Regulation of neuregulin/ErbB signaling by contractile activity in skeletal muscle

Nathan K. Lebrasseur1,2, Gregory M. Coté1, Thomas A. Miller1, Roger A. Fielding2, and Douglas B. Sawyer1

1 Myocardial Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston 02218; and 2 Human Physiology Laboratory, Department of Health Sciences, Sargent College of Health and Rehabilitation Sciences, Boston University, Boston, Massachusetts 02215


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Putative roles of neuregulin (NRG) and the ErbB receptors in skeletal muscle biology include myogenesis, ACh receptor expression, and glucose transport. To date, however, the physiological regulation of NRG/ErbB signaling has not been examined. We tested the hypothesis that contractile activity in vivo induces NRG/ErbB activation. Rat hindlimb muscle contraction was elicited with a single bout of electrical stimulation (RX) or treadmill running (EX). Western blot and immunofluorescence confirmed the expression of multiple NRG isoforms and the ErbB2, ErbB3, and ErbB4 receptors in adult skeletal muscle. Both RX and EX significantly increased phosphorylation of all NRG receptors. Furthermore, contraction induced a shift in the expression profile of NRG, consistent with proteolytic processing of a transmembrane isoform. Thus two distinct modes of exercise activated processing of NRG with concomitant stimulation of ErbB2, ErbB3, and ErbB4 signaling in vivo. To our knowledge, this is the first demonstration of physiological regulation of NRG/ErbB signaling in any organ and implicates this pathway in the metabolic and proliferative responses of skeletal muscle to exercise.

exercise; growth factor; receptor tyrosine kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROLIFERATIVE AND METABOLIC responses of skeletal muscle to contractile activity involve the complex integration of both intra- and intercellular signaling pathways. The activation of diverse intracellular kinase cascades has been demonstrated in skeletal muscle, and these have been associated with fiber hypertrophy and alterations in metabolism. The extracellular stimuli and cell-cell interactions that trigger the activation of these pathways during and after exercise, however, have not been elucidated. In multiple cell types, growth factors relay key extracellular signals to trigger diverse cellular events via receptor tyrosine kinases (RTKs) (9). One such candidate system in the regulation of exercise signaling that has pleiotrophic effects in skeletal muscle is neuregulin (NRG) and its cognate RTKs ErbB2, ErbB3, and ErbB4.

The NRGs (also known as heregulin, neu differentiation factor, ACh receptor-inducing activity, glial growth factor II, and sensory motor neuron-derived factor) are a complex family of proteins structurally related to the classical polypeptide mitogen-epidermal growth factor (EGF; see Ref. 19). More than 15 distinct soluble and membrane-anchored NRG isoforms result from alternative splicing of mRNA from one of four known NRG genes (21). All NRGs identified to date feature an EGF-like motif that is both necessary and sufficient for biological activity (6, 29). NRG signals are mediated via activation of the type I subfamily RTKs: ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4) (7, 13, 30, 41). Heteromeric complexes of ErbB2, ErbB3, and ErbB4 and homomeric ErbB4 are activated by NRG binding and lead to phosphorylation of cytoplasmic tyrosine residues that initiate a diverse array of downstream signaling events (5, 46).

NRGs activate growth, differentiation, and survival signaling pathways in multiple cell types, including epithelium (44), nerve (23), cardiac (47), and skeletal muscle. Targeted mutations in mice have demonstrated an essential role for NRG and ErbBs in the development of the nervous and cardiovascular systems (11, 18, 23). In skeletal muscle, NRG has been studied extensively in vitro as a regulator of differentiation and development of the neuromuscular junction. Nerve- and muscle-derived members of the NRG family have been observed to stimulate myotube formation and muscle-specific gene expression (10, 16), induce ACh receptor expression (AchR; see Refs. 8, 15, and 22), and regulate formation and maintenance of chemical synapses (34). Generation of a muscle-specific ErbB2 deletion resulted in mice with impaired motor coordination and poor body condition. Isolated ErbB2-deficient myoblasts suffered extensive apoptosis upon differentiation to myofibers (3). Furthermore, NRG appears to play a role in regulating metabolism, in that NRG stimulation of skeletal muscle facilitates glucose uptake in a manner that is additive to the effects of insulin (38).

