Am J Physiol Cell Physiol 304: C128–C136, 2013.

First published October 31, 2012; doi:10.1152/ajpcell.00025.2012.

Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle

cells: role of the STAT3 pathway
Miriam Hoene,1,3 Heike Runge,1 Hans Ulrich Häring,2,3 Erwin D. Schleicher,1,3 and Cora Weigert1,3
1
Division of Pathobiochemistry and Clinical Chemistry, University Tuebingen, Tuebingen, Germany; 2Division of
Endocrinology, Diabetology, Angiology, Nephrology, and Clinical Chemistry, Department of Internal Medicine, University
Tuebingen, Tuebingen, Germany; and 3Paul Langerhans Institute Tuebingen, Institute for Diabetes Research and Metabolic
Diseases of the Helmholtz Centre Munich, University Tuebingen, Tuebingen, Germany
Submitted 20 January 2012; accepted in final form 19 October 2012

Hoene M, Runge H, Häring HU, Schleicher ED, Weigert C.
Interleukin-6 promotes myogenic differentiation of mouse skeletal
muscle cells: role of the STAT3 pathway. Am J Physiol Cell
Physiol 304: C128 –C136, 2013. First published October 31, 2012;
doi:10.1152/ajpcell.00025.2012.—Myogenic differentiation of skel-
etal muscle cells is characterized by a sequence of events that in-
clude activation of signal transducer and activator of transcription 3
(STAT3) and enhanced expression of its target gene Socs3. Autocrine
effects of IL-6 may contribute to the activation of the STAT3-Socs3
cascade and thus to myogenic differentiation. The importance of IL-6
and STAT3 for the differentiation process was studied in C2C12 cells
and in primary mouse wild-type and IL-6⫺/⫺ skeletal muscle cells. In
differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA
expression and protein secretion started after increased phosphoryla-
tion of STAT3 on tyrosine 705 and increased mRNA expression of
Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in
differentiating C2C12 myoblasts impaired the expression of the myo-
genic markers myogenin and MyHC IIb and subsequently myotube
fusion. However, the knockdown of IL-6 did not prevent the induction
of STAT3 tyrosine phosphorylation. The IL-6-independent activation
of STAT3 was verified in differentiating primary IL-6⫺/⫺ myoblasts.
The phosphorylation of STAT3 and the expression levels of STAT3,
Socs3, and myogenin during differentiation were comparable in the
primary myoblasts independent of the genotype. However, IL-6⫺/⫺
cells failed to induce MyHC IIb expression to the same level as in
wild-type cells and showed reduced myotube formation. Supplemen-
tation of IL-6 could partially restore the fusion of IL-6⫺/⫺ cells. These
data demonstrate that IL-6 depletion during myogenic differentiation
does not reduce the activation of the STAT3-Socs3 cascade, while
IL-6 and STAT3 are both necessary to promote myotube fusion.
Socs3; STAT3; differentiation; IGF-I; IL-6⫺/⫺ myoblasts
INTERLEUKIN-6 (IL-6) IS CLASSICALLY known as a central player in
the regulation of inflammation, hematopoesis, immune re-
sponse, and host defense mechanisms. During the last decade,
IL-6 has also been recognized as being a myokine (20), with
the working skeletal muscle as predominant producer of IL-6
during exercise (9, 25). While strenuous physical activity can
be accompanied by an up to 100-fold increase of plasma IL-6
(19, 21), short-term moderate intensity exercise leads to high
interstitial IL-6 concentrations within the muscle without a
significant rise in circulating levels (22). This implies that
skeletal muscle per se is an important target for IL-6. Subse-
quent studies in human and rat L6 myotubes, in human muscle
Address for reprint requests and other correspondence: C. Weigert, Dept. of
Internal Medicine, Division of Pathobiochemistry and Clinical Chemistry,
Univ. of Tuebingen, Otfried-Mueller-Stra␤e 10, D-72076 Tuebingen, Ger-
many (e-mail: Cora.Weigert@med.uni-tuebingen.de).
C128 0363-6143/13 Copyright © 2013 the American Physiological Society
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strips, and in rodent muscle demonstrate that IL-6 may pro-
mote fuel uptake and utilization by the working muscle (1, 4,
5, 7, 34). Studies in IL-6-deficient mice show impaired recov-
ery of their muscle mass from atrophy (33), and a detailed
investigation of these mice could provide evidence that mus-
cle-produced IL-6 regulates muscle growth in vivo due to
impaired proliferation and migration of satellite cells (23). IL-6
is also implicated in the activation and proliferation of human
muscle satellite cells following eccentric muscle contractions
(15, 28). Thus there is good evidence that IL-6 is important for
skeletal muscle hypertrophy.
Whether IL-6 also regulates the differentiation and fusion of
skeletal muscle cells is less clear. Stimulation of C2C12 myo-
blasts with recombinant IL-6 induces the expression of the tran-
scription factor myogenin, an important initiator of the differen-
tiation program (3). In contrast, other members of the IL-6
