1 Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02215; and 2 Department of Molecular Endocrinology, Merck Research Laboratories, Rahway, New Jersey 07065
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ABSTRACT |
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To study the effects of contractile activity on mitogen-activated protein kinase (MAP kinase), p70 S6 kinase (p70S6K), and Akt kinase signaling in rat skeletal muscle, hindlimb muscles were contracted by electrical stimulation of the sciatic nerve for periods of 15 s to 60 min. Contraction resulted in a rapid and transient activation of Raf-1 and MAP kinase kinase 1, a rapid and more sustained activation of MAP kinase and the 90-kDa ribosomal S6 kinase 2, and a dramatic increase in c-fos mRNA expression. Contraction also resulted in an apparent increase in the association of Raf-1 with p21Ras, although stimulation of MAP kinase signaling occurred independent of Shc, IRS1, and IRS2 tyrosine phosphorylation or the formation of Shc/Grb2 or IRS1/Grb2 complexes. Insulin was considerably less effective than contraction in stimulating the MAP kinase pathway. However, insulin, but not contraction, increased p70S6K and Akt activities in the muscle. These results demonstrate that contraction-induced activation of the MAP kinase pathway is independent of proximal steps in insulin and/or growth factor-mediated signaling, and that contraction and insulin have discordant effects with respect to the activation of the MAP kinase pathway vs. p70S6K and Akt. Of the numerous stimulators of MAP kinase in skeletal muscle, contractile activity emerges as a potent and physiologically relevant activator of MAP kinase signaling, and thus activation of this pathway is likely to be an important molecular mechanism by which skeletal muscle cells transduce mechanical and/or biochemical signals into downstream biological responses.
signal transduction; muscle contraction; exercise
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INTRODUCTION |
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CONTRACTILE ACTIVITY plays a critical role in determining the biochemical and morphological characteristics of skeletal muscle. Chronic increases in contractile activity can lead to muscle hypertrophy and increased expression of numerous muscle proteins (13). These activity-induced changes in muscle phenotype may be regulated in part by the induction of "immediate-early" genes such as c-fos, c-jun, and egr-1 (22). Contractile activity can also alter the metabolic and morphological profile of the muscle by directly increasing rates of protein synthesis (4). Despite these profound effects of contractile activity in regulating muscle phenotype, little is known about the intracellular signaling mechanisms that link the biochemical and mechanical events of muscle contraction with the activation of translation and transcription.
Signaling through the classical mitogen-activated protein (MAP) kinase cascade involves the sequential phosphorylation and increased catalytic activity of the Raf-1 kinase, the MAP kinase kinases (MEK1 and MEK2), the MAP kinase isoforms p44MAPK (ERK1) and p42MAPK (ERK2), and the cytosolic p90 ribosomal S6 kinase (RSK) (26). In rat hindlimb skeletal muscle, insulin has been shown to modestly increase MAP kinase activity (12, 41), whereas in rat diaphragm muscle epidermal growth factor (EGF) is more effective than insulin in stimulating MAP kinase activity (3). Furthermore, we have demonstrated that physical exercise can increase MAP kinase signaling in both rat (8) and human (2) skeletal muscle. The physiological consequences of insulin, EGF, or exercise-stimulated increases in MAP kinase activity in skeletal muscle are not known. However, because a role for growth factor-induced MAP kinase stimulation in controlling carbohydrate metabolism and protein synthesis (3) has been ruled out, a primary function of MAP kinase signaling in skeletal muscle may be to regulate transcription, which in turn may regulate the expression of critical muscle proteins. In cultured cardiac myocytes, mechanical stretch can stimulate MAP kinase signaling (30, 38, 39), with some studies providing evidence that this increase in activity plays a role in stretch-induced hypertrophy (7, 39).
