1Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02215; 2Department of Pharmacology, Osaka City University Medical School, Osaka 545-8585; 3Gene Experiment Center, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba-City 305-8572, Japan; and 4Howard Hughes Medical Institute, Chevy Chase, Maryland 20815
Submitted 29 September 2003 ; accepted in final form 6 March 2004
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ABSTRACT |
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electroporation; gene delivery; muscle contraction; exercise
In other cells and tissues, JNK signaling has been implicated in the regulation of numerous cellular processes, including apoptosis and survival signaling, T-cell maturation, brain development, cardiac hypertrophy, and ischemic or ischemia-reperfusion injury (17, 30). Depletion of JNK1 activity in knockout mice is protective against obesity and insulin resistance, probably because of chronic regulation of adipose cell size and distribution (26). Only one study has focused on the role of JNK in skeletal muscle, and the group that conducted it proposed that JNK is involved in the regulation of glycogen metabolism (36).
Glycogen content in skeletal muscle is controlled by the coordinated regulation of glycogen synthase and glycogen phosphorylase activities. It is well established that glycogen synthase activity is regulated by both allosteric and phosphorylation-dependent mechanisms. Insulin stimulation of glycogen synthesis may involve phosphorylation and activation of protein kinase B/Akt (Akt), serine phosphorylation, and deactivation of glycogen synthase kinase-3 (GSK-3), leading to dephosphorylation and activation of glycogen synthase (16). Moxham et al. (36) demonstrated that activation of JNK by anisomycin, a protein synthesis inhibitor and JNK activator, mimics insulin's action on glycogen synthesis in mouse skeletal muscle in vivo. This group concluded that insulin-induced JNK activation increases glycogen synthase activity through the activation of p90 ribosomal S6 kinase (RSK-3) and subsequent deactivation of GSK-3. Because muscle contraction can increase RSK activity and decrease GSK-3 activity in skeletal muscle (19, 31, 44), contraction-stimulated JNK activation may stimulate glycogen synthesis via a RSK-3-GSK-3-glycogen synthase signaling cascade.
Studies of signaling protein function in contracting skeletal muscle have been limited by the lack of a satisfactory model that allows the expression of foreign genes. However, direct intramuscular DNA injection in combination with electrical stimulation (in vivo electroporation) has recently received considerable attention as an effective gene delivery method in the field of gene therapy (4, 38). In vivo electroporation has several advantages for studying intracellular signaling in contracting skeletal muscle. With the use of this technique, gene delivery and subsequent protein expression occur in adult animals without an immune response to the expression vector, making it less likely for an immunological response to dictate the observed physiological outcome (4, 34, 41). Other advantages include restricted localization and expression within a specific muscle, resulting in high reproducibility in a controlled experimental paradigm. Gene delivery by this method does not cause a disruption of genomic function by incorporation of the gene into the chromosome. In addition, time-consuming processes such as virus-based vector construction or transgenic animal generation can be eliminated.
In this study, we applied existing electroporation methodologies to overexpress JNK in mouse skeletal muscles. Moreover, JNK-deficient (JNK/) mice were used as an ablation model of JNK. Using these approaches, we assessed the role of JNK signaling in the regulation of glycogen synthase activation in contracting skeletal muscle. Our data show that both JNK overexpression and ablation do not alter contraction-induced glycogen synthase activation in skeletal muscle in vivo. Moreover, JNK overexpression results in alterations in the phosphorylation state of several signaling proteins associated with extracellular signal-regulated kinase (ERK) and Akt signaling.
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MATERIALS AND METHODS |
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Expression vector construction.
Human JNK1 cDNA was cloned by performing PCR with a 5'-oligonucleotide (5'-cgggatccagcagaagcaagcgtgacaacaatttttatagtg-3') encoding recognition sequences for BamHI and JNK1 5'-end coding sequence and with a 3'-oligonucleotide (5'-gcccaagtagtcatctacagcagcccagag-3') containing a 3'-untranslated region of JNK1 (this region contains a XbaI site). The PCR product was subcloned into the BamHI and XbaI sites of the pcDNA3-HAN vector, which has a Kozac sequence and a hemagglutinin (HA) epitope sequence upstream of the BamHI site of the vector (45). HA-tagged JNK1 cDNA was excised with HindIII and XhoI and transferred to the XhoI site between the CAG promoter and a 3'-flanking region of a rabbit -globin gene of pCAGGS expression vector after blunt end treatment (35). Plasmid DNA was prepared according to a standard procedure and dissolved in saline.
