Extracellular-regulated protein kinase cascades are activated
in response to injury in human skeletal muscle
Doron
Aronson1,
Jørgen F. P.
Wojtaszewski2,
Anders
Thorell3,
Jonas
Nygren3,
David
Zangen1,
Erik A.
Richter2,
Olle
Ljungqvist3,
Roger A.
Fielding4, and
Laurie J.
Goodyear1
1 Research Division, Joslin Diabetes
Center, Department of Medicine, Brigham and Women's Hospital
and Harvard Medical School, and 4 Department of
Health Sciences, Sargent College of Allied Health Professions, Boston
University, Boston, Massachusetts 02215;
2 Copenhagen Muscle Research Center, August Krogh
Institute, University of Copenhagen, DK-2100 Copenhagen, Denmark;
and 3 Department of Surgery, Karolinska Hospital
and Institute, S-10401 Stockholm, Sweden
 |
ABSTRACT |
The
mitogen-activated protein (MAP) kinase signaling pathways are believed
to act as critical signal transducers between stress stimuli and
transcriptional responses in mammalian cells. However, it is not known
whether these signaling cascades also participate in the response to
injury in human tissues. To determine whether injury to the vastus
lateralis muscle activates MAP kinase signaling in human subjects, two
needle biopsies or open muscle biopsies were taken from the same
incision site 30-60 min apart. The muscle biopsy procedures
resulted in striking increases in dual phosphorylation of the
extracellular-regulated kinases (ERK1 and ERK2) and in activity of the
downstream substrate, the p90 ribosomal S6 kinase. Raf-1 kinase and MAP
kinase kinase, upstream activators of ERK, were also markedly
stimulated in all subjects. In addition, c-Jun NH2-terminal kinase and p38
kinase, components of two parallel MAP kinase pathways, were activated
following muscle injury. The stimulation of the three MAP kinase
cascades was present only in the immediate vicinity of the injury, a
finding consistent with a local rather than systemic activation of
these signaling cascades in response to injury. These data demonstrate
that muscle injury induces the stimulation of the three MAP kinase
cascades in human skeletal muscle, suggesting a physiological relevance of these protein kinases in the immediate response to tissue injury and
possibly in the initiation of wound healing.
mitogen-activated protein kinase; signal transduction; stress
enzymology
 |
INTRODUCTION |
EXPOSURE OF CELLS to environmental stressors such as
ultraviolet (UV) irradiation or osmotic shock evokes a series of
phosphorylation events leading to the modification of transcription
factors and altered gene expression (5, 21, 29). In mammalian cells, there are at least three related but distinct mitogen-activated protein
(MAP) kinase cascades that are activated by diverse environmental stresses. The extracellular-regulated kinase (ERK) pathway involves the
sequential phosphorylation and activation of the serine kinase Raf-1,
the MAP kinase kinases (MEK1/2), and two ERK isoforms (ERK1/ERK2) (34).
The ERKs can phosphorylate and activate cytosolic substrates such as
the p90 ribosomal S6 kinase (RSK) (36). A fraction of the activated ERK
population translocates into the nucleus and activates several nuclear
transcription factors including c-myc, c-fos, and Elk-1 (34, 37). The ERK
pathway is activated by growth factors (34) and by cellular stresses
such as hyperosmolarity (23) and reperfusion injury (20).
A second pathway uses the c-Jun
NH2-terminal kinases (JNKs) for
transmitting stress signals (21). JNKs are phosphorylated and activated
by MAP kinase kinase 4 (MKK4) (32), which in turn is
phosphorylated and activated by MAP kinase kinase kinase 1 (MEKK1)
(38). JNKs can be stimulated by a variety of cellular stresses such as
UV radiation (9) and osmotic and heat shock (5), as well as by
proinflammatory cytokines including tumor necrosis factor-
(TNF-
)
and interleukin-1 (IL-1) (21, 22). The third member of the MAP kinase
family, the p38 kinase, resembles the yeast high-osmolarity glycerol
response 1 kinase (HOG1 kinase). The p38 kinase is activated by MKK3
(10) and MKK6 (17). In mammalian cells p38 kinase is also regulated by
stress signals such as UV light, heat shock (31), proinflammatory
cytokines (29), and endotoxin from gram-negative bacteria (16). An
important physiological substrate of p38 kinase is the MAP
kinase-activated protein kinase 2, an enzyme that plays a role in the
regulation of heat shock proteins as part of the cellular response to
stress (31).
Most of our knowledge concerning the stress-induced activation of these
MAP kinase signaling cascades has come from studies of cultured cells.
However, stresses inflicted on cells in culture are frequently
different from those that are encountered by animals or humans. It has
recently been shown that stress-induced activation of MAP kinase may
play a role in the context of wound healing in plants (35). The purpose
of the current study was to test the hypothesis that wounding of
skeletal muscle activates ERK, JNK, and p38 kinase signaling. We
demonstrate that injury, caused by two different biopsy procedures,
leads to a striking activation of these pathways in human skeletal
muscle.
 |
METHODS |
Experimental protocols. The study
population included eight healthy volunteers (6 males and 2 females).
