1Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada; 2Equipe Différenciation Neuromusculaire, Unité Mixte de Recherche 5161, Centre National de la Recherche Scientifique/Ecole Normale Supérieure de Lyon, Lyon, France; 3Neuromuscular Research Laboratory, Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada; 4Molecular Medicine Program, Ottawa Health Research Institute, Ottawa Hospital, Ottawa, Ontario, Canada; 5Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, San Diego, California
Submitted 26 April 2005 ; accepted in final form 27 May 2005
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ABSTRACT |
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synaptic gene expression; Duchenne muscular dystrophy; nuclear respiratory factor 2
Recently, calcineurin-NFAT (nuclear factors of activated T cells) signaling has emerged as a key pathway controlling the expression of contractile proteins and oxidative enzymes along muscle fibers, leading to the notion that calcineurin-NFAT signaling controls the slower, more highly oxidative and metabolically efficient myogenic program (for review, see Refs. 4, 40, 43, 48, 53, 54). In this context, utrophin is a large cytoskeletal protein that displays a high degree of sequence identity with dystrophin (for review, see Refs. 5, 23, 30). In contrast to dystrophin, which is present at the sarcolemma along muscle fibers, utrophin accumulates preferentially at the NMJ, where it appears to participate in the maturation of the postsynaptic apparatus. In a series of recent studies, we documented the expression of utrophin in extrasynaptic regions of muscle fibers and furthermore demonstrated that such expression occurred specifically in slower (types I and IIa), more highly oxidative fibers. In addition, we showed that expression of utrophin outside the junctional compartments is under the direct influence of calcineurin-NFAT signaling (Ref. 10; see also Refs. 12 and 33).
On the basis of these observations, we decided to examine whether the calcineurin-NFAT pathway plays a role in specifically regulating utrophin gene expression at the NMJ. In addition, we investigated whether calcineurin cooperates with the well-characterized GABP/N-box pathway to further stimulate utrophin gene expression in skeletal muscle.
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MATERIALS AND METHODS |
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In the present study, CD-1 mice were treated twice daily for 14 days with either cyclosporin A (CsA; 25 mg/kg administered subcutaneously), FK506 (5 mg/kg administered subcutaneously), or vehicle (control) as previously described (16). To verify that drug treatments were effective, plasma levels of CsA or FK506 were measured (Toronto Medical Laboratories) after 2 wk of administration. In CsA-treated animals, plasma levels were 808 ± 70 ng/ml, in contrast to vehicle-treated animals, in which plasma levels were <30 ng/ml. In FK506-treated animals, plasma levels were 26.9 ± 1.7 ng/ml , in contrast to vehicle-treated animals, in which plasma levels were <2 ng/ml. Open field testing was also performed, and all animals, regardless of treatment, showed comparable levels of locomotor activity. Two weeks later, muscles were excised, frozen, and subsequently processed for immunofluorescence (see below). Muscles were also harvested from transgenic mice in which a constitutively active form of calcineurin (CnA*) was overexpressed (17), and these muscles were similarly processed for immunofluorescence. In these experiments, all surgical procedures were performed while the animals were under pentobarbital sodium anesthesia.
In separate experiments, diaphragm muscles from OF1 mice were isolated, and synaptic vs. extrasynaptic regions were visualized using AChE staining (25). Synaptic and extrasynaptic regions from stained tissues were microdissected under binocular magnification, and total RNA was extracted as described below. For these experimental procedures, mice were anesthetized using ketamine (100 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally. In these experiments, similar observations were obtained using mice of different strains and after procedures performed at the different contributing laboratories.
In vivo electroporation.
