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Am J Physiol Cell Physiol 310: C66–C79, 2016.
First published October 21, 2015; doi:10.1152/ajpcell.00052.2015.
Lewis lung carcinoma regulation of mechanical stretch-induced protein
synthesis in cultured myotubes
Song Gao1 and James A. Carson1,2
1
Integrative Muscle Biology Laboratory, Department of Exercise Science, University of South Carolina, Columbia, South
Carolina; and 2Center for Colon Cancer Research, University of South Carolina, Columbia, South Carolina
Submitted 24 February 2015; accepted in final form 19 October 2015
cachexia; mTORC1; gp130; MAP kinase; AMPK
CACHEXIA, A CONDITION THAT
involves the unintentional loss of
body weight, including muscle and fat mass, is associated with
many types of cancer (16). Fundamentally, cachexia-associated
skeletal muscle loss includes the activation of skeletal muscle
protein degradation and the suppression of muscle protein
synthesis (66). Implantation of Lewis lung carcinoma (LLC)
cells into mice has been used widely to study mechanisms of
cancer-induced muscle wasting (46). Studies with LLC cells
have implicated cellular signaling involving TNF-␣ (33),
NF-␬B (9), glycoprotein 130 (gp130)/STAT3 (7, 49), and p38
in muscle wasting (75, 76). LLC cells can induce cultured
myotube wasting directly in vitro by disrupting the regulation
of protein degradation and protein synthesis (49, 75). Whereas
Address for reprint requests and other correspondence: J. A. Carson,
Dept. of Exercise Science, Public Health Research Center, Univ. of South
Carolina, Rm. 405, 921 Assembly St., Columbia, SC 29208 (e-mail: carsonj
@mailbox.sc.edu).
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LLC-induced gp130/STAT3, p38, and CCAAT enhancer-binding protein-␤ signaling pathways have been identified as important regulators of protein degradation (49, 75, 76), gaps
remain in our understanding of the mechanisms of protein
synthesis suppression by LLC-derived cachectic factors.
Mechanical stimuli such as loading or stretch induce an
anabolic response in skeletal muscle that is important for the
maintenance of mass and function (65). Therefore, it is plausible that disease-induced disruptions to mechanical signaling
could contribute to muscle wasting. Cultured myotubes respond to chronic or intermittent stretch through increased
protein synthesis, morphological maturation, and development
of the contractile apparatus (13, 43, 67). Overload and stretch
can stimulate skeletal muscle hypertrophy by inducing mammalian target of rapamycin (mTOR) complex 1 (mTORC1)
(27, 74). The subsequent phosphorylation of p70 ribosomal
protein S6 kinase (p70S6K), S6 ribosomal protein (S6RP), and
eukaryotic initiation factor-4E binding protein 1 (4EBP1) results in ribosomal RNA and protein transcription, ribosomal
biogenesis, translation initiation, and the induction of protein
synthesis (35). Activation of mTOR signaling by mechanical
stimulation can occur independent of Akt, and there is evidence that signaling involving phospholipase D (25, 73) and
extracellular signal-regulated kinases 1/2 (ERK1/2) (34, 38) is
involved in the process. Mechanical stimuli can activate muscle ERK1/2 and p38 signaling (37, 44), which can both induce
mTORC1 (38, 42, 72). Chronic systemic inflammation has the
potential to inhibit muscle protein synthesis activation. The
muscle inflammatory response can attenuate IGF-I activation
of mTOR (14, 62) and leucine induction of protein synthesis
(29). Interestingly, muscle pathways associated with cachexiainduced skeletal muscle wasting also can be activated by
mechanical stimuli to produce growth (31, 47, 49, 75). While
muscle basal mTOR activity and protein synthesis are suppressed in several mouse models of cancer cachexia (49, 68),
the ability of the cachectic environment to interact with muscle
mechanical signaling is in need of further investigation. Further work is needed to determine whether cachexia-induced
inflammatory signaling can negatively regulate the mechanical
induction of protein synthesis.
The functional receptor complexes for the IL-6 family of
cytokines involve gp130, which can induce several intracellular signal pathways, including STAT3 and MAPK (63). The
IL-6 family of cytokines has a documented role in skeletal
muscle mass regulation (39). Skeletal muscle expression of
IL-6 and leukemia inhibitory factor (LIF) are induced by
exercise (8, 45), and both IL-6 and LIF can regulate overloadinduced muscle hypertrophy (5, 59). A role of gp130-initiated
signaling for mechanical regulation of muscle protein synthesis
has not been established. Cancer-induced systemic elevation of
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Gao S, Carson JA. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes. Am J
Physiol Cell Physiol 310: C66 –C79, 2016. First published October
21, 2015; doi:10.1152/ajpcell.00052.2015.—Mechanical stretch can
activate muscle and myotube protein synthesis through mammalian
target of rapamycin complex 1 (mTORC1) signaling. While it has
been established that tumor-derived cachectic factors can induce
myotube wasting, the effect of this catabolic environment on myotube
mechanical signaling has not been determined. We investigated
whether media containing cachectic factors derived from Lewis lung
carcinoma (LLC) can regulate the stretch induction of myotube
protein synthesis. C2C12 myotubes preincubated in control or LLCderived media were chronically stretched. Protein synthesis regulation
by anabolic and catabolic signaling was then examined. In the control
condition, stretch increased mTORC1 activity and protein synthesis.
The LLC treatment decreased basal mTORC1 activity and protein
synthesis and attenuated the stretch induction of protein synthesis.
LLC media increased STAT3 and AMP-activated protein kinase
phosphorylation in myotubes, independent of stretch. Both stretch and
LLC independently increased ERK1/2, p38, and NF-␬B phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38
rescued the stretch induction of protein synthesis. Interestingly, either
leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein
synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived
cachectic factors can dissociate stretch-induced signaling from protein
synthesis through ERK1/2 and p38 signaling, and that glycoprotein
130 signaling is associated with the basal stretch response in myotubes.
LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
METHODS
C2C12 myotube culture. C2C12 myoblasts and LLC (American
Type Culture Collection, Manassas, VA) were cultured in DMEM,
supplemented with 10% FBS, 50 U/ml penicillin, and 50 ␮g/ml
streptomycin. To induce C2C12 myoblast differentiation, C2C12
myoblasts were incubated in DMEM supplemented with 2% heatinactivated horse serum, 50 U/ml penicillin, and 50 ␮g/ml streptomycin for 72 h after they reached ⬃95% confluence.