To date, the physiological regulation of NRG/ErbB signaling has not been examined. Given the evidence of NRG mediation of skeletal muscle growth and metabolism in vitro, we tested the hypothesis that contractile activity in vivo elicits NRG/ErbB activation. We employed two distinct modes of contractile activity that initiate specific intracellular signaling pathways (26) and induce unique functional and morphological properties in skeletal muscle. We used electrical stimulation as a model of resistance exercise, which is known to enhance muscle strength through fiber hypertrophy and contractile characteristics (20), and treadmill running as a model of endurance exercise, which is known to enhance muscle oxidative capacity through changes in key metabolic enzymes, mitochondrial density, and capillary supply with no resultant hypertrophy (12).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. ErbB2, ErbB4, and extracellular NRG [Heregulin Ab1 (clone 7D5)] antibodies were purchased from NeoMarkers (Fremont, CA). ErbB3, intracellular NRG [HRG-alpha (C20): sc-348] specific to the carboxy terminus of the NRG precursor and PY99 phosphotyrosine antibodies, and protein A/G-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). Molecular mass markers were from Invitrogen Life Technologies (Carlsbad, CA). Horseradish peroxidase-conjugated anti-rabbit, anti-goat, and anti-mouse IgG whole antibodies were also from Santa Cruz Biotechnology, and an enhanced chemiluminescence system was purchased from Pierce Biotechnology (Rockford, IL). Dye reagent for determination of protein concentrations was from Bio-Rad Laboratories (Hercules, CA). Secondary antibodies for immunofluorescence (Alexa Fluor 488 goat anti-rabbit and anti-mouse IgG) and alpha -bungarotoxin were from Molecular Probes (Eugene, OR). Actin antibody and all other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Animals. Protocols for animal use were reviewed and approved by the Institutional Animal Care and Use Committee of the Boston University Medical Center and were in accordance with National Institutes of Health guidelines. Male Sprague-Dawley rats weighing 180-220 g obtained from Charles River Laboratories (Wilmington, MA) were fed standard laboratory chow and water ad libitum. Rats were fasted overnight (10:00 PM to 8:00 AM) before the experiment.

In vivo muscle contraction. To examine the acute effects of a single bout of an in vivo model of resistance exercise (RX), a protocol was chosen based on its efficacy in eliciting contractile activity and inducing skeletal muscle hypertrophy (4). In this model, hindlimb muscles of the posterior compartment (gastrocnemius, soleus, and plantaris) perform concentric contractions, and muscles of the anterior compartment [tibialis anterior and extensor digitorum longus (EDL)] undergo high-resistance eccentric contractions facilitating hypertrophy. Briefly, animals (n = 5) were anesthetized using an intraperitoneal injection of a ketamine (75 mg/kg) and xylazine (25 mg/kg) cocktail. The sciatic nerves to both legs were surgically exposed, and two fine-wire platinum electrodes were placed around the nerves. Muscle contractions were produced in one hindlimb using a Grass S48 stimulator (Grass Instruments, Quincy, MA) at a frequency of 100 Hz to recruit both fast- and slow-twitch muscle fibers (6-12 V, 3-s duration, and 10-s delay, for 10 sets of 10 repetitions). A 1-min recovery was given between sets, resulting in a protocol time of 45 min. The contralateral hindlimb served as a sham-operated control.

To analyze the acute effects of a single bout of endurance exercise (EX) in vivo, a 45-min treadmill running protocol was employed (32). Briefly, control (n = 5) and experimental (n = 5) animals were acclimated to a motor-driven treadmill (Quinton Instruments, Seattle, WA) for 3 days (5 min at 21.7 m/min). On the 4th day, experimental animals were subjected to a three-stage exhaustive bout of exercise. The protocol consisted of 15 min at 21.7 m/min and a 15% incline and 15 min at 26.7 m/min and a 20% incline and concluded with 15 min at 31.7 m/min and a 25% incline, or until exhaustion, whichever came first. For the equivalent duration, control animals were placed on an adjacent treadmill, and the belt remained stationary.

After RX and EX, a lethal dose of pentobarbital sodium was administered to experimental and control animals, and gastrocnemius (medial and lateral head), soleus, plantaris, tibialis anterior, and EDL muscles were removed rapidly and quick-frozen in liquid nitrogen. For immunofluorescence studies, a portion of soleus and EDL muscles was oriented transversely and mounted in optimum-cutting temperature compound (Sakura, Torrance, CA) and quick-frozen in isopentane cooled to the temperature of liquid nitrogen. Tissues were stored at -80°C until analyses were performed.