Five micrograms of total plasmid DNA (pcDNA3) used as a control vector or the same vector containing a cDNA for the peroxisome proliferator-activated receptor- coactivator-1
(PGC-1
) also carrying a hemagglutinin (HA) tag, termed pcDNA3-HA-hPGC-1
(29), and 2 µg of pECFP-Nuc (Clontech, Palo Alto, CA) resuspended in 30 µl of 0.9% NaCl, were injected into the tibialis anterior (TA) muscles of 6-wk-old male OF1 mice anesthetized with intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). One-cm2 plaque electrodes were then placed on each side of the leg, and eight 200-V/cm pulses of 20-ms duration were applied to the injected muscles at 2 Hz. Seven days after injection, the injected muscles were microdissected under a fluorescence binocular microscope (SZX12; Olympus) to isolate cyan fluorescent protein (CFP)-positive fibers, i.e., those successfully transduced and expressing the plasmids, and to determine the levels of several mRNA-encoding synaptic proteins in these single muscle fibers (see below). Specifically, electroporated TA muscles were quickly dissected in 1x PBS. Groups of CFP-positive fibers containing 1020 fibers were isolated using ultrafine forceps (no. 5; Moria). Nonpositive extremities of the fibers were eliminated, and CFP-positive portions of muscle were placed into RLT buffer (RNeasy Mini kit; Qiagen, Chatsworth, CA) to prevent RNA degradation.
RNA extraction and real-time RT-PCR.
Real-time RT-PCR was performed using total RNA isolated from synaptic and extrasynaptic regions of diaphragm muscles and from microdissected CFP-positive fibers with the RNeasy Mini RNA extraction kit (Qiagen) according to the manufacturer's recommendations with the addition of proteinase K and DNase treatments. Reverse transcription was performed using the SuperScript II kit (Invitrogen Life Technologies, Carlsbad, CA) with random hexamer primers. A 1-µl sample of cDNA (1:10 dilution) was analyzed using real-time quantitative PCR (Light Cycler; Roche) using the QuantiTect SYBR Green PCR kit (Qiagen) as described by the manufacturer in the presence of 1 µM concentration of the respective forward and reverse primers. The sequences of the primers were as follows: -actin forward primer, 5'-CCCTGTATGCCTCTGGTCGT-3'; reverse primer, 5'-ATGGCGTGAGGGAGAGCAT-3'; ErbB3 forward primer, 5'-CTTACGGGACACAATGCTGA-3'; reverse primer, 5'-GCATGGCTGGAGTTGGTATT-3'; AChR
-subunit forward primer, 5'-ACCTGGACCTATGACGGCTCT-3'; reverse primer, 5'-AGTTACTCAGGTCGGGCTGGT-3'; AChR
-subunit forward primer, 5'-CTTGGTGCTGCTCGCTTACTT-3', reverse primer, 5'-CGTTGATAGAGACCGTGCATTTC-3'; calcineurin A forward primer, 5'-CGATTCTCCGACAGGAAAAA-3', reverse primer, 5'-AAGGCCCACAAATACAGCAC-3'; GABP-
forward primer, 5'-GGGGAACAGAACAGGAAACA-3', reverse primer, 5'-CCGTAATGCACGGCTAAGTT-3'; GABP-
forward primer, 5'-CTCCGAGCCGGTGTAAGTAG-3', reverse primer, 5'-CCTCCAGCTTCTGCCTGTAG-3'; uncoupling protein 3 (UCP3) forward primer, 5'-ATGAGTTTTGCCTCCATTCG-3', reverse primer, 5'-CCAGTTCCCAAGCGTATCAT-3'; cytochrome c forward primer, 5'-CCAAATCTCCACGGTCTGTT-3', reverse primer, 5'-GTCTGCCCTTTCTCCCTTTCT-3'; cytochrome oxidase IV forward primer, 5'-ACTACCCCTTGCCTGATGTG-3', reverse primer, 5'-GCCCACAACTGTCTTCCATT-3'; utrophin A forward primer, 5'-GGCAGGAAGATTGCACAAGT-3', reverse primer, 5'-CTGCTAGCCAAGTCCCAGAG-3'; and heat shock protein 48 (HSP48) forward primer, 5'-CGGGAAAGAGCTGAAAATTG-3', reverse primer, 5'-AGAATCCGACACCAAACTGC-3'.