family, namely leukemia inhibitory factor (LIF; Refs. 2, 11)
and cardiotrophin-1 (CT-1; Ref. 17), have been reported to
inhibit myogenic differentiation in skeletal muscle cells. The
signal transduction induced by IL-6 and its related cytokines is
mediated by homo- or heterodimerization of the gp130 recep-
tor, subsequent activation of Janus kinases (JAK), and phos-
phorylation of signal transducers and activators of transcription
(STAT) (8). Studies in C2C12 cells and mouse primary myo-
blasts showed that a JAK1/STAT1 pathway promotes prolif-
eration and prevents differentiation (26), while a JAK2/
STAT2/STAT3 pathway is required for myogenic differentia-
tion (31). A recent publication of the same group added more
details to this field: activation of the negative feedback regu-
lators suppressor of cytokine signaling (Socs)1, Socs3, and
protein inhibitor of activated STAT (PIAS)1 downregulates the
JAK1/STAT1/STAT3 pathway, an event apparently essential
for myogenic differentiation (6). Notably, overexpression of
Socs3 has been shown to induce myoblast differentiation (24).
The specific function of STAT3 in the differentiation program
of skeletal muscle cells remains unclear, since STAT3 activity
could be necessary for the induction of Socs3, while on the
other hand STAT3 can directly interact with and inhibit the
activity of the muscle-specific transcription factor MyoD,
which plays an essential role in differentiation (12). Further-
more, it is unknown whether IL-6 as an activator of STAT3
and subsequent Socs3 expression is necessary for the differen-
tiation and fusion of skeletal muscle cells.
In conclusion, there is good evidence from previous studies
that components of the IL-6-STAT3-Socs3 signaling cascade
are regulators of myogenic differentiation, but the function of
endogenous IL-6 production in muscle cells herein is un-
known.
http://www.ajpcell.org
 IL-6 AND STAT3 REGULATE MYOGENIC DIFFERENTIATION
We focused here on the importance of IL-6 and its down-
stream signaling molecules STAT3 and Socs3 in the differen-
tiation of C2C12 muscle cells and of primary mouse muscle
cells. We demonstrate that activation of STAT3 and Socs3 is
an early, but IL-6-independent, event that induces the myo-
genic differentiation program, while lack of IL-6 also impairs
myotube fusion.
EXPERIMENTAL PROCEDURES
Materials. LightCycler system was from Roche (Mannheim, Ger-
many). Oligonucleotides were from Qiagen (Hilden, Germany) or
synthesized by Invitrogen (Karlsruhe, Germany). Antibodies against
phospho-STAT3 (no. 9131), STAT3 (no. 9132), and ␤-actin (no.
4970) were from Cell Signaling (Frankfurt, Germany), and the pro-
tease inhibitor mixture was from Roche. IL-6 (mouse, recombinant
protein) was from R&D Systems (Wiesbaden-Nordenstadt, Ger-
many).
Cell culture. C2C12 myoblasts were obtained from ATCC and
cultured in growth medium (DMEM; Lonza) containing 25 mM
glucose, 2 mM glutamine, and 1% penicillin/streptomycin and sup-
plemented with 10% FBS (GIBCO). Cells were seeded in a density of
1 ⫻ 105 cells/well of a six-well dish. Myotube formation was induced
after 48 h by switch to fusion medium (FM) consisting of DMEM
containing 25 mM glucose and 2% FBS. Medium was replaced every
other day.
The mouse studies experiments were conducted in accordance with
the national guidelines of laboratory animal care and were approved
by the local governmental commission for animal research (Regier-
ungspraesidium Tuebingen, Baden-Wuerttemberg, Germany). Pri-
mary mouse myoblasts were isolated according to a method described
for rat myoblasts (13). Briefly, C57/Bl6 mice and IL-6⫺/⫺ (B6.129S2-
Il6tmlKopf/J) maintained on a C57Bl/6J-background (purchased from
Jackson Laboratory, Bar Harbor, ME) were anesthetized with an
intraperitoneal injection of ketamine (150 mg/kg body wt) and xyla-
zine (10 mg/kg body wt) and killed by decapitation. Quadriceps
muscles were dissected out and placed in ice-cold Dulbecco’s PBS
containing 1% glucose and 0.5% penicillin/streptomycin. Muscles
were carefully minced and rotated in DMEM containing 25 mM
glucose, 1% penicillin/streptomycin, and 0.2% collagenase (CLS,
Worthington) for 90 min at 37°C. After centrifugation at 300 g for 15
min, the pellet was resuspended in DMEM containing 25 mM glu-
cose, 1% penicillin/streptomycin, 0.2% collagenase, and 0.25% tryp-
sin and rotated for 30 min at 37°C. The cell suspension was diluted
with primary growth medium [PGM; DMEM with 25 mM glucose,
10% horse serum (GIBCO), 10% FBS, 2 mM glutamine and 1%
penicillin/streptomycin], centrifugated for 15 min at 300 g, resus-
pended in PGM and filtered through a 70-␮m sterile filter. Equal
number of cells were seeded onto dishes precoated with Matrigel (no.
356234; Becton-Dickinson) in a 1:6 dilution with DMEM. Medium
was replaced every other day. Primary mouse myoblasts started
spontaneously to differentiate in PGM and FM which was set as day
0 of differentiation. Sufficient myotube maturation was only observed
