Effect of Contraction on Mitogen-activated Protein Kinase Signal Transduction in Skeletal Muscle

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38MAPK phosphorylation. Phosphorylation of ERK1/2 or p38MAPK was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38MAPK was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38MAPKinhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90Rsk), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90Rsk and MAPKAP-K2 most closely reflects ERK and p38MAPK stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38MAPK. These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.

Chronic exercise leads to changes in expression of key proteins involved in the regulation of fuel metabolism in skeletal muscle (1,2). Muscle contraction/exercise has been shown to increase the activity of various members of the mitogen-activated protein kinase (MAPK) 1 family, including the extracel-lular signal-regulated kinases 1/2 (ERK1/2) (3-7), the c-Jun N-terminal kinase (3,5,8), and p38 MAPK (6). Because MAPK activation is associated with increased transcriptional activity (9), increased MAPK signaling could mediate exercise-induced changes in gene expression and alterations in muscle metabolism. Although exercise results in the activation of MAPK signaling, very little is known of the mechanism by which MAPK mediates signal transduction in direct response to muscle contraction.
ERK1/2 activation is mediated via a number of signaling molecules including growth factor receptor tyrosine kinases (9 -11) and protein kinase C (PKC) (12,13). Growth factor receptor tyrosine kinases activate ERK1/2 through the Shc/ Grb2/SOS pathway via activation of Ras (9 -11, 14). Ras associates with Raf, which phosphorylates MAPK Kinase (MEK) (9). Subsequent growth factor activation of ERK1/2 occurs through phosphorylation by MEK. Activation of the p38 MAPK signaling cascade is initiated via a number of stimuli including inflammatory cytokines (15), ultraviolet light exposure (16), hyperosmolarity (17), and exercise (6). Direct activation of p38 MAPK occurs via phosphorylation by MEK3 or MEK6 (9). Similar to the ERK pathway, PKC has also been implicated in p38 MAPK activation, as endothelin-1 activation of p38 MAPK in rat ventricular myocytes is inhibited by phorbol ester downregulation of PKC or by treatment with a PKC inhibitor GF109203X (18). However, the role of PKC in the exerciseinduced activation of ERK1/2 or p38 MAPK signaling pathways has not been determined.
Activated MAPK interacts with a number of downstream targets, including the p90 ribosomal S6 kinase (p90 Rsk ) (19) and MAPK-activated protein kinase-2 (MAPKAP-K2) (20,21), which are activated by ERK1/2 and p38 MAPK , respectively. Additionally, the recently identified mitogen-and stress-activated protein kinase 1 (MSK1) is activated by a mechanism involving an interaction with either ERK or p38 MAPK (22). To date, the role of MSK1 in exercise-induced signal transduction is unknown.
The aim of this study was to first assess whether PKC is involved in MAPK activation in response to muscle contraction. Secondly, we investigated the effect of ex vivo muscle contraction on MAPK signaling to the downstream targets p90 Rsk , MAPKAP-K2, and MSK1. Here we show that contractile activity activates p90 Rsk and MAPKAP-K2. Activation of p90 Rsk and MAPKAP-K2 by muscle contraction most closely reflects ERK and p38 MAPK activity, respectively. Additionally, we show that contractile activity stimulates the recently identified MSK1. The use of an ex vivo model of muscle contraction demonstrates that local effects of muscle contraction lead to the activation of the MAPK signaling cascade, independent of systemic influence.  (23), MAPKAP-K2 (24), and MSK1 (22). Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G were from Bio-Rad. Chemiluminescence reagents were from Amersham Pharmacia Biotech. PD98059, SB203580, and GF109203X were from Cal-Biochem (La Jolla, CA). All other reagents were of analytical grade (Sigma).

Animals-Male
Muscle Incubations-All incubation media were prepared from a pregassed (95%O 2 /5%CO 2 ) stock of Krebs Henseleit buffer (KHB) supplemented with 5 mM HEPES and 0.1% bovine serum albumin (RIA grade). Rats were anesthetized with sodium pentobarbital (5 mg/100 g body weight), and epitrochlearis muscles were carefully dissected out and incubated in a shaking water bath (30°C) for 60 min in 2 ml KHB containing 5 mM glucose and 15 mM mannitol. When pharmacological inhibitors were used, an additional 30-min incubation was employed to pre-expose the muscle to the inhibitor. Once added, inhibitors remained present for the duration of the experiment. Final concentration of dimethyl sulfoxide was adjusted to 0.1% for each group (without or with inhibitor) in the experiments using GF109203X or 0.35% in experiments using PD98059 and SB203580. In some experiments muscles were incubated for 20 min in 12-O-tetradecanoylphorbol 13-acetate (TPA) as described in the figure legends.