The procedures involved were fully described, and informed consent was
obtained from each subject. Injury was caused to the vastus lateralis
muscle by either needle biopsy or a surgical open muscle biopsy
procedure. For studies examining the effects of the percutaneous needle
biopsy technique (2), the biopsy needle was inserted through an
incision made in the skin under local anesthesia (2-3 ml, 2%
Lidocaine) using sterile conditions. The incision penetrated the
underlying fascia and allowed easy entry of the biopsy needle to the
desired depth of ~3 cm. Once the muscle tissue was removed, it was
quickly dissected free from the surrounding fat and connective tissue and was immediately placed in liquid nitrogen and stored at
80°C.
For the first protocol, three needle biopsies were taken from each
subject (n = 4). The initial biopsy
was obtained, and a second biopsy was taken from the same incision 30 min later. A third biopsy was taken ~5 min after the second biopsy
from the contralateral vastus lateralis. For the second protocol, four muscle biopsies were obtained from two additional subjects. The first
two biopsies were taken as described above, i.e., the first biopsy was
taken and 30 min later a second biopsy was obtained from the same
incision site. This was followed by a third biopsy obtained ~10 min
later from a separate incision spaced 5 cm apart from the first
incision site. A forth biopsy was taken from the second incision 30 min
after the third biopsy.
To control for the possibility that only a specific form of muscle
injury affects the MAP kinase pathways (e.g., crush of muscle cells
induced by the needle biopsy), two subjects underwent open muscle
biopsies. For the open biopsy, a local anesthetic (15-20 ml 2%
Lidocaine) was infiltrated subcutaneously down to but not into the
muscle. An incision was made and the subcutaneous tissue was dissected
down to the muscle fascia. The fascia was opened and the vastus
lateralis fibers carefully separated. A bundle of muscle fiber was
dissected and clamped with a forked hemostat, cut, and immediately
placed in liquid nitrogen. The fascia and incision were loosely sutured
and covered with a sterile dressing. For the second biopsy, the
incision site was reopened 60 min following the initial biopsy, and a
second bundle of muscle fibers was removed. For this open biopsy
procedure, the second sample was taken from an area of the muscle that
was adjacent but separate from fibers handled in the first biopsy,
ensuring that the sample was macroscopically intact.
Reagents.
[
-32P]ATP (3,000 Ci/mmol) was purchased from Du Pont-New England Nuclear (Boston, MA). A
PGEX vector designed to express a glutathione
S-transferase (GST)-c-Jun fusion
protein (NH2-terminal residues
1-135) was provided by Dr. John Kyriakis, Massachusetts General
Hospital. Anti-RSK2, 3R S6 RSK substrate peptide, and GST-MEK1 were
from Upstate Biotechnology (Lake Placid, NY); anti-JNK1, anti-p38
kinase, GST-activating transcription factor 2 (ATF-2), and recombinant
ERK2 were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-MEK1 and
anti-Raf-1 monoclonal antibodies were from Transduction Laboratories
(Lexington, KY); and phosphospecific anti-ERKs were from Quality
Controlled Biochemicals (Hopkinton, MA). Protein A-agarose and protein
G-agarose were from Pierce (Rockford, IL). Protein concentrations were
determined using a dye reagent from Bio-Rad (Hercules, CA), and all
other chemicals were purchased from Sigma Chemical (St. Louis, MO).
Muscle processing. Muscle samples were
Polytron homogenized (Brinkmann, Westbury, NY) in ice-cold lysis buffer
containing 20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM
-glycerophosphate,
1 mM dithiothreitol (DTT), 1 mM
Na3VO4,
1% (vol/vol) Triton X-100, 10% (vol/vol) glycerol, 10 µM leupeptin,
3 mM benzamidine, 5 µM pepstatin A, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulphonyl fluoride (PMSF). Homogenates were rotated for 1 h
at 4°C and centrifuged at 14,000 g
for 68 min at 4°C to remove insoluble matter. Protein concentrations were estimated by the Bradford method (4).
Immunoblotting. Muscle proteins (200 µg) were solubilized in Laemmli buffer and boiled for 5 min. Samples
were then resolved on a 10% SDS-PAGE gel, transferred to
nitrocellulose paper, and immunoblotted with anti-MEK1, anti-Raf-1,
anti-RSK, or a phosphospecific anti-ERK. The filters were then
incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG
(1:2,500), and antibody binding was detected via the method of enhanced
chemiluminescence.