Cell stretch. Cell stretch experiments were conducted using methods previously described (11, 58). Static stretching device consists of
a small frame with two axles (part 1 in Fig. 1, A and B) separated by
lateral supports (part 2 in Fig. 1, A and B). The axles were threaded to
accept screw nuts (part 3 in Fig. 1, A and B). Silastic membranes
(GLOSS/GLOSS, 0.02 in., Speciality Manufacturing, Saginaw, MI,
part 5 in Fig. 1, A and B) were mounted onto the devices by two
friction-fit C-clamps (part 4 in Fig. 1, A and B). The assembled
stretching device was then immersed into distilled H2O, autoclaved,
and transferred into a sterile 100-mm petri dish (Fig. 1A). The screw
nuts were set to 30 cycles from baseline when the stretching device
was assembled (Fig. 1B, left). Approximately 175 ␮l type I collagen
solution (Advanced Biomatrix, San Diego, CA; diluted into 1 mg/ml
by serum-free DMEM) was applied onto one edge of Silastic membrane on each stretching device. The collagen solution was then drawn
across the substrate with a sterile cell scraper. Excess collagen was
aspirated, and the culture dish containing the stretching device was
transferred to a 37°C incubator for 1 h. After the incubation period,
stretching device was washed by sterile distilled H2O twice and air
dried under UV overnight. C2C12 myoblasts were suspended (⬃1 ⫻
106 cells/ml) and plated onto Silastic membrane mounted in a stretching device (⬃1.5–2 ⫻ 105 cells/stretching device). The cells were
grown to ⬃95% confluence and differentiated into myotubes. To
induced 5% stretch, screw nuts on both axles were rotated by 1.5
cycles using sterile forceps (Fig. 1B, right). Myotubes were constantly
stretched by for 4 or 24 h.
Cell stretch in LLC conditioned media. C2C12 myotubes were
treated with control or LLC media for 72 h, as previously described
(75). Briefly, ⬃2 ⫻ 105 LLC cells were plated into 10-cm-diameter
culture dish. LLC cells were grown for 48 h, and the final density of
LLC cells was 0.8 –11 ⫻ 106 cells per culture dish. The LLC cell
culture media was then collected by centrifuge (3,000 rpm for 5 min).
One volume of LLC cell culture media was mixed with three volumes
of serum-free DMEM to form 25% LLC media. Seventy-two hour
differentiated myotubes were incubated in LLC media for 72 h. LLC
media was refreshed every 24 h. DMEM media supplemented with
2.5% FBS, 50 U/ml penicillin, and 50 ␮g/ml streptomycin was used
as control media. Myotubes were stretched by 5% in last 4 or 24 h of
control/LLC media incubation.
gp130 signaling and ERK1/2 signaling inhibition. To inhibit LLCinduced gp130 dependent signaling, myotubes were incubated in
LLC media supplemented with gp130 antibody (1:1,000, Santa
Cruz Biotechnology, Santa Cruz, CA; dialyzed in PBS at 4°C
overnight) for 72 h. To inhibit gp130 downstream targets, myotubes were incubated in LLC media supplemented with NF-␬B and
STAT3 inhibitor ammonium pyrrolidinecarbodithioate (PDTC) (50
␮M; Sigma-Aldrich, St. Louis, MO) and STAT3 specific inhibitor,
LLL12 (100 nM, BioVision, Milpitas, CA) for 72 h. To inhibit
LLC-induced ERK1/2 signaling, myotubes were incubated in LLC
media supplemented with PD-98059 (20 ␮M, Cell Signaling Technology, Danvers, MA) for 72 h.
AMPK and p38 MAPK signaling inhibition. To inhibit LLCinduced AMPK and p38 MAPK signaling, myotubes were incubated
in LLC media for 72 h. AMPK-specific inhibitor, compound C (20
␮M, Sigma-Aldrich), or p38 MAPK-specific inhibitor, SB-203580
(10 ␮M, Cell Signaling Technology), was added into LLC media, and
myotubes were stretched by 5% in the last 4 h of LLC media
incubation.
Cytokine analysis of LLC media. The level of 10 inflammatory
cytokines [IL-1␤, IL-6, IL-10, IL-17, IFN-␥, monocyte chemoattractant protein (MCP)-1, regulated on activation normal T-expressed and
presumably secreted (RANTES), TNF-␣, LIF, and macrophage colony-stimulating factor (M-CSF)] in culture media from three independent LLC cell cultures was measured by Bio-Plex multiplex
analysis kit (Mouse Cytokine Th17 Panel A 6-Plex Group l and 2,
Bio-Rad, Hercules, CA), following the manufacturer’s instructions.
The beads in a 96-well filter plate were analyzed by Bio-Plex 200
system (Bio-Rad). The growth media (DMEM supplemented with
10% FBS, 50 U/ml penicillin, and 50 ␮g/ml streptomycin) used in
those three LLC cell cultures were used as controls.
LIF signaling inhibition. To inhibit the LLC-induced LIF/gp130
signaling, myotubes were incubated in LLC media supplemented with
LIF antibody (1.5 ␮g/ml, Novus Biologicals, Littleton, CO) for 72 h.
Myotubes were stretched by 5% in last 24 h. The dosage of LIF
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the IL-6 family of cytokines is also associated with muscle
wasting (10). We have demonstrated that gp130 is necessary
for LLC to induce muscle STAT3, p38 MAPK, and NF-␬B
signaling pathways, muscle protein degradation, and muscle
wasting (49). Interestingly, IL-6 suppression had no effect on
LLC-induced gp130 signaling (49). Additionally, IL-6 or
gp130 antibody administration could not rescue LLC suppression of protein synthesis (49). Further work is needed to
determine gp130’s role in LLC suppression of muscle protein
synthesis.
AMP-activated protein kinase (AMPK) is an intracellular
sensor of energy stress that can negatively regulate muscle
protein synthesis in response to nutrient deficiency (54).
AMPK can inhibit mTORC1 through the phosphorylation of
tuberous sclerosis complex 2 and raptor, resulting in suppressed protein synthesis (22). Furthermore, AMPK activation
is sufficient to prevent mTORC1 induction by anabolic signaling involving IGF-I, leucine, glucose, and electrically stimulated muscle contractions (41, 53, 64). In the mouse muscle,
AMPK signaling is activated as cachexia progresses (49, 68).
IL-6, an important inducer of muscle wasting in some cancer
cachexia models, can activate AMPK (52). Additionally,
AMPK inhibition rescues IL-6 suppression of mTOR signaling
in myotubes (69), which suggests a role for AMPK in the basal
suppression of muscle protein synthesis with some types of
cancer. LLC-derived cachectic factors, independent of IL-6 or
gp130 signaling, also induce myotube AMPK signaling (49).
However, it is not known if LLC-induced AMPK signaling can
alter the mechanical induction of protein synthesis.