Activation of the MAP kinase pathway can occur through ras-dependent and -independent mechanisms, including G protein-coupled receptors (28) and numerous growth factors (26). Growth factors stimulate this signaling cascade through activation of tyrosine kinase receptors, Shc phosphorylation, association of phosphorylated Shc with the adapter protein Grb2, leading to Sos-mediated activation of Ras (32). Insulin-induced activation of the MAP kinase pathway can also occur through the association of tyrosine-phosphorylated insulin receptor substrate 1 (IRS1) with Grb2 (36). It is not known whether these molecules are involved in exercise-stimulated MAP kinase signaling.
The p70 S6 kinase (p70S6K) and Akt kinase (also referred to as RAC/PKB) are alternative signaling proteins that could potentially link the biochemical and mechanical events of muscle contraction with downstream biological responses. These two serine/threonine kinases can be activated by growth factors via complex mechanisms involving phosphoinositide 3-kinase (PI-kinase) and 3-phosphoinositide-dependent kinase (PDK1) (6). Interestingly, certain stimuli such as hyperosmolarity (31), growth hormone (31), heat shock (21), and reagents that increase cAMP (29) can increase Akt activity through a PI 3-kinase-independent mechanism. p70S6K is thought to play an important role in regulating protein synthesis by controlling the translation of numerous mRNA transcripts that encode components of the translational apparatus (18, 24). Akt mediates diverse cellular functions, including the inhibition of apoptosis (6) and possibly the stimulation of glucose transport and GLUT-4 translocation (33). Muscle contraction also increases glucose transport through the translocation of GLUT-4, although the signaling mechanism that mediates this response is not known (11).
In the current investigation, we studied the effects of contractile activity on MAP kinase, p70S6K, and Akt signaling in rat skeletal muscle. Our results demonstrate that neither p70S6K nor Akt is altered by muscle contractions. In contrast, contractile activity rapidly and potently stimulates the MAP kinase signaling cascade in rat skeletal muscle, and this stimulation occurs immediately before an increase in expression of c-fos mRNA. Because the MAP kinase pathway has been implicated in the regulation of protein synthesis and gene transcription in numerous cell types, activation of this cascade in response to contraction is likely to be a key mechanism in the regulation of these processes in skeletal muscle.
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MATERIALS AND METHODS |
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Animals.
For insulin treatment, male Sprague-Dawley rats (Taconic) were fasted
for 16 h and then injected intraperitoneally with 20 U of porcine
insulin (maximal dose). Control (basal) animals were injected with
saline. Rats were decapitated, and hindlimb muscles, including biceps
femoris, gastrocnemius, and soleus, were rapidly dissected and frozen
in liquid nitrogen. For contraction studies, both control (basal) and
treated (contraction) rats were anesthetized with sodium amobarbital
(50 mg/kg body wt ip). The sciatic nerves to both hindlimbs were
exposed, and electrodes were attached. For contraction treatment,
hindlimb muscles were stimulated for 15 s or for 2, 5, 10, 15, 30, or
60 min with one 500-ms train/s. Each train consisted of one to three
repeated V pulses of 0.1 ms duration delivered at 100 Hz. When the
stimulation period exceeded 5 min, each additional 5-min period was
separated by 2 min of rest. Hindlimb skeletal muscles were removed and
frozen in liquid nitrogen. For
p70S6K mobility shift studies,
hindlimb muscles were powdered and then homogenized in 50 mM MOPS (pH
7.4), 5 mM EGTA, 2 mM EDTA, 75 mM -glycerophosphate, 2 mM
dithiothreitol (DTT), 1 mM
Na3VO4,
10 µM leupeptin, 3 mM benzamidine, 5 mM pepstatin A, 10 mg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 200 µg/ml
soybean trypsin inhibitor. For all other assays, powdered muscle was
homogenized in lysis buffer containing 20 mM HEPES (pH 7.4), 2 mM EGTA,
50 mM
-glycerophosphate, 1 mM DTT, 1 mM
Na3VO4,
1% Triton X-100, 10% glycerol, 10 µM leupeptin, 3 mM benzamidine, 5 µM pepstatin A, 10 µg/ml aprotinin, 1 mM PMSF, and 200 µg/ml
soybean trypsin inhibitor. For all studies, homogenates were mixed for
1 h at 4°C and centrifuged at 15,000 g for 1 h at 4°C. Protein
concentration was determined on the supernatants by use of the Bradford
dye-binding procedure, with BSA as standard (Bio-Rad).