DNA injection into skeletal muscle and in vivo electroporation. DNA injection and in vivo electroporation were performed by using a modification of the method of Aihara and Miyazaki (4). Mice were anesthetized with pentobarbital sodium (90 mg/kg body wt ip), and 100 µg of HA-JNK/pCAGGS plasmid in 25 µl of saline were injected into the tibialis anterior muscle of one leg (JNK O/E) with an insulin syringe and a 29-gauge needle. For control, lacZ/pCAGGS was injected into the opposite leg. Square-wave electrical pulses (200 V/cm) were applied eight times with an electrical pulse generator at a rate of one pulse per second, with each pulse being 20 ms in duration. The electrodes were a pair of stainless steel needles inserted into the tibialis anterior muscles and fixed 5 mm apart. Ten days after gene delivery, the muscles were removed and prepared for analysis.
In situ muscle contraction. For the in situ muscle contraction experiments, mice were anesthetized with pentobarbital sodium (90 mg/kg body wt ip), the sciatic nerves of both hindlimbs were exposed, and subminiature electrodes were attached to the nerves (23, 42). Hindlimb muscles were electrically stimulated to contract for 15 min (train rate 1/s, train duration 500 ms, rate 100 pulses/s, duration 0.1 ms, 13 V). Immediately after contraction, the tibialis anterior muscle was rapidly dissected and frozen in liquid nitrogen for biochemical analysis or fixed with 4% paraformaldehyde for X-gal staining.
JNK1/ and JNK2/ mice. JNK1/ and JNK2/ mice were generated as reported by Dong et al. (18) and Yang et al. (51). Homozygous mutant (/) and wild-type mice were generated from intercrosses between heterozygous (+/) mice, and treatment groups were derived from littermates. No experimental mice were backcrossed (i.e., all JNK/ mice had mixed genetic backgrounds).
Analysis of -galactosidase activity.
Histochemical detection of
-galactosidase activity was performed according to standard procedures (28). Briefly, tibialis anterior muscles were dissected, rinsed with phosphate-buffered saline (PBS), and immediately fixed with 4% paraformaldehyde on a rocker for 2 h at 4°C. The muscles were washed twice with PBS for 30 min each time and incubated at 37°C with a solution of 1 mg/ml X-gal stain containing 5 mM K4Fe(CN)6, 2 mM MgCl2, and 0.04% Igepal CA-630 for 12 h. X-gal-stained muscles were dissected, and portions that appeared blue were separated from those that appeared unstained. The fraction of tissue that expressed
-galactosidase activity was estimated by measuring weight.
Immunohistochemistry. Immunohistochemical analysis was performed according to standard procedures (29). Immediately after dissection, muscle was frozen in isopentane precooled by liquid nitrogen. Frozen sections (8 µm) were obtained, fixed in cold acetone (20°C) for 10 min, and blocked with PBS (pH 7.4) containing 0.5% bovine serum albumin (BSA) and 0.5% Triton-X for 20 min. The sections were then incubated with FITC-conjugated hemagglutinin antibody (1:100) for 1 h at room temperature and rinsed in PBS. Muscle fiber nuclei also were stained with propidium iodide (10 µg/ml). After a final wash in PBS, and sections were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and visualized with the use of a confocal microscope (Carl Zeiss, Thornwood, NY) at x250 magnification.
Muscle processing. Tibialis anterior muscles were homogenized (Polytron; Brinkmann Instruments, Westbury, NY) in ice-cold lysis buffer containing 20 mM Tris·HCl, pH 7.5, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM NaVO4, 1% NP-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 3 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were rotated for 1 h at 4°C and centrifuged at 14,000 g for 10 min at 4°C. Samples were quickly frozen and stored in liquid nitrogen. Supernatants were collected, and protein concentrations of the muscle lysates were measured according to the Bradford method (13).
JNK activity assay.