Preparation of skeletal muscle tissue lysates. For protein studies, isolated hindlimb muscles were homogenized on ice in a buffer containing 1% Nonidet P-40, 50 mM Tris, pH 7.4, 0.25% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, 1 mM NaF, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3Vo4. Homogenates were rotated end over end for 1 h at 4°C and then centrifuged at 14,000 g for 10 min at 4°C. The protein concentrations of the supernatants were determined by the Bradford method using dye reagent from Bio-Rad Laboratories and BSA as the standard. Muscle lysates were frozen in liquid nitrogen and stored in aliquots at -80°C.

Analysis of NRG/ErbB expression and activation. To characterize the expression of NRG and ErbB receptor subtypes in whole muscle lysates, aliquots (50 µg) of muscle protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). Membranes were blocked in Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 (TBST) and 5% BSA for 1 h at room temperature and then probed with NRG antibodies for intracellular- and extracellular-specific sequences (1:1,000) or ErbB receptor subtypes with antibodies specific to ErbB2, ErbB3, or ErbB4 (1:1,000) in 2% BSA overnight at 4°C. To determine if ErbB receptors were activated in response to contractile activity, aliquots (500 µg) of muscle protein were incubated with antibodies specific to ErbB2, ErbB3, or ErbB4 (1:100) overnight at 4°C and precipitated with protein A/G plus agarose. Immunoprecipitates were collected and released by boiling in Laemmli buffer. Samples were fractionated by SDS-PAGE, transferred to PVDF membranes, and blocked in TBST and 2% BSA. Membranes were probed with a PY99 anti-phosphotyrosine antibody (1:1,000) in TBST and 5% BSA for 1 h at room temperature.

Bound antibodies were detected with anti-mouse horseradish peroxidase-linked whole antibody (1:5,000). Protein immunoblots were visualized by enhanced chemiluminescence, and bands were quantified with scanning densitometry using Molecular Analyst Software (Bio-Rad Laboratories). The sizes of the antibody-bound proteins were verified using standard molecular mass markers.

Immunofluorescence studies. To determine the localization of NRG and its receptors, control soleus and EDL muscle serial tissue cross sections (8 µm) were cut and mounted on glass slides using a cryostat microtome (Leica Microsystems) and fixed in cold acetone at -20°C. After rehydration in PBS (pH = 7.4), samples were blocked in 10% BSA for 1 h at room temperature and then incubated overnight at 4°C in a humidified chamber with anti-ErbB2 (1:100), -ErB3 (1:100), -ErbB4 (1:100), or -NRG (EGF-like domain; 1:300) in PBS with 3% BSA. To identify neuromuscular junctions (NMJ), sections were coincubated in Texas red-conjugated alpha -bungarotoxin (1:1,000). Control sections were incubated in 3% BSA. Slides were washed in PBS and then incubated for 1 h at room temperature in FITC-labeled secondary antibody (1:300) diluted in PBS with 3% BSA. Sections were rinsed in PBS, mounted in Vectashield medium (Vector Laboratories, Burlingame, CA), and examined using a Nikon Eclipse E400 (Melville, NY) and either ×40 or ×100 magnification. Images were captured using the Bioquant Nova image analysis system (Bioquant Image Analysis, Nashville, TN). Overlay images were processed in Adobe Photoshop.

Statistics. Data are expressed as means ± SE. Statistical analysis was undertaken using a paired Student's t-test and one-way ANOVA. When ANOVA revealed significant differences, further analysis was performed using Tukey's post hoc test for multiple comparisons. Differences between groups were considered statistically significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression and localization of ErbB2, ErbB3, and ErbB4 in skeletal muscle. To quantify the relative expression of ErbB receptors in adult skeletal muscle, we probed lysates from multiple hindlimb muscle types using specific ErbB2, ErbB3, and ErbB4 antibodies. As shown in Fig. 1A, ErbB2 and ErbB4 are abundant in soleus and EDL muscles. Interestingly, ErbB3 expression appears to be decreased in the soleus relative to the EDL. Immunofluorescence analysis of muscle cross sections showed that ErbB2, ErbB3, and ErbB4 were localized clearly to the myocyte, and ErbB3 immunoreactivity was decreased in the soleus compared with the EDL (Fig. 1B). All three receptors colocalized to the NMJ (Fig. 2), although there was staining for each receptor on the myocyte membrane remote from the NMJ.


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Fig. 1.   ErbB2, ErbB3, and ErbB4 receptors are expressed in adult skeletal muscle. A: soleus (SOL) and extensor digitorum longus (EDL) muscle lysates were resolved on SDS-PAGE and immunoblotted with specific antibodies for ErbB2, ErbB3, and ErbB4 (representative of 6 experiments). B: immunofluorescent staining of ErbB2, ErbB3, and ErbB4 in soleus and extensor digitorum longus muscle cross sections (sections representative of 5 animals, ×40 magnification).



