Nrf2 1.0600.078 GSTA3, NQO1, MAF, SLC35A2, DNAJC13, DNAJC3, CLPP, TXNRD1, GSR, AKT1,
JUN, ERP29, KEAP1, ACTA1, EPHX1, PRKCB
TGF-b Signaling0.920 0.091 JUN, GRB2, HDAC1, TGFB3, ACVR2B, SMAD5, SERPINE1
Mitochondrial Dysfunction0.8640.080 GSR, NDUFB7, UQCRC2, CYB5R3, COX3, CYCS (includes EG:54205), OGDH,
UQCRC1, COX7A1, TXNRD2
Cholesterol Biosynthesis 0.674 0.125ACAT1, HMGCR
Anti-Apoptosis0.6080.094 HDAC1, TNFAIP3, BCL2L10
G2/M Transition of the Cell Cycle 0.558 0.088WEE1, BRCA1, SKP1
Hepatic Cholestasis0.5180.067ADCY9, MAP3K14, RELA, JUN, NFKBIA, ADCY3, NFKB2, NFKB1, PRKCB
LPS/IL-1 Mediated Inhibition of RXR Function0.5120.064 GSTA3, SLC27A5, LIPC, JUN, CHST7, FABP4, SLC35A2, CHST12, CES2 (includes
EG:8824), FABP3, ALDH3A1, ALDH9A1
Hypoxia-Inducible Factor Signaling0.4880.071 AKT1, JUN, NQO1, ELAVL1, PRKCB
Cytochrome P450 Panel - Substrate is
a Fatty Acid (Human)
CAR/RXR Activation0.3300.069GSTA3, CCND1
Positive Acute Phase Response Proteins0.2840.063C3, SERPINE1
Fatty Acid Metabolism0.262 0.054SLC27A5, ACAA1, ACAT1, ALDH3A1, ALDH9A1, GCDH, ADH4
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org7January 2010 | Volume 5 | Issue 1 | e8760
molecular relationships between genes developed by IPA is
presented in Figure 6 and Figure 7. Based on the input
information, the genes that are down-regulated are shown in
green and the up-regulated genes are shown in red (Figure 6
Several transcription factors and protein kinases such as NF-
kB2, FoxS1, Notch1, Wnt10A, MMP-9, PSMB10, colony
stimulating factor 1 (CSF1), and MAP3K14 were found to be
involved in the network related to inflammation, proteolysis, cell
survival, proliferation and differentiation (Figure 6). This network
also showed that many of these genes are regulated by each other
either directly or indirectly. The networks related to cellular
development and connective tissue disorder showed that enzymes
such as phosphoglycerate 12 mutase (glycolysis), muscle proteins
such as myosin heavy chain 4 (Actin cytoskeleton signaling),
nebulin-related anchoring protein (Actin binding protein) were
significantly down regulated by TWEAK (Figure 7).
Since the targets of many of the miRNAs that are regulated by
TWEAK in skeletal muscle are not yet known, we identified the
putative target genes and the cellular processes which these micro
RNAs affect using miRDB online tool. As shown in Table 3, the
microRNAs regulated by TWEAK are involved in regulation of
various genes and distinct cellular responses with target score $90.
Using these miRs, we also generated a network of pathways from
differentially regulated genes in cDNA microarray data set.
Interestingly, miRNAs network was found to considerably overlap
with mRNA networks suggesting that miRNAs may play
important roles in the regulation of gene expression in muscle
cells (Figures 6 and 7). Collectively, our in vitro, in vivo and in silico
experiments show that TWEAK differentially affects the expres-
sion of various miRNAs which regulate distinct cellular responses.
TWEAK Active Multiple Signaling Pathways in Skeletal
Microarray analysis of TWEAK-treated myotubes has
revealed that TWEAK differentially regulates the expression
of approximately 25% (p,0.05) of total 25,000 genes probed.
However, the number of genes which showed major changes
(.1.5 fold) were only in the range of hundred and the
functions of some of the differentially regulated genes is not yet
Figure 3. Differential expression of miRNAs in TWEAK-treated C2C12 myotubes measured by low-density miRNA array. A) C2C12
myotubes were treated with 10ng/ml of TWEAK for 18h following isolation of total RNA enriched with small RNAs. Untreated C2C12 myotubes under
exactly similar conditions served as control. The normalized expression ratios were plotted for each miRNA are mean 6 SD (n=3). Low-density miRNA
array of TWEAK-treated C2C12 myotubes showed down-regulation of miR-1, miR-133a, miR-133b, miR-206, miR-27, miR-23, miR-93, miR-199, miR-107,
and miR-192. The numbers above the bar represents the fold changes with TWEAK treatment against control with p-values #0.05. B). TWEAK
increased the expression of miR-715, miR-146a, miR-455, miR-322, mir-98, and miR-470 in C2C12 myotubes. The relative expression values from
the QRT-PCR analysis were plotted for each gene are mean 6 SD (n=3). The values significantly different from corresponding untreated control
(p-value #0.01) were represented with ‘*’.