In the case of microdissected CFP-positive fibers obtained after in vivo electroporation, relative cDNA measurements of synapse-specific genes, i.e., utrophin and the AChR - and
-subunits, were normalized to presynaptic ErbB3 cDNA. The expression of all other transcripts was normalized to
-actin cDNA. In the case of synaptic and extrasynaptic whole diaphragm samples, relative cDNA measurements were also normalized to
-actin cDNA levels.
Immunofluorescence staining.
Cryostat sections of TA or plantaris muscles from CD-1 or C57BL/6 mice were labeled for detection of calcineurin A (AB1698; Chemicon International, Temecula, CA), NFATc1 (sc-7294; Santa Cruz Biotechnology, Santa Cruz, CA), utrophin A (10), or PGC-1 (sc-13067; Santa Cruz Biotechnology) using appropriate antibodies as previously described (9, 10). To localize NMJs, AChR were labeled with Alexa 488-conjugated
-bungarotoxin (B-13422; Molecular Probes, Eugene, OR). Quantitation of protein expression at the NMJ was performed using muscle cryostat sections from three different animals as described in detail elsewhere (1). Briefly, longitudinal sections of plantaris muscles taken from wild-type as well as CnA*, vehicle-, CsA-, and FK506-treated animals were mounted on the same slide to ensure that the fluorescence intensities were obtained at comparable exposures with similar backgrounds. The intensity of utrophin A staining was quantified using the Northern Eclipse imaging system, and values were standardized to the area occupied by the NMJ as determined using
-bungarotoxin staining. These procedures were repeated on three separate occasions using muscles from three different animals per experimental group to account for differences in variability.
Cell culture. Mouse C2C12 muscle cells (American Type Culture Collection, Manassas, VA) were cultured on Matrigel (Collaborative Biomedical Products, Bedford, MA)-coated plates and maintained in Dulbecco's modified Eagle's medium (DMEM; Life Sciences/Life Technologies, Burlington, ON, Canada) supplemented with 10% fetal bovine serum, 20% horse serum, and 100 U/ml penicillin-streptomycin at 37°C in a 5% CO2 atmosphere. Culture media were changed every 48 h.
Stable expression of PGC-1 in myogenic cells.
C2C12 cells were transfected with 1 µg of pcDNA3-HA-hPGC-1
using LipofectAMINE (Invitrogen Life Technologies) according to the manufacturer's instructions. Pooled, stable transfectants were selected in DMEM containing 1 mg/ml G-418 (GIBCO-BRL, Grand Island, NY). Cells designated as controls were stably transfected with an empty vector. Western blot analysis was performed using anti-HA antibody (H9658; Sigma, St. Louis, MO) to confirm that the cells were overexpressing PGC-1
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In vitro transfections.
C2C12 myoblasts were transfected using LipofectAMINE. The utrophin plasmids used for transfection included a 1.3-kb, utrophin A (UA)-specific promoter region linked to a lacZ reporter gene (see Ref. 22) and a mutated version of UA (mutUA) in which the previously identified upstream NFAT binding site (10) was mutated by site-directed mutagenesis such that TATTGGAAAACA was altered to TATTGCTTAACA (mutation underlined). Mutagenesis was performed using the GeneTailor site-directed mutagenesis system (Invitrogen Life Technologies). Expression vectors for GABP-, GABP-
, PGC-1
, CnA*, and a nuclear localized form of NFATc1 (nNFATc1) were cotransfected in various combinations with UA or mutUA. These plasmids have been described previously (10, 17, 29, 52). To control for the efficiency of transfection, a plasmid encoding the chloramphenicol transferase (CAT) gene driven by the SV40 promoter (Promega, Madison, WI) was always cotransfected. After transfection, the myoblasts were maintained for 24 h in growth medium and then harvested for analysis of reporter gene expression using quantitative RT-PCR analysis.
In additional experiments, cells were transfected with the CnA* expression vector and treated with 5 µM CsA for 48 h before being harvested to demonstrate that activity of CnA is necessary for the observed effects.