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Fig. 2.   ErbB receptors and neuregulin (NRG) are enriched at the neuromuscular junction (NMJ) in adult skeletal muscle. Soleus muscle cross sections were labeled with specific ErbB2, ErbB3, ErbB4, and NRG antibodies. Sections were counterstained using alpha -bungarotoxin (alpha BTX) to label the postsynaptic membrane (representative of 5 animals, ×100 magnification).

Multiple NRG isoforms are expressed in skeletal muscle. In multiple cell types, ErbB2, ErbB3, and ErbB4 receptor interaction and phosphorylation is triggered by the ligand NRG (7, 17, 30, 31, 37, 41). We examined NRG expression in skeletal muscle using antibodies specific to intracellular vs. extracellular domains of NRG. Under basal conditions, several bands were detected in whole skeletal muscle with both NRG antibodies (Fig. 3, A and B, representative blots), consistent with the expression of multiple NRG splice variants. The molecular size of several of these isoforms corresponds to known masses of recombinant NRG proteins (1, 24, 35, 43), including the 115-kDa species that is immunoreactive to both the intracellular and extracellular domain antibodies. The 183-kDa band (Fig. 3A) is also detectable with the extracellular domain antibody with prolonged exposure (data not shown) and may represent "mature" type III NRG (43). The reduced sensitivity compared with its detection using the intracellular antibody may represent epitope modification during protein maturation. As shown in Fig. 2, examination of muscle cross sections revealed that the distribution of NRG was enriched but not confined to the NMJ.


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Fig. 3.   Multiple NRG isoforms are expressed in skeletal muscle. Contractile activity initiates NRG processing. Muscle lysates (representative blot of soleus) were subjected to Western blotting and probed for NRG isoforms using intracellular (A) and extracellular (B; EGF) specific NRG antibodies (representative of 10 experiments). C: to identify alterations in NRG after an acute bout of treadmill running (EX), control (C), EX soleus, and extensor digitorum longus muscle lysates were resolved on SDS-PAGE and immunoblotted with the specific intracellular (top) and extracellular (middle) NRG antibodies. Membranes were eluted and probed for actin to control for protein loading (representative of 5 experiments).

Contractile activity stimulates NRG processing in vivo. Alternatively spliced transcripts of NRG generate both secreted and membrane-anchored isoforms. Cleavage of transmembrane isoforms and the release of their extracellular EGF-like domain may be critical for NRG activation (21, 45). To test the hypothesis that contractile activity initiates NRG processing, we used intracellular and extracellular NRG antibodies to detect a shift in the expression profile of NRG. In response to both forms of exercise (RX and EX), the relative distribution of NRG changed markedly in all muscles examined. Most notably, we observed the disappearance of the 183-kDa band (Fig. 3C, top, representative blot) and the concurrent increase in the 64- and 48-kDa bands (Fig. 3C, middle) in exercised vs. control muscles. These changes arguably represent acute proteolytic processing of mature NRG transmembrane protein to soluble ligand (24, 35, 43).

Skeletal muscle contractile activity stimulates ErbB2, ErbB3, and ErbB4 phosphorylation. To test the hypothesis that NRG/ErbB signaling is activated in response to contractile activity, we stimulated contractile activity either by sciatic nerve stimulation (RX) or treadmill running (EX) and rapidly isolated hindlimb skeletal muscles. ErbB immunoprecipitates from lysates were probed with an anti-phosphotyrosine antibody as an indicator of receptor activation. Both RX and EX resulted in significant activation of skeletal muscle ErbB2, ErbB3, and ErbB4. Activation of ErbB receptors was comparable among muscles despite decreased ErbB3 expression in the soleus. Compared with sham-operated muscles, RX resulted in an approximately fourfold increases in ErbB2, ErbB3, and ErbB4 phosphorylation for all muscle groups examined (Fig. 4, A and C). Similarly, compared with nonexercised control muscles, EX resulted in more than twofold increases in ErbB2, ErbB3, and ErbB4 phosphorylation (Fig. 4, B and C). Moreover, RX significantly increased ErbB2 (P < 0.02) and ErbB4 (P < 0.04) receptor phosphorylation compared with EX; however, differences in ErbB3 phosphorylation were not significant (P < 0.08).