Suppressed mechanical signaling in muscle has the potential
to contribute to the overall rate of muscle wasting in cancer
patients by disrupting anabolic signaling related to daily activity. It has not been established if intracellular signaling induced
by tumor-derived cachectic factors can regulate stretch induction of myotube protein synthesis. The purpose of this study
was to examine if the stretch induction of myotube protein
synthesis could be inhibited by the administration of LLC
media that induces wasting. We hypothesized that LLC-secreted factors would attenuate the mechanical stretch induction
of skeletal muscle protein synthesis through chronic activation
of MAPK, gp130/STAT3, and AMPK signaling. We examined
the effect of chronic stretch on C2C12 myotube protein synthesis in the presence or absence of LLC media. The role of
LLC-induced gp130-dependent STAT3, NF-␬B, MAPK, and
gp130 independent AMPK signaling was investigated with the
administration of specific inhibitors.
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LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
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Fig. 1. Cell stretch regulation on myotube hypertrophy. A: cell
stretch device. Static stretching device is a small frame with
two threaded axles (part 1) and two lateral supports (part 2).
The distance between two lateral supports was adjusted by
rotating screw nuts (part 3) on both axles. The Silastic membrane where cells were grown (part 5) was fixed on lateral
supported by two friction-fit C-clamps (part 4). B: representative image of cell stretch. The screw nuts were set to 30 cycles
from the baseline when stretch device was assembled (left) and
were rotated by another 1.5 cycles when cells were stretched
(right). C: pictures (left, bar: 100 ␮M) and mean diameter of
0% stretch control and 4 h/24 h stretched myotubes (right). All
values are means ⫾ SE. *P ⬍ 0.05 vs. control, one-way
ANOVA.
antibody was determined by treating myotubes with 10 ng/ml mouse
recombinant LIF (Thermo Fisher Scientific, Waltham, MA) and
different doses of LIF antibody for 30 min.
Western blots. Western blot analysis was performed as previously
described (24, 69). Briefly, cells were scraped into ice-cold RIPA
buffer [50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA,
0.5% sodium deoxycholate, 0.1% SDS, 5 mM NaF, 1 mM NaVO4, 1
mM ␤-glycerophosphate, and 1% protease inhibitor cocktail (SigmaAldrich)]. Cell lysates were homogenized on ice and centrifuged at
4°C, and supernatants were collected. Protein concentrations were
determined by the Bradford method. Homogenates were fractionated
in SDS-polyacrylamide gels and transferred to polyvinylidene fluoride
membranes. After the membranes were blocked, antibodies for phosphorylated/total ERK1/2, STAT3, NF-␬B p65, AMPK, MAPK-activated protein kinase 2 (MAPKAPK-2), p70S6K, S6RP, 4EBP1,
GAPDH (Cell Signaling Technology), p38 MAPK (Santa Cruz Biotechnology), and puromycin (Millipore, Billerica, MA) were incubated at dilutions from 1:2,000 to 1:8,000 overnight at 4°C in 2%
Tris-buffered saline-Tween 20 milk. Anti-rabbit or anti-mouse IgGconjugated secondary antibodies (Cell Signaling Technology) were
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LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
incubated with the membranes at 1:2,000 to 1:5,000 dilutions for 2 h
in 2% Tris-buffered saline-Tween 20 milk. Enhanced chemiluminescence (Advansta, Menlo Park, CA) developed by autoradiography
was used to visualize the antibody-antigen interactions. Blots were
analyzed by measuring the integrated optical density (IOD) of each
band with ImageJ software (National Institutes of Health, Bethesda,
MD).
Protein synthesis measurement. The rate of protein synthesis rate in
myotubes was determined by puromycin, as previously described
(21). In brief, puromycin (EMD Chemicals, San Diego, CA) was
added into cell culture media (1 ␮M final concentration) 30 min
before protein collection. The amount of puromycin incorporated into
newly synthesized protein was determined by Western blots.
Myotube diameter measurement. C2C12 myotube diameter was
quantified as previously published (69). Briefly, the diameter of each
myotube was determined by the average value of diameters at three
points randomly distributed on whole myotube. For each condition, all
myotubes in 15 randomly chosen fields were measured. All measurements were conducted in a blinded fashion.
Statistical analysis. Student’s t-test, one-way ANOVA, and twoway ANOVA (indicated in figure legends) were used to examine the
effects of stretch and culture conditions. Post hoc analyses were
performed with Student-Newman-Keuls method. For all comparisons,
six replicates from two independent experiments were included. P
values ⬍ 0.05 were considered significant.
RESULTS
Mechanical stretch induces C2C12 myotube protein synthesis and hypertrophy. Differentiated C2C12 myotubes were
subjected to 5% stretch for either 4 or 24 h. Stretch for 24 h
significantly increased C2C12 myotube mean diameter (Fig.
1C). Related to growth signaling, both 4- and 24-h stretch
induced p70S6K and S6RP phosphorylation (Fig. 2A). However, neither stretch time point demonstrated an increase
4EBP1 phosphorylation (Fig. 2A). Myotube protein synthesis
rate was also significantly increased by both 4- and 24-h stretch
(Fig. 2B). These results demonstrate stretch can induce a strong
anabolic stimulus to cultured myotubes.
LLC attenuates stretch induction of C2C12 myotube protein
synthesis. Previously, we have demonstrated that LLC secreted
factors can suppress myotube protein synthesis and mTOR
signaling (49). To investigate whether LLC secreted factors
can suppress the stretch induction of protein synthesis, we
compared stretch effect on protein synthesis signaling in
C2C12 myotubes preincubated with control and LLC media
(Fig. 3A). As previously reported (49), LLC media resulted in
myotube atrophy (Fig. 3B). Stretch was able to increase myotube diameter in LLC media, but stretched myotubes in LLC
media had a decreased mean diameter compared with stretched
myotubes in control media (Fig. 3B). Stretch for 24 h increased
the phosphorylation of p70S6K and S6RP, but not the phosphorylation of 4EBP1, in myotubes that were incubated in
control media (Fig. 3C). LLC media decreased the basal
expression level of p70S6K, S6RP, and 4EBP1 phosphorylation and prevented the stretch induction of p70S6K and S6RP
phosphorylation (Fig. 3C). Stretch significantly increased myotube protein synthesis in control media. In addition to the LLC
media suppression of basal protein synthesis, the stretch induction of protein synthesis was prevented (Fig. 3D). These results
demonstrate LLC-derived cachectic factors can dissociate
stretch-induced mechanical signaling from mTORC1 signaling
and protein synthesis induction.