Kinase activity assays.
For RSK2 activity assays, 1 mg of rat muscle protein was precleared
with protein A-Sepharose beads for 1 h at 4°C. Supernatants were
incubated with 3.5 µg/ml of -RSK2 [Upstate
Biotechnology (UBI) 06-300] for 1 h at 4°C,
followed by addition of fresh protein A beads and overnight incubation
at 4°C. RSK2 activity was measured in the immune complexes using
the 3R S6 peptide substrate (RRRLSSLRA) as previously described (8).
Immunoblotting.
Phosphorylation of MAP kinase was assessed by immunoblotting muscle
lysates with an antibody (-MAPK-PP) that recognizes only the
phosphorylated forms of p44MAPK
and p42MAPK (Quality Controlled
Biochemicals). Immunoblotting was also used to detect changes in the
electrophoretic mobility of the RSK2, Raf-1, and
p70S6K proteins. Muscle proteins
(300 µg for RSK2 and p70S6K, 100 µg for Raf-1 and
-MAPK-PP) were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and blocked in Tris-buffered saline plus NaN3 (TNA) containing
10% BSA (RSK2, Raf-1, p70S6K)
or 5% milk (
-MAPK-PP) for 1 h at room temperature. The membranes were incubated overnight at 4°C with
-RSK2 (0.4 mg/ml),
-Raf-1 (2 µg/ml),
-p70S6K (1 µg/ml), or
-MAPK-PP (1 µg/ml). Bound antibodies were detected with rabbit
Ig-horseradish peroxidase, and the membranes were washed in
Tris-buffered saline plus 0.05% Tween 20 and then incubated for 1 min
with enhanced chemiluminescence (ECL) reagents.
Immunoprecipitation/immunoblotting.
To measure tyrosine phosphorylation of IRS1, IRS2, and Shc, aliquots of
muscle proteins were incubated with -IRS1 [5 µg (10)],
-IRS2 [10 µl (23)], or
-Shc [5 µl
(Transduction Laboratories S14630)] for 16 h at 4°C.
Immunoprecipitated proteins were separated by SDS-PAGE (10% gel), and
tyrosine-phosphorylated proteins were detected by immunoblotting with
an anti-phosphotyrosine antibody preparation (
-PY) [2 µg/ml
(9)]. To assess Grb2-associated proteins, muscle lysates (1 mg)
were incubated with 20 µg of GST-Grb2 fusion protein coupled to
agarose beads for 4 h at 4°C, or with 1 µg/ml of
-Grb2
(Transduction Laboratories G16720) for 16 h at 4°C followed by
incubation with protein A-Sepharose beads for 2 h at 4°C. The beads
were washed two times in lysis buffer. Proteins were separated by
SDS-PAGE (10% gel) and immunoblotted with
-Shc or
-IRS1. To
study Raf-1/Ras association, aliquots of muscle protein (2 mg) were
incubated with 1 µg/ml of
-Raf-1 for 16 h at 4°C. The samples
were then incubated with protein A-Ssepharose beads for 2 h at 4°C,
and the immune complexes were washed 2 times in 50 mM HEPES, 100 mM
NaF, 1% Triton X-100, 2 mM
Na3VO4,
and 0.1% SDS. Immunoprecipitated proteins were separated by SDS-PAGE (10% gel), transferred to nitrocellulose, immunoblotted with
-Ras (1 µg/ml; Santa Cruz SC-35, Oncogene Science OP23), and
antibody binding was detected using ECL.