JNK activity was assessed with the use of an in vitro immune complex assay as previously described in detail (6). Briefly, muscle lysates (250 µg) were immunoprecipitated with 1 µg of anti-JNK1 and 50 µl of prewashed protein A beads. After immunoprecipitation, the JNK immune complexes were washed and then suspended in 30 µl of kinase assay buffer containing 3 µg of inactive glutathione S-transferase (GST)-c-Jun(1135) as substrate, 3.75 mM MgCl2, 50 µM ATP, and 10 µCi [32P]ATP. The kinase reaction was performed at 30°C for 30 min and terminated with Laemmli buffer. The reaction products were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the phosphorylated GST-c-Jun was visualized and quantified with a PhosphorImager and ImageQuant software (Molecular Dynamics).
Immunoblotting.
Muscle proteins (40 µg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked in Tris-buffered saline with either 5% milk or 5% BSA and immunoblotted using antibodies against HA, JNK1, phospho-JNK Thr183/Tyr185, phospho-c-Jun Ser63, ERK1/2, phospho-ERK1/2 Thr202/Tyr204, RSK, phospho-RSK-1RSK-3 Ser380, RSK-3, phospho-RSK-3 Thr353/356, GSK-3, phospho-GSK-3 Ser21/
-Ser9, Akt, phospho-Akt Ser473, p70S6K, and phospho-p70S6K Thr389. The blots were then incubated with secondary antibody for 1 h at room temperature followed by enhanced chemiluminescence. The intensity of the bands was quantified by densitometry.
Immunoprecipitation. Muscle lysates (500 µg) were incubated with 10 µl of IRS-1 antiserum for 2 h at 4°C. Immune complexes were precipitated with protein A beads during 1 h of incubation at 4°C. The beads were washed three times with washing buffer (50 mM Tris, pH 7.5, 1% NP-40, 150 mM NaCl, and 2 mM EGTA) and boiled for 5 min with Laemmli buffer. The proteins were separated by SDS-PAGE and immunoblotted to detect IRS-1 phosphotyrosine or IRS-1 phospho-Ser307.
Glycogen synthase activity assay. Muscle was homogenized in 19 volumes of ice-cold glycogen synthase buffer (50 mM Tris, pH 7.8, 100 mM NaF, 5 mM EDTA), and glycogen synthase activity was measured as described previously (48). Glycogen synthase activity was expressed as %I form of total activity (i.e., enzyme activity in the absence of glucose-6-phosphate divided by activity in the presence of 6.7 mM glucose 6-phosphate).
Statistics. Data are expressed as means ± SE. Groups in Figs. 36 were compared by one-way ANOVA, and statistically significant differences were localized by the Bonferroni t-test. Statistical analysis of data in Fig. 7 was performed with a paired Student's t-test.
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RESULTS |
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Downregulation of protein phosphorylation with JNK overexpression.
We next determined the effects of JNK overexpression on the phosphorylation state of several muscle signaling proteins on sites critical for the kinase activity of each protein. Interestingly, the threefold increase in JNK activity in the basal state was associated with significant decreases in the basal phosphorylation state of several proteins, including ERK1 (56% decrease from lacZ injected basal), ERK2 (58%), RSK-3 (51%), RSK-1RSK-3 (50%), GSK-3 (34%), Akt (43%), and p70S6K (76%) (Fig. 7). The decreases in phosphorylation were not due to decreases in protein expression levels, because immunoblotting experiments revealed no differences in the detection of all proteins (data not shown). The effect of JNK overexpression in altering the basal phosphorylation state of cellular proteins was not indiscriminate, because increased basal JNK activity in JNK O/E muscles had no effect on the phosphorylation state of several other skeletal muscle proteins (e.g., GSK-3
, p38, AMP kinase; see Fig. 7). The lower levels of the RSKs and GSK-3
phosphorylation were unexpected because JNK has been proposed to activate RSK-3 and GSK-3 signaling in mouse skeletal muscle (36). Contractile activity, a potent activator of ERK and RSK signaling (24, 25, 50), resulted in normal activation of these proteins in JNK O/E muscles (data not shown). Thus muscle contractile activity can overcome the downregulation of the ERK signaling cascade induced by JNK overexpression.
IRS-1 Ser307 phosphorylation and tyrosine phosphorylation are not changed by JNK overexpression.