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org8January 2010 | Volume 5 | Issue 1 | e8760
known (Table 1). Interestingly, some of the genes affected by
TWEAK are also similarly regulated in atrophying muscles in
response starvation and unloading [25,26]. Accumulating
evidence suggests that NF-kB is one the most important
signaling pathways, activation of which leads to skeletal muscle
wastage . The activation of NF-kB can occur through two
parallel pathways. The canonical NF-kB signaling pathway
involves the upstream activation of IkB kinase-b (IKKb) and
subsequent phosphorylation and degradation of IkB proteins
[46,47,48]. In contrast, the activation of the alternative NF-kB
pathway requires the upstream activation of NF-kB-inducing
kinase (NIK or MAP3K14) and IKKa and the proteolytic
processing of NFkB2 (p100 subunit) into p52 protein [47,48].
Our study suggests that TWEAK augments the expression of
both NIK (i.e. MAP3K14) and NFkB2 in myotubes (Figure 1B,
D, and Table S1). Similarly, increased expression of NIK and
NFkB2 were also noticeable in skeletal muscle of TWEAK-Tg
mice (Figure 2A and C) further confirming that TWEAK
increases the expression of the components of alternative NF-
kB signaling pathway. Interestingly, our microarray experi-
ment did not show any increase in the levels of Nfkb1 (e.g.
p105 or p50) and RelA, the major components of classical NF-
kB pathway [47,48]. However, we can not articulate that
TWEAK does not activate classical NF-kB pathway in skeletal
muscle cells. NFkB1 is present in abundance in cytoplasm of
the cell and it is rapidly activated in response to various
extracellular stimuli through upstream activation of a series of
protein kinases [47,48]. Previously published reports from our
group and others have demonstrated that TWEAK increases
the activation of both classical and alternative NF-kB pathways
[49,50]. Indeed, the increased expression of Nfkbia (i.e. IkBa),
cyclinD1, and MMP-9, which are predominately regulated
through classical NF-kB pathway  in our microarray and
QRT-PCR assays (Figure 1) is suggestive of the fact that
TWEAK also augments the activity of classical NF-kB
signaling pathway in muscle cells. However, it is important
to consider that while TWEAK increases the expression of a
number of NF-kB-related genes, all of them may not be
involved in TWEAK-induced skeletal muscle-wasting.
Our microarray analysis also revealed that TWEAK inhibits
the expression of Notch-1 which was confirmed by performing
QRT-PCR and Western blot (Figure 1C and 1D). Similar,
reduction in Notch1 levels was observed in skeletal muscle of
TWEAK-Tg mice (Figure 2B and 2C). Notch-1 receptors are
Figure 4. Validation of differentially expressed miRs and their regulatory enzymes by QRT-PCR in TWEAK-treated C2C12
myoblasts. A). TaqMan qRT-PCR analysis of miR-1-1, miR-1-2, miR-133a, miR-133b, miR-206, miR-146a, miR-206, miR146a, and miR-455 in TWEAK-
treated C2C12 cells. The normalized expression ratios were plotted for each miRNA are mean 6 SD (n=3). The numbers above the bar represents the
fold changes with TWEAK treatment against control ‘*’ represents the statistical significance (p-value #0.01). B). QRT-PCR analysis showed no
significant difference in the expression ratio of Dicer, Dorsha and Exportin-5 between control (n=3) and TWEAK-treated myotubes (n=3). C). TWEAK-
treatment significantly reduced the expression of MEF2C transcription factor. The relative expression values were plotted for MEF2C are mean 6 SD
(n=3). D). C2C12 myotubes were treated with 10 ng/ml of TWEAK for 18h followed by isolation of total protein and performing Western blot. Equal
amounts of proteins were loaded on 10% SDS-PAGE gel. Representative immunoblots from two independent experiments presented here showed
that TWEAK significantly reduced the protein level of TRAF6 in C2C12 myotubes.