Northern blotting.
Total muscle RNA was extracted by TRI REAGENT according to the
manufacturer's protocol (Molecular Research Center). Twenty micrograms
of total RNA per sample were electrophoresed in a 1% agarose
formaldehyde gel and transferred to a nylon membrane. RNA was
immobilized by ultraviolet cross-linking, and the blots were hybridized
with a 32P-labeled rat
c-fos probe labeled with
[-32P]dCTP to
~109 dpm/mg with a random
hexamer priming kit (Amersham).
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RESULTS |
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The stimulation of MAP kinase can occur in response to the sequential
phosphorylation and activation of Raf-1 and MEK (26, 40), although
under some conditions MAP kinase and RSK2 activation may not correlate
with Raf-1 and MEK activation (40). Figure 1 shows the time course of activation of
Raf-1, MEK1, and MAP kinase in response to contraction. To study the
effects of contraction on Raf-1, we assessed Raf-1 activation by
electrophoretic mobility shift of the protein by immunoblotting with an
anti-Raf-1 antibody. The representative immunoblot in Fig.
1A shows that with 15 s of
contraction there was reduced electrophoretic mobility of the Raf-1
protein, and with prolonged periods of contraction the change in
mobility was no longer present. Similarly, when Raf-1 activity was
measured using an immune complex assay, contraction resulted in a rapid
increase in enzyme activity that was no longer present with longer
periods of contraction (data not shown). Similar to the rapid
activation of Raf-1, MEK1 activity was stimulated with only 15 s of
muscle contraction and rapidly returned to baseline (Fig.
1B). The effects of contractile
activity on MAP kinase phosphorylation was measured by immunoblotting
muscle lysates with MAPK-PP, an antibody that recognizes only the
phosphorylated MAP kinase proteins (Fig.
1C). Similar to Raf-1 and MEK1
activity, MAP kinase phosphorylation was significantly increased with
15 s of contraction. However, in contrast to Raf-1 and MEK1 activity,
there was a more sustained activation of MAP kinase in the muscle.
Contraction significantly increased phosphorylation of
p42MAPK and
p44MAPK, indicating that both
isoforms are activated in response to contraction in skeletal muscle.
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Of the three known isoforms of the mammalian p90 RSK, RSK2 is
abundantly expressed in muscle and has been implicated as a direct
substrate of the p44 and p42 MAP kinases (34). RSK2 activity toward a
S6 protein peptide substrate (3R S6 peptide) was measured in RSK2
immune complexes from the muscle lysates. Figure
2 shows the time course of RSK2 activity in
response to muscle contractions. By 2 min of contraction, RSK2 activity
was increased by more than twofold above basal, with peak kinase
activity occurring with 10 min of contraction. Thereafter, RSK2
activity decreased, although there was still significant stimulation
with 30 min of contraction. Insulin also increased RSK2 activity as
early as 5 min after injection (1.5-fold above basal), with peak
activity occurring 20 min after insulin injection. Figure 2 shows that
the maximal insulin response was considerably less than the maximal
contraction effect. The inset of Fig.
2 shows the effects of contraction on RSK phosphorylation as assessed
by gel mobility shift. Skeletal muscle contraction caused a clear
retardation in the electrophoretic mobility of the RSK protein,
suggesting increased phosphorylation and confirming the increase in RSK
activity by the contraction stimulus.
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Because MAP kinase signaling has been implicated in the activation of
immediate-early genes, we determined whether the stimulation of the MAP
kinase cascade induced by this contraction protocol was also effective
in increasing c-fos mRNA. Figure
3 shows that contraction dramatically
increased c-fos mRNA by 25-fold.
Increased c-fos expression was present
after only 15 min of contraction and was still elevated with 30 and 60 min of contraction. In contrast, insulin only increased
c-fos mRNA by 4.5-fold above basal,
and this effect was maximal 30 min after insulin injection.