Kinase assays. For the MEK1 kinase
assay, 500 µg of muscle extracts were incubated with 2 µg anti-MEK1
or 1.0 µg purified rabbit IgG and adsorbed to protein G beads. The
immunoprecipitates were washed three times in buffer
A (150 mM NaCl, 20 mM Tris, pH 8, 10 mM NaF, 100 µM
Na3VO4,
2 mM EDTA, 1 mM PMSF, 10% glycerol, 1% Nonidet P-40, 2 µg/ml
leupeptin) and three times with MEK1 kinase buffer containing 25 mM
HEPES, pH 7.5, 10 mM MgCl2, 2 mM DTT, and 100 µM
Na3VO4.
The immunoprecipitates were resuspended in 80 µl MEK1 kinase buffer
containing 50 µM ATP and 100 ng recombinant ERK2. Reactions were
incubated for 30 min at 30°C and were terminated by adding Laemmli
sample buffer. The products were resolved on 10% SDS-PAGE gel followed
by transfer to nitrocellulose paper. ERK2 phosphorylation was then
detected using the phosphospecific anti-ERK antibodies as described
above. For the MEK1 assay and all subsequent kinase assays, control
values (obtained by immunoprecipitation of lysates using preimmune IgG)
were subtracted from all bands using features of the ImageQuant
software.
For the Raf-1 kinase assay, an aliquot of muscle extracts (500 µg)
was incubated with 2 µg anti-Raf-1 and adsorbed to protein G beads.
The immunoprecipitates were washed three times in
buffer A and three times with Raf-1
kinase buffer (30 mM HEPES, pH 7.4, 10 mM
MnCl2, 5 mM
MgCl2, 100 µM
Na3VO4,
25 mM
-glycerophosphate, 1 mM DTT, and 0.003% Brij 35). The
immunoprecipitates were resuspended in 50 µl Raf-1 kinase buffer
containing 20 µM ATP, 10 µCi
[
-32P]ATP, and 1 µl recombinant kinase inactive GST-MEK1. Reactions were incubated for
30 min at 30°C and were terminated by adding Laemmli sample buffer.
Products were boiled for 5 min and resolved on 10% SDS-PAGE. The
phosphorylated GST-MEK1 was quantified by PhosphorImager analysis of
the dried gels (Molecular Dynamics).
For the RSK assay, 400 µg of muscle extracts were incubated with 2.5 µg of anti-RSK2 antibody and adsorbed to protein A beads. The immune
complexes were washed three times with lysis buffer, three times with
LiCl buffer, and three times with RSK kinase buffer (30 mM Tris, pH
7.4, 10 mM MgCl2, 0.1 mM EGTA, 1 mM DTT) and were then resuspended in 50 µl RSK kinase buffer
containing 50 µg S6 peptide, 40 µM ATP, and 10 µCi
[
-32P]ATP.
Reactions were carried out at 30°C for 15 min, terminated by adding
10 µl of stopping solution containing 0.6% HCl, 1 mM ATP, and 1%
BSA, and spotted onto P81 phosphocellulose papers. The papers were
washed five times with 175 mM phosphoric acid and counted by the
Cerenkov method.
For the JNK assay, 250 µg of muscle extracts were incubated with 10 µg anti-JNK1 and adsorbed to protein A beads. The immunoprecipitates were washed twice with lysis buffer, twice with LiCl buffer (500 mM
LiCl, 100 mM Tris · HCl, pH 7.6, and 0.1% Triton
X-100, 1 mM DTT), and twice with JNK buffer (20 mM MOPS, pH 7.2, 2 mM
EGTA, 10 mM MgCl2, 1 mM DTT, 0.1%
Triton X-100). The immunoprecipitates were resuspended in 50 µl JNK
buffer containing 3 µg GST-Jun-(1-135), 30 µM ATP, and 10 µCi [
-32P]ATP.
The kinase reaction was performed at 30°C for 30 min and was
terminated with Laemmli sample buffer. The reaction products were
subjected to 10% SDS-PAGE, and the incorporation of
32P into GST-c-Jun was
quantitated.
For the p38 kinase assay, 250 µg of muscle extracts were incubated
with 2 µg of anti-p38 kinase adsorbed to protein A beads. The
immunoprecipitates were washed twice with lysis buffer and four times
with p38 kinase buffer (20 mM HEPES, pH 7.6, 20 mM
-glycerophosphate, 1 mM
Na3VO4,
1 mM DTT, 10 mM MgCl2). The
immunoprecipitates were resuspended in 30 µl of p38 kinase buffer
containing 20 µM ATP, 10 µCi
[
-32P]ATP, and 1 µg GST-ATF-2. Reactions were incubated for 30 min at 30°C and
were terminated by the addition of Laemmli buffer. Products were
resolved using 10% SDS-PAGE, and the phosphorylated GST-ATF-2 was
quantified.
 |
RESULTS |
Muscle injury stimulates the ERK signaling
cascade. Studies in cultured cells have shown that the
maximal activation of ERK (23), JNK (9, 12, 22), and p38 kinase (29)
frequently occurs several minutes following the stress exposure.