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org9 January 2010 | Volume 5 | Issue 1 | e8760
transmembrane proteins which are expressed in a broad range
of tissues and function in diverse developmental and cell
maturation processes [52,53]. Besides its role in regulation of
the activity of many other transcription factors, recent studies
have shown that constitutively Notch-1 functions as a novel
IkB-like molecule and regulates NF-kB-mediated gene expres-
sion through a direct interaction with the NFkB1 (i.e. p50)
subunit [54,55]. This interaction prevents NF-kB from binding
to NF-kB recognition sites in DNA to regulate NF-kB-
dependent gene expression [54,55]. Therefore, the reduced
levels of Notch1 (Figure 1C and 1D) may be responsible, at
least in part, for sustained activation of NF-kB in skeletal
muscle cells in response to TWEAK. It is also of interest to
note that the expression levels of Notch1 are significantly
reduced in atrophying skeletal muscle in response to unloading
or denervation  suggesting that down-regulation of Notch1
may be a common phenomenon in muscle atrophy in response
to different stimuli including TWEAK.
In addition to NF-kB, our microarray analysis indicates that
TWEAK affects the expression of several genes involved in
different cell signaling pathways such as Wnt, MAPK, PI3K/Akt,
TGF-b, interferon-c (IFN-c), and ubiquitin-proteasome system
(Figure 6). While there are published reports demonstrating that
TWEAK affects the activation of NF-kB MAPK, PI3K/Akt, and
ubiquitin-proteasome systems [17,20,21,22,49,50], the present
study provides the first evidence regarding a potential role of
TWEAK in regulation of Wnt, TGF-b and IFN-c pathways
(Figure 6). The Wnt gene family consists of structurally related
genes which encode secretary signaling proteins . These
proteins have been implicated in oncogenesis and in several
developmental processes, including regulation of cell fate and
patterning during embryogenesis . Interestingly, NF-kB is the
key pathway for the transactivation of Wnt10a [58,59]. In a recent
study, Brack et al have demonstrated that in aged animals which
show significant muscle wasting, the conversion of satellite cells
from a myogenic to a fibrogenic lineage occurs due to the
activation of the canonical Wnt signaling pathway . It is
important to note that the levels of proinflammatory cytokines are
increased in skeletal muscle and in circulation during aging and in
several chronic diseases . Therefore, it is possible that
proinflammatory cytokines such as TWEAK mediates the loss of
skeletal muscle mass and causes fibrosis through the activation of
Wnt pathway. Indeed, our analysis of differentially regulated genes
in TWEAK-treated myotubes by Ingenuity pathway analysis also
indicates that TWEAK affects the activation of several toxic
pathways including those involved in initiation and manifestation
of fibrosis, oxidative stress, and mitochondrial dysfunction
Table 3. List of differentially expressed miRNAs with known targets/cellular processes.
DetectorFold Target/cellular process
226.467 SOS2, Endoribonuclease Dicer
23.1953 Endoribonuclease Dicer, SOS2, decrease proapoptotic signaling, proliferation and remodeling of muscles
23.2Increase cell growth, proliferation and remodeling of muscles
23.4278Increase cell growth, proliferation and remodeling of muscles
22.8 Increase cell growth, proliferation and remodeling of muscles
miR-146a18.6416 Traf6, Delays differentiation through Numb.
22.6183 decrease apoptosis
22.3341Increase cell growth
26.2545 Bcl2, N-myc
23.5756 proliferation and remodeling of muscles
22.2Binds to Ubiquitin-specific protease 46, increase cell growth
22.4871 Increase cell growth
21.9 Activin receptor IIA, Map3k4
22.2887Increase cell growth
22.1623 Increase Cell growth, proliferation
22.4565decrease proapoptotic signaling
22.7Ubiquitin-conjugating enzyme E2N
23Ubiquitin-conjugating enzyme E2N
27.3378Increase cell growth, decrease apoptosis
miR-3228.7 Hydroxysteroid (17-beta) dehydrogenase 7
miR-455129.249Up-regulated brown adipocyte differentiation
miR-4703.2TGFB-induced factor homeobox 1
miR-71518.25 Fucosyltransferase 1
26.2174Increase cell growth, decrease apoptosis
248.423 Map3k14 (NIK)
miR-981.8 Tripartite motif-containing 6, insulin-like growth factor 2 mRNA binding protein 1
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org10January 2010 | Volume 5 | Issue 1 | e8760
TWEAK Modulates the Expression of Select Cytoskeleton
Molecules in Skeletal Muscle
The interaction between myosin and actin is the molecular basis
of muscle contraction and ATP hydrolyzed by myosin is the
energy source for mechanical power output. Myosin heavy chain
(MyHC) isoforms determine the contractile properties of the
myosin molecule and are considered as molecular markers of the
fiber type . So far nine myosin isoforms, each coded by a
distinct gene, have been found to be expressed in striated muscles
and incorporated in the thick filaments . Among them, three
fast type MyHC isoforms expressed in adult fast skeletal muscle
fibers (called 2A, 2X, and 2B, coded by the genes MYH2, MYH1,
and MYH4, respectively) have been found to be the major
proteins that undergo proteolytic degradation in various atrophy
conditions [1,11,62,63]. Using an antibody that recognizes all the
three fast isoforms of MyHC (MyHCf), we have previously
reported that TWEAK induces the degradation of MyHCf
through the activation of NF-kB and ubiquitin-proteolytic systems
. However, there are no reports of TWEAK directly
regulating any of these MyHCf isoforms. Our microarray analysis
and independent QRT-PCR in this study has suggested that
TWEAK may also reduces the expression of MYH4 (i.e. MYHC-
2B) in myotubes. The reduced expression of MYH4 also suggests a
possibility that TWEAK may inhibit the differentiation and/or
dedifferentiation of C2C12 cells. Indeed, we have previously
reported that TWEAK inhibits the expression of MyHC and
differentiation of C2C12 myoblasts in to myotubes .