in PGM but not in serum-reduced DMEM. C2C12 myoblasts also
differentiate in PGM with no obvious difference in the number of
fused myotubes, but a defined start of differentiation could only be
achieved by switching from growth medium containing 10% FBS to
the serum reduced FM.
RNA isolation and quantitative PCR analysis. RNA was extracted
with the RNeasy kit (Qiagen) according to the manufacturer’s instruc-
tions. Reverse transcription of total RNA (1 ␮g) was performed in a
volume of 20 ␮l using random hexamer primers with the first strand
cDNA synthesis kit for RT-PCR (Roche, Mannheim, Germany).
Aliquots (2 ␮l) of the reverse transcription reactions were then
submitted to online quantitative PCR with the Light Cycler system
(Roche) with SYBR green using FastStart DNA-MasterSYBR Green
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C129
I (Roche) or the QuantiFast SYBR Green PCR kit (Qiagen). The
following primer pairs were used: IL-6 sense: gat gct acc aaa ctg gat
ata atc, antisense: ggt cct tag cca ctc ctt ctg tg, product of 268 bp;
myogenin sense: act ccc tta cgt cca tcg tg, antisense: cag gac agc ccc
act taa aa, product of 174 bp; MyHC IIb sense: agc ttg aaa acg agg tgg
aa, antisense: cct cct cag cct gtc tct tg, product of 191 bp; Socs3 sense:
gct ggc caa aga aat aac ca, antisense: agc tca cca gcc tca tct gt, product
of 224 bp; and ␤-actin sense: agc cat gta cgt agc cat cc, antisense: ctc
tca gct gtg gtg gtg aa, product of 227 bp. The quantitative PCR was
performed in a volume of 20 ␮l: 2 ␮l FastStart DNA-MasterSYBR
Green I, 4 mmol/l MgCl2, and primers at a concentration of 1 ␮mol/l.
The instrument settings were: After denaturing at 95°C for 10 min, 50
cycles were performed by denaturing at 95°C for 15 s, annealing at
65°C for 5 s, elongation for 11 s for IL-6; annealing at 64°C for 10 s,
elongation for 8 s for myogenin; annealing at 66°C for 10 s, elonga-
tion for 15 s for MyHC IIb; annealing at 66°C for 10 s, elongation for
9 s for mouse Socs3; annealing at 69°C for 10 s, elongation for 10 s
for mouse ␤-actin. Primers for STAT3: Mm Stat3_1_SG and insulin-
like growth factor-I (IGF-I): Mm_Igf1_2_SG were from Qiagen, and
instrument settings were as indicated by the manufacturer.
Small interfering RNA. Small interfering (si)RNA targeting IL-6
and STAT3 were designed and synthesized and annealed at Dharma-
con Research. An unrelated siRNA targeting firefly luciferase was
used as control in all experiments. Transfection was performed with
CellPhect (Amersham Biosciences, Buckinghamshere, England) with
200 nM siRNA according to the instructions of the manufacturer.
Briefly, 1 ⫻ 105 cells/well were seeded in six-well plates and
transfected in DMEM containing 25 mM glucose and 10% FCS
without antibiotics followed by glycerol shock after 24 h. Cells
received growth medium or differentiation medium afterwards for the
indicated time points.
Tissue lysates and Western blotting. Proteins were separated by
7.5% SDS-PAGE, and Western blotting was performed as previously
described (34). Subcellular fractionation to obtain cytoplasmic and
nuclear proteins was performed according to Wang et al. (32).
Immunostaining. Primary mouse myoblasts were seeded on glass
coverslips coated with Matrigel (diluted 1:10 in DMEM) and cultured
in PGM as described above until day 10 of differentiation. Cells on
coverslips were washed twice in PBS, fixed in PBS containing 4%
formaldehyde (pH 7.4) for 20 min, and quenched with 150 mM
glycine in PBS for 10 min before being washed again with PBS.
Blocking was performed in 5% normal goat serum and 0.05% Tween
20 in PBS for 30 min. Coverslips were incubated with antibody
recognizing fast-type skeletal myosin MyHC II (M4276; Sigma-
Aldrich, Deisenhofen, Germany) in blocking solution for 2 h at room
temperature, washed three times in PBS and incubated with the Alexa
488-labeled secondary antibody (Invitrogen) in blocking solution for
another 1 h and washed again. Nuclei were stained using TO-PRO3
(Invitrogen) diluted 1:1,000 in PBS for 10 min before mounting in
Mowiol 4-88 (Calbiochem, Bad Soden, Germany).
Statistical analysis. Data were calculated as means ⫾ SE, and
groups of data were compared using Student’s t-test. Statistical sig-
nificance was set at P ⬍ 0.05.
RESULTS
Enhanced expression and secretion of IL-6 in differentiating
C2C12 cells. C2C12 skeletal muscle cells are a well-estab-
lished cell culture system to study the regulation of myotube
formation (3, 17, 18, 31). Myogenic differentiation is induced
in these cells by replacing the growth medium containing 10%
serum with a serum-reduced medium. First, we studied the
regulation of IL-6 mRNA expression during differentiation and
compared it with the mRNA expression of the muscle differ-
entiation marker myogenin and myosin heavy chain MyH4,
also known as fast MyHC IIb. Figure 1 shows the characteristic
C130 IL-6 AND STAT3 REGULATE MYOGENIC DIFFERENTIATION