Stretch- and LLC-induced C2C12 myotube signaling
pathways. To understand the potential mechanisms underlying
the LLC suppression on stretch induction of myotube protein
synthesis, we investigated the effects of stretch and LLC media
on stress and inflammatory signaling pathways involved in
muscle protein turnover, including STAT3 (49), NF-␬B (49),
ERK1/2 (38), p38 MAPK (17), and AMPK (22). Both mechanical stretch alone and LLC alone increased p38, ERK1/2, and
NF-␬B p65 phosphorylation (Fig. 4). However, stretch in the
presence of LLC media significantly attenuated the LLC induction of p38 and ERK1/2 phosphorylation (Fig. 4). LLC
increased AMPK and STAT3 phosphorylation, independent of
stretch, while stretch alone or in the presences of LLC media
did not increase AMPK or STAT3 phosphorylation (Fig. 4).
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Fig. 2. Effect of mechanical stretch on myotube mammalian target of rapamycin (mTOR) signaling pathway and protein synthesis. A: phosphorylation
[ratio between phosphorylated (p) and total] of p70 ribosomal protein S6
kinase (p70S6K), S6 ribosomal protein (S6RP), and eukaryotic initiation
factor-4E binding protein 1 (4EBP1) was measured by Western blot. B: protein
synthesis rate was measured by Western blot against puromycin. NC is the
negative control, the protein sample from C2C12 myotubes without puromycin
treatment. IOD, integrated optical density. Bar graphs represent fold changes
relative to control group. All values are means ⫾ SE. *P ⬍ 0.05 vs. control,
one-way ANOVA.
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LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
Inflammatory cytokines in LLC media. The levels of 10
well-described inflammatory cytokines (IL-1␤, IL-17, IL-6,
IFN-␥, IL-10, MCP-1, TNF-␣, LIF, M-CSF, and RANTES)
were measured in media collected from LLC cell culture.
M-CSF, LIF, and RANTES were only detected in media
collected from LLC cell cultures (M-CSF: 12.40 ⫾ 3.34 pg/ml;
LIF: 9.93 ⫾ 2.55 pg/ml, RANTES: 0.58 ⫾ 0.33 pg/ml) (Table.
1). MCP-1 was significantly elevated in LLC media (353.90 ⫾
179.60 pg/ml) compared to control media (6.83 ⫾ 5.10 pg/ml).
No significant differences in IL-1␤, IL-6, IL-10, or TNF-␣
levels were found between control and LLC media. IL-17 and
IFN-␥ were not detected in either control or LLC media.
The role of LIF in LLC regulation of myotube protein
turnover/stretch-induced protein synthesis. LLC media can
activate myotube gp130/STAT3 signaling pathway through
IL-6-independent mechanisms (49). We found no significant
difference in IL-6 level between control and LLC media, while
another gp130-dependent cytokine, LIF, was only detected in
LLC media (Table 1). LIF/STAT3 signaling is required for
colon-26-induced cancer cachexia (57). Administration of a
mouse LIF antibody was used to inhibit LIF signaling in LLC
media (57). Myotubes were treated with recombinant mouse
LIF (10 ng/ml) and different doses of LIF antibody for 30 min
to examine LIF antibody dose effects. As previously reported
(57), LIF administration induced STAT3 and ERK1/2 phosphorylation (Fig. 5A). LIF did not induce STAT3 or ERK1/2
phosphorylation in the presence of 1.5 ␮g/ml LIF antibody,
and 2 ␮g/ml LIF antibody showed no further inhibitory effects
on STAT3 or ERK1/2 phosphorylation (Fig. 5A). These results
demonstrate 1.5 ␮g/ml LIF antibody was sufficient to attenuate
STAT3 and ERK1/2 induction by 10 ng/ml LIF, which was
⬃1,000-fold higher than LIF level in LLC media. However,
LIF antibody (1.5 ␮g/ml) with LLC media did not attenuate the
induction of STAT3 or AMPK, whereas ERK1/2 phosphorylation was attenuated (Fig. 5B). LIF antibody administration in
LLC media increased p70S6K phosphorylation (Fig. 5C), but
did not change S6RP phosphorylation (Fig. 5C) or protein
synthesis (Fig. 5D). Stretch in LLC media with LIF antibody
had no effect on p70S6K phosphorylation (Fig. 5C), but
decreased S6RP phosphorylation (Fig. 5C) and protein synthesis (Fig. 5D).
The role of gp130 in LLC regulation of stretch-induced
myotube protein synthesis. We investigated the role of gp130
receptor-dependent signaling in the LLC suppression of
stretch-induced myotube protein synthesis. Inhibition of gp130
signaling was achieved by administration of a gp130 antibody
to the media. We have reported that gp130 is required for
LLC-induced STAT3, NF-␬B, and p38 signaling, but gp130
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Fig. 3. Lewis lung carcinoma (LLC) regulation on stretch-induced myotube protein synthesis activation and hypertrophy. A: experiment design. C2C12 myotubes
were incubated in control or 25% LLC media for 72 h and stretched (5%) in last 24-h period. B: diameter of C2C12 myotubes after 24 h of 0% or 5% stretch
in control or LLC media. C: phosphorylation of p70S6K, S6RP, and 4EBP1 was measured by Western blot. Blots shown on separate panels are from different
areas of the same gel. D: protein synthesis was measured by Western blot against puromycin. Dashed lines indicate different areas on the same gel. Bar graphs
represent fold changes relative to control group (B–D). All values are means ⫾ SE. *P ⬍ 0.05 vs. control; $P ⬍ 0.05 vs. LLC; #P ⬍ 0.05 vs. control ⫹ stretch: two-way
ANOVA.
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LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
signaling inhibition is not sufficient to rescue LLC suppression
of mTORC1 signaling or protein synthesis in LLC tumorbearing mice or LLC media-treated myotubes (49). Compared
with LLC-treated myotubes, gp130 antibody administration
decreased STAT3 phosphorylation, which was independent of
stretch (Fig. 6A). Stretch in LLC media with the gp130 antibody decreased S6RP phosphorylation, but had no influence on
4EBP1 phosphorylation (Fig. 6A). Consistent with the change
in S6RP phosphorylation, stretch in LLC media with gp130
antibody further reduced myotube protein synthesis (Fig. 6B).
To further investigate the gp130 role in stretch induction of
myotube protein synthesis, we stretched myotubes in control
media supplemented with gp130 antibody. Stretch in control
media with gp130 antibody significantly increased p70S6K and
S6RP phosphorylation (Fig. 6C). Basal p70S6K phosphorylation was decreased by gp130 antibody administration in control
media, but there was no effect on S6RP phosphorylation (Fig.
