Department of Anatomy, University of Bern, CH-3012 Bern, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is believed that the induction of the fos and jun gene family of transcription factors might be at the origin of genetic events leading to the differential regulation of muscle-specific genes. We have investigated the effect of a 30-min running bout in untrained subjects on the expression of the mRNAs of all members of the fos and jun gene families, including c-fos, fosB, fosBdel, fra-1, and fra-2 as well as c-jun, junB, and junD. While the fos family members were transiently upregulated 10- to 20-fold (an exception being fra-2), the induction of the jun family members was up to 3-fold only. The induction of c-fos could also be demonstrated at the protein level. Both c-fos and c-jun mRNAs were coinduced in muscle fiber nuclei. The induction was not restricted to a particular fiber type, as expected from established muscle fiber recruitment schemes, but followed a "patchy" pattern confined to certain regions of the muscle. The signals leading to the expression of these immediate early genes are therefore unclear.
immediate early gene expression; AP-1; stress; running training
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SKELETAL MUSCLE is a highly malleable tissue with the capability of adapting to diverse challenges. In response to endurance training, it undergoes adaptive changes of structural and metabolic properties, such as isoform switches of myofibrillar components, increased density of the capillary network, and increased capacities of membrane transport processes and the oxidative metabolism (17). In recent years, it has become evident that most of these changes are accompanied by changes in the expression of the corresponding genes; e.g., mitochondrial mRNAs increase in proportion to the mitochondrial density (29; for review see Ref. 7).
Such adaptations are specific to the type of exercise and therefore require appropriate control mechanisms. The signals and early cellular events that exert this control are poorly understood, however. In general, it is observed that any disturbances in the homeostasis of a cell will eventually result in changes of gene expression in an attempt to adapt to altered demands (6). We have recently shown that the mRNA for the 70-kDa heat shock protein is upregulated in skeletal muscle immediately after a 30-min running bout in untrained subjects (30), possibly resulting from the appearance of denatured peptides, either as a direct consequence of the cellular stress or in response to unfolded parts of proteins during translation.
In the present study, we have focused on genes that are directly involved in the control and induction of gene expression and are usually induced very early in response to a variety of intra- and extracellular signals. For this reason, they are called immediate early genes (IEGs; Ref. 2). It is believed that particular sets of IEGs act in specific biological responses (24). These properties make these genes primary candidates to be induced in a specific way in response to a single exercise bout.
The most prominent and extensively studied IEGs are the fos and jun gene families. Several members of each family have been described: c-fos, fosB, fosBdel (a human homologue of murine deleted form of murine fosB has not been described so far), fos-related antigens fra-1 and fra-2, as well as c-jun, junB, and junD. Fos and Jun proteins constitute the transcription factor complex AP-1, whereby fos family members combine with Jun proteins to form heterodimers. Jun members can additionally form homo- or heterodimers with other Jun proteins. Depending on its composition the AP-1 dimer is more or less transcriptionally active and the target gene specificity of the complex may be affected (reviewed in Refs. 2, 24).
It is commonly believed that the induction of these protooncogenes might be at the origin of genetic events leading to the differential regulation of muscle-specific genes (5). In fact, the expression of c-fos and c-jun as well as other IEGs was found to have changed in skeletal muscle in rodents after denervation (1, 5, 33), tenotomy (34), and electrical stimulation and stretch (1, 11, 23, 28). Because the composition of the AP-1 complex determines its transcriptional activity and is therefore a key control mechanism for AP-1 activity and specificity, it is important to consider the expression of all members of the fos/jun gene families simultaneously.