Activation of STAT3 in differentiating C2C12 cells. Next, we

A 30 investigated the activation of the signal transducer of IL-6,
myogenin mRNA

#
* STAT3 in the early differentiation state. Phosphorylation of
20 * * * * STAT3 on tyrosine 705 was detected after 6 h in differentiation
(x 10-2)

medium (Fig. 2A), and STAT3 remained phosphorylated after

# 24 and 48 h (Fig. 2B). The phosphorylated form was only
*
10
found in the nuclear extracts of the cells, further indicating a
* very rapid activation and nuclear translocation of STAT3 in
0
0 1 2 3 4 5 6 7 day myogenic conditions. The observed slight increase in STAT3
B protein levels after 24 h in Fig. 2A was accompanied by a
5
significant increase in STAT3 mRNA expression in differen-
*
MyHC IIb mRNA

4 tiating cells (Fig. 2C).

# * Knockdown of STAT3 blocks differentiation. Activation of
(x 10-2)

3
* STAT3 was confirmed to be responsible for the increased
#
*
2
#
mRNA expression levels of Socs3 during the differentiation
1 # process. Silencing STAT3 mRNA by transfecting the cells
* * *
0 with siRNA oligonucleotides targeting STAT3 blocked Socs3
C 0 1 2 3 4 5 6 7 day
10 # IL-6 protein secretion
* total lysate
IL-6 mRNA (x 10-4)

8 * * 20
A
*
(pg/ml)

6 15 pY-705
#
*
4 10
STAT3
2 5

0 0 β-actin
D 0 1 2 3 4 5 6 7 day
SOCS3 mRNA (x 10-2)

4 - 6 24 h
* * FM
* * * *
2 * B cytosol nucleus

pY-705
0
0 1 2 3 4 5 6 7 day
STAT3
Fig. 1. Enhanced expression and secretion of IL-6 in differentiating C2C12
cells. Expression of myogenin (A), MyHC IIb (B), IL-6 (C), and suppressor of
cytokine signaling 3 (Socs3; D) was measured by quantitative (q)PCR in RNA β-actin
obtained from C2C12 cells at the indicated day of differentiation. Switch to
differentiation medium was set as day 0. Values are shown as arbitrary units
related to ␤-actin expression as means ⫾ SE (n ⫽ 6 for myogenin, MyHC IIb,
- + 6 24 48 h - + 6 24 48 h
and IL-6; n ⫽ 4 for Socs3). * P ⬍ 0.05 vs. day 0; #P ⬍ 0.05 vs. the preceding ActD FM ActD FM
day. IL-6 protein production for 24 h was measured in the supernatant of cells
at days 1, 3, 5, and 7 of differentiation (C). Values are shown as means ⫾ SE
pg/ml (n ⫽ 6).
C
6 *
STAT3 mRNA

expression kinetics of the myogenic marker genes with an early

induction of myogenin mRNA on day 1 reaching plateau *
expression levels on day 4 to day 7 (⬎100-fold induction; Fig. 4
*
1A) and a subsequent increase in MyHC IIb expression with
2
the highest expression on day 6 (⬎7,000-fold induction; Fig.
1B). The mRNA expression of IL-6 was first significantly
increased on day 3 (2.8-fold) and reached a maximum induc- 0
0 1 2 4
tion on day 5 (9.1-fold; Fig. 1C). This upregulation of IL-6 day
expression led to accumulation of IL-6 protein in the superna-
tant of the cells, 5.5 ⫾ 0.2 pg/ml on day 3 and 21.3 ⫾ 3.9 pg/ml Fig. 2. Activation of signal transducers and activators of transcription 3
(STAT3) in differentiating C2C12 cells. Phosphorylation of STAT3 on ty-
on day 6 vs. 3.3 ⫾ 0.2 pg/ml on day 1 (Fig. 1C). After the first rosine 705 was detected by immunoblotting total lysates (A) or cytosolic and
48 h in the differentiation medium, no increases in IL-6 mRNA nuclear fractions (B) of C2C12 after 6, 24, or 48 h of differentiation [fusion
expression or protein were detected. Of note, the IL-6 target medium (FM)]. Actinomycin D (ActD) served as positive control. ␤-Actin was
gene Socs3 was significantly induced on day 1 with no further probed on the same membrane, and STAT3 protein was probed on a different
membrane obtained with the same lysates. C: expression of STAT3 mRNA in
increases during the differentiation process (Fig. 1D). Thus differentiating C2C12 cells was measured by qPCR. Values are shown as
Socs3 and IL-6 showed divergent expression kinetics in dif- arbitrary units related to ␤-actin expression as means ⫾ SE (n ⫽ 4). Day 0 was
ferentiating C2C12 myocytes. set as 1. *P ⬍ 0.05 vs. day 0.