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One mechanism for the activation of the MAP kinase signaling cascade is
via growth factor receptor stimulation and subsequent formation of
Shc/Grb2/Sos and IRS1/Grb2/Sos complexes, resulting in increased
Ras-GTP exchange and activation of Raf-1. We have assessed whether
these proteins are mediators of the contraction-induced increase in MAP
kinase signaling. The standard method to measure Ras activation
involves the loading of cells with radiolabeled GTP. Because it is not
possible to use this technique in intact animals, we used
immunoprecipitation and immunoblotting techniques to assess the
association of Raf-1 with Ras as a surrogate indication of Ras
activation. Muscle lysates were immunoprecipitated with -Raf-1, and
the immune complexes were immunoblotted with
-Ras. For these
experiments, two different antibodies made to unique epitopes of Ras
were used. There was an increase in the abundance of p21 Ras in the
Raf-1 immunocomplexes from the insulin and contracted samples (Fig.
4A).
Interestingly, immunoblotting of the Raf-1 immunoprecipitates with this
Ras antibody also recognized a 30-kDa protein that was significantly
stimulated by insulin, contraction, and acute treatment of anesthetized
rats with EGF (Fig. 4B).
Immunoblotting of muscle lysates with the Ras antibody (with no prior
immunoprecipitation) also revealed two bands with molecular masses of
~21 and 30 kDa (Fig. 4C). The
identity of this 30-kDa protein is not known, but on the basis of its
size and cross-reactivity with two different Ras antibodies, it may be
a Ras-related GTPase. The ability of contractile activity to
stimulate the association of Raf-1 with Ras and a putative Ras-related
molecule suggests that Ras is involved in the activation of MAP kinase
signaling by contraction in skeletal muscle.
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Activation of MAP kinase signaling with contractile activity could
result from tyrosine phosphorylation of one or more muscle tyrosine
kinase receptors. A precedent for this hypothesis comes from studies of
PC12 cells, where activation of MAP kinase signaling in response to
calcium influx was shown to be mediated by tyrosine phosphorylation of
EGF receptors (27). In skeletal muscle, there are transient increases
in intracellular calcium concentrations with the initiation of each
contraction. Therefore, we tested the possibility that tyrosine
phosphorylation of the EGF receptor was involved in the
contraction-induced stimulation of MAP kinase signaling in skeletal
muscle. Lysates from rat muscles contracted for various periods of time
were first immunoprecipitated with an EGF receptor antibody, and the
immune complexes were immunoblotted with -PY, an
anti-phosphotyrosine antibody. Control experiments were done by
injecting rats with a maximal dose of EGF (200 µg/kg body wt) and
dissecting hindlimb skeletal muscles 15 min later. Figure
5A is a
representative phosphorimage showing minimal, if any, effects of muscle
contraction on EGF receptor phosphorylation. In contrast, EGF
administration, which stimulated RSK2 activity and MAP kinase
phosphorylation by considerably less than muscle contraction (<2-fold
above basal; data not shown), caused a dramatic increase in EGF
receptor phosphorylation (Fig. 5A).
To determine whether another tyrosine kinase receptor may be involved
in the stimulation of MAP kinase signaling by contraction, similar
experiments were performed using insulin receptor antibodies. As with
the EGF experiments, there was no effect of contraction on insulin receptor tyrosine phosphorylation (data not shown). The lack of an
effect of contraction on EGF receptor and insulin receptor tyrosine
phosphorylation is consistent with our previous study showing no effect
of contraction on tyrosine phosphorylation of proteins in the molecular
mass range of 80-200 kDa, when measured in
-PY
immunoprecipitates (9). Taken together, these studies strongly suggest
that secondary activation of receptor tyrosine kinases is unlikely to
represent a mechanism for the stimulation of MAP kinase signaling by
contraction in skeletal muscle.