Both the ERK and Akt signaling pathways can be regulated by IRS-1, via the Grb2-Sos-Ras complex and phosphatidylinositol 3-kinase (PI3-K), respectively. Ser307 is a major site of JNK phosphorylation on IRS-1, and phosphorylation of this site has been proposed to mediate the inhibitory effect of proinflammatory cytokines such as TNF- on IRS-1 function in Chinese hamster ovary cells (2) and 3T3-L1 preadipocytes and adipocytes (43), as well as in rat, mouse, and human skeletal muscles (43). This raises the possibility that IRS-1 could be an upstream molecule responsible for the suppression of basal phosphorylation levels of the ERK and Akt pathways by JNK overexpression. However, the levels of Ser307 phosphorylation of IRS-1 and tyrosine phosphorylation were not changed by JNK overexpression (Fig. 8). Therefore, IRS-1 can be excluded from the list of candidates that may mediate the suppressed phosphorylation levels of molecules in the ERK and Akt pathways.
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DISCUSSION |
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We used this gene transfer system to overexpress JNK1 in skeletal muscle, with the initial goal of determining whether JNK can regulate glycogen synthase in contracting skeletal muscle. During muscle contraction, glycogen breakdown is a major source of ATP regeneration for the working muscle. Simultaneously, glycogen resynthesis is activated by contractile stimuli (15, 40), providing a mechanism that protects the myofibers from declining energy stores under conditions of cellular stress. The finding that anisomycin treatment increases both JNK and glycogen synthase activities in skeletal muscle (36) raises the possibility that JNK is the stress signal that mediates glycogen synthase activation. Additional studies of L6 myotubes and rat adipocytes have suggested that the protein kinase C inhibitor Ro-31-8220 increases glycogen synthase activity and that this increase may be mediated by JNK activation (46). With the use of several cell lines, JNK1 has also been shown to be activated by an increase in oxidant release from mitochondria caused by stimulated pyruvate metabolism, and redox-dependent JNK1 activation has been demonstrated to increase glycogen synthase activity through RSK-3 activation and the subsequent inactivation of GSK-3 (37). On the basis of these observations, we sought to determine whether JNK mediates glycogen synthase activity in both resting and contracting skeletal muscle in vivo. In contrast to the studies cited above, our data provide no support for the hypothesis that JNK activation is sufficient to activate glycogen synthase activity, because increased basal levels of JNK activity did not increase glycogen synthase activity. Furthermore, despite a large (15-fold over basal) increase in JNK activity associated with contraction in the JNK O/E mice, the increase in contraction-induced glycogen synthase activation was similar to that seen in control mice. In addition, muscle contraction-induced glycogen synthase activation was completely intact in both JNK1/ and JNK2/ mice. It may be possible that the disruption of one isoform could functionally compensate for the other isoform, although we have also demonstrated that the ablation of one isoform did not change the expression level of the other isoform. These findings are consistent with the JNK overexpression data and suggest that JNK signaling is unlikely to be involved in glycogen synthase regulation in contracting muscle.
RSK-3 has been proposed to be an intermediate in JNK-regulated glycogen synthase activity in mouse skeletal muscle in vivo (36) and in HeLa cells (37), and the mechanism by which RSK-3 regulates glycogen synthase may involve GSK-3 (36, 37). To determine whether JNK activation is sufficient to increase RSK-3 and GSK-3 phosphorylation in skeletal muscle, we examined the phosphorylation state of RSK-3 and GSK-3 in the JNK O/E muscles. Our results show that in resting muscle, JNK overexpression and the associated threefold increase in JNK activity are associated with decreases in RSK-3 and GSK-3 phosphorylation. Muscle contraction, which increased JNK activity in both the control (3-fold) and JNK O/E muscles (15-fold), did not increase RSK-3 phosphorylation (data not shown). These results demonstrate that JNK activation is not sufficient for RSK-3 and GSK-3 phosphorylation in adult skeletal muscle tissue and suggest that subsequent phosphorylation and activation (or deactivation) of JNK, RSK-3, and GSK-3 are not mechanisms leading to increased glycogen synthase activity in contracting skeletal muscle. Instead, we think that other signaling molecules are involved, because we have shown that the regulatory subunit (RGL or GM) of the protein phosphatase 1G is necessary for muscle contraction- and exercise-stimulated glycogen synthase activity (7).