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 IL-6 AND STAT3 REGULATE MYOGENIC DIFFERENTIATION
induction completely (Fig. 3A). Moreover, the mRNA levels of
myogenin were strongly reduced on days 1, 2, and 3 of
differentiation (Fig. 3B). Subsequently, the induction of MyHC
IIb expression was blocked (Fig. 3C). These data clearly
indicate that the early activation of STAT3 is important for the
initiation of myotube formation in C2C12 cells.
Knockdown of IL-6 reduces differentiation but does not
block phosphorylation of STAT3. The next question was
whether IL-6 is responsible for the activation of STAT3 and
thus gives the starting signal for the myogenic program. A
knockdown approach targeting IL-6 mRNA resulted in a clear
reduction of IL-6 mRNA levels during the differentiation
process (Fig. 4A). The C2C12 cells with silenced IL-6 expres-
sion also showed reduced myogenin expression on day 2 and
reduced MyHC IIb expression on days 2, 4, and 6 of differen-
tiation (Fig. 4, B and C). Myogenin protein expression levels
were decreased by the knockdown of STAT3 and IL-6 on day
2 of differentiation (Fig. 4D). On day 7 of differentiation,
myotube fusion was detected as number of MyHC II-positive
cells (Fig. 4E). Knockdown of STAT3 resulted in a strong
reduction of myotube fusion (fusion index of cells with si-
lenced STAT3 expression was 15 ⫾ 4% of control cells). The
effect in C2C12 cells with silenced IL-6 expression was less
pronounced but clearly visible (fusion index was 53 ⫾ 20% of
control cells). Noteably, knockdown of IL-6 did not prevent the
phosphorylation of STAT3 after 24 h in differentiation medium
(Fig. 4F). These data indicate that the early activation of
STAT3 is independent of IL-6.
SOCS-3 mRNA
C131
Differentiation of IL-6-deficient mouse primary myoblasts is
impaired. To clarify the importance of endogenous IL-6 produc-
tion for the myogenic differentiation, primary mouse skeletal
muscle cells obtained from quadriceps muscle of wild-type and
IL-6-deficient mice were used. Compared with C2C12 cells,
primary wild-type cells produced large amounts of IL-6 protein
with no further increase during differentiation (Fig. 5A). IL-6 was
not detectable in the supernatant of IL-6⫺/⫺ cells despite the
presence of Matrigel in the primary cell culture. When seeded
at the same density, wild-type and IL-6⫺/⫺ cells showed a
similar growth rate with comparable number of cells when the
differentiation of cells started spontaneously (65 ⫾ 15 ⫻ 103 in
wild-type vs. 75 ⫾ 26 ⫻ 103 in IL-6⫺/⫺ cells, day 0) and 24 h
thereafter (127 ⫾ 11 ⫻ 103 in wild-type vs. 133 ⫾ 19 ⫻ 103
in IL-6⫺/⫺ cells). The spontaneous start of differentiation
without being confluent can be explained by the high mRNA
expression levels of myogenin in both primary wild-type and
IL-6⫺/⫺ cells, which were ⬃100-fold higher than in C2C12
cells at day 0 of differentiation (0.36 ⫾ 0.08 myogenin/␤-actin
in wild-type and 0.24 ⫾ 0.04 in IL-6⫺/⫺ cells vs. 0.002 ⫾
0.0007 in C2C12 cells). It cannot be excluded that this differ-
ence between primary cells and C2C12 is caused by the
presence of growth factors in the Matrigel. The continuous
increase in myogenin expression in C2C12 cells during differ-
entiation led to comparable expression levels at day 4 (0.19 ⫾
0.06 myogenin/␤-actin in wild-type and 0.15 ⫾ 0.04 in
IL-6⫺/⫺ cells vs. 0.18 ⫾ 0.04 in C2C12 cells). No significant
difference in the mRNA levels of myogenin was detected
between wild-type and IL-6⫺/⫺ cells (Fig. 5B). No reduction in
the phosphorylation of STAT3 on tyrosine 705 was observed in
IL-6⫺/⫺ cells (Fig. 5C). Moreover, the increase in STAT3
mRNA expression was not prevented in the IL-6⫺/⫺ cells (Fig.

5 5D). The mRNA expression of Socs3 was slightly reduced on

A 4 days 0 and 1 in IL-6⫺/⫺ cells, but not different in later stages
3
of cell differentiation (Fig. 5E). In contrast, the induction of
* * * *
2
MyHC IIb mRNA expression was impaired in IL-6⫺/⫺ cells on
1
day 6 of differentiation (Fig. 5F). This effect was clearly
0
0 1 2 3 6 0 1 2 3 6 day visible in IL-6⫺/⫺ cells on day 10 of differentiation after
con si-STAT3 immunostaining for MyHC II protein. IL-6⫺/⫺ cells showed a
B 100
pronounced reduction in the number of MyHC II-positive cells
myogenin mRNA

400 indicating impairment to form myotubes (Fig. 5, G and H).

75 Determination of IGF-I expression revealed a strong upregu-
50
200
* lation during differentiation independent of the genotype (Fig.
25 * 5I). In quadriceps, plantaris, and tibialis muscle of adult wild-
type and IL-6⫺/⫺ mice, MyHC IIb mRNA expression was
*0 *1
3 6 3 6
0
0 1 2 3 6 2 3 6 day con si-STAT3 comparable (Fig. 5J).
con si-STAT3 Supplementation of IL-6 partially restores differentiation of
C 100 6000 IL-6⫺/⫺ cells. To study whether exogenous IL-6 can restore
MyHC IIb mRNA

75 4000 * myotube fusion, IL-6 was added to the medium of IL-6⫺/⫺

cells during the differentiation process at every other day. IL-6
50 * 2000 induced the expression of Socs3 indicating the effectiveness of
25 * this treatment (Fig. 6A). The IL-6 treatment also enhanced the
* 3 6 3 6
expression of MyHC IIb mRNA on days 1, 2, and 4 of
0 con si-STAT3
0 1 2 3 6 0 1 2 3 6 day differentiation but failed to increase the expression further on
con si-STAT3 day 6 (Fig. 6B). Compared with wild-type cells, the MyHC IIb
Fig. 3. Knockdown of STAT3 blocks differentiation. Expression of Socs3 (A), expression levels of IL-6 treated IL-6⫺/⫺ cells were still
myogenin (B), and MyHC IIb (C) was measured by qPCR in RNA obtained significantly reduced on days 4 and 6 (P ⫽ 0.001 on day 4; P ⫽
from C2C12 cells transfected with control small interfering (si)RNA (con) or 0.01 on day 6). A partial recovery of the impaired fusion of
siRNA oligonucleotides targeting STAT3. Values are shown as arbitrary units
related to ␤-actin expression as means ⫾ SE (n ⫽ 8 for days 0, 1, and 2 or n ⫽
IL-6⫺/⫺ cells by IL-6 treatment was also observed on day 10 of
4 for days 3 and 6). Day 0 was set as 1. *P ⬍ 0.05 vs. control transfected cells. differentiation after immunostaining for MyHC II protein (Fig.
Data of days 3 and 6 are also shown in a separate histogram. 6C). IL-6-treated IL-6⫺/⫺ cells showed a higher fusion index