Therefore, we obtained the second muscle sample 30-60 min
following the first (injury-inducing) biopsy. We first examined the
effects of muscle injury on the phosphorylation of the ERK1 and ERK2
proteins using a phosphospecific antibody. This antibody recognizes
only the phosphorylated form of the mammalian ERKs at Thr-183 and
Tyr-185, which are required for full enzymatic activity (27). Muscle injury resulted in a striking increase in ERK phosphorylation (11.1 ± 2.6-fold above basal; means ± SE; Fig.
1). In contrast, when a third biopsy was
obtained from a second incision site from the contralateral muscle,
there was no effect on ERK phosphorylation (Fig.
1A). This finding suggests that
the effect of muscle injury on ERK phosphorylation is localized to the
injured muscle and is not a systemic response to the first muscle
biopsy. Furthermore, when the third biopsy was taken from the same
vastus lateralis muscle at a second incision site 5 cm proximal to the
first incision, there was no effect on ERK phosphorylation (Fig.
1B). A forth biopsy, taken from the
second incision site 30 min after the third biopsy, showed a similar
pattern of activation observed for the first and second biopsy (Fig.
1B). These data demonstrate that the
increase in ERK phosphorylation is a local phenomenon, restricted only
to the injury site. Open muscle biopsies also resulted in a marked
increase in the dual phosphorylation of ERK (Fig.
1C), indicating that ERK
phosphorylation occurred in the macroscopically intact tissue
surrounding the initial injury site.

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Fig. 1.
Effects of muscle injury on extracellular-regulated kinase (ERK)
phosphorylation. Muscle lysates were separated by SDS-PAGE and
transferred to nitrocellulose membrane, and phosphorylation of the ERK
isoforms was demonstrated by immunoblotting with phosphospecific
antibodies that recognize only dual-phosphorylated ERK. Arrows, ERK1
and ERK2 isoforms. A: representative
immunoblot from a subject who underwent 3 needle biopsies, showing a
marked increase in ERK phosphorylation when second biopsy was done, 30 min after initial biopsy from same incision site, but showing no change
in ERK phosphorylation in the contralateral leg.
B: representative immunoblot from a
subject who underwent 4 muscle biopsies taken from 2 separate incisions
in the same leg. Effect of muscle injury on ERK phosphorylation was
observed only when repeat biopsy was taken from same incision site.
C: immunoblot of injury-induced ERK
phosphorylation in 2 subjects who underwent open biopsies. Bx,
biopsy.
|
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Raf-1 kinase and MEK1 are upstream activators of ERK (34). To determine
whether these enzymes are also activated by muscle injury, we assayed
MEK1 and Raf-1 activities in the muscle extracts. MEK1 is a
dual-specificity enzyme that activates ERK by phosphorylating both
Thr-183 and Tyr-185 residues (27). The ability of MEK1 immunoprecipitates to phosphorylate ERK2 on these specific residues was
determined by immunoblotting the reaction products with a phosphospecific anti-ERK. The phosphorylation of ERK2 increased by 3.2 ± 0.3-fold above basal following muscle injury (means ± SE,
combined needle and open biopsies data), indicating that MEK1 was
activated and was able to function as a dual-specificity enzyme in ERK2
phosphorylation (Fig.
2A).
Figure 2B demonstrates that equivalent
amounts of the MEK1 protein were immunoprecipitated from all samples.

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Fig. 2.
Effects of muscle injury on MEK1 activity.
A: mitogen-activated protein kinase
kinase 1 (MEK1) immunoprecipitates were assayed for kinase activities
using ERK2 as substrate. Phosphorylated proteins were separated by 10%
SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted
with phosphospecific anti-ERK antibodies. Immunoreactive bands were
visualized by enhanced chemiluminescence (ECL).
A: representative immunoblot from 2 subjects who underwent 3 needle biopsies. Muscle injury resulted in an
increase in MEK1 activity only at the injury site.
B: membranes were subsequently
reprobed with anti-MEK1, to confirm that equivalent amounts of MEK1
were immunoprecipitated from each sample.
|
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Muscle injury also resulted in a marked increase in Raf-1 activity, as
assessed by the ability of immunoprecipitated Raf-1 to phosphorylate
its downstream substrate MEK1 (6.9 ± 2.8 above basal, means ± SE; Fig.
3A). In
addition, muscle injury caused a marked decrease in the relative
electrophoretic mobility of Raf-1 (Fig.
3B), suggesting increased
phosphorylation and confirming the increase in kinase activity. Similar
to ERK phosphorylation, the injury-induced increases in MEK1 (Fig.
2A) and Raf-1 (Fig. 3,
A and
B) activities were restricted to the
injury site.

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Fig. 3.
Effect of muscle injury on Raf-1 activity.
A: Raf-1 was immunoprecipitated from
muscle extracts and assayed for its ability to phosphorylate
kinase-inactive glutathione
S-transferase (GST)-MEK1 as described
in METHODS. Representative
autoradiogram shows 71-kDa band corresponding to phosphorylated
GST-MEK1 in a subject who underwent 3 needle biopsies.