Interestingly, we have also found that TWEAK down regulates
the expression of ankyrin repeat domain 2 (Ankrd2), a structural
constituent of striated muscle  and Tcap, a 19 kDa sarcomeric
protein that is located in the periphery of Z-discs . Mutations
in TCap lead to autosomal recessive girdle muscular dystrophy or
LGMD type 2 and severe weakness of leg muscle [66,67]. It has
been recently demonstrated that knockdown of TCap in C2C12
myoblasts using siRNA reduces the expression of myogenic
regulatory factors MyoD and myogenin suggesting that TCap
might be required for the differentiation and maintenance of
skeletal muscle mass . Interestingly, we have previously
reported that TWEAK reduces the expression of MyoD and
myogenin in C2C12 cultures . Since TWEAK significantly
reduced the expression of TCap in cultured myotubes within 18h,
these data suggest that TWEAK may also be affecting the levels of
MyoD and myogenin through inhibiting the expression of TCap.
We have also found that the levels of TCap and Ankrd2 are
significantly reduced in skeletal muscle of TWEAK-Tg mice
(Figure 2B). Furthermore, Ingenuity Pathway Analysis (IPA)
showed that Ankrd2 and TCap interact with each other
(Figure 7) which is in agreement with a previously published
In addition to structural proteins, skeletal muscle also expresses
many metabolic enzymes required for energy production. The
muscle-specific isoform (type M, PGAM2) of phosphoglycerate
mutase (PGAM) is a housekeeping enzyme which catalyzes the
conversion of 3-phosphoglycerate into 2-phosphoglycerate in the
glycolysis process to release energy . PGAM2 is developmen-
tally regulated during myogenesis. Mutations in human have been
shown to cause PGAM2 deficiency, which results in acute muscle
dysfunction with exercise intolerance and muscle breakdown .
Our experiments suggest that TWEAK significantly reduces the
expression of PGAM2 not only in cultured myotubes (Figure 1C)
but also in skeletal muscle of TWEAK-Tg mice (Figure 2B).
Furthermore, our microarray data has confirmed our previous
findings  that TWEAK augments the expression of extracel-
lular protease matrix metalloproteinase-9 (MMP-9) and reduces
the levels of MMP-2 and TIMP2 (Figure 1). Altered expression
and production of these molecules in response to TWEAK may
cause extracellular matrix abnormalities during muscle-wasting
TWEAK Regulates the Expression of Several MicroRNAs
(miRs) in Skeletal Muscle
The low-density miR array revealed that TWEAK reduces the
expression of a large number of miRs which coincidently is in
directional correspondence with the up-regulation of majority of
genes in our cDNA microarray data with the selected stringent p-
values and fold changes (Table 1). Differential expression of
relatively fewer miRs when compared to the large number of
differentially regulated genes in microarray data also suggests the
possibility of targeting more than one gene by each miRNA.
Additionally, because miRNAs target genes can directly influence
the expression of many other genes indirectly, many of the
miRNAs differentially expressed could also be involved in the
regulation of some non-target genes . Recently, a few muscle-
specific miRs such as miR-1, miR-133a, miR-133b, and miR-206
(also called myomiRs) have been identified which are essential for
muscle cell proliferation, differentiation, and maintenance .
Figure 5. Expression profiles of select microRNAs and TRAF-6
proteins in skeletal muscle of TWEAK-Tg mice. A) TaqMan QRT-
PCR analysis of miR-1-1, miR-133a, miR-133b, and miR-146a in skeletal
muscles of TWEAK-Tg mice. Gastrocnemius muscle from 6 months old
TWEAK-Tg mice and littermate control mice were taken and total RNA
enriched with small RNAs was isolated for TaqMan qRT-PCR analysis.