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C132 IL-6 AND STAT3 REGULATE MYOGENIC DIFFERENTIATION

A
6
E
*
IL-6 mRNA

MyHC II
4 con +
nuclei
2 *
0
* * *
0 1 2 4 6 0 1 2 4 6 day
con si-IL-6
B
100 400
myogenin mRNA

si-STAT3
75
200
50

25
* 4 6 4 6
0 con si-IL-6 1

Fusion index
0 1 2 4 6 0 1 2 4 6 day

C con si-IL-6
si-IL6
*
6000 0.5
100
*
MyHC IIb mRNA

75 4000 * 0
con si-STAT3 si-IL6
50 2000
†
25 *
4 6 4 6
0
0 1 2 4 6 0 1 2 4 6 day con si-IL-6 F con si-IL-6

con si-IL-6 pY-705

STAT-3

GM FM GM FM GM FM GM FM
D
3

3
AT

AT

AT

AT

6h 24 h 6h 24 h
IL6

IL6

IL6

IL6
ST

ST

ST

ST
n

n
si-
si-
co

co

si-
si-

si-
si-
co

co

si-
si-

STAT3

myogenin

β-actin

2
β-actin protein
myogenin/

* *
0
0 2 4 6 day
3

3
3

n
n

IL6

IL6

IL6
IL6

AT

AT
AT

AT

co

co
co

co

si-

si-

si-
si-

ST

ST
ST

ST

si-

si-
si-

si-

Fig. 4. Knockdown of IL-6 does not block phosphorylation of STAT3. Expression of IL-6 (A), myogenin (B), and MyHC IIb (C) was measured by qPCR in RNA
obtained from C2C12 cells transfected with control siRNA (con) or siRNA oligonucleotides targeting IL-6. Values are shown as arbitrary units related to ␤-actin
expression as means ⫾ SE (n ⫽ 4). Day 0 was set as 1. *P ⬍ 0.05, †P ⬍ 0.1 vs. control transfected cells. Data of days 4 and 6 are also shown in a separate
histogram. D: STAT3, myogenin, and ␤-actin protein expression was detected by immunoblotting of total lysates of C2C12 cells. Histogram shows the results
of the densitometric quantification as means ⫾ SE (n ⫽ 4) *P ⬍ 0.05 vs. control transfected cells. E: immunostaining of MyHC II protein in C2C12 cells
transfected with control siRNA (con), siRNA oligonucleotides targeting STAT-3 or IL-6 on day 7 of differentiation (⫻10 magnification). Nuclei are shown in
red. Fusion index was determined by dividing the number of nuclei in MyHC II-positive myotubes by the total number of nuclei analyzed and is shown as
means ⫾ SE (n ⫽ 4). Fusion index of control cells was set as 1. *P ⬍ 0.05 vs. con. F: phosphorylation of STAT3 on tyrosine 705 was detected by immunoblotting
of total lysates of C2C12 cells after 6 or 24 h of differentiation (GM, growth medium; FM, fusion medium). STAT3 protein was detected on a separate membrane
prepared with the same lysates.

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 IL-6 AND STAT3 REGULATE MYOGENIC DIFFERENTIATION C133

A 40
day 1 day 4 day 7 F 120

MyHC IIb mRNA

IL-6 pg/µg RNA
30
80
20 *
40
1
$ $ $
0
C2C12 wt ko C2C12 wt ko C2C12 wt ko 0
B 0 1 2 4 6 0 1 2 4 6 day
myogenin mRNA

wt IL-6 ko
2
G wt IL-6 ko H
1

1
0

Fusion index
0 1 2 4 6 0 1 2 4 6 day
wt IL-6 ko MyHC II

*
+
C 0 1 2 0 1 2 day
nuclei 0.5

pY-705
STAT-3 0
wt IL-6 ko
β-actin
day 10 day 10
wt IL-6 ko
I
50
D 12
IGF1 mRNA
STAT3 mRNA

40
8 30
20
4
1
0 0
0 1 2 4 6 0 1 2 4 6 day 0 1 2 4 6 0 1 2 4 6 day
wt IL-6 ko wt IL-6 ko
E J
SOCS3 mRNA