B: muscle proteins were
electrophoresed on an 8% polyacrylamide gel and transferred to
nitrocellulose filters. Protein blots were probed with anti-Raf-1
antibody, and bands were visualized by ECL. Muscle injury resulted in a
decrease in the electrophoretic mobility of Raf-1.
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RSK2 is a cytosolic kinase that can be phosphorylated and activated by
ERK (34, 36) and may act as a mediator between signal transduction
pathways and intranuclear events (3). We determined whether this
downstream signaling molecule is also activated in response to muscle
injury. RSK2 activity increased in all subjects following muscle injury
(11.7 ± 2.5-fold; means ± SE; Fig.
4A). A
slow migrating form of RSK was the predominant protein species
following muscle injury (Fig. 4B),
indicating increased phosphorylation. The increase in RSK2 activity was
confined to the injury site (Fig. 4).

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Fig. 4.
Effect of muscle injury on p90 ribosomal S6 kinase (RSK) activity.
A: muscle proteins were
immunoprecipitated with RSK, and RSK activity was measured in
immunoprecipitates using 3R S6 peptide as substrate. RSK activity in 4 subjects who underwent 3 needle biopsies is depicted. Data are
expressed as means ± SE counts/min (cpm) of
32P incorporated into the
substrate. B: RSK mobility shift assay
in a subject who underwent 3 needle biopsies. Muscle homogenates were
resolved on an 8% SDS-polyacrylamide gel, transferred to
nitrocellulose paper, and immunoblotted with anti-RSK. RSK
phosphorylation in postinjury samples was demonstrated by a decrease in
the electrophoretic mobility of the RSK protein.
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|
Muscle injury stimulates JNK and p38
activities. Although one of the MAP kinase signaling
pathways may be preferentially activated in response to a specific
environmental stress (e.g., JNK for UV irradiation), numerous cellular
stresses result in the simultaneous activation of two or more MAP
kinase cascades (5). Parallel activation of multiple signaling cascades
may be particularly likely in the context of tissue injury in vivo,
where several different stimuli coexist. To determine whether muscle
injury activates JNK, enzyme activity was measured by an immune complex kinase assay, using c-Jun-(1-135) as substrate. As shown in Fig. 4, JNK activity increased following both needle (Fig.
5, A and B) and open (Fig.
5C) biopsy (6.0 ± 3.3-fold above
basal; means ± SE).

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Fig. 5.
Effect of muscle injury on c-Jun
NH2-terminal kinase (JNK)
activity. Muscle extracts were immunoprecipitated with anti-JNK1, and
the immunoprecipitates were subjected to in vitro kinase reactions
containing GST-c-Jun-(1-135) as substrate. The phosphorylated
proteins were resolved by SDS-PAGE, and bands corresponding to
phosphorylated GST-c-Jun (42-kD) were quantitated by
PhosphorImager. A: representative
autoradiogram from a subject who underwent 3 needle biopsies shows a
marked increase in JNK activity when repeat biopsy was obtained from
same incision site, and no change in activity in contralateral leg.
B: representative autoradiogram from a
subject from whom muscle biopsies were taken from 2 separate incisions
separated by 5 cm. Increase in JNK activity was observed only when the
repeat biopsy was taken from the same incision site.
C: autoradiogram showing
injury-induced JNK activation in 2 subjects who underwent open
biopsies.
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|
Similar to JNK, the p38 kinase pathway is implicated in the
transduction of stress signals (5, 16, 29). JNK and p38 kinase have
similar activation profiles by environmental stress and proinflammatory
cytokines, and most extracellular stimulants investigated thus far
activate both JNK and p38 kinase (5, 9, 16, 21, 22). To determine
whether muscle injury also activates p38 kinase signaling, p38 kinase
activity was assayed in muscle extracts using GST-ATF-2 as substrate
(29). The p38 kinase activity increased after needle and open biopsies
by 3.0 ± 1.0-fold above basal (Fig. 6).
Similar to the local activation of the MAP kinase signaling cascade,
there was no increase in JNK or p38 kinase activities in the
contralateral muscle (Figs. 5A and
6A) or when the muscle samples were
taken from a separate site in the same vastus lateralis (Figs.
5B and
6B).

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Fig. 6.
Effect of muscle injury on p38 kinase activity. Muscle extracts were
immunoprecipitated with anti-p38 antibody, and in vitro immune complex
kinase reactions were performed using GST-activating transcription
factor 2 (GST-ATF-2) fusion protein as substrate. Proteins were
resolved by SDS-PAGE, and bands corresponding to phosphorylated
GST-ATF-2 were quantitated by PhosphorImager.
A: autoradiogram showing increased p38
activity only when repeat biopsy was obtained from same incision site.