The normalized expression ratios were plotted for each miRNA are
mean 6 SD (n=3). ‘*’ represents the statistical significance (p-value #
0.01). B). Gastrocnemius muscle of 6 months old TWEAK-Tg mice and
littermate control mice were taken and total protein was isolated for
Western blotting analysis. Representative immunoblot presented here
show that the levels of TRAF-6 are considerably reduced in skeletal
muscle of TWEAK-Tg (n=4) mice compared to control (n=4) mice.
Equal amounts of protein loading were ensured by the expression
levels of b-actin.
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org11January 2010 | Volume 5 | Issue 1 | e8760
Expression of miR-1 and miR-133a in embryonic stem cells and
other non-muscle cell types showed that they promote the
differentiation into the skeletal muscle lineage . Unlike other
myomiRs which are also expressed in cardiac tissues, miR-133b
and miR-206 are specifically expressed in skeletal muscle though
their biological functions are yet to be established. Interestingly,
our low density miRs array and independent TaqMan QRT-PCR
assays demonstrate that TWEAK reduces the expression levels of
miR-1, miR-133a, miR-133b, and miR-206 in skeletal muscle cells
(Figure 3A and Figure 4A). Furthermore, the level of at least miR-
1 was also found to be significantly reduced in skeletal muscle of
TWEAK-Tg mice (Figure 5A). Recent studies have demonstrated
that myogenic transcription factors such as serum response factor
(SRF), MEF2c, and MyoD control the expression of myomiRs in
skeletal and cardiac muscles (reviewed in ). We have
previously demonstrated that TWEAK reduces the levels of
MyoD and myogenin in differentiating C2C12 cultures . Our
microarray and QRT-PCR assays in this study have also shown
that TWEAK inhibits the expression of MEF2c transcription
factor in cultured myotubes (Figure 4C). MEF2C is particularly
important for miR-1 and miR-133a and miR-1 further regulates
MEF2C levels [42,43].
In addition to MyomiRs, TWEAK also down-regulated a few
more miRNAs such as miR-27a and b, miR-93, miR-199a-3p,
miR-107, miR-192, and miR-23b (Fig. 5A). Though miRNAs
have been explored extensively in recent years, the targets of many
miRNAs are yet to be identified. For this purpose, we have utilized
miRNA database (http://mirdb.org/miRDB/) to identify the
putative targets of selected miRNAs. From the miRNA database,
we identified that miR-27a and b targets ubiquitin-conjugating
enzyme E2N with target score above 90. Ubiquitin-conjugating
enzyme is an important component of ubiquitin-proteasome
pathway, which causes muscle protein degradation in various
atrophy conditions [1,4,73,74]. Interestingly, TWEAK has been
found to induce the ubiquitination of muscle proteins both in vivo
and in vitro . The miRNA database also identified that miR-
93 can target MAP3K14 (i.e. NIK) which is involved in NF-kB
activation. This suggests that the down-regulation of miR-27a & b
and miR-93 leads to activation of ubiquitin-conjugating enzyme
leading to up-regulation of ubiquitin-proteasome pathway and
NIK in alternative NF-kB signaling pathway thereby inducing
muscle atrophy (Figure 6). From the miRNA database, we also
observed that TWEAK down-regulates miRNAs that are targeting
proliferation and remodeling of muscles (miR-107), matrix
Figure 6. Network of genes up-regulated by TWEAK in microarray experiment. NF-kB and proteasome pathways are major pathways
affected by differentially regulated genes by TWEAK. Although many of the microRNAs differentially expressed by TWEAK may not be targeting the
differentially regulated genes directly, they can regulate indirectly through other intermediary molecules. For example let-7a and miR-98 may have an
indirect effect on expression of Nocth1 by regulating Akt pathway. The solid lines connecting molecules here represent a direct relation and dotted
lines an indirect relation. The gene network presented here was adopted from Ingenuity pathway analysis tool with differentially regulated genes by
TWEAK with p-values #0.05 and $1.5-fold. The genes shown in red are up-regulated in microarray data whereas down-regulated genes are shown in
green color. Differentially expressed miRNAs (in blue colored boxes) having their putative targets are superimposed on the network.