Our finding that JNK overexpression resulted in decreased RSK-3 and GSK-3 phosphorylation led us to examine whether JNK overexpression suppressed the phosphorylation state of other cellular protein kinases. Similarly to RSK-3 and GSK-3, JNK overexpression decreased ERK, RSK, Akt, and p70S6K phosphorylation at sites that are important for the regulation of kinase activities. For the downregulation of the ERK signaling cascade, it is possible that JNK did not directly affect RSK-3 and GSK-3 phosphorylation but instead was a consequence of attenuated ERK phosphorylation. ERK1/2 can directly phosphorylate RSK isoforms (21), and, at least in vitro, RSK can phosphorylate and inactivate GSK-3 and GSK-3
(47). In addition, GSK-3 phosphorylation and inactivation are mediated by the ERK/RSK signaling pathway in NIH/3T3 cells in response to EGF (20). Therefore, it is possible that decreased basal phosphorylation levels of ERK led to sequential decreases in basal phosphorylation levels of RSK and GSK-3
. Likewise, downregulation of p70S6K may be a consequence of decreased Akt phosphorylation.
One possible mechanism that could explain the suppressed phosphorylation of molecules in both the ERK and Akt pathways is the inhibition of IRS function by JNK overexpression. Phosphorylation of Ser307 in IRS-1 is critical for the inhibitory effect of anisomycin and TNF- on IRS-1-dependent signaling in Chinese hamster ovary cells, which is mediated by the association of activated JNK and IRS-1 (2, 43). Ser307 phosphorylation triggers a decrease in the phosphorylation level of tyrosine residues on IRS-1 itself and promotes general inhibition of IRS-1 signaling, as revealed by reduced activation of both the PI3-K and ERK cascades (3). We thus hypothesized that IRS-1 might be an upstream regulator that inhibits the ERK and Akt cascades by JNK overexpression. As shown in Fig. 7, however, we saw no effect of JNK overexpression on IRS-1 Ser307 phosphorylation or tyrosine phosphorylation in the muscles we studied. Therefore, IRS-1 Ser307 phosphorylation and its subsequent decrease in IRS-1 signaling do not contribute to the inhibition of ERK and Akt signaling associated with JNK overexpression. This does not rule out the possibility that IRS-2 could initiate this downregulation, because IRS-2 is also phosphorylated by serine during anisomycin or TNF-
stimulation, which inhibits insulin-stimulated tyrosine phosphorylation. A residue analogous to Ser307 of IRS-1 does not exist in IRS-2 (3), nor do phosphospecific antibodies to serine sites on IRS-2. Therefore, the possible role of IRS-2 in the inhibition of ERK and Akt signaling remains to be examined.
In NIH/3T3 fibroblast, JNK activation increases the expression of MAP kinase phosphatase-1 (MKP-1), a phosphatase specific for ERK. This results in decreased phosphorylation of ERK without a change in ERK protein expression in these cells (9). On the basis of these data, we recently measured MKP-1 protein expression by immunoblotting but saw no effect of JNK overexpression in the hindlimb muscles (Fujii N, Sakamoto K, and Goodyear LG, unpublished data). At this time, the mechanism for the downregulation of these multiple signaling molecules is unknown and is an important area of future investigation.
In summary, to explore the role of JNK signaling in skeletal muscle, we overexpressed wild-type JNK in mouse skeletal muscle with the use of an in vivo electroporation technique. JNK overexpression did not affect contraction-induced glycogen synthase activation in muscle, even though JNK activity and phosphorylation of c-Jun were dramatically increased. Moreover, contraction-induced glycogen synthase activation was normal in the muscles of JNK1/ and JNK2/ mice. These results suggest that JNK does not play a major role in the pathway leading to contraction-induced glycogen synthase activation in skeletal muscle. Overexpression-induced increases in basal JNK activity were associated with inhibition of the ERK and Akt signaling pathways. Elucidation of the mechanism whereby JNK downregulates ERK and Akt signaling in skeletal muscle is of great interest. Because JNK regulates apoptotic events (17, 30) and ERK and Akt regulate cell growth events (8, 14) in various cell types, future studies should investigate the possibility that sustained JNK activation regulates apoptosis and/or atrophy cooperatively with suppression of the ERK and Akt signaling pathways in mature skeletal muscle.
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GRANTS |
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FOOTNOTES |
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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.
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