6C). gp130 antibody administration decreased p70S6K and
S6RP phosphorylation in stretched myotubes (Fig. 6C). Basal
myotube protein synthesis was not affected by gp130 antibody
Table 1. Concentration of cytokines in control and LLC
media
Cytokine
Increased
MCP-1
RANTES
LIF
M-CSF
No change
IL-1␤
IL-6
IL-10
TNF-␣
Not detected
IL-17
IFN-␥
Control
LLC
6.83 ⫾ 5.10
ND
ND
ND
353.90 ⫾ 179.60*
0.58 ⫾ 0.33*
9.93 ⫾ 2.55*
12.40 ⫾ 3.34*
94.25 ⫾ 1.88
3.38 ⫾ 0.48
8.07 ⫾ 0.18
42.97 ⫾ 1.46
ND
ND
103.30 ⫾ 1.90
2.60 ⫾ 0.65
6.28 ⫾ 0.54
45.86 ⫾ 1.33
ND
ND
Values are means ⫾ SE in pg/ml. The levels of 10 inflammatory cytokines
in control medium (DMEM with 10% FBS) and culture medium from 3
independent LLC cell cultures were measured by Bio-Plex multiplex analysis.
LLC, Lewis lung carcinoma; MCP-1, monocyte chemoattractant protein;
RANTES, regulated on activation normal T-expressed and presumably secreted; LIF, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor; ND, not detected. Student’s t-test was performed to determine the
difference between levels of cytokines. *Statistical significance was set at P ⬍
0.05.
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Fig. 4. Effect of stretch and LLC on stress and inflammatory signaling
pathways in myotubes. C2C12 myotubes were incubated in control or 25%
LLC media for 72 h and stretched (5%) in last 24-h period. Phosphorylation of
p38 MAPK, ERK1/2, NF-␬B p65, STAT3, and AMP-activated protein kinase
(AMPK) was measured by Western blot. A: blots shown on separate panels are
from different areas of the same gel. B: bar graphs represent fold changes
relative to control group. All values are means ⫾ SE. *P ⬍ 0.05 vs. control;
#P ⬍ 0.05 vs. control ⫹ stretch; †P ⬍ 0.05 vs. LLC: two-way ANOVA.
administration, but did abrogate the stretch induction of protein
synthesis in control media (Fig. 6D).
We used PDTC (a NF-␬B and STAT3 inhibitor) and LLL12
(a STAT3-specific inhibitor) to exam the role of gp130 downstream targets, STAT3 and NF-␬B. As previously reported
(49), PDTC administration decreased both NF-␬B and STAT3
phosphorylation, whereas LLL12 administration specifically
decreased STAT3 phosphorylation (Fig. 7, A–C). The PDTC
and LLL12 suppression of STAT3 and NF-␬B p65 phosphorylation was independent of stretch (Fig. 7, A–C). Interestingly,
stretch did not downregulate LLC-induced ERK1/2 phosphorylation in the presence of LLL12 (Fig. 7, A and C). Stretch in
LLC media containing PDTC or LLL12 did not increase
myotube S6RP phosphorylation (Fig. 7D) or protein synthesis
(Fig. 7E). These results demonstrate gp130-dependent signaling is involved in stretch induction of myotube protein synthesis. However, it is not involved in LLC suppression effect on
stretch induction of myotube protein synthesis.
The role of ERK1/2 in LLC regulation of stretch-induced
myotube protein synthesis. To investigate the role of ERK1/2
signaling in LLC suppression of stretch-induced myotube protein synthesis, we used PD-98059 to inhibit LLC-induced
ERK1/2 phosphorylation. Myotubes were incubated in 25%
LLC media for 72 h in the presence of PD-98059 and were
stretched during last 24 h. PD-98059 administration in LLC
media decreased ERK1/2 phosphorylation but had no influence
on MAPKAPK-2 phosphorylation, a marker of p38 MAPK
activity (Fig. 8A). PD98059 administration in LLC media did
not change phosphorylation of p70S6K, S6RP, and 4EBP1 in
nonstretched myotubes (Fig. 8B). Stretch in either LLC media
or LLC media with PD-98059 did not increase p70S6K or
4EBP1 phosphorylation (Fig. 8B). However, stretch significantly increased protein synthesis in LLC media supplemented
with PD-98059 (Fig. 8C). These results demonstrate a novel
role of ERK1/2 in the mechanical regulation of protein synthesis in LLC-induced atrophic myotubes, which may be independent of p70S6K or 4EBP1.
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The role of AMPK in LLC regulation of stretch-induced
myotube protein synthesis. Previously, we have demonstrated
that LLC secreted factors can induce myotube AMPK signaling (49), which is an upstream suppressor of mTOR and is
involved in muscle anabolic resistance (22, 53). To investigate
the role of AMPK signaling in the LLC inhibition of stretchinduced protein synthesis, we stretched myotubes in LLC
media containing AMPK pharmaceutical inhibitor, compound
C. Because 24 h or more of compound C treatment resulted in
myotube death (data not shown), we did stretch and compound
C administration on myotubes in last 4 h of LLC media
incubation. First, we compared 4-h stretch effect on mTORC1
signaling pathway in control and LLC media incubated myotubes. Four-hour stretch induced p70S6K phosphorylation in
both control and LLC media incubated myotubes (Fig. 9A).
However, stretched myotubes in LLC media showed less
p70S6K phosphorylation than stretched myotubes in control
media (Fig. 9A). Furthermore, 4-h stretch was not sufficient to
increase S6RP, 4EBP1 phosphorylation (Fig. 9A), or protein
synthesis in LLC media incubated myotubes (Fig. 9D). The
administration of compound C decreased AMPK phosphorylation independent of stretch (Fig. 9B) and increased phosphorylation of p70S6K, S6RP, and 4EBP1 in both nonstretched and
stretched myotubes (Fig. 9C). However, the increase in
p70S6K, S6RP, or 4EBP1 phosphorylation due to compound C
was not further increased by stretch (Fig. 9C). Compound C
administration in LLC media increased protein synthesis in
both nonstretched and stretched myotubes. However, stretch
did not further increase the compound C-induced protein synthesis (Fig. 9D). These results demonstrate LLC-induced
AMPK signaling is an upstream suppressor of myotube
mTORC1 signaling. However, AMPK signaling is not involved in LLC suppression of stretch induction of myotube
mTORC1 signaling and protein synthesis.