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org12 January 2010 | Volume 5 | Issue 1 | e8760
metalloprotease such as aggrecanase-2 (miR-192), adamtsl-3 (miR-
199-3p), and genes involved in increasing cell growth and
proliferation, and microtubule-associated proteins (miR-23b)
The Low-density miRNA arrays of TWEAK-treated C2C12
myotubes also showed upregulation of a few select miRs. Out of
the up-regulated miRs, miR-146a and miR-455 have known
targets (Table 3). A recent study by Kuang et al showed that
miR-146a targets Numb, which promotes satellite cell differen-
tiation towards muscle cells and inhibition of miR-146a by
antago-miR146a rescued the expression of Numb and facilitated
the differentiation of C2C12 cells . The miRNA database
also suggests that miR-146a has a putative target TRAF6 (with
target score $95). TRAF6 belongs to E3 ubiquitin ligase family
which induces the activation of multiple signaling proteins
including Akt through formation of Lysine-63-linked poly-
ubiquitin chains . The up-regulation of miR-146a in
TWEAK-treated C2C12 suggests that one of the potential
mechanisms by which TWEAK might be inducing loss of
skeletal muscle mass is through down-regulation of Numb and
TRAF6. Indeed, our Western blot data suggest that the levels of
TRAF6 are reduced in TWEAK-treated myotubes (Figure 4D)
and in skeletal muscle of TWEAK-Tg mice (Figure 5B). It is also
noteworthy that the expression of miR-146a is regulated
through the activation of NF-kB , which is activated in
response to TWEAK-treatment. Since TRAF6 contributes to
the phosphorylation and activation of Akt , reduced level of
TRAF6 in TWEAK-treated myotubes is consistent with our
previous findings that TWEAK inhibits the activation of Akt in
C2C12 myotubes .
Reduction in skeletal muscle mass and increase of adipocytes
(body fat) are the common features of atrophying skeletal muscle
[1,2,3]. Recent studies have suggested that miR-455 is linked to
the up-regulation of brown adipocyte formation. deCastro
Rodrigues et al  showed the occurrence of fat cell invasion
in long-term denervated skeletal muscle. In addition, Eisenberg
et al  have reported that the levels of miR-455 were increased
about two fold in dystrophic muscle of facioscapulohumeral
muscular dystrophy, limb girdle muscular dystrophy 2A, and
nemaline myopathy. The up-regulation of this miR-455 in our
miRNA-array and QRT-PCR assays further signifies its potential
role in TWEAK-induced skeletal muscle-wasting (Figure 4A).
Figure 7. Gene network ofdown-regulated genes by TWEAK andtheir possible regulatory miRNAs. Notch1 signaling is major pathway down
regulated by TWEAK in C2C12 myotubes. Genes represented in green boxes are those which were found to be significantly down-regulated in our
microarray experiment. The genes shown without color are intermediate to the network and are not found in our microarray data. The solid lines
connecting molecules here represents a direct relation and dotted lines an indirect relation. This network was obtained from IPA using differentially
regulated genes by TWEAK with p-values #0.05 and $1.5-fold and was superimposed with the miRNAs (blue colored boxes) having their putative targets.
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org13 January 2010 | Volume 5 | Issue 1 | e8760
The data presented in this study suggest that TWEAK affects
the expression of several genes and related miRs in skeletal muscle
cells. These genes and miRs are involved in the regulation of
various molecular pathways/processes including ubiquitin-protea-
some pathway, extracellular matrix degradation, brown adipocyte
formation, and muscle cell proliferation and differentiation. The
study has also identified several important genes and miRs that are
differentially expressed in skeletal muscle in response to TWEAK.
Similar molecules might be involved in skeletal muscle wasting in
response to other catabolic stimuli.
Materials and Methods
Horse serum was purchased from Sigma Chemical Company
(St. Louis, MO). Recombinant mouse TWEAK protein and
antibodies against MMP-9 and MMP2 were purchased from R&D
Systems (Minneapolis, MN). Antibodies against IkBa and Notch1
were purchased from Santa Cruz Biotechnology (San Diego, CA).
Tubulin and NFkB2 antibodies were obtained from Cell Signaling
Technology (Beverly, MA). TRAF6 antibody was obtained from
Millipore (Bedford, MA). Primers for PCR were synthesized by
Integrated DNA Technologies (Coralville, IA) or Sigma-Genosys
C2C12 myoblastic cell line was obtained from American Type
Culture Collection (Rockville, MD). These cells were grown in
Dulbecco’s modified Eagle’s Medium (DMEM) containing 20%
fetal bovine serum. C2C12 myoblasts were differentiated into
myotubes by incubation in differentiation medium (DM, 2% horse
serum in DMEM) for 96h as described [21,22]. Myotubes were
maintained in DM and medium was changed every 48h.