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Fig. 4.   Contractile activity stimulates ErbB receptor phosphorylation. After an acute bout of sciatic nerve stimulation (RX) or treadmill running (EX), muscle lysates were immunoprecipitated (IP) with anti-ErbB2, -ErbB3, or -ErbB4 antibody and blotted with anti-phosphotyrosine (pY) antibody. Representative blots of receptor phosphorylation in sham control and RX gastrocnemius muscle (A; n = 5) and control (C) and EX (B; n = 5) gastrocnemius muscle. C: mean activation of ErbB2, ErbB3, and ErbB4 in multiple hindlimb muscles after RX (filled bars) and EX (hatched bars) relative to controls (gray bars). *P < 0.01 vs. control. dagger P < 0.05 vs. EX.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NRG and its receptors have been implicated in multiple aspects of skeletal muscle development (10, 16) and in the regulation of skeletal muscle metabolism (38). In the present study, we have shown that ErbB2, ErbB3, and ErbB4 receptors and multiple NRG isoforms are expressed at high levels in adult skeletal muscle and are activated in response to contractile activity. Two distinct modes of exercise activated proteolytic processing of NRG with concomitant stimulation of ErbB2, ErbB3, and ErbB4 signaling in vivo. To our knowledge, this is the first demonstration of physiological regulation of NRG/ErbB signaling in any organ and implicates this pathway in the metabolic and proliferative responses of skeletal muscle to exercise.

Consistent with other investigators, we have demonstrated the expression and localization of ErbB2, ErbB3, and ErbB4 proteins in skeletal muscle and the enrichment of these receptors at the NMJ (2, 14, 15, 25, 40, 48). A novel finding in our study is the increased expression of ErbB3 in the EDL, a predominantly fast-twitch, glycolytic muscle compared with the soleus, a predominantly slow-twitch, oxidative muscle. Whether this differential ErbB expression confers distinct metabolic and/or morphological properties in muscle fibers warrants further investigation. It is interesting that ErbB3 is not expressed in adult cardiac myocytes (47), which have a similar phenotype to slow-twitch skeletal muscle.

There appear to be multiple products of the NRG-1 gene expressed in skeletal muscle. The use of NRG antibodies to extracellular and intracellular domains reveals that there are multiple NRG isoforms in whole muscle lysates that likely represent both precursor pro-NRG and mature NRG (13, 44). Immunohistochemistry suggests NRG expression in skeletal muscle and motor neurons. Currently available tools have not permitted us to resolve potential differences in nerve- and muscle-derived NRG isoform expression. Previous reports have also shown that NRG mRNA is highly expressed in motor neurons (8, 21) and is detectable in muscle cells (NK LeBrasseur, unpublished observation, and Refs. 25 and 27). These studies provide a framework for both autocrine and paracrine NRG signaling. Cleavage of transmembrane NRG isoforms may be essential for function given that the ErbB receptors could be located a significant distance from the ligand. We observed a marked shift in NRG isoform distribution in response to contractile activity, consistent with proteolytic cleavage. After RX and EX, we detected a significant increase in the relative amounts of 64- and 48-kDa NRG isoforms, which are consistent with the predicted molecular sizes of the soluble and excreted species (8, 13, 28, 42). The lower molecular mass bands seen may represent truncated NRG fragments (8). Definitive identification of these awaits improved antibody reagent and/or mass spectroscopic methods. The proteolytic mechanisms responsible for the solubilization of membrane-anchored NRGs are not known, although prior studies of NRG and other transmembrane growth factors suggest a role for the ADAM (a disintegrin and metalloprotease) family of proteases (24, 35). Our data suggest that, with exercise, there is rapid activation of these enzymes as a proximal step in the activation of NRG/ErbB signaling.

In muscle and various other cells, ErbB3 and ErbB4 bind NRG ligand and dimerize with ErbB2 to initiate signaling by phosphorylation of intrinsic tyrosine residues (7, 17, 30, 31, 37, 41). In this study, we observed significant phosphorylation of ErbB2, ErbB3, and ErbB4 in response to two distinct modes of exercise (RX and EX). The relatively similar degree of ErbB2, ErbB3, and ErbB4 expression and activation supports the possibility of multiple receptor interactions (i.e., ErbB3/ErbB2 or ErbB4/ErbB2 heterodimers and ErbB4/ErbB4 homodimers) and underscores the likelihood of this system playing multiple biological roles in skeletal muscle.