20
15 quadriceps plantaris tibialis
12
MyHC IIb mRNA

10 6
8
5
0 0 1 2 4 6
*0 *1 2 4 6 day
8
4
4
wt IL-6 ko 4 2

0 0 0
wt IL-6 ko wt IL-6 ko wt IL-6 ko

Fig. 5. Differentiation of IL-6-deficient mouse primary myoblasts is impaired. A: IL-6 protein concentration in the supernatant of primary muscle cells obtained
from wild-type mice (wt), IL-6-deficient mice (ko), or C2C12 cells cultivated in FM for C2C12 cells during differentiation related to total RNA content as pg
IL-6/ ␮g total RNA (means ⫾ SE; n ⫽ 6). $IL-6 protein concentration was below detection limit. B, D, E, F, and I: expression of myogenin, STAT3, Socs3,
MyHC IIb, and IGF-I mRNA in differentiating primary cells. Values are shown as arbitrary units related to ␤-actin expression as means ⫾ SE (n ⫽ 4). Day 0
was set as 1. *P ⬍ 0.05 vs. wild type at the same day. C: phosphorylation of STAT3 on tyrosine 705 and ␤-actin were detected by immunoblotting of total lysates
of primary cells. STAT3 protein was detected on a separate membrane prepared with the same lysates. G: immunostaining of MyHC II protein in primary cells
on day 10 of differentiation (⫻10 magnification). Nuclei are shown in red. H: fusion index was determined by dividing the number of nuclei in MyHC II-positive
myotubes by the total number of nuclei analyzed and shown as means ⫾ SE (n ⫽ 5). Fusion index of wild-type cells was set as 1. J: expression of MyHC IIb
mRNA in quadriceps, plantaris, and tibialis muscle of 15-wk-old wild-type or IL-6 ko mice. Values are shown as arbitrary units related to ␤-actin expression
as means ⫾ SE (n ⫽ 5).

compared with untreated IL-6⫺/⫺ cells, but it was still reduced
compared with wild-type cells.
DISCUSSION
The principal finding of this study is that two components of
the IL-6 signaling cascade, IL-6 itself and STAT3, are inde-
pendently involved in the differentiation and fusion of skeletal
muscle cells. This conclusion is supported by the following
results: The expression of both IL-6 and STAT3 was increased
during the differentiation of C2C12 cells and of STAT3 during
the differentiation of primary mouse skeletal muscle cells.
Knockdown of IL-6 and STAT3 in C2C12 cells resulted in
impaired expression of the myogenic markers myogenin and
MyHC IIb and reduced myotube fusion. Primary muscle cells
obtained from IL-6⫺/⫺ mice showed impaired expression of
MyHC IIb and a clear reduction in myotube formation. Our
data demonstrating the importance of STAT3 are well in line
with previous results obtained in C2C12 cells and primary
mouse myoblasts (31): the authors describe enhanced protein
level and tyrosine phosphorylation of STAT3 when C2C12
cells start to differentiate. They found that inhibition of STAT3
activation by the JAK2 inhibitor AG490 and by knockdown of
JAK2 by siRNA or the knockdown of STAT3 itself block the
myogenic differentiation of C2C12 cells and primary mouse
myoblasts. A recent study supports the activation of the JAK2/
STAT3 pathway during myogenic differentiation since it

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C134 IL-6 AND STAT3 REGULATE MYOGENIC DIFFERENTIATION

# # visible after 6 h in differentiation medium. Knockdown of IL-6

A 12 in C2C12 cells did not impair the increased phosphorylation of
SOCS3 mRNA

STAT3 observed during the first 24 h in differentiation me-

6
# dium. In IL-6⫺/⫺ primary mouse muscle cells, the phosphor-
# ylation of STAT3 was not impaired and the STAT3 expression
levels during the differentiation were not reduced. These data
0 clearly indicate that in our cell culture system the phosphory-
0 1 2 4 6 0 1 2 4 6 1 2 4 6 day
wt IL-6 ko IL-6 ko + IL-6
lation and signal transduction activity of STAT3 are not de-
pendent on IL-6. Of note, IL-6 was not detectable in the
B supernatant of IL-6⫺/⫺ cells grown on Matrigel-coated cell
MyHC IIb mRNA

80 culture dishes.
# This raises the question about the mechanism responsible for
40 the activation of STAT3 or more precisely: which mechanism
allows or maintains the activity of STAT3 during myogenic
# #
0 differentiation? It should be noted that one explanation for the
0 1 2 4 6 0 1 2 4 6 1 2 4 6 day apparent increase in the tyrosine phosphorylation of STAT3 is
wt IL-6 ko IL-6 ko + IL-6
the upregulation of STAT3 protein levels during differentiation
(31 and data from the present study), which is due to enhanced
C wt IL-6 ko RNA expression. Importantly, increased STAT3 mRNA levels
were found in both C2C12 cells and primary mouse muscle
cells 24 h after the start of differentiation with further increases
MyHC II
+
during the later phase. The kinases responsible for the detected
nuclei tyrosine phosphorylation of STAT3 belong to the Janus family
of tyrosine kinases. The data of Wang et al. (31) clearly
showed that JAK2 activity is necessary for the phosphorylation
and activation of STAT3 during differentiation. JAK2 can be
IL-6 ko + IL-6 activated by ligands of the IL-6 family of cytokines binding to
1
a gp130/heterodimer receptor complex. In addition, previous
Fusion index