B: autoradiogram showing
injury-induced p38 stimulation in 2 subjects who underwent open
biopsies.
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 |
DISCUSSION |
The MAP kinases comprise a ubiquitous group of enzymes that are
activated in response to a wide variety of cellular stresses (5, 21).
Studies of MAP kinase signaling and cellular stress have almost
exclusively been carried out using cultured cell systems. Thus it is
critical to determine whether these biological phenomena are relevant
to understanding the physiological responses of higher organisms to
tissue injury. Therefore, we determined whether the induction of
similar signaling events occurs in response to injuries akin to those
humans frequently encounter. In the present study, we demonstrate that
injury to skeletal muscle results in the activation of multiple
components of the MAP kinase signaling cascade (Raf-1/MEK1/ERK/RSK) and
increases the activity of both JNK and p38 kinase.
The marked activation of the MAP kinase cascades 30 min following
tissue injury may initiate adaptive responses that render cells more
resistant to the stress of the wound environment. In NIH/3T3 and PC-12
cells, Guyton et al. (15) have shown that the potential for cell
survival following oxidant injury correlated with the capacity for ERK
activation. MAP kinases may trigger a beneficial stress response by
activating genes coding for proteins that confer protection against
environmental insults. For example, recent work by Schreiber et al.
(33) indicates that both c-fos and
c-jun induction are part of the
natural defense mechanism that increases the ability of mammalian cells
to withstand UV irradiation. Several studies have demonstrated that
tissue injury also causes a rapid induction of early-response genes
(18, 26), which are among the major nuclear targets of MAP kinases (13, 32, 34, 47). Hengerer et al. (18) have shown that transection of the
sciatic nerve rapidly increases c-fos
and c-jun mRNA, with peak levels 2 h
following injury. Similarly, Pawar et al. (26) have demonstrated a
rapid induction of c-fos and Egr-1
after scrape wounding of renal epithelial cells. Hence activation of
MAP kinases following tissue injury may represent a prototype stress
response that results in an increased ability of cells to survive by
activating genes coding for proteins that confer protection against
stress, or that facilitates the repair of stress-damaged cells. In
addition, MAP kinases (especially the p38 kinase) can also regulate
heat shock proteins, which have been shown to be involved in the
response to tissue injury and human wound healing (25). Interestingly, wounding of plants rapidly activates a 46-kDa protein kinase, which
appears to be a plant member of the MAP kinase family (35). Activation
of this kinase has been hypothesized to be involved in wound healing
(35).
In the current study, the stimulation of the MAP kinase pathways was
restricted to the area of injured muscle, indicating a local activation
rather than a systemic response to injury. One mechanism for the local
activation of MAP kinases could involve growth factor and inflammatory
cytokines that participate in normal wound healing (8, 11, 24, 28).
However, many of these mediators are delivered locally at the wound
site by recruited inflammatory cells (7, 28), with peak levels in human
wound fluids several hours to days following injury (7, 11, 14). This
temporal appearance cannot explain the rapid activation of MAP kinases
observed in our study. Furthermore, in animal studies we have observed
activation of ERK phosphorylation as early as 2 min following tissue
injury (D. Garrel and L. J. Goodyear, unpublished observations).
A more immediate source of growth factors is platelet degranulation,
resulting in the release of these mediators (7). However, it is
noteworthy that Northern blot data from adult skeletal muscle has shown
no measurable mRNA for platelet-derived growth factor receptor, the
major platelet mitogen (19). In addition, systemic administration of
large doses of growth factors, such as epidermal growth factor (EGF) to
rats, results only in a modest (~2-fold) increase in the activity of
ERK in skeletal muscle (D. J. Sherwood and L. J. Goodyear, unpublished
observations). These data imply that the rapid activation of the MAP
kinase cascades does not require a paracrine action of stroma or
platelet-derived growth factors.
Alternatively, activation of the MAP kinase signaling cascades under
stress conditions may not require the presence of growth factor or
cytokines in the wound environment. For example, Rosette and Karin (30)
have recently shown that the initial signaling event that activates the
JNK cascade in response to either UV irradiation or osmotic stress is
multimerization and clustering of cell surface receptors such as EGF,
TNF, and IL-1, an event that occurs in the absence of ligand binding to
the receptor. Furthermore, heat shock-induced JNK activation is not
dependent on membrane-associated components (1). Thus similar growth factor/cytokine-independent mechanisms may also be responsible for the
activation of MAP kinases following muscle injury.
In summary, we have shown that injury to skeletal muscle robustly
stimulates ERK, JNK, and p38 kinase signaling. These findings suggest
that these signaling cascades may be involved in the immediate response
to tissue injury. Although we have shown that this response occurs in
skeletal muscle, a similar response is likely after wounding of other
tissues. In this context, activation of MAP kinases can serve as a
convergence point that integrates diverse stress-induced signals that
are relevant to the wound healing process in human tissues.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-42238 and by a grant from
the Juvenile Diabetes Foundation (L. J. Goodyear).
 |
FOOTNOTES |
Address for reprint requests: L. J. Goodyear, Joslin Diabetes Center,
One Joslin Place, Boston MA 02215.