In vitro studies have demonstrated multiple effects of recombinant NRG on myoblasts, myotubes, and isolated skeletal muscle (10, 16, 38). Development of the NMJ remains the most extensively studied target of NRG action. NRG-stimulated homo- and heteromeric ErbB complexes activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways and subsequently upregulate transcription of various AchR genes in cultured cells (36, 39). More recently, evidence for a metabolic role of NRG has been recognized. In muscle cells and tissue, NRG stimulates glucose transport by PI-3K- and/or Akt/protein kinase B-dependent translocation of GLUT4, GLUT1, and GLUT3 transporters to the cell membrane (38). The effect of NRG on glucose uptake was comparable with and additive to insulin. Furthermore, in vivo studies support an essential role for NRG/ErbB signaling in skeletal muscle. Muscle-specific ErbB2 deletion in mice resulted in impaired motor coordination and poor body condition, whereas isolated myoblasts lacking ErbB2 suffered extensive apoptosis upon differentiation to myofibers (3). It is now well established that specific intracellular signaling pathways (e.g., MAPK and PI3-K) are activated by contractile activity and orchestrate alterations in muscle growth and metabolism that occur after exercise (for review, see Ref. 33). Our finding that exercise is a potent activator of NRG/ErbB signaling suggests that NRG may in part mediate some of these biological effects of exercise on skeletal muscle.

In summary, physical exercise activates NRG/ErbB signaling in skeletal muscle. To our knowledge, this is the first demonstration of physiological regulation of NRG/ErbB signaling in adult tissue. On the basis of our work and in vitro work by other investigators, activation of NRG/ErbB signaling may mediate one or more adaptive growth and metabolic responses of skeletal muscle to exercise. To better understand this complex system and its contributions to skeletal muscle physiology, future work is needed in characterizing the signaling events, identifying age- or disease-associated alterations, and examining the consequences of manipulating the NRG/ErbB system.


    ACKNOWLEDGEMENTS

This research was supported by National Institutes of Health Grants HL-68144 (D. B. Sawyer) and AG-18844 (R. A. Fielding), an American College of Sports Medicine Foundation Research Grant (N. K. LeBrasseur), the Dudley Allan Sargent Research Fund (N. K. LeBrasseur), and the American Federation for Aging Research (N. K. LeBrasseur).


    FOOTNOTES

Address for reprint requests and other correspondence: Douglas B. Sawyer, 650 Albany St., X-704, Boston, MA 02218 (E-mail: douglas.sawyer{at}bmc.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 2, 2003;10.1152/ajpcell.00487.2002

Received 21 October 2002; accepted in final form 27 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aguilar, Z, and Slamon DJ. The transmembrane heregulin precursor is functionally active. J Biol Chem 276: 44099-44107, 2001[Abstract/Free Full Text].

2.   Altiok, N, Bessereau JL, and Changeux JP. ErbB3 and ErbB2/neu mediate the effect of heregulin on acetylcholine receptor gene expression in muscle: differential expression at the endplate. EMBO J 14: 4258-4266, 1995[Abstract].

3.   Andrechek, ER, Hardy WR, Girgis-Gabardo AA, Perry RL, Butler R, Graham FL, Kahn RC, Rudnicki MA, and Muller WJ. ErbB2 is required for muscle spindle and myoblast cell survival. Mol Cell Biol 22: 4714-4722, 2002[Abstract/Free Full Text].

4.   Baar, K, and Esser K. Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. Am J Physiol Cell Physiol 276: C120-C127, 1999[Abstract/Free Full Text].

5.   Burden, S, and Yarden Y. Neuregulins and their receptors: a versatile signaling module in organogenesis and oncogenesis. Neuron 18: 847-855, 1997[ISI][Medline].

6.   Carraway, KL, III, and Burden SJ. Neuregulins and their receptors. Curr Opin Neurobiol 5: 606-612, 1995[ISI][Medline].

7.   Carraway, KL, III, Sliwkowski MX, Akita R, Platko JV, Guy PM, Nuijens A, Diamonti AJ, Vandlen RL, Cantley LC, and Cerione RA. The erbB3 gene product is a receptor for heregulin. J Biol Chem 269: 14303-14306, 1994[Abstract/Free Full Text].

8.   Falls, DL, Rosen KM, Corfas G, Lane WS, and Fischbach GD. ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family. Cell 72: 801-815, 1993[ISI][Medline].

9.   Fambrough, D, McClure K, Kazlauskas A, and Lander ES. Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes. Cell 97: 727-741, 1999[ISI][Medline].

10.   Florini, JR, Samuel DS, Ewton DZ, Kirk C, and Sklar RM. Stimulation of myogenic differentiation by a neuregulin, glial growth factor 2. Are neuregulins the long-sought muscle trophic factors secreted by nerves? J Biol Chem 271: 12699-12702, 1996[Abstract/Free Full Text].