The role of p38 MAPK in LLC regulation of stretch-induced
myotube protein synthesis. p38 MAPK plays both an anabolic
and catabolic role in skeletal muscle protein turnover regulation (17, 76) and was upregulated by both stretch and LLC
media (Fig. 4). To investigate the role of p38 MAPK signaling
in LLC suppression of stretch-induced myotube protein synthesis, we used p38 MAPK-specific inhibitor SB-203580,
which blocks downstream signaling initiated by p38 without
affecting p38 phosphorylation. Chronic (24 h or more) SB203580 administration in LLC media resulted in myotube
death (data not shown). Therefore, we stretched myotubes for
4 h in LLC media containing SB-203580. SB-203580 administration in LLC media decreased phosphorylation of p38
MAPK downstream target, MAPKAPK-2, in both non-
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Fig. 5. Leukemia inhibitory factor (LIF) role in LLC regulation of stretch-induced myotube protein synthesis. A: myotubes were administrated with LIF (10
ng/ml) and different doses of LIF antibody (Ab). Phosphorylation of STAT3 and ERK1/2 were determined by Western blot. *P ⬍ 0.05 vs. control; #P ⬍ 0.05
vs. LIF: one-way ANOVA. B: myotubes were administrated with LLC media and 1.5 ␮g/ml LIF Ab for 72 h. Phosphorylation of STAT3, AMPK, and ERK1/2
was measured by Western blot. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. LLC: one-way ANOVA. C: mytoubes were stretched for 24 h in LLC media with LIF
Ab. Phosphorylation of p70S6K and S6RP was measured by Western blot. D: protein synthesis was measured by Western blot against puromycin. Bar graphs
represent fold changes relative to control group (A and B) and LLC group (C and D). All values are means ⫾ SE. *P ⬍ 0.05 vs. LLC; #P ⬍ 0.05 vs. LLC with
LIF Ab: one-way ANOVA.
LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
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stretched and stretched myotubes, whereas SB-203580 did not
influence the downregulation of stretch on ERK1/2 phosphorylation in LLC media (Fig. 10A). SB-203580 administration in
LLC media did not change phosphorylation of p70S6K, S6RP,
and 4EBP1 induced by LLC alone and did not influence 4-h
stretch induction of p70S6K phosphorylation (Fig. 10B). However, stretch significantly increased protein synthesis in LLC
media supplemented with SB-203580 (Fig. 10C). These results
demonstrate that LLC-induced p38 MAPK signaling is a suppressor of stretch induction of myotube protein synthesis
through mTORC1-independent mechanisms.
DISCUSSION
Increased mechanical signaling, such as overload, has been
theorized to have potential for attenuating cancer-induced muscle mass loss. However, it is not clear if the mechanical
induction of muscle protein synthesis can be regulated by
cachectic factors. Disrupted mechanical signaling in cachectic
skeletal muscle would have the potential to accelerate the
wasting process and negatively impact the therapeutic potential
of exercise and physical activity interventions. We report the
important and novel finding that media derived from LLC cells
can negatively affect the stretch induction of mTORC1 signaling and protein synthesis in myotubes. While both STAT3 and
NF-␬B signaling have been implicated in the regulation of
myotube and muscle catabolism, we report that these pathways
are not necessary for the LLC suppression of stretch-regulated
protein synthesis in myotubes. LLC-induced AMPK signaling,
an established suppressor of basal myotube protein synthesis,
is also not involved in the LLC attenuation of the stretch
response. However, the inhibition of either ERK1/2 or p38
MAPK in LLC-derived media restored myotube protein synthesis stretch responsiveness. LLC media expressed high levels
of LIF, which is involved in LLC induction of ERK1/2 in
myotubes. Interestingly, we report the novel finding that
gp130-initiated signaling plays a role in the mechanical induction of myotube protein synthesis. In untreated myotubes, the
stretch induction of protein synthesis was inhibited by gp130
antibody administration, while stretch in the presence of LLC
media caused a further suppression of myotube protein synthesis suppression. Overall, these results demonstrate that
LLC-derived cachectic factors can dissociate mechanical sig-
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Fig. 6. Glycoprotein 130 (gp130) role in LLC regulation of stretch-induced myotube protein synthesis. A: phosphorylation of STAT3, S6RP, and 4EBP1 in
myotubes stretched in LLC media with gp130 Ab. *P ⬍ 0.05 vs. LLC, one-way ANOVA. B: protein synthesis was measured by Western blot against puromycin.
*P ⬍ 0.05, Student’s t-test. C: phosphorylation of p70S6K and S6RP in myotubes stretched in control media with/without gp130 Ab. D: protein synthesis was
measured by Western blot against puromycin. Bar graphs represent fold changes relative to LLC group (A), 0% stretch group (B), and control group (C and D).
All values are means ⫾ SE. *P ⬍ 0.05 vs. control; $P ⬍ 0.05 vs. gp130 Ab; #P ⬍ 0.05 vs. control ⫹ stretch: two-way ANOVA.
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LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
naling from myotube protein synthesis, possibly through the
hyperactivation of ERK1/2 and p38, and that the mechanical
stretch-induced signaling involves gp130 in both control and
LLC-treated myotubes (Fig. 11).
While cancer-induced skeletal muscle wasting involves the
disrupted homeostatic regulation of muscle protein turnover
(66), our understanding of protein degradation regulation during the wasting process exceeds our understanding of protein
synthesis. The integration of muscle anabolic processes
through mTOR signaling is an acknowledged control point of
skeletal muscle mass (6), which also can be targeted by the
cancer environment (55). Paradoxically, we have reported that,
during cachexia, mTOR signaling and protein synthesis suppression are accompanied by increased Akt phosphorylation
(49, 68, 69), suggesting a disconnection between Akt and
mTOR in cancer cachexia. Mechanical signaling can activate
muscle mTOR signaling and protein synthesis through Aktindependent mechanisms (26). We report that LLC-derived
cachectic factors are sufficient to attenuate stretch-induced
mTOR signaling involving the phosphorylation of p70S6K and
S6RP. Interestingly, mTOR inhibition occurred while ERK1/2
and p38 MAPK, mediators of stretch-induced protein synthesis, were activated. MAPKs, including ERK1/2 and p38, are
part of an important skeletal muscle signal transduction nexus
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Fig. 7. STAT3 and NF-␬B role in LLC regulation of stretch-induced myotube protein synthesis. A: representative blots. B and C: phosphorylation of STAT3,
NF-␬B p65, ERK, and p38 MAPK in myotubes stretched in LLC media with/without inhibitors [ammonium pyrrolidinecarbodithioate (PDTC; B); LLL12 (C)].