Ex Vivo Muscle Contraction-Following preincubation, epitrochlearis muscles were placed inside a temperature controlled (30°C) stimulation chamber and immersed in 4 ml of KHB identical to preincubation condition. Each muscle was positioned between two platinum electrodes with the distal tendon mounted to the bottom of the chamber. The proximal tendon was connected to a jeweler's chain, which was fixed to an isometric force transducer (Harvard Apparatus, Inc., South Natick, MA). Resting tension was adjusted to 0.5 g. The isometric tension development during the contraction protocol was recorded using a compact 2-Channel Student Oscillograph (Harvard Apparatus, Inc., South Natick, MA). Isometric muscle contraction was achieved via electrical stimulation. Muscles were stimulated at a frequency of 100 Hz (pulse duration, 0.2 ms; amplitude, 10 V) delivered at a rate of one 0.2-s contraction every 2 s (0.2 s/2 s) or one 10-s contraction every 60 s (10 s/60 s) for 10 min. The pulses were generated by a Tektronix TM 503 Power Module (Beaverton, OR) and amplified on a 4-Channel Power Amplifier (Somedic, Inc., Sollentuna, Sweden). Basal muscles were treated as described above minus electrical stimulation. Muscles were then frozen immediately between aluminum tongs cooled to the temperature of liquid nitrogen or further incubated for the assessment of glucose transport activity. Additional experiments were performed examining the effects of electrical stimulation with various voltages or time points as described in the figure legends.
Glucose Transport Activity-Muscles were transferred to vials containing glucose free KHB without or with the addition of 120 nM insulin and incubated for 10 min. Muscles were then transferred to KHB containing 8 mM [ 3 H]3-O-methylglucose (438 Ci/mmol) and 12 mM [ 14 C]mannitol (42 Ci/mmol) without or with insulin, and glucose transport activity was assessed as described by Wallberg-Henriksson et al. (25). Glucose transport activity was assessed for 20 (basal) or 12 (insulin or contraction) min. Glucose transport activity is expressed as mol/ml of intracellular water/h. Glycogen Analysis-Glycogen content was determined fluorometrically on HCl extracts as described by Lowery and Passonneau (26). Results are expressed as mol glucose/g wet weight.
Western Blot Analysis-Aliquots of muscle homogenates containing 20 g of protein were suspended in Laemmli buffer. Proteins were separated by SDS-PAGE (10% resolving gel), transferred to polyvinylidenedifluoride membranes (Millipore, Bedford, MA) and blocked with 5% nonfat milk. Membranes were incubated in primary antibody overnight at 4°C as described in the figure legends. Membranes were washed in TBST (10 mM Tris, 140 mM NaCl, 0.02% Tween 20, pH 7.6), incubated with appropriate secondary antibody, and washed in TBST again. Proteins were visualized by enhanced chemiluminescence and quantified by densitometry.
Statistics-Data are presented as the means Ϯ S.E. Differences were determined using a one-way analysis of variation with a subsequent Fisher's LSD post hoc analysis. Significance was accepted at p Ͻ 0.05.