RNA extraction and RT-PCR. RNA was extracted from transfected cells using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer's instructions. Briefly, cells were lysed with 1 ml of TRIzol per 35-mm dish and mixed with chloroform before centrifugation at 12,000 g. Isopropanol was then used to precipitate the RNA from the aqueous layer, and subsequent washes of the RNA pellets with ethanol were performed. To eliminate contaminating traces of plasmid DNA (20), the RNA samples were treated with DNase I (Fermentas Life Sciences, Burlington, ON, Canada) as recommended by the manufacturer. Briefly, aliquots of RNA were treated with DNase I for 2.5 h at 37°C and then reisolated using phenol-chloroform extraction. Total RNA was quantified using the Amersham Pharmacia Biotech GeneQuant II RNA/DNA spectrophotometer and subjected to quantitative RT-PCR as described previously (10, 20). Briefly, RT was performed for 45 min at 42°C with 100 ng of total RNA using random hexamer primers. The resulting lacZ and CAT cDNA were then amplified by performing PCR as described previously (10, 20). The relative abundance of the PCR products was assessed using agarose gel electrophoresis, ethidium bromide staining for visualization, and the fluorescent dye VistraGreen (Amersham Pharmacia Biotech, Arlington Heights, IL) for quantification. The efficiency of all transfections was assessed by analyzing the expression of a cotransfected plasmid encoding the CAT gene and normalizing lacZ reporter gene expression to CAT mRNA expression. In all experiments, negative controls, consisting of RNase-free water instead of RNA, as well as samples in which the reverse transcriptase was omitted, were always included and never revealed the presence of contaminating transcripts or cDNA.
Direct gene transfer.
Direct gene transfer experiments were performed as previously described (21, 22). Briefly, 50 µg of plasmid DNA containing either the UA or mutUA promoter-reporter gene constructs were directly injected into the TA muscle of 4-wk-old C57BL/6 mice. During this experimental procedure, mice were anesthetized by exposure to halothane gas and were continuously monitored for a response to tail and toe pinch to ensure that an acceptable level of anesthesia was maintained. Seven days after this procedure, the mice were euthanized with a lethal dose of pentobarbital sodium (Somnotol; MTC Pharmaceuticals, Cambridge, ON, Canada), and the muscles were excised and frozen in melting isopentane. Cryostat sections were later processed for -galactosidase (
-gal) and AChE activity to identify expression of the reporter gene and to localize NMJs, respectively. The percentage of synaptic events was quantified as previously described elsewhere in detail (15, 22).
Statistical analysis. Statistical analysis was performed using ANOVA or Student's t-test, and statistical significance was set at P < 0.05. A minimum of three independent experiments were performed, and the data are presented as means ± SE.
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RESULTS |
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Next, we sought to determine whether the calcineurin pathway actively regulates utrophin expression at the NMJ. To this end, we pharmacologically inhibited the calcineurin pathway and subsequently examined the pattern of utrophin expression within the postsynaptic sarcoplasm using immunofluorescence. Mice were thus treated with the calcineurin inhibitors CsA or FK506, and 2 wk later, plantaris muscles were excised and the synaptic expression of utrophin A was assessed using immunofluorescence. Immunofluorescence experiments rather than Western blot analyses were necessary in this case because 1) we were interested specifically in examining the subcellular localization of utrophin after these drug treatments and 2) postsynaptic membrane domains occupy <0.1% of the total surface of muscle fibers.
As expected, we observed a strong accumulation of utrophin A at the NMJs in plantaris muscles obtained from vehicle-treated control animals (Fig. 2A). In sharp contrast, however, the synaptic expression of utrophin A was markedly reduced in muscles from either CsA- or FK506-treated mice (Fig. 2A). Quantitative analyses revealed that the intensity of utrophin A staining at the NMJ was decreased by 3550% (P < 0.05) under these two conditions (Fig. 2B). Of note, a similar reduction in the expression of AChR- protein was also observed at the NMJ under these conditions (2530%; P < 0.05). Additional experiments showed that the reduction in utrophin A staining after these drug treatments was paralleled by a 3035% reduction (P < 0.05) in the relative abundance of utrophin A transcripts in the synaptic compartment of treated vs. control (vehicle) diaphragm samples as determined by performing quantitative RT-PCR assays. Finally, in this case, immunofluorescence experiments conducted in muscles from transgenic mice overexpressing CnA* showed, in agreement with the calcineurin inhibition experiments, an increase in utrophin A staining at the NMJ (65%; P < 0.05) (Fig. 2B). Thus perturbations of the calcineurin pathway clearly affect the expression of utrophin A at the mammalian NMJ.