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Activation of the MAP kinase pathway by
ras-dependent mechanisms can involve
the association of Grb2 with tyrosine-phosphorylated IRS or Shc
molecules. In addition to these complexes being formed in response to
stimulation of receptor tyrosine kinases, some classes of G
protein-coupled receptors can also activate the association of Shc with
Grb2 (28). To determine whether these molecules are involved in the
increase in MAP kinase signaling by contraction, we studied the effects
of contractile activity on IRS1, IRS2, and Shc tyrosine
phosphorylation, Shc/Grb2 association, and IRS1/Grb2 association.
Immunoprecipitation of muscle lysates with -IRS1 or
-IRS2 and
immunoblotting with
-PY also showed no increase in IRS tyrosine
phosphorylation in response to contraction (Fig. 5,
B and
C). In fact, there was a decrease in
tyrosine phosphorylation of IRS1 with longer periods of contraction
(Fig. 5B). Similar to the lack of
effect of contraction on IRS tyrosine phosphorylation, none of the Shc
isoforms were tyrosine phosphorylated in response to contraction (Fig.
5D). To determine whether muscle
contraction increased the association of Shc with Grb2, muscle lysates
were incubated with an antibody to Grb2 or with a GST-Grb2 fusion
protein coupled to agarose beads and immunoblotted with
-Shc. Both
of these experiments demonstrated that there was no increase in
immunodetectable Shc in the Grb2 immunoprecipitates from the contracted
muscle, suggesting that muscle contractile activity did not increase
Shc/Grb2 (data not shown). Furthermore, contraction did not increase
immunodetectable Grb2 in IRS1 immunoprecipitates (data not shown).
These results demonstrate that contraction activates MAP kinase
signaling by a mechanism that is independent of IRS and Shc tyrosine
phosphorylation and of formation of Shc/Grb2 or IRS1/Grb2 complexes.
Because p70S6K has been implicated
in the regulation of protein synthesis (14, 19), and contraction can
alter rates of protein synthesis (13), we hypothesized that this enzyme
may also be activated by contractile activity in skeletal muscle. The
effects of muscle contraction on
p70S6K activity were assessed by
molecular weight shift of the protein and by measuring
p70S6K activity toward a GST-S6
protein substrate in p70S6K immune
complexes. Figure
6A shows
that muscle contractile activity for periods ranging from 15 s to 30 min did not alter the mobility of the
p70S6K protein. In contrast,
insulin induced a marked shift in the electrophoretic mobility of
p70S6K. Muscle contraction had no
effect on phosphorylation of the GST-S6 protein, whereas 20 min of
insulin treatment significantly increased p70S6K-mediated GST-S6 protein
phosphorylation by ~75% (Fig. 6, B
and C).
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Recent studies have suggested that Akt is part of the mechanism by
which insulin increases glucose transport. Physical exercise can
increase glucose transport in the contracting skeletal muscles, and
therefore we determined whether muscle contraction can increase Akt
activity. Figure 7 demonstrates that 5 min
after insulin injection there was an approximately fivefold increase in
Akt activity in the skeletal muscle. In contrast, contraction for as
little as 15 s, or as long as 60 min, did not increase Akt activity.
Similar to these results, we have also shown that exercising rats on a rodent treadmill for periods up to 60 min has no effect on Akt activity
in hindlimb skeletal muscle (Markuns and Goodyear, unpublished observation).