RESULTS
Glucose Transport Activity and Muscle Glycogen Content-To test the efficacy of two different electrical stimulation protocols with respect to altering muscle metabolism, glucose transport and muscle glycogen content were analyzed following either insulin exposure or ex vivo muscle contraction. Muscle contraction was delivered at a rate of one 0.2-s contraction every 2 s or one 10-s contraction every 60 s for a total of 10 min. Glucose transport activity increased 7-fold (p Ͻ 0.001) in response to insulin stimulation (120 nM) (Table I). Ex vivo contraction resulted in a similar 5-6-fold (p Ͻ 0.001) increase in 3-O-methylglucose transport activity (Table I), with no statistical difference observed between the two contraction protocols. Muscle glycogen utilization was also similar between the two contraction protocols, with glycogen stores reduced Ͼ 60% (p Ͻ TABLE I Glucose transport activity and glycogen content in rat epitrochlearis muscle following ex vivo contraction Rat epitrochlearis muscles were incubated in KHB (basal), exposed to insulin (120 nM), or forced to contract via electrical stimulation for 10 min as described under "Experimental Procedures." Muscle contraction was delivered at a rate of one 0.2-s contraction every 2 s (0.2 s/2 s) or one 10-s contraction every 60 s (10 s/60 s) for 10 min. Thereafter, muscles were rinsed free of extracellular glucose, and 3-O-methylglucose transport activity was assessed. Some muscles were frozen immediately after muscle contraction for muscle glycogen analysis. Values are presented as the means Ϯ S.E. Number of muscles is shown in parentheses (n). Stimulus
Ex Vivo Muscle Contraction Increases Phosphorylation of ERK1/2 and p38 MAPK -Muscle contraction, evoked by electrical stimulation, led to the induction of ERK1/2 phosphorylation. ERK phosphorylation, as assessed by phospho-specific immunoblot analysis, was increased 2-3-fold (p Ͻ 0.05), with the greatest effect observed after impulses were delivered for 0.2 s at a rate of one contraction every 2 s for 10 min (Fig. 1A). Protein expression of ERK1 and ERK2 was not altered by electrical stimulation (Fig. 1, B and C). Muscle contraction also stimulated the phosphorylation of p38 MAPK (Fig. 1D). Under basal conditions, p38 MAPK phosphorylation was nearly undetectable (Fig. 1D). After 10 min of muscle contraction via electrical stimulation, phospho-p38 MAPK immunoreactivity was markedly increased (Fig. 1D, p Ͻ 0.005). Protein expression of p38 MAPK was not altered by muscle contraction (Fig. 1E). Similar to the results noted for ERK1/2, the greatest effect of muscle contraction upon p38 MAPK signaling was observed when impulses were delivered for 0.2 s at a rate of one contraction every 2 s for 10 min, therefore in subsequent experiments we utilized this protocol. To assess weather muscle contraction or electrical stimulation directly account for increased MAPK phosphorylation, epitrochlearis muscles were stimulated with 0 (basal), 1, 2, or 10 V for 10 min. Tension development was undetectable (Ͻ0.02 g) when 1 V was applied. In contrast, initial tension development was 17 Ϯ 3 and 25 Ϯ 3 g of tension in response to stimulation with 2 and 10 V, respectively. Increased phosphorylation of ERK1/2 and p38 MAPK was only detectable in contracting muscles (Fig. 2, A and B). Furthermore, contraction-induced phosphorylation of ERK1/2 and p38 MAPK occurred in a time-dependent manner. Maximal phosphorylation was observed after 10 min of muscle contraction and activation was sustained at 20 min (Fig. 2, C and D).
Effect of Protein Kinase C Inhibition on ERK1/2 and p38 Phosphorylation-Following 60-min recovery period, muscles were incubated for an additional 30 min in the absence or presence of 5 M GF109203X (bisindoylamalamide I), a highly selective PKC inhibitor (27). When muscles were pre-exposed to GF109203X, the 2-fold increase in TPA stimulated (5 M) ERK phosphorylation was reduced 50% (Fig. 3A). TPA did not increase p38 MAPK phosphorylation (data not shown). To determine whether PKC mediates MAPK phosporylation in response to muscle contraction, muscles were incubated in the absence or presence of 5 M GF109203X. Thereafter, muscles were incubated in KHB (basal) or forced to contract via electrical stimulation as described in Fig. 3. Exposure to GF109203X did not alter contraction-induced ERK1/2 phosphorylation (Fig. 3B). Similarly GF109203X was without effect upon p38 MAPK phosphorylation in response to muscle contraction (Fig. 3C).
Effect of PD98059 and SB203580 on ERK1/2 and p38 MAPK Phosphorylation-Muscles were incubated for 30 min in the absence or presence of 50 M PD98059, 10 M SB203580, or a

FIG. 2. Effect of increasing voltage, and time course for ERK1/2 and p38 MAPK phosphorylation.