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Additive effects of calcineurin and GABP on utrophin gene expression.
Both the calcineurin-NFAT and GABP/N-box pathways are known to regulate the expression of the utrophin gene transcriptionally (see the introduction). Because both pathways are active at the level of the postsynaptic membrane domain (our present study and Refs. 19, 27), we sought to determine whether these pathways cooperate in the induction of utrophin gene expression. Toward this end, we capitalized on the use of cultured C2C12 myogenic cells, which have been used extensively in the past to study synaptic gene regulation and postsynaptic membrane differentiation. To address this issue, cultured myoblasts were first cotransfected with the UA promoter-reporter gene construct and expression vectors encoding CnA* or nNFATc1. As observed previously (see Ref. 10), CnA* or nNFATc1 expressed alone resulted in an 3.0- to 5.5-fold increase (P < 0.05) in UA promoter-reporter gene expression (Fig. 3). We subsequently performed several additional experiments to further highlight the importance of the calcineurin-NFAT signaling cascade in the regulation of utrophin gene expression. As described above, we first mutated the NFAT site previously identified within the promoter region of the utrophin A gene and confirmed that incorporation of the mutation inhibited the expression of the lacZ reporter gene after NFAT overexpression in C2C12 myoblasts. Moreover, we observed that the enzymatic activity of calcineurin was necessary for these effects, because cyclosporin treatment of cells cotransfected with the CnA* expression vector and the utrophin A promoter-reporter construct prevented this transcriptional induction. Taken together, these results further demonstrate the involvement of the calcineurin-NFAT signaling cascade in the regulation of utrophin A gene expression and show the direct involvement of these cis- and trans-acting elements.
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PGC-1 is synaptically expressed and stimulates expression of utrophin.
The mechanisms by which the coactivator PCG-1
mediates gene expression have been shown to involve both GABP and the calcineurin signaling cascade (35, 62). Given that utrophin expression is under the control of both calcineurin- and GABP-mediated pathways (our present study and Refs. 10, 19, 27), we tested whether PGC-1
is also involved in the regulation of gene expression at the NMJ. To address this issue, we first examined the expression pattern of PGC-1
along muscle fibers using immunofluorescence. As expected (see Ref. 35), PGC-1
was expressed throughout the muscle fibers (Fig. 4A). However, we detected a clear accumulation of PGC-1
at the NMJ (Fig. 4B), suggesting an enhancement of PGC-1
function within the postsynaptic sarcoplasm.
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Several mechanisms may be envisaged to explain the effect of PGC-1 on GABP-mediated gene expression, including a direct effect of PGC-1
on GABP gene expression and/or interaction of PGC-1
with GABP. To address these possibilities, we performed two sets of experiments. First, we generated stable myogenic cell lines expressing PGC-1
and examined whether the endogenous mRNA levels of GABP-
were increased. Expression of PGC-1
in stably transfected cells was confirmed using Western blot analysis with an antibody that allowed us to recognize the HA tag (Fig. 5A). Quantitative RT-PCR assays indicated that stable expression of PGC-1
in myogenic cells increased the expression of GABP-
transcripts approximately twofold (P < 0.05) compared with control cells stably transfected with the empty vector (Fig. 5, B and C). As expected on the basis of the promoter-reporter data shown in Fig. 4, stable expression of PGC-1
also increased the endogenous expression of utrophin A transcripts (Fig. 5, B and C).