11.   Gassmann, M, Casagranda F, Orioli D, Simon H, Lai C, Klein R, and Lemke G. Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature 378: 390-394, 1995[ISI][Medline].

12.   Holloszy, JO, and Booth FW. Biochemical adaptations to endurance exercise in muscle. Annu Rev Physiol 38: 273-291, 1976[ISI][Medline].

13.   Holmes, WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, Park JW, Yansura D, Abadi N, Raab H, and Lewis GD. Identification of heregulin, a specific activator of p185erbB2. Science 256: 1205-1210, 1992[ISI][Medline].

14.   Huang, Y, Wang Q, Won S, Luo Z, Xiong W, and Mei L. Compartmentalized NRG signaling and PDZ domain-containing proteins in synapse structure and function (Abstract). Int J Dev Neurosci 20: 173, 2002[ISI][Medline].

15.   Jo, SA, Zhu X, Marchionni MA, and Burden SJ. Neuregulins are concentrated at nerve-muscle synapses and activate ACh receptor gene expression. Nature 373: 158-161, 1995[ISI][Medline].

16.   Kim, D, Chi S, Lee KH, Rhee S, Kwon YK, Chung CH, Kwon H, and Kang MS. Neuregulin stimulates myogenic differentiation in an autocrine manner. J Biol Chem 274: 15395-15400, 1999[Abstract/Free Full Text].

17.   Kita, YA, Barff J, Luo Y, Wen D, Brankow D, Hu S, Liu N, Prigent SA, Gullick WJ, and Nicolson M. NDF/heregulin stimulates the phosphorylation of Her3/erbB3. FEBS Lett 349: 139-143, 1994[ISI][Medline].

18.   Lee, KF, Simon H, Chen H, Bates B, Hung MC, and Hauser C. Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378: 394-398, 1995[ISI][Medline].

19.   Lemke, G. Neuregulins in development. Mol Cell Neurosci 7: 247-262, 1996[ISI][Medline].

20.   MacDougall, J. Adaptability of muscle to strength training: a cellular approach. In: Biochemistry of Exercise VI, edited by Saltin B.. Champaign, IL: Human Kinetics, 1986, p. 501-513.

21.   Marchionni, MA, Goodearl AD, Chen MS, Bermingham-McDonogh O, Kirk C, Hendricks M, Danehy F, Misumi D, Sudhalter J, and Kobayashi K. Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system. Nature 362: 312-318, 1993[ISI][Medline].

22.   Martinou, JC, Falls DL, Fischbach GD, and Merlie JP. Acetylcholine receptor-inducing activity stimulates expression of the epsilon-subunit gene of the muscle acetylcholine receptor. Proc Natl Acad Sci USA 88: 7669-7673, 1991[Abstract].

23.   Meyer, D, and Birchmeier C. Multiple essential functions of neuregulin in development. Nature 378: 386-390, 1995[ISI][Medline].

24.   Montero, JC, Yuste L, Diaz-Rodriguez E, Esparis-Ogando A, and Pandiella A. Differential shedding of transmembrane neuregulin isoforms by the tumor necrosis factor-alpha-converting enzyme. Mol Cell Neurosci 16: 631-648, 2000[ISI][Medline].

25.   Moscoso, LM, Chu GC, Gautam M, Noakes PG, Merlie JP, and Sanes JR. Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/heregulin, and its putative receptors, ErbB2 and ErbB3, in developing mammalian muscle. Dev Biol 172: 158-169, 1995[ISI][Medline].

26.   Nader, GA, and Esser KA. Intracellular signaling specificity in skeletal muscle in response to different modes of exercise. J Appl Physiol 90: 1936-1942, 2001[Abstract/Free Full Text].

27.   Ng, YP, Pun S, Yang JF, Ip NY, and Tsim KW. Chick muscle expresses various ARIA isoforms: regulation during development, denervation, and regeneration. Mol Cell Neurosci 9: 132-143, 1997[ISI][Medline].

28.   Peles, E, Bacus SS, Koski RA, Lu HS, Wen D, Ogden SG, Levy RB, and Yarden Y. Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells. Cell 69: 205-216, 1992[ISI][Medline].

29.   Peles, E, and Yarden Y. Neu and its ligands: from an oncogene to neural factors. Bioessays 15: 815-824, 1993[ISI][Medline].

30.   Plowman, GD, Culouscou JM, Whitney GS, Green JM, Carlton GW, Foy L, Neubauer MG, and Shoyab M. Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family. Proc Natl Acad Sci USA 90: 1746-1750, 1993[Abstract].