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DISCUSSION |
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Contractile activity is a critical regulator of protein synthesis (4) and gene transcription (22) in skeletal muscle. Chronic changes in the level of contractile activity lead to structural remodeling of muscle, resulting in profound effects on the functional capacity of the tissue (13). To determine the underlying molecular mechanisms through which contraction causes these important changes in skeletal muscle, the signaling molecules that convert the mechanical/biochemical contraction stimulus into intracellular responses must be defined. The MAP kinase pathway is a candidate for such a signaling system in contracting skeletal muscle. This pathway is extensively used for transcytoplasmic signaling to the nucleus and is involved in a wide variety of cellular responses to external stimuli (26). Furthermore, several studies have shown that the MAP kinase pathway is activated in cultured cardiac myocytes in response to stretch (30, 38, 39). It is noteworthy, however, that treatment of cultured cardiac myocytes in vitro does not mimic the mechanical loading present in skeletal muscles contracting in the intact animal, nor does it replicate the hormonal and neural environment of the contracting skeletal muscle in situ.
The current findings demonstrate that sciatic nerve stimulation to produce muscle contractions in intact rat skeletal muscle causes a rapid and potent stimulation of the MAP kinase signaling cascade. The temporal activation of these molecules suggests that Raf-1, MEK1, MAP kinase, and RSK2 are sequentially phosphorylated and activated in response to contractile activity in the muscle. Our MAP kinase and RSK2 data also suggest that contractile activity is a considerably more potent activator of MAP kinase signaling in skeletal muscle than growth factor stimulation by EGF or insulin. Furthermore, contraction of isolated muscles in vitro is a much more potent stimulator of MAP kinase phosphorylation than incubation of isolated skeletal muscles with a maximal dose of insulin (T. Hayashi and L. J. Goodyear, unpublished data). Thus the major function of this cascade of signaling proteins in adult, fully differentiated skeletal muscle may be to mediate the biological responses to increased muscle activity rather than responses to growth factors.
Components of the MAP kinase signaling pathway are expressed in smooth (1, 37), cardiac (30, 39), and skeletal muscles (8). In cardiac myocytes, MAP kinase signaling has been shown to be involved in adaptations to stretch (30, 39), and emerging data indicate a similar response of MAP kinase in arterial smooth muscle cells subjected to mechanical load or hypertension (1, 37). Our studies of rat (8) and human (2) skeletal muscle have shown that physical exercise can also stimulate MAP kinase phosphorylation. Thus, although there are considerable differences in the nature of the mechanical stimuli to which different muscle cell types are normally exposed, the MAP kinase pathway emerges as a signaling system for the conversion of mechanical stimuli into intracellular signals, a system that is shared by all (skeletal, smooth, and cardiac) muscle cell types.
The physiological consequences of increased MAP kinase signaling in skeletal muscle in response to insulin (12, 41), EGF (3), and contraction (current study) are not known. In cardiac myocytes, activation of the MAP kinase signaling pathway by mechanical stretch may regulate the stretch-induced increases in protein synthesis and the transcriptional activation of several immediate-early genes (16). In skeletal muscle, the induction of the immediate-early genes c-fos, c-jun, and egr-1 has been shown to occur 4 h after the onset of sustained muscle contractile activity caused by low-voltage electrical stimulation (22). In the current study, we show that a period of only 15 min of tetanic contractions is necessary for a dramatic increase in c-fos mRNA in skeletal muscle. The temporal relationship of the peak activation of the MAP kinase signaling intermediaries and c-fos expression (Raf-1 = 15 s; MEK1 = 15 s; MAP kinase = 5-10 min; RSK2 = 10 min; c-fos = 15 min) suggests that MAP kinase signaling could be involved in the activation of this immediate-early gene. It is also interesting that, compared with the modest effects of insulin to increase both RSK2 activity and c-fos mRNA, contraction results in a much greater activation of both RSK2 and c-fos mRNA. Because the MAP kinase pathway has been implicated in the regulation of gene transcription in numerous cell types (26), it is likely that transcriptional regulation is a consequence of increased MAP kinase signaling with muscle contraction. Thus the control of gene transcription through the MAP kinase signaling pathway could be a major factor in the ability of repeated bouts of muscle contraction to alter muscle phenotype.