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Finally, we confirmed the above-described findings in vivo and extended the influence of PGC-1 to other synaptically expressed genes. For this analysis, the expression vector for PGC-1
was transduced into mouse muscle fibers using electroporation, and individual muscle fibers were microdissected and subsequently analyzed using real time RT-PCR for changes in the levels of endogenous transcripts encoding GABP-
and GABP-
utrophin A and the model synaptic genes AChR-
and AChR-
. In control experiments, we analyzed transcript levels in single fibers transduced with the empty pcDNA3 plasmid. Figure 6A confirms that the expression of PGC-1
induced (P < 0.05) the expression of genes involved in oxidative metabolism such as cytochrome c and cytochrome oxidase IV, both of which are well-known targets of PGC-1
(35, 62). As expected, transcript levels of UCP3 remained unchanged (P > 0.05), further confirming the specificity of our approach. Examination of the levels of GABP-
and GABP-
mRNA in these transduced muscle fibers showed a specific induction of GABP-
after expression of PGC-1
(Fig. 6B). Furthermore, PGC-1
stimulated expression of utrophin A mRNA as well as transcripts encoding other synaptically expressed genes, i.e., AChR-
and AChR-
(see Fig. 6C). Collectively, these results show that PGC-1
mediates the expression of utrophin at the NMJ via GABP and that this effect can be extended to the regulation of other synaptically expressed genes.
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DISCUSSION |
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A key question arising from these observations deals with the mechanisms leading to the activation of calcineurin within the postsynaptic sarcoplasm. Calcineurin is a Ca2+- and calmodulin-dependent phosphatase, and its activation relies on Ca2+ fluxes typically observed in slower, highly oxidative myofibers (for review, see Refs. 4, 40, 48, 53, 54). Ca2+ transients have been described at the NMJs of mouse muscle and are known to occur in response to incoming electrical activity. Previous studies describing these slow Ca2+ transients have suggested that they arise from AChR channels or from inositol 1,4,5-trisphosphate receptors localized in the sarcoplasmic reticulum of NMJs and are distinct from muscle contractile events (14, 44). While the role of Ca2+ transients within the postsynaptic sarcoplasm has yet to be resolved, it has been suggested that they are involved in the stabilization of the postsynaptic apparatus and in the mechanisms regulating local synaptic gene expression (8, 38, 44). Together, these observations suggest that Ca2+ waves likely occur at the NMJ, where they activate calcineurin, which in turn regulates the expression of synaptic genes such as utrophin.
We recently proposed a model in which utrophin gene expression in muscle is regulated by different mechanisms along muscle fibers (10). In this model, extrasynaptic expression of utrophin is regulated by the calcineurin-NFAT pathway in slower (types I and IIa myosin heavy chains), highly oxidative fibers, whereas utrophin gene expression occurs at the NMJ of both fast and slow fibers via the activation of GABP and the N-box motif. In support of this model, we previously demonstrated that 1) utrophin A transcript and protein levels are higher in extrasynaptic regions of slow vs. fast muscle fibers (20) and 2) this difference in utrophin A expression correlates with the contractile speed and oxidative status of individual muscle fibers (10).
In the present study, we have refined this model to include the novel and additional contribution of the calcineurin-NFAT pathway as a positive regulator of synaptic gene expression (Fig. 7). Furthermore, our data lead us to propose that the calcineurin-NFAT and GABP/N-box pathways cooperate to induce the transcriptional activity of utrophin at the NMJ. In this context, a recent study showed that calcineurin plays a role in the redistribution of presynthesized AChR molecules after agrin treatment of cultured myotubes (37). Collectively, the findings from these studies clearly indicate that calcineurin can assume multiple functions according to the state of differentiation, maturation, and innervation of the postsynaptic apparatus.
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NRF2 was initially identified as a transcriptional regulator of cytochrome oxidase genes and has since been shown to regulate the expression of numerous genes involved in oxidative metabolism (2, 26, 59, 60, 62). Further investigations have indicated that NRF2 can bind to known Ets sites on promoter regions of various genes and that it is a human homolog of the Ets-related transcription factor GABP (51, 59, 61). This point is particularly interesting and important because, as described above, GABP is also a key transacting factor at the NMJ and is known to direct the synaptic expression of several genes (see also Ref. 52). Within the postsynaptic compartment, GABP binds to a sequence present in the regulatory regions of several synaptic genes, termed the N-box element, which contains the core Ets binding sequence GGA that is also recognized by NRF2. Thus regulation of oxidative and synaptic gene expression in skeletal muscle appears to be mediated by the same transacting factor, namely, GABP/NRF2.