# * work of Spangenburg (24) has highlighted a role for an

0.5 * IGF-I-mediated activation of STAT3 in myogenic differentia-
0
wt IL-6 ko IL-6 ko
+ IL-6
Fig. 6. Application of IL-6 partially restores cell fusion of primary IL-6-
deficient myoblasts. Primary wild-type and IL-6⫺/⫺cells were cultured and
differentiated as described in EXPERIMENTAL PROCEDURES. Starting from day 0
of differentiation IL-6⫺/⫺ cells received every other day mouse recombinant
IL-6 (10 ng/ml). A and B: mRNA expression of Socs3 and MyHC IIb. Values
are shown as arbitrary units related to ␤-actin expression as means ⫾ SE (n ⫽
4). Day 0 was set as 1. #P ⬍ 0.05 vs. IL-6⫺/⫺ without IL-6 supplementation
at the same day. C: Immunostaining of MyHC II protein in primary cells on
day 10 of differentiation (⫻10 magnification). Nuclei are shown in red. Fusion
index was determined by dividing the number of nuclei in MyHC II-positive
myotubes by the total number of nuclei analyzed and shown as means ⫾ SE
(n ⫽ 4). Fusion index of wild-type cells was set as 1. *P ⬍ 0.05 vs. wild-type;
#P ⬍ 0.05 vs. IL-6⫺/⫺ without IL-6 supplementation.
shows enhanced phosphorylation of JAK2 and STAT3 as well
as increased STAT3 protein levels in differentiating human
skeletal muscle cells (30).
At first sight, IL-6 would be a good candidate for the
activation of STAT3 since the knockdown of IL-6 or STAT3 in
our study similarly repressed the RNA levels of myogenic
markers. Moreover, knockdown of IL-6 previously has been
shown to reduce the expression of myogenin in C2C12 cells
after 24 h in differentiation medium (3). However, our study
provides evidence for a regulation and function of STAT3
independent of IL-6. This IL-6-independent activation of
STAT3 is supported by the following data: Increased expres-
sion and secretion of IL-6 are late events during differentiation
of C2C12 cells while increased phosphorylation of STAT3 was
tion. Spangenburg found that the increased expression and
secretion of IGF-I that occurred in differentiating skeletal
muscle cells (27) are responsible for the STAT3-mediated
Socs3 expression. Moreover, activation of STAT3 by IGF-I-
mediated IGF-I receptor activation has not only been observed
in C2C12 cells but also in mouse muscle tissue in vivo (36).
Based on these findings, we hypothesize that this pathway
might also be of importance for the IL-6-independent activa-
tion of STAT3 found in the present study. Notably, primary
mouse IL-6⫺/⫺ myoblasts showed similar expression levels of
IGF-I as wild-type cells.
A novel pathway described as an activator of the JAK2-
STAT3 axis involves the Eph family of receptor tyrosine
kinases and their membrane bound ligands, the ephrins (14).
Although there is currently no experimental proof for the
hypothesis that ephrin/Eph signals are involved in the activa-
tion of STAT3 during differentiation, this idea is intriguing
because it opens the possibility that cell-cell contact rather than
circulating factors are necessary for the start of myogenic
differentiation and would be another explanation for an IL-6-
independent activation of STAT3. Notably, the ephrinA/EphA
signal has recently been involved in the facilitation of myo-
genic differentiation by IGF-I (16).
Our data point to an important function of IL-6 for the
complete differentiation of muscle cells independent of STAT3
since the fusion of the IL-6⫺/⫺ muscle cells was clearly
disturbed despite the fact that the activation of STAT3 was not
different from that in wild-type cells. This function is also
independent of Socs3, since the expression level of Socs3 was
similar in wild-type and IL-6⫺/⫺ cells in the later phase of
differentiation when the defect in the IL-6⫺/⫺ cells became

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 IL-6 AND STAT3 REGULATE MYOGENIC DIFFERENTIATION
evident. The difference in cell fusion could also not be ex-
plained by differences in IGF-I expression which is similar in
wild-type and IL-6⫺/⫺ cells at later time points. The sponta-
neous start of differentiation of the primary myoblasts can also
be due to other factors present in the Matrigel that can activate
pathways known to promote myogenic differentiation, namely
the Akt/PKB, ERK, or p38 MAPK pathway. Reduced activa-
tion of these pathways in IL-6⫺/⫺ cells may also contribute to
the impaired differentiation of these cells, since the pathways
can also be activated by IL-6 (3, 10, 35).
Our data clearly indicate that IL-6 is important for the
fusion of skeletal muscle cells. In vivo data suggest that
IL-6 deficiency could be compensated during development
since IL-6⫺/⫺ mice exhibit no reduced myofiber number or
myofiber cross-sectional area in plantaris muscle (23) and
MyHC IIb RNA expression was not reduced in adult muscle
tissue of these mice as shown in the present study. However,
IL-6 might be of relevance when formation of myofibers is
necessary in adults after functional muscle overloading or
tissue injury. Under these conditions, increased muscular ex-
pression of IL-6 is detected in mice and humans as well as
phosphorylation of STAT3 particularly in activated satellite
cells and proliferating myoblasts (15, 23, 28, 29). Furthermore,
Serrano et al. (23) could show that in IL-6⫺/⫺ mice the
compensatory muscle hypertrophy after overloading is re-
duced. To conclude, there is good evidence that IL-6 and
STAT3 are important for the activation and proliferation of
satellite cells and myoblasts during postnatal skeletal muscle
growth. By using primary and C2C12 cell culture models for
myogenic differentiation, we can now demonstrate that IL-6
also has a crucial function for the differentiation of myoblasts
that is not mediated via STAT3.
GRANTS
This study was supported by grants from the Deutsche Forschungsgemein-
schaft (WE 4176/1-2 and GRK1302/2; to C. Weigert and GRK 1302/1; to E.
Schleicher). This study was supported in part by a grant from the German
Federal Ministry of Education and Research (BMBF) to the German Center for
Diabetes Research (DZD e.V.).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.H. and H.R. performed experiments; M.H. ana-
lyzed data; M.H. drafted manuscript; H.-U.H., E.D.S., and C.W. interpreted
results of experiments; C.W. conception and design of research; C.W. prepared
figures; C.W. edited and revised manuscript; C.W. approved final version of
manuscript.
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