We hypothesized that the members of the fos and jun gene families are upregulated transiently in a specific manner after a single exercise bout of 30-min duration in previously untrained humans. This was tested by quantitative polymerase chain reaction (PCR). In addition, IEGs have previously served as markers for cell activation (18). We therefore expected fos and jun mRNA probes to label only a subset of muscle fibers in situ, namely, those recruited for contraction during the exercise session. Also, we assumed that, in general, stressed fibers would coexpress fos and jun mRNAs. Thus the demonstration of an activation of fos and jun genes could serve as a useful early marker for muscle fiber recruitment. In addition, Western blotting and immunocytochemistry were used to determine whether changes of the mRNAs of c-fos were also reflected at the protein level.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subjects
Five untrained subjects [1 female, 4 male; age 26 ± 4 (mean ± SE) yr, weight 72 ± 7 kg, height 180 ± 2 cm] volunteered to participate in this study. Written informed consent was obtained from all subjects. The study protocol was approved by the Ethics Committee of the Faculty of Medicine, University of Bern.Exercise Protocol
Each subject performed one exercise bout at his or her individual anaerobic threshold for 30 min on a treadmill. The anaerobic threshold was estimated by an extrapolation procedure, which is based on the blood lactate concentrations during the warmup (17). During the run, running velocities were adjusted to hold blood lactate concentrations constant in the 5 mM range. Mean blood lactate concentrations after 20 min of running were 5.1 ± 0.6 mM.Muscle Biopsy Sampling
Biopsies were taken at midthigh level from vastus lateralis using the Bergström technique (4). Control samples were taken 2 wk before the exercise bout. Postexercise samples were taken 4 min (from same leg as control), 30 min (from contralateral leg), and 3 h (from control leg) after the end of the exercise. Samples were immediately frozen in isopentane cooled in liquid nitrogen and subsequently stored in liquid nitrogen.RNA Extraction and Reverse Transcription
Total RNA was prepared by the acid-phenol method (8) as described previously (29).Total RNA was reverse transcribed by Superscript ribonuclease (RNase) H-reverse transcriptase (RT; GIBCO/BRL) using random hexamer priming according to the manufacturer's specifications. After 1 h at 37°C, the enzyme was inactivated by incubation at 95°C for 10 min. Thereafter, the solution was diluted to 200 µl with 10 mM tris(hydroxymethyl)aminomethane (Tris)-1 mM EDTA (TE; pH 7.4), yielding the RT mix used in the PCR.
PCR Primers
The following primer locations were chosen.c-fos. Human fos protooncogene (c-fos, GenEMBL AC M16287); 5'-primer: bases 2509-2535; 3'-primer: bases 3010-2985; expected PCR fragment: 388 base pairs (bp); 5'-biotin probe: bases 2763-2792.
fosB. Homo sapiens G0S3 mRNA, the human homologue of the murine fosB gene (GenEMBL AC L49169); 5'-primer: bases 1291-1311; 3'-primer: bases 1589-1568; expected PCR fragment: 299 bp; 5'-biotin probe: bases 1476-1495.
fosBdel. H. sapiens G0S3 mRNA, the human homologue of the murine fosB gene (GenEMBL AC L49169); 5'-primer: bases 1291-1304 and 1445-1452 (5'-GCCCGCTGGCGGAGTGAAGTTC-3'); 3'-primer: bases 1589-1568; expected PCR fragment: 159 bp; 5'-biotin probe: bases 1476-1495.
Note that a human homologue of the murine truncated or deleted form of murine fosB (12, 25, 26, 35) has not yet been described to our knowledge. On the basis of the high sequence homologies of fosB between human and mouse, we expected an identical splice variant in humans as well. The 5'-primer was therefore designed to span the deleted region. The specificity of this PCR was demonstrated by amplification of human genomic DNA, which gave no PCR product, in contrast to fosB, which has the same 3'-primer and a 5'-primer running into the deleted sequence.fra-1. Human fra-1 mRNA (GenEMBL AC X16707); 5'-primer: bases 616-638; 3'-primer: bases 888-863; expected PCR fragment: 273 bp; 5'-biotin probe: bases 789-808.
fra-2. Human fra-2 mRNA (GenEMBL AC X16706); 5'-primer: bases 198-223; 3'-primer: bases 391-365; expected PCR fragment: 194 bp; 5'-biotin probe: bases 324-343.
c-jun. Human c-jun protooncogene encoding Jun (GenEMBL AC J04111); 5'-primer: bases 2514-2540; 3'-primer: bases 2675-2651; expected PCR fragment: 162 bp; 5'-biotin probe: bases 2561-2580.
junB. Human junB mRNA (GenEMBL AC X51345); 5'-primer: bases 283-310; 3'-primer: bases 468-442; expected PCR fragment: 185 bp; 5'-biotin probe: bases 341-360.
junD. Human junD mRNA for JunD protein (GenEMBL AC X51346); 5'-primer: bases 1271-1294; 3'-primer: bases 1562-1539; expected PCR fragment: 292 bp; 5'-biotin probe: bases 1371-1390.