Given that PGC-1 is known to exert its effects through GABP/NRF2 as well as the calcineurin pathway, which, as described previously, are both important regulators of utrophin gene expression (Fig. 7), we examined whether this coactivator also participates in the regulation of utrophin. Toward this end, we showed that PGC-1
expression is enhanced in synaptic regions of muscle fibers and that PGC-1
increases utrophin A gene expression both in cultured myogenic cells and in muscle in vivo. These results show that utrophin is a downstream target gene of PGC-1
signaling. These observations can be extended to include the regulation of other synaptically expressed genes because transcripts encoding AChR
- and
-subunits are also induced after ectopic expression of PGC-1
in muscle fibers.
On the basis of our findings, PGC-1 appears to mediate its most dramatic effect on utrophin expression by synergizing with GABP/NRF2 rather than by coactivating the calcineurin-NFAT pathway. Specifically, we have shown that PGC-1
preferentially stimulates expression of GABP-
, and not GABP-
, in cultured myogenic cells and muscle fibers. These results are in excellent agreement with previous studies showing that NRF2a (known also as GABP-
) is induced by PGC-1
(42, 62). Because GABP-
transcripts accumulate at the NMJ (52), this preferential induction of GABP-
by PGC-1
is not surprising. Given the ability of GABP to induce utrophin gene expression, these findings collectively indicate that induction of GABP-
by PGC-1
represents a key mechanism by which PGC-1
contributes to utrophin expression at the NMJ. This scenario further supports previous observations that GABP is an important mediator and coactivator of PGC-1
-induced gene expression (42).
While PGC-1 and GABP are expressed throughout muscle fibers (see RESULTS and Refs. 35, 52), we propose that their important synergistic effect on utrophin gene expression likely occurs only at the NMJ. PGC-1
and GABP-
both show enhanced expression within synaptic regions of muscle fibers (see RESULTS and Ref. 52). Furthermore, the phosphorylation events that confer transactivating potential to GABP are likely confined to the NMJ, because they are initiated by the release of nerve-derived trophic agents such as heregulin and the binding of such agents to the appropriate receptors (18, 52, 56). Finally, PGC-1
activity is regulated by p38 MAPK, and the activation of p38 MAPK in response to neuregulin was recently described in myogenic cells (7), which further supports a physiological role for PGC-1
at the NMJ.
The regulation of utrophin gene expression at the NMJ can now be envisioned to include induction or coactivation through additional factors of GABP by PGC-1. A key denominator arising from these findings is that synaptic gene expression appears to occur via molecules localized to the NMJ that also control the expression of genes involved in oxidative metabolism (PGC-1
, GABP, calcineurin, and NFAT) (see Fig. 7). Consistent with this notion, the postsynaptic region of muscle fibers is known to be rich in mitochondria and oxidative enzymes (see Ref. 24 and references therein). An attractive possibility is that the mechanisms driving utrophin expression at the NMJ involve multiple signaling pathways that cross-talk to coordinately regulate synaptic gene expression and oxidative metabolism to ensure an adequate supply of key proteins within the postsynaptic membrane domain, allowing for efficient and sustained neurotransmission. A recent study (36) described the reduced expression of brain-specific and mitochondrial genes in PGC-1
-deficient mice and associated abnormalities in neuronal function, suggesting that PGC-1
also mediates the coordinated expression of genes in the central nervous system.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of R. N. Michel: Department of Exercise Science, Concordia University, The Richard J. Renaud Science Complex, 7141 Sherbrooke Street W., Montreal, QC, Canada H4B 1R6.
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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.
* L. M. Angus and J. V. Chakkalakal contributed equally to this study.
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