A and B, rat epitrochlearis muscles were incubated under basal conditions (nonstimulated) or electrically stimulated with 1, 2, or 10 V for a total of 10 min. Electrical stimulation was delivered for 0.2 s every 2 s for 10 min. C and D, muscles were incubated in KHB (basal; nonstimulated) or forced to contract via electrical stimulation for 2, 5, 10, or 20 min at a rate of one 0.2-s contraction every 2 s. Therafter, ERK1/2 or p38 MAPK phosphorylation was determined by Western blot analysis as described in Fig. 1.   FIG. 3. Effect of GF109203X on contraction-stimulated ERK1/2 and p38 MAPK phosphorylation. Rat epitrochlearis muscles were incubated in the absence (Ϫ) or presence (ϩ) of 5 M PKC inhibitor GF109203X for 30 min. Thereafter, muscles were incubated without or with 5 M TPA for 20 min (A). Additional muscles incubated in KHB (basal) or forced to contract via electrical stimulation for 10 min at a rate of one 0.2 s contraction every 2 s (B and C). Muscle proteins were separated by SDS-PAGE, and phospho-ERK1/2 (B) and phospho-p38 MAPK (C) were detected via Western blot analysis using phosphospecific antibodies as described in Fig. 1. combination of PD98059 and SB203580. The utilities of PD98059 (23,28) and SB203580 (20,29) as respective inhibitors of MEK1 and p38 MAPK have previously been described. After initial exposure to the inhibitor(s), ex vivo muscle contraction was evoked for 10 min in identical media. Ex vivo muscle contraction led to 3.2-(p Ͻ 0.001) and 2.0-fold (p Ͻ 0.001) increases in ERK1 and ERK2 phosphorylation, respectively (Fig. 4A). Addition of 50 M PD98059 reduced ERK1/2 activity 80 -95% (p Ͻ 0.001) in contracting muscles. Basal phosphorylation of ERK1/2 was also decreased 70% by prior exposure to PD98059 (data not shown). Exposure of muscles to the p38 MAPK inhibitor SB203580 (10 M) reduced contractioninduced phosphorylation of ERK1/2 25% (p Ͻ 0.05). Nevertheless, ERK1/2 phosphorylation was still significantly increased above basal levels (p Ͻ 0.05). Contraction-induced p38 MAPK phosphorylation was not significantly reduced by 50 M PD98059 (Fig. 4B). In contrast, SB203580 reduced p38 phosphorylation 90% (p Ͻ 0.001). Alterations in contraction-stimulated MAPK signal transduction cannot be accounted for by an inhibition of muscle contractile activity, because initial tension development and time to 50% fatigue were not altered by PD98059 or SB203580 (Table II).
p90 Rsk (MAPKAP-K1) and MAPKAP-K2 Activity Is Increased by Muscle Contraction-Ex vivo muscle contraction increased p90 Rsk activity 5-fold (Fig. 5A, p Ͻ 0.001). The contractioninduced increase in p90 Rsk activity was completely prevented by the addition of 50 M PD98059. Similar to the results noted above for ERK1/2 phosphorylation, SB203580 led to a 48% reduction in contraction-stimulated p90 Rsk activity (p Ͻ 0.001). Nevertheless, contraction-induced p90 Rsk activity was elevated 2.5-fold (p Ͻ 0.001) with respect to basal levels after SB203580 exposure. MAPKAP-K2 activity was increased 10-fold by muscle contraction (Fig. 5B, p Ͻ 0.005). This effect was unaffected by PD98059 but completely inhibited by the presence of SB203580, suggesting that p38 MAPK mediates the activation of MAPKAP-K2 in response to muscle contraction. Muscle Contraction Activates MSK1 Activity-Recently, MSK1 has been shown to be activated by both ERK and p38 MAPK pathways. Ex vivo muscle contraction led to a 5-fold increase in MSK1 activity (Fig. 6, p Ͻ 0.001). This effect was completely inhibited by the addition of either PD98059 or SB203580, suggesting that muscle contraction stimulates MSK1 by both ERK and p38 MAPK mediated pathways. DISCUSSION Exercise and contractile activity stimulate ERK (3-7), c-Jun N-terminal kinase (3,5,8), and p38 MAPK (6). We have utilized an ex vivo system to achieve muscle contraction to examine the direct role of muscle contraction upon stimulation of MAPK signal transduction. This system allows us to study the effects of muscle contraction on signal transduction independent of systemic factors or influence of surrounding tissue. Contractile activity increased muscle glucose transport 5-6-fold, and decreased muscle glycogen stores Ͼ 60%, demonstrating a profound effect of contractile activity on carbohydrate metabolism in skeletal muscle. This system of muscle contraction greatly influenced signaling by the MAPK pathways. Here we show that ex vivo muscle contraction, void of any systemic factors, is sufficient for ERK1/2 and p38 MAPK phosphorylation. Increased phosphorylation of ERK1/2 and p38 MAPK occurred only at voltages that elicit measurable muscle contraction. This suggests that muscle contraction rather than electrical stimulation is directly responsible for initiating MAPK signaling in skeletal muscle. Furthermore, we show that ex vivo contraction activates the downstream MAPK targets p90 Rsk , MAPKAP-K2, and MSK1.