31.   Plowman, GD, Green JM, Culouscou JM, Carlton GW, Rothwell VM, and Buckley S. Heregulin induces tyrosine phosphorylation of HER4/p180erbB4. Nature 366: 473-475, 1993[ISI][Medline].

32.   Roberts, CK, Barnard RJ, Scheck SH, and Balon TW. Exercise-stimulated glucose transport in skeletal muscle is nitric oxide dependent. Am J Physiol Endocrinol Metab 273: E220-E225, 1997[Abstract/Free Full Text].

33.   Sakamoto, K, and Goodyear LJ. Invited review: intracellular signaling in contracting skeletal muscle. J Appl Physiol 93: 369-383, 2002[Abstract/Free Full Text].

34.   Sandrock, AW, Jr, Dryer SE, Rosen KM, Gozani SN, Kramer R, Theill LE, and Fischbach GD. Maintenance of acetylcholine receptor number by neuregulins at the neuromuscular junction in vivo. Science 276: 599-603, 1997[Abstract/Free Full Text].

35.   Shirakabe, K, Wakatsuki S, Kurisaki T, and Fujisawa-Sehara A. Roles of Meltrin beta /ADAM19 in the processing of neuregulin. J Biol Chem 276: 9352-9358, 2001[Abstract/Free Full Text].

36.   Si, J, Tanowitz M, Won S, and Mei L. Regulation by ARIA/neuregulin of acetylcholine receptor gene transcription at the neuromuscular junction. Life Sci 62: 1497-1502, 1998[ISI][Medline].

37.   Sliwkowski, MX, Schaefer G, Akita RW, Lofgren JA, Fitzpatrick VD, Nuijens A, Fendly BM, Cerione RA, Vandlen RL, and Carraway KL, III. Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin. J Biol Chem 269: 14661-14665, 1994[Abstract/Free Full Text].

38.   Suarez, E, Bach D, Cadefau J, Palacin M, Zorzano A, and Guma A. A novel role of neuregulin in skeletal muscle. Neuregulin stimulates glucose uptake, glucose transporter translocation, and transporter expression in muscle cells. J Biol Chem 276: 18257-18264, 2001[Abstract/Free Full Text].

39.   Tansey, MG, Chu GC, and Merlie JP. ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway. J Cell Biol 134: 465-476, 1996[Abstract].

40.   Trinidad, JC, Fischbach GD, and Cohen JB. The Agrin/MuSK signaling pathway is spatially segregated from the neuregulin/ErbB receptor signaling pathway at the neuromuscular junction. J Neurosci 20: 8762-8770, 2000[Abstract/Free Full Text].

41.   Tzahar, E, Levkowitz G, Karunagaran D, Yi L, Peles E, Lavi S, Chang D, Liu N, Yayon A, Wen D, and Yarden Y. ErbB-3 and ErbB-4 function as the respective low and high affinity receptors of all Neu differentiation factor/heregulin isoforms. J Biol Chem 269: 25226-25233, 1994[Abstract/Free Full Text].

42.   Wang, HG, Rapp UR, and Reed JC. Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell 87: 629-638, 1996[ISI][Medline].

43.   Wang, JY, Miller SJ, and Falls DL. The N-terminal region of neuregulin isoforms determines the accumulation of cell surface and released neuregulin ectodomain. J Biol Chem 276: 2841-2851, 2001[Abstract/Free Full Text].

44.   Wen, D, Peles E, Cupples R, Suggs SV, Bacus SS, Luo Y, Trail G, Hu S, Silbiger SM, and Levy RB. Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell 69: 559-572, 1992[ISI][Medline].

45.   Wen, D, Suggs SV, Karunagaran D, Liu N, Cupples RL, Luo Y, Janssen AM, Ben-Baruch N, Trollinger DB, Jacobsen VL, Meng S-Y, Lu HS, Hu S, Chang D, Yang W, Yanigahara D, Koski RA, and Yarden Y. Structural and functional aspects of the multiplicity of Neu differentiation factors. Mol Cell Biol 14: 1909-1919, 1994[Abstract].

46.   Yarden, Y, and Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2: 127-137, 2001[ISI][Medline].

47.   Zhao, YY, Sawyer DR, Baliga RR, Opel DJ, Han X, Marchionni MA, and Kelly RA. Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J Biol Chem 273: 10261-10269, 1998[Abstract/Free Full Text].

48.   Zhu, X, Lai C, Thomas S, and Burden SJ. Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses. EMBO J 14: 5842-5848, 1995[Abstract].


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