*P ⬍ 0.05 vs. LLC; #P ⬍ 0.05 vs. LLC ⫹ stretch; $P ⬍ 0.05 vs. LLC with PDTC/LLL12: two-way ANOVA. D: phosphorylation of S6RP in myotubes stretched
in LLC media with/without inhibitors. No significant difference was detected. P ⬍ 0.05, two-way ANOVA. E: protein synthesis was measured by Western blot
against puromycin. Bar graphs represent fold changes relative to LLC group (B–E). All values are means ⫾ SE. No significant difference was detected in D or
E. P ⬍ 0.05, two-way ANOVA.
LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
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that mediates a muscle’s response to a variety of physiological
and pathological stressors (70). Several studies provide support
for ERK1/2’s role in the mechanical induction of skeletal
muscle mTOR signaling (38, 71). Additionally, mechanical
activation of p38 signaling has been associated with peroxisome proliferator-activated receptor-␣ coactivator-transcription (2), glucose uptake (12), and mTOR signaling activation
(17, 72). However, chronic inflammatory diseases also activate
muscle MAPK signaling, resulting in increased protein degradation and muscle wasting (51, 76). LLC-released factors can
induce a strong inflammatory response in skeletal muscle (49),
and our results are consistent with the current understanding of
mechanical stimuli and inflammation regulation on MAPK
signaling (4, 32). Interestingly, stretch attenuated LLC-induced
ERK1/2 phosphorylation, which suggests that mechanical
stimuli can counter the LLC-induced stress response. However,
the paradoxical activation of MAPK signaling during both
wasting and growth conditions requires further examination.
Our results demonstrate that the suppression of mechanical
signaling by LLC can be rescued. Inhibition of either ERK1/2
or p38 during LLC treatment restored the stretch induction of
myotube protein synthesis. Unexpectedly, ERK1/2 or p38
MAPK inhibition did not restore the stretch regulation to
mTOR signaling. Stretch for 24 h in the presence of LLC did
not increase the phosphorylation of p70S6K, S6RP, or 4EBP1.
In fact, some studies have demonstrated that mTORC1 is not
the only mediator of mechanical stimuli-induced muscle protein synthesis. For example, synergistic ablation-induced muscle rRNA accumulation is only partially attenuated by
mTORC1 suppression (20). In denervated skeletal muscles,
protein synthesis is also decreased due to the downregulation
of ribosomal biogenesis, even though mTORC1 signaling is
activated (36). MAPKs, such as ERK1/2, can directly activate
rRNA transcription and ribosomal biogenesis through the
phosphorylation of rRNA transcriptional factor, transportation
infrastructure financing and innovation act and upstream binding factor, independent of mTORC1 signaling (61, 78). Interestingly, hyperphosphorylation of the ERK1/2 phosphorylation
site on upstream binding factor can decrease rRNA transcriptional activity (61), suggesting a dual role for ERK1/2 signaling in ribosomal biogenesis and protein synthesis regulation.
The IL-6 family of cytokines regulates intracellular signal
transducers, such as STAT3 and MAPK, through interaction
with their specific cytokine receptors and the gp130 transmembrane protein (63). The IL-6 cytokines play an enigmatic role
in skeletal mass regulation (39). They are secreted by muscle
fibers during exercise or overload and are involved in muscle
hypertrophy and regeneration by stimulating satellite cell proliferation (5, 45, 56, 60). However, they are involved in muscle
wasting during cancer cachexia. Inhibition of IL-6/STAT3 signaling can attenuate muscle wasting in different cachexia models,
including ApcMin/⫹ mice and C26 tumor-bearing mice (7, 68).
Interestingly, we did not find elevated IL-6 levels in our LLC
media, but another member of this cytokine family, LIF, was
significantly increased. It was recently reported that LIF signaling
is required for C26-induced muscle atrophy (57). We have reported that LLC-induced muscle protein degradation involves
signaling though the muscle gp130, both in vivo and in vitro (49).
Additionally, LLC activation of NF-␬B, STAT3, and the ubiquitin
protein degradation pathway requires the muscle gp130 receptor
(49). Our present results demonstrate that LIF is highly expressed
in LLC media and plays a role in the induction of ERK1/2 by
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Fig. 8. ERK1/2 role in LLC regulation of stretch-induced myotube protein synthesis. A: phosphorylation of ERK1/2 and MAPK-activated protein kinase 2
(MAPKAPK-2) was measured by Western blot. Blots shown on separate panels are from different areas of the same gel. *P ⬍ 0.05 vs. LLC; #P ⬍ 0.05 vs.
LLC ⫹ stretch: two-way ANOVA. B: phosphorylation of p70S6K, S6RP, and 4EBP1 was measured by Western blot. Protein sample from nonstretched myotubes
in control media was used for p-p70S6K positive control. Blots shown on separate panels are from different areas of the same gel. C: protein synthesis was
measured by Western blot against puromycin. Bar graphs represent fold changes relative to LLC group. All values are means ⫾ SE. *P ⬍ 0.05 vs. LLC ⫹ stretch;
#P ⬍ 0.05 vs. LLC with PD-98059 (PD): two-way ANOVA.
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LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
LLC, while LLC induction of STAT3 appears LIF independent.
However, LLC signaling through the gp130 receptor is not required for basal mTOR suppression in either wasting myotubes or
skeletal muscle (49).
Mechanical stimulation can induce the muscle expression of
some members of the IL-6 cytokine family, and these cytokines play a role in muscle mass regulation related to both
hypertrophy and atrophy (39). However, despite significant
interest in these cytokines, relatively little is known about
gp130-dependent signaling in mechanical induction of muscle
protein synthesis. We report that inhibition of gp130 signaling
prevented the stretch induction of protein synthesis in control
condition. Furthermore, inhibition of gp130 or LIF, but not
STAT3 or NF-␬B, in stretched myotubes resulted in further
LLC suppression of mTOR signaling and protein synthesis.
These findings suggest that gp130-dependent cytokines play an
important role in mechanical stretch regulation of protein
synthesis. Additional study is needed to determine whether
LIF/gp130 signaling has a permissive role in maintaining
mechanical signaling. However, our results support previous
research demonstrating that IL-6 and LIF are required for
overload-induced muscle hypertrophy (56, 59). We have previously reported that IL-6 administration can cause a dosedependent increase in myotube p70S6K phosphorylation, paradoxically being most effective at low doses (69). Taken
together, these results suggest that gp130 may play a dual role
in the regulation of muscle protein turnover that is based on the
muscle’s microenvironment, which involves expression of circulating and local cytokines. However, future research is still
needed to investigate the downstream targets of gp130 signaling that play a role in protein synthesis regulation by mechanical and cachectic signaling.
NF-␬B and STAT3 are important mediators of muscle catabolism. The activation of NF-␬B during cachexia increases
muscle RING finger-1 transcription, inducible nitric oxide
synthase expression, and ubiquitin-proteasome-mediated protein degradation and suppresses MyoD mRNA expression (9,
15). STAT3 signaling is also an important regulator of muscle
wasting and increases atrogin-1 (7, 49) and myostatin expression (77). Furthermore, NF-␬B and STAT3 have the potential
to disrupt anabolic activation of muscle protein synthesis (19).