Classic growth factor-mediated activation of ERK1/2 involves the Ras/Raf/MEK signaling cascade (9 -11). Activated MEK1/2 phosphorylates and activates ERK1/2. Exercise/contraction-induced ERK1/2 signaling appears to involve similar signal transduction (4, 7). However, the mechanism by which muscle contraction initiates the Ras-Raf association remains undetermined (7). PKC activation is also known to lead to Ras FIG. 4. Effect of PD98059 and SB203580 on contraction-stimulated ERK1/2 and p38 MAPK phosphorylation. Rat epitrochlearis muscles were incubated in the absence (Ϫ) or presence (ϩ) of 50 M PD98059, 10 M SB203580, or a combination of both inhibitors for 30 min. Thereafter, muscles were incubated in KHB (basal; nonstimulated) or forced to contract via electrical stimulation for 10 min at a rate of one 0.2 s contraction every 2 s. Muscle proteins were separated by SDS-PAGE, and phospho-ERK1/2 and phospho-p38 MAPK were detected via Western blot analysis using phospho-specific antibodies as described in Fig. 1. A, ERK1 (open bars) and ERK2 (closed bars) phosphorylation quantified by densitometry. Representative autoradiograph is presented, and order of sample loading is identical to histogram order. B, p38 MAPK phosphorylation quantified by densitometry. A representative autoradiograph is presented, and order of sample loading is identical to histogram order. Results are presented as the means Ϯ S.E. for 5-9 muscles/group. *, p Ͻ 0.05 versus basal.

TABLE II
Initial tension development and time to 50% fatigue in contracting epitrochlearis muscles pre-exposed to PD98059 and/or SB203580 Muscles were incubated for 30 min in KHB with or without the addition of 50 M PD98059 or 10 M SB203580. Thereafter, muscles were forced to contract via electrical stimulation at a rate of one 0.2-s contraction every 2 s for 10 min. Tension development was measured with an isometric force transducer as described under "Experimental Procedures." Values are presented as the means Ϯ S.E. Number of muscles is shown in parentheses (n).

Condition
Initial tension development activation and thus stimulate MAPK activtity (12). Additionally, PKC can mediate p38 MAPK activation by an unidentified mechanism (18). Muscle contraction has been suggested to activate PKC in response to electrical stimulation (30,31). Sciatic nerve stimulation in rat hind limb is associated with a rapid (2-5 min) shift of PKC activity, as measured by 32 P incorporation to histone IIIS, from the cytosol to the membrane fraction (30,31). Therefore, we determined whether PKC mediates MAPK signal transduction in response to ex vivo muscle contraction. Pre-exposure of rat epitrochlearis muscles to the PKC inhibitor GF109203X did not alter ERK1/2 or p38 MAPK phosphorylation in contracting skeletal muscle. These results suggest that GF109203X-sensitive PKC isoforms (i.e. conventional and novel) are not involved in contraction-induced MAPK kinase signaling. The mechanism by which muscle contraction initiates Ras-Raf association (7) remains elusive. The uses of the specific inhibitors PD98059 and SB203580 have been powerful tools in the delineation of downstream MAPK signaling in response to numerous stimuli. PD98059 (23) and SB203580 (20) have been reported to be highly selective inhibitors of MEK1 and p38 MAPK activity, respectively, with very little inhibition reported on other members of the MAPK family. We show that PD98059 completely inhibited contraction-stimulated ERK1/2 phosphorylation, with ERK2 phosphorylation reduced to 50% of basal activity. This indicates that MEK1 is the primary kinase involved in ERK1/2 phosphorylation in response to muscle contraction. Unexpectedly, SB203580 also inhibited ERK1/2 phosphorylation in contracting muscle, although to a lesser degree than observed with PD98059. Contraction-stimulated ERK1/2 phoshorylation was reduced by 25% by SB203580; however, ERK1/2 phosphorylation was still significantly elevated over basal levels. The activity of p90 Rsk activity in contracting skeletal muscle paralleled ERK phosphorylation. Muscle contraction induced a 5-fold increase in p90 Rsk activity. This effect was completely abolished in the presence of PD98059, indicating that muscle contraction simulates p90 Rsk activity via an ERK dependent mechanism. Similar to our findings for ERK phosphorylation, exposure of skeletal muscle to SB203580 resulted in an intermediate degree of inhibition on p90 Rsk activity. To our knowledge, these results are the first to show an inhibitory effect of SB203580 on the ERK signaling pathway. Previously, SB203580 has been reported to be without inhibitory effect on MEK1, ERK2, or p90 Rsk activity (20). Our results could suggest that there is cross-talk between the activation of the p38 MAPK pathway and the ERK pathway, which may be promoting the activation of ERK in skeletal muscle. Although we observe no inhibition of force development in contracting muscles exposed to SB203580, the possibility remains that SB203580 inhibits unidentified signaling molecules involved in the coupling of muscle contraction to the MAPK signaling pathway.