Transgenic (Tg) mice expressing TWEAK in skeletal muscle
(TWEAK-Tg) have been described previously . Since
TWEAK-Tg mice were generated in B6D2F1 background, these
mice were crossed with C57BL/6 mice for 7 generations before
using for this study. All the experiments with animals were
approved by the Institutional Animal Care and Use Committee of
the University of Louisville.
Total RNA was isolated from control and TWEAK-treated
C2C12 myotubes using the Agilent total RNA isolation kit (Agilent
Technologies, Palo Alto, CA). Any contaminating DNA was
removed using DNA-freeTMkit from Ambion (Ambion, Austin,
TX). The total RNA concentration was determined by NanoDrop
spectrophotometer, and RNA quality was determined by 18 S/28
S ribosomal peak intensity on an Agilent Bioanalyzer. Each
experiment was performed with a minimum of five replicates.
Custom cDNA slides were spotted with Oligator ‘‘MEEBO’’
mouse genome set with 38,467 cDNA probes (Illumina, Inc., San
Diego, CA), which allows interrogation of 25,000 genes. A Q-
Array2 robot (Genetix) was used for spotting. The array includes
positive controls, doped sequences, and random sequences to
insure correct gene expression values were obtained from each
array. A total of 250 ng RNA was used to synthesize double
stranded cDNA using the Low RNA Input Fluorescent Linear
Application Kit (Agilent). The microarray slides were scanned
using a GSI Lumonics ScanArray 4200A Genepix scanner (Axon).
The image intensities were analyzed using the ImaGene 5.6
software (Biodiscovery, Inc., El Segundo, CA). Expression analysis
of microarray experiments was performed with GeneSpring 7.1
(Silicon Genetics, Palo Alto, CA) using the raw intensity data
generated by the ImaGene software. Local background was
subtracted from total signal intensities and was used as intensity
measures. The data were normalized using per spot and per chip
LOWESS normalization. Data analysis was performed using SAS
(SAS Institute, Cary, NC), R and Q value software. The probe sets
with absent calls across all samples were removed to reduce the
multiple-testing problem. The expression levels were normalized
to the chip median and log transformed. Two–way ANOVA tests
were carried out to identify differentially expressed genes. For each
probe set, the model yijk~mzaizbjzcijzeijkwas fit, where yijk
is the log-transformed expression level of the kthchip in the ith
treatment and the jthreplicate. The variable m represents the
grand mean expression, aiis the effect due to the treatment, bjis
the effect due to the replicate, cijis the interaction effect between
treatment and replicate, and eijkis an error term, which is assumed
to be normally distributed with mean 0 and variance s2. Q values
computed using Q value software indicates the false detection rate
for each probe set. Ratio comparison was performed by dividing
expression levels in TWEAK-treated myotubes with the expression
levels in untreated myotubes. Functional classification of select
probe sets was performed at NIH DAVID server (http://apps1.
niaid.nih.gov/david/upload.asp). Volcano plots were prepared
using the R program. The complete raw and normalized
microarray data have been submitted in MIAME compliant
base with accession number E-MEXP-2432.
MicroRNA (miR) Array Analyses
For miRNA array experiments, total RNA along with small
RNAs was isolated from control and 10ng/ml TWEAK-treated
C2C12 myotubes using mirVANA miRNA isolation Kit (Ambion,
Austin, TX). cDNA was synthesized using Megaplex RT primers
(Applied Biosystems, Foster City, CA) which are a set of two
predefined pools (pool A and Pool B) of up to 380 stem-looped
reverse transcription primers that specifically binds to miRNAs
and synthesize cDNA from mature miRNAs. For this we used
500ng of total RNA and 4.5ml of RT reaction mix in a total
volume of 7.5ml at the following cycle conditions: 16uC for 2 min,
42uC for 1 min and 50uC for 1 min for total of 40 cycles followed
by 85uC for 5 min and bringing the contents to 4uC. The contents
were stored at 220uC until further use. The mouse Low Density
miRNA array system (Applied Biosystems, Foster City, CA) was
used for the miRNA profiling of TWEAK treated C2C12 cells.
This miRNA-array kit consists of four plates of plate A and four
plates of plate B which contain around 384 miRNAs including
four internal controls. For this we used 6ml of cDNA synthesized
by using Megaplex RT and 450 ml of TaqMan universal PCR
master mix in a total of 900 ml of reaction volume and 100 ml of
the reaction mixture was loaded into each port provided in the
card (which has 8 ports for each card). Each cDNA was run on
both plate A and plate B according to the manufactures protocol.
