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
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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-alpha (TNF-alpha ) 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
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Abstract
Introduction
Methods
Results
Discussion
References

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. [gamma -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 beta -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 beta -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 [gamma -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 [gamma -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 [gamma -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 beta -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 [gamma -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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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.

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.

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 alpha 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.

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.

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.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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