For example, anabolic resistance to amino acid and resistance
exercise caused by either sarcopenia or sepsis is associated
with NF-␬B activation (23). Muscle contraction-induced
mTORC1 signaling is attenuated in severely cachectic
ApcMin/⫹ mice, which exhibit chronic activation of muscle
STAT3 (50). Interestingly, PDTC administration to these cachectic mice can rescue contraction-induced mTORC1 activation (50). We found that both anabolic and catabolic stimuli
can activate muscle NF-␬B signaling, which may be related to
differential functions of NF-␬B in muscle as it relates to
inflammatory and mechanical signals (28). NF-␬B and STAT3
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Fig. 9. AMPK role in LLC regulation of stretch-induced myotube protein synthesis. A: 4-h stretch effect on control or LLC-treated myotubes p70S6K, S6RP,
and 4EBP1 phosphorylation were measured by Western blot. Blots shown on separate panels are from different areas of the same gel. *P ⬍ 0.05 vs. control;
#P ⬍ 0.05 vs. control ⫹ stretch; $P ⬍ 0.05 vs. LLC: two-way ANOVA. B: phosphorylation of AMPK in myotubes stretched in LLC media with or without
compound C. Blots shown on separate panels are from different areas of the same gel. C: phosphorylation of p70S6K, S6RP, and 4EBP1 in myotubes stretched
in LLC media with or without compound C. Blots shown on separate panels are from different areas of the same gel. D: protein synthesis was measured by
Western blot against puromycin. Bar graphs represent fold changes relative to control group (A) and LLC group (B–D). All values are means ⫾ SE. *P ⬍ 0.05
vs. LLC; #P ⬍ 0.05 vs. LLC ⫹ stretch: two-way ANOVA.
LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
C77
signaling pathways are acutely activated by exercise, but these
signaling pathways have not been clearly linked to the mechanical induction of protein synthesis (30). We did not observe any effect of stretch on STAT3 phosphorylation, and
stretch did not alter LLC induction of STAT3. The inhibition
of STAT3 or NF-␬B does not appear sufficient to restore the
stretch induction of myotube protein synthesis in LLC media.
An energy deficiency or cachectic environment can induce a
shift toward muscle catabolism. AMPK signaling plays a role in
this shift through mTORC1 inhibition via raptor and tuberous
sclerosis complex 2 phosphorylation (22), induction of FOXOdependent E3 ubiquitin ligase transcription (40), and increased
Fig. 11. Speculated pathways of LLC regulation on stretch-induced myotube
protein synthesis in myotubes. Mechanical stretch induces mTOR-p70S6K
signaling activation and subsequent protein synthesis increase. gp130 also
mediated stretch-induced protein synthesis, which is independent of mTORp70S6K. LLC media suppress stretch induction of protein synthesis through
the hyperactivation of ERK1/2 and p38 MAPK signaling, which is independent
of mTOR-p70S6K. LLC media also induces AMPK signaling activation,
which is an additional suppressor of mTOR-p70S6K signaling, independent of
stretch.
autophagy processes (3). Muscle AMPK signaling is chronically
activated in cachectic ApcMIN/⫹ mice (49, 68), but it remains to be
determined whether energy status, metabolic regulation related to
oxidative metabolism, or other inflammatory signaling is responsible for the induction. Exercise and repetitive muscle contractions
also induce a transient activation of AMPK signaling (18). Interestingly, contracted cachectic muscle has an induction of AMPK
signaling, which goes above an already elevated baseline level
(50). Here, we report that the inhibition of myotube AMPK
activity during LLC treatment can rescue suppressed mTORC1
signaling and protein synthesis. Clearly AMPK signaling has a
role in the LLC suppression of basal protein synthesis. However,
chronic stretch does not increase AMPK phosphorylation or alter
LLC activation of AMPK. Furthermore, AMPK inhibition in
myotubes treated with LLC media does not rescue the stretch
regulation of myotube protein synthesis. These results suggest
limited cross talk between AMPK and mechanical signaling in
myotubes. The relationship between the AMPK and mechanical
signaling on the regulation of muscle protein synthesis needs to be
further defined.
In summary, we examined whether tumor-derived cachectic
factors could disrupt mechanical stretch regulation of protein
synthesis in cultured myotubes. Chronic stretch in basal conditions induced myotube protein synthesis and hypertrophy,
while the addition of LLC-derived media attenuated the stretch
induction of protein synthesis. LLC-induced p38 and ERK1/2
signaling appear critical for disrupted mechanical signaling, as
their inhibition restored stretch induction of protein synthesis
in the presence of LLC media. Interestingly, we demonstrated
that the stretch induction of myotube protein synthesis involves
gp130-initiated signaling in myotubes cultured in both untreated and LLC media. AMPK signaling plays a regulatory
role in the inhibition of basal myotube protein synthesis by
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Fig. 10. p38 MAPK role in LLC regulation of stretch-induced myotube protein synthesis. A: phosphorylation of ERK1/2 and MAPKAPK-2 was measured by
Western blot. B: phosphorylation of p70S6K, S6RP, and 4EBP1 was measured by Western blot. C: relative protein synthesis was measured by Western blot
against puromycin. ND, not detected. Bar graphs represent fold changes relative to LLC group. All values are means ⫾ SE. *P ⬍ 0.05 vs. LLC; #P ⬍ 0.05 vs.
LLC with SB-203580 (SB); $P ⬍ 0.05 vs. LLC ⫹ stretch: two-way ANOVA.
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LLC REGULATION OF STRETCH-INDUCED MYOTUBE PROTEIN SYNTHESIS
LLC, but inhibition did not restore stretch sensitivity. Our work
provides a new insight into the mechanisms by which cancer
cachexia suppresses muscle protein synthesis. Further work is
necessary to determine whether anti-inflammatory therapies
combined with mechanical stimulation are useful to alleviate
muscle protein synthesis suppression in cancer cachexia.
13.
14.
ACKNOWLEDGMENTS
The authors thank Gaye Christmas, Justin P. Hardee, and Kimbell L.
Hetzler for editorial review of the manuscript.
15.
GRANTS
The research described in this report was supported by research grant R01
CA121249 to J. A. Carson from the National Cancer Institute, and National
Center for Research Resources Grant P20 RR-017698 to J. A. Carson.
16.
No conflicts of interest, financial or otherwise, are declared by the author(s).
17.
AUTHOR CONTRIBUTIONS
18.
Author contributions: S.G. and J.A.C. conception and design of research;
S.G. performed experiments; S.G. analyzed data; S.G. and J.A.C. interpreted
results of experiments; S.G. and J.A.C. prepared figures; S.G. and J.A.C.
drafted manuscript; S.G. and J.A.C. approved final version of manuscript;
J.A.C. edited and revised manuscript.
19.
20.
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