The plates were run in Applied Biosystems Real-time PCR system
(7900 HT) by selecting relative quantification (DDCt) and 384 well
TaqMan low density array cards. All the samples were run in
triplicates. Finally, all the raw data from each plate set was
retrieved from the 7900HT machine and was run on RQ manager
ver.1.2 (Applied Biosystems, Foster City, CA). The samples were
named as Control for control plates and Treatment for TWEAK-
treated samples and were checked for their threshold values and
peaks. The samples with many peaks or inconsistent peaks were
deleted before calculating DDCT and RQ values. The mean
Effects of TWEAK on Muscles
PLoS ONE | www.plosone.org 14January 2010 | Volume 5 | Issue 1 | e8760
values for RQ (which is fold values of treatment compared to
control) were used to plot the bar diagrams. The miRNAs with p-
value #0.05 and fold value of $2 were considered for further
The selected miRNAs were searched for their known targets
and those miRNAs with unknown targets were used to identify
their putative targets by using miRDB web site (http://mirdb.org/
miRDB/) with target score $90. The targets/putative targets of
selected miRNAs were also analyzed by Ingenuity pathway
analysis software to generate interactive pathways and were
compared with the pathways obtained from cDNA microarray
Quantitative Real-Time-PCR (QRT-PCR)
The expression of the differentially regulated genes from the
microarray data was validated using QRT-PCR using a method as
described . The sequence of the primers used is described in
The selected miRNAs from the miRNA array were validated
using TaqMan QRT-PCR analysis by using their specific primers
from Applied Biosystems (Foster City, CA). For this the cDNA for
each selected miRNA was synthesized by using the miRNA
specific primers supplied by the manufacturer (Applied Biosys-
tems). Briefly, 100 ng of total RNA was taken for cDNA synthesis
of each miRNA in a final volume of 20ml by using the miRNA
specific primers (Part number 4427975; assay IDs, 002455,
000468, 002222, 002247, 000510, 001637, and 002882) to ensure
cDNA synthesis of mature miRNAs as given in the manufactures
protocol. U6 was used as an internal control for miRNA in
Pathways and Networks Analyses
Relative levels of gene expression were first computed with
GeneSpring 7.1 to obtain data sets of differentially regulated genes
based on cut-off values of 5% error rate (p,0.05, determined by t-
test with Benjamini and Hochberg Multiple Testing Correction).
These data sets included up and down regulated genes when
C2C12 myotubes were treated with TWEAK. The second step of
analysis consisted of identifying canonical pathways. Tab separat-
ed (txt) files containing Accession IDs and symbols derived from
MEEBO genome set and the normalized expression ratios were
then uploaded to Ingenuity Pathways Analysis. Ingenuity Path-
ways Analysis is a web-delivered bioinformatics tool (IPA 5.0,
http://www.ingenuity.com) to identify pathways and functional
networks. IPA knowledge database is generated from the peer-
reviewed scientific publications that enables discovery. The
Accession IDs and symbols in each data set were queried against
all genes stored in the IPA knowledge database for pathway
analysis. Canonical pathways analysis identified the pathways from
IPA library of canonical pathways that were most significant to the
data set. The significance of the association between the data set
and the canonical pathways was measured in 2 ways: 1) A ratio of
the number of genes from the data set that map to the pathway
divided by the total number of genes that map to the canonical
pathway is displayed. 2) Fisher’s exact test was used to calculate a
p-value determining the probability that the association between
the genes in the data set and the canonical pathway is explained by
Immunoblotting was performed to measure the levels of various
proteins in C2C12 myotubes or skeletal muscle tissues of
TWEAK-Tg mice using a protocol as described .
Methods used for statistical analysis of the cDNA microarray
and microRNA arrays data has been described above in their
respective sections. For all other studies, results were expressed as
mean 6 SD. The Student’s t test was used to compare quantitative
data populations with normal distributions and equal variance. A
value of P ,0.05 was considered statistically significant unless
down-regulated by TWEAK in microarray experiment.
Found at: doi:10.1371/journal.pone.0008760.s001 (0.12 MB
Extreme 50 known genes that are up-regulated or
time PCR assay.
Found at: doi:10.1371/journal.pone.0008760.s002 (0.04 MB
Sequence of the primers used for quantitative real-
Authors are thankful to Dr. Ron Gregg, Director, Nucleic Acid Core
Facility of University of Louisville for providing access to Applied
Biosystems 7900HT real-time PCR system.
Conceived and designed the experiments: SKP NGC RFL AK. Performed
the experiments: SKP SB AK. Analyzed the data: SKP SB AKS NGC
RFL AK. Contributed reagents/materials/analysis tools: JJM. Wrote the
paper: SKP SB AK.
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