28S. Human 28S ribosomal RNA gene (GenEMBL AC M11167); 5'-primer: bases 4535-4564; 3'-primer: bases 4667-4638; expected PCR fragment: 133 bp; 5'-biotin probe: bases 4283-5006.
The specificities of the primers were tested by diagnostic restriction cuts of the PCR products.PCR
Quantification of specific RNAs was performed using a statistical PCR approach as described previously (29).For every PCR run, a master mix was prepared on ice using the ×10 buffer supplied by the manufacturer, 0.2 µM each primer, 40 µM dATP, dCTP, dGTP, 38 µM dTTP, and 2 µM digoxigenin-dUTP (PCR DIG labeling mix, Boehringer Mannheim), and 1.6 U/100 µl of DynaZyme DNA polymerase (Finnzymes Oy, Finland). RT mix (2 µl; further diluted 1:10 for all 28S rRNA measurements) was pipetted to 0.5-ml Eppendorf tubes on ice, and 38 µl of the master mix plus 2 drops of mineral oil (Sigma) were added. In each PCR run, a duplicate reference sample (see Ref. 29) was amplified in parallel. A control without template was also run each time. Mixtures containing 1 µl of two samples each were amplified in parallel during the c-fos PCR run to control for systemic differences between samples that could affect amplification efficiencies. No such differences were found between the samples (data not shown). The tubes were transferred into the preheated (95°C) thermocycler (Perkin-Elmer), and DNA was denatured for 2 min. PCR steps were denaturation at 95°C for 10 s, annealing at the appropriate temperature (28S 60°C, c-fos 68°C, c-jun 65°C, fosB 65°C) for 90 s and extension at 72°C for 10 s. A two-step PCR (90°C for 10 s, 70°C for 90 s) was performed in the cases of fosBdel, fra-1, fra-2, junB and junD. Cycle numbers were 28S ×16, c-fos ×29, fosB ×29, fosBdel ×37, fra-1 ×34, fra-2 ×27, c-jun ×33, junB ×34, and junD ×34. After the amplification, 30 µl of the reaction mixture were transferred into a new tube to get rid of the mineral oil.
Enzyme-Linked Immunosorbent Assay Quantification of PCR Products
The amounts of PCR products were quantitated by hybridization of specific biotinylated probes and detection by enzyme-linked immunosorbent assay (ELISA).PCR product (10 µl) was denatured by adding 40 µl of Bio-probe [0.1 µM biotinylated probe (see PCR Primers) in 0.1 M NaOH] for 10 min at room temperature in a 96-well microplate. Then 50 µl of hybridization solution were added [6× saline-sodium citrate (SSC), 10 µM Tris · HCl (pH 7.4), 10 mM EDTA, and 0.2 M HCl], and samples were incubated at room temperature for 1 h. Hybrids (50 µl) were then transferred to a streptavidin-coated microplate [coating: 100 µl of 10 µg/ml of streptavidin (Boehringer) in phosphate-buffered saline (PBS) at 4°C overnight or longer], which had been washed four times with 100 µl of TBST [20 mM Tris (pH 7.6), 192 mM glycine, and 0.9% Tween 20]. Hybrids were bound to streptavidin by incubation for 30 min at room temperature. Plates were again washed four times with 100 µl of TBST and then incubated for 30 min at room temperature with 50 µl of an alkaline phosphatase-conjugated anti-digoxigenin antibody (Fab fragments, Boehringer), 1:1,000 diluted in TBST. Plates were washed four times with 100 µl of TBST, and 50 µl of freshly prepared substrate were added: 10% (vol/vol) diethanolamine, 0.5 mM MgCl2, 0.08 M HCl, and 4 mg/ml of p-nitrophenyl phosphate (Boehringer). After incubation for 10-90 min, absorbance at 405 nm