Received 3 December 1997; accepted in final form 7 May 1998.
 |
REFERENCES |
1.
Adler, V.,
A. Schaffer,
J. Kim,
L. Dolan,
and
Z. Ronai.
UV irradiation and heat shock mediate JNK activation via alternate pathways.
J. Biol. Chem.
270:
26071-26077,
1995[Abstract/Free Full Text].
2.
Bergström, J.
Muscle electrolytes in man. Determined by neutron activation analysis on needle biopsy specimens.
Scand. J. Clin. Lab. Invest. Suppl.
68:
1-110,
1962.
3.
Blenis, J.
Signal transduction via the MAP kinases: proceed at your own RSK.
Proc. Natl. Acad. Sci. USA
90:
5889-5892,
1993[Abstract].
4.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
5.
Cano, E.,
and
L. C. Mahadevan.
Parallel signal processing among mammalian MAPKs.
Trends Biochem. Sci.
20:
117-122,
1995[Medline].
6.
Currie, R. W.,
and
F. P. White.
Trauma-induced proteins in rat tissues: a physiological role for a "heat shock" protein?
Science
214:
72-73,
1981[Medline].
7.
Davidson, J. M.,
and
S. I. Benn.
Biochemical and molecular regulation of angiogenesis and wound repair.
In: Cellular and Molecular Pathogenesis, edited by A. E. Sirica. New York: Raven, 1995, p. 79-108.
8.
De Boer, W. I.,
A. G. Schuller,
M. Vermey,
and
T. H. van der Kwast.
Expression of growth factors and receptors during specific phases in regenerating urothelium after acute injury in vivo.
Am. J. Pathol.
145:
1199-1207,
1994[Abstract].
9.
Dérijard, B.,
M. Hibi,
I.-H. Wu,
T. Barrett,
B. Su,
T. Deng,
M. Karin,
and
R. J. Davies.
JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-JUN activation domain.
Cell
76:
1025-1037,
1994[Medline].
10.
Dérijard, B.,
J. Raingeaud,
T. Barrett,
T. Wu,
I.-H. Han,
J. Ulevitch,
and
R. J. Davis.
Independent human MAP kinase signal transduction pathways defined by MEK and MKK isoforms.
Science
267:
682-685,
1995[Medline].
11.
Fahey, T. J.,
B. Sherry,
K. J. Tracey,
S. van Deventer,
W. G. Jones,
J. P. Minei,
S. Morgello,
G. T. Shires,
and
A. Cerami.
Cytokine production in a model of wound healing: the appearance of MIP 1, MIP 2, cachectin/TNF and IL 1.
Cytokine
2:
92-99,
1990[Medline].
12.
Galcheva-Gargova, Z.,
B. Dérijard,
I.-H. Wu,
and
R. J. Davies.
An osmosensing signal transduction pathway in mammalian cells.
Science
265:
806-808,
1994[Medline].
13.
Gille, H.,
A. D. Sharrocks,
and
P. E. Shaw.
Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation on c-fos promoter.
Nature
358:
414-417,
1992[Medline].
14.
Grayson, L. S.,
J. F. Hansbrough,
R. L. Zapata-Silvert,
C. A. Dore,
J. L. Morgan,
and
M. A. Nicolson.
Quantitation of cytokine levels in skin graft donor site wound fluid.
Burns
19:
401-405,
1993[Medline].
15.
Guyton, K. Z.,
Y. Liu,
M. Gorospe,
Q. Xu,
and
N. J. Holbrook.
Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.
J. Biol. Chem.
271:
4138-4142,
1996[Abstract/Free Full Text].
16.
Han, J.,
J.-D. Lee,
L. Bibbs,
and
R. J. Ulevitch.
A MAP targeted by endotoxin and hyperosmolarity in mammalian cells.
Science
265:
808-811,
1994[Medline].
17.
Han, J.,
J.-D. Lee,
Y. Jiang,
Z. Li,
L. Feng,
and
R. J. Ulevitch.
Characterization of the structure and function of a novel MAP kinase kinase (MKK6).
J. Biol. Chem.
271:
2886-2891,
1996[Abstract/Free Full Text].
18.
Hengerer, B.,
D. Lindholm,
R. Heumann,
U. Ruther,
E. F. Wagner,
and
H. Thoenen.
Lesion-induced increase in nerve growth factor mRNA is mediated by c-fos.
Proc. Natl. Acad. Sci. USA
87:
3899-3903,
1990[Abstract].
19.
Jin, P.,
M. Rahm,
L. Claesson-Welsh,
C. H. Heldin,
and
T. Sejersen.
Expression of PDGF A-chain and
-receptor genes during rat myoblast differentiation.