Activation of the MAP kinase signaling cascade by contraction could be mediated by systemic effects that occur in response to increased muscle contractile activity, local tissue-specific effects that are associated with the mechanical or biochemical events of muscle contraction, or a combination of both. We believe that there are limited, if any, systemic contributions to the activation of this pathway, because we have shown that one-legged exercise in human subjects stimulates MAP kinase signaling only in the exercising leg (2). Thus it is probably a local effect of contractile activity that results in increased MAP kinase signaling. Intrinsic changes in the skeletal muscle that could lead to increased MAP kinase activation include alterations in the energy status of the muscle fibers, osmotic changes, and mechanical or shear stresses. It is also possible that local activation of MAP kinase signaling occurs via an autocrine or paracrine mechanism. The current studies have made the important observation that contractile activity does not act via tyrosine kinase receptors Shc, IRS1, IRS2, and Grb2. These findings also suggest that G protein-coupled receptors that utilize Shc for the activation of MAP kinase signaling are not a mechanism for the effects of muscle contraction. One potential mechanism for activation of the MAP kinase pathway involves protein kinase C. Protein kinase C can activate the MAP kinase cascade in several cells types (15, 17), and muscle contraction has been shown to increase protein kinase C activity in rat skeletal muscle (25). Future investigations using isoform-specific protein kinase C inhibitors will be required to determine whether this enzyme mediates the effects of contraction to stimulate MAP kinase in skeletal muscle.
Contractile activity increases skeletal muscle glucose transport through a PI 3-kinase-independent mechanism (9, 11). Certain stress-activated stimuli have been shown to activate Akt in a PI 3-kinase-independent manner (21, 31), and several recent studies have implicated Akt in the regulation of glucose transport (33). However, the current data clearly demonstrate no effect of contractile activity on Akt activity in skeletal muscle, suggesting that this molecule does not play a role in mediating contraction-stimulated glucose transport. While our manuscript was in preparation, two other groups reported that contraction of isolated skeletal muscles contracted in vitro had no effect on Akt activity (5, 20). Similar to the lack of effect of muscle contraction on Akt activity, contraction had no effect on p70S6K activity or IRS2 tyrosine phosphorylation. The lack of stimulation of IRS2 phosphorylation by contraction in skeletal muscle is in contrast to a recent report suggesting that contraction of ventricular cardiomyocytes in vitro increases IRS2 tyrosine phosphorylation (35). Thus, although some contraction-activated intracellular signaling responses (e.g., MAP kinase) are similar among different muscle types, the regulation of other signaling molecules is tissue specific.
In conclusion, contractile activity rapidly and potently stimulates the MAP kinase signaling cascade in skeletal muscle. Our data suggest that the activation of MAP kinase signaling in skeletal muscle involves Ras, whether by contraction, insulin, or EGF. On the other hand, in contrast to the mechanisms by which insulin and EGF stimulate the MAP kinase pathway in skeletal muscle, contraction-induced stimulation of this cascade occurs by an IRS-, Shc-, and Grb2-independent mechanism. The temporal relationship for the activation of the MAP kinase cascade and c-fos expression suggests that signaling through this pathway may regulate the expression of immediate-early genes. These findings provide direction for future studies intended to determine the molecular basis for the acute regulation of gene transcription and subsequent changes in phenotype that occur in skeletal muscle in response to contractile activity.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert J. Smith (Joslin Diabetes Center) for the
anti-phosphotyrosine (PY) and IRS-1 antibodies and the
c-fos probe, and Drs. Jesse Chow and
Robert J. Smith for supplying the EGF-stimulated muscle lysates.
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FOOTNOTES |
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This work was supported by National Institute on Aging Grant AR-42238 and a grant from the Juvenile Diabetes Foundation International (both to L. J. Goodyear).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: L. J. Goodyear, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 (E-mail: goodyeal{at}joslab.harvard.edu).
Received 27 August 1998; accepted in final form 22 January 1999.
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