In the present study, we show that p38 MAPK phosphorylation is dramatically increased in direct response to ex vivo muscle contraction. Exposure to PD98059 did not prevent contractioninduced phosphorylation of p38 MAPK . In contrast, p38 MAPK phosphorylation was reduced by 90% in muscle exposed to SB203580. Similar to the observed effects of contraction on p38 MAPK activation, MAPKAP-K2 activity was increased 10fold in response to muscle contraction. This response was also unaffected by PD98059 but completely inhibited in the presence of SB203580. The mechanism of SB203580 inhibition is through binding to the ATP pocket of p38 MAPK (32,33), therefore the finding that SB203580 inhibited p38 MAPK phosphorylation was unexpected. However, this is not the first report to show SB203580-induced inhibition of p38 MAPK phosphorylation, because SB203580 exposure to ischemic cardiomyocytes also leads to reduced p38 MAPK phosphorylation (34). Interestingly, in epitrochlearis muscles exposed to osmotic shock (KHB supplemented with 600 mM mannitol) for 20 min we do not observe an inhibition of p38 MAPK phosphorylation by SB203580 exposure. 2  Muscles were pre-exposed to 50 M PD98059 or 10 M SB203580 and forced to contract as described in Fig. 4. MSK1 activity was measured by 32 P incorporation into peptide substrate as described under "Experimental Procedures." Inset is a representative phosphorimage. Order of sample loading is identical to histogram order. Values are reported in arbitrary units. Results are presented as the means Ϯ S.E. for 4 -5 muscles/ group. *, p Ͻ 0.01 versus basal. MAPK signal transduction coupling, with the greatest effects elicited upon the p38 MAPK cascade. Nevertheless, p38 MAPK inhibition is evident from the complete ablation of contractioninduced MAPKAP-K2 activity.
Recently a new MAPK target protein, MSK1, has been identified (22). Here we provide evidence that contractile activity activates MSK1 in skeletal muscle. Thus, muscle contraction is the first identified stimulus of MSK1 activity in primary tissue. In 293 cells, MSK1 is activated in an ERK-dependent manner by stimulation with TPA or epidermal growth factor (22). Interestingly, ultraviolet light, arsenite, or H 2 O 2 also activate MSK1 through p38 MAPK . These results suggest that either ERK or p38 MAPK activity are sufficient for activation of MSK1 (22). Accordingly, because muscle contraction leads to the activation of ERK1/2 and p38 MAPK kinase, simultaneous inhibition of ERK and p38 MAPK activity would be expected to fully inhibit MSK1 activity in response to muscle contraction. Surprisingly, PD98059 and SB203580 independently and completely inhibited contraction-induced MSK1 activity. Inhibition of MSK1 occurred despite the observation that contractionstimulated MAPKAP-K2 and p90 Rsk activities were significantly increased following PD98059 and SB203580 exposure, respectively. Thus, both ERK and p38 MAPK activation appear to be required for MSK1 activation in skeletal muscle in response to muscle contraction.
MAPK activation plays an important role in the regulation of gene transcription. Here we provide evidence that muscle contraction stimulates activation of ERK and p38 MAPK pathways, separate from any endocrine or paracrine influence. This activation appears to occur independently of PKC. Moreover, we show that contraction-induced p90 Rsk and MAPKAP-K2 activation occur largely via separate MAPK signaling pathways. Importantly, we provide evidence demonstrating that MSK1 is directly activated by ex vivo muscle contraction and that this activation requires the simultaneous activation of both the ERK and p38 MAPK pathways.