J. Cell Biol.
110:
1665-1672,
1990[Abstract].
20.
Knight, R. J.,
and
D. B. Buxton.
Stimulation of c-Jun kinase and mitogen-activated protein kinase by ischemia and reperfusion in the perfused rat heart.
Biochem. Biophys. Res. Commun.
218:
83-88,
1996[Medline].
21.
Kyriakis, J. M.,
and
J. Avruch.
Sounding the alarm: protein kinase cascades activated by stress and inflammation.
J. Biol. Chem.
271:
24313-24316,
1996[Free Full Text].
22.
Kyriakis, J. M.,
P. Banerjee,
E. Nikolakaki,
T. Dai,
E. A. Rubie,
M. F. Ahmad,
J. Avruch,
and
J. W. Woodgett.
The stress-activated protein kinase subfamily of c-Jun kinases.
Nature
369:
156-160,
1994[Medline].
23.
Matsuda, S.,
H. Kawasaki,
T. Moriguchi,
Y. Gotoh,
and
E. Nishida.
Activation of protein kinase cascades by osmotic shock.
J. Biol. Chem.
270:
12781-12786,
1995[Abstract/Free Full Text].
24.
Matsuoka, J.,
and
G. R. Grotenddorst.
Two peptides related to platelet derived growth factor are found in human wound fluid.
Proc. Natl. Acad. Sci. USA
86:
4416-4420,
1989[Abstract].
25.
Oberringer, M.,
H. P. Baum,
V. Jung,
C. Welter,
J. Frank,
M. Kuhlmann,
W. Mutschler,
and
R. G. Hanselmann.
Differential expression of heat shock protein 70 in well healing and chronic human wound tissue.
Biochem. Biophys. Res. Commun.
214:
1009-1014,
1995[Medline].
26.
Pawar, S.,
S. Kartha,
and
F. G. Toback.
Differential gene expression in migrating renal epithelial cells after wounding.
J. Cell. Physiol.
165:
556-565,
1995[Medline].
27.
Payne, D. M.,
A. J. Rossamando,
P. Martino,
A. K. Erickson,
J.-H. Her,
J. Shabanowitz,
D. F. Hunt,
M. J. Weber,
and
T. W. Sturgill.
Identification of the regulatory phosphorylation sites in pp42/mitogen activated protein kinase (MAP kinase).
EMBO J.
10:
885-892,
1991[Abstract].
28.
Pierce, G. F.,
and
T. A. Mustone.
Pharmacologic enhancement of wound healing.
Annu. Rev. Med.
46:
467-481,
1995[Medline].
29.
Raingeaud, J.,
S. Gupta,
J. S. Rogers,
M. Dickens,
J. Han,
R. J. Ulevitch,
and
R. J. Davis.
Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine.
J. Biol. Chem.
270:
7420-7426,
1995[Abstract/Free Full Text].
30.
Rosette, C.,
and
M. Karin.
Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors.
Science
274:
1194-1197,
1996[Abstract/Free Full Text].
31.
Rouse, J.,
P. Cohen,
S. Trigon,
M. Morange,
A. Alonso-Llamazares,
D. Zamanillo,
T. Hunt,
and
A. R. Nebreda.
A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.
Cell
78:
1027-1037,
1994[Medline].
32.
Sánchez, I.,
R. T. Hughes,
B. J. Mayer,
K. Yee,
J. R. Woodgett,
J. Avruch,
J. M. Kyriakis,
and
L. I. Zon.
Role of SAPK/ERK kinase 1 in the stress activated pathway regulating transcription factor c-Jun.
Nature
372:
794-798,
1994[Medline].
33.
Schreiber, M.,
B. Baumann,
M. Cotten,
P. Angel,
and
E. F. Wagner.
Fos is an essential component of the mammalian UV response.
EMBO J.
14:
5338-5349,
1995[Abstract].
34.
Seger, R.,
and
E. G. Krebs.
The MAPK signaling cascade.
FASEB J.
9:
726-735,
1995[Abstract/Free Full Text].
35.
Seo, S.,
M. Okamoto,
H. Seto,
K. Ishizuka,
H. Sano,
and
Y. Ohasi.
Tobacco MAP kinase: a possible mediator of wound signal transduction pathways.
Science
270:
1988-1991,
1995[Abstract].
36.
Sturgill, T. W.,
L. B. Ray,
E. Erikson,
and
J. L. Maller.
Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II.
Nature
334:
715-718,
1988[Medline].
37.
Whitmarsh, A. J.,
P. Shore,
A. D. Sharrocks,
and
R. J. Davis.
Integration of MAP kinase signal transduction pathways at the serum response element.
Science
269:
403-407,
1995[Medline].
38.
Yan, M.,
T. Dai,
J. C. Deak,
J. M. Kyriakis,
L. I. Zon,
J. R. Woodgett,
and
D. J. Templeton.
Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1.
Nature
372:
798-800,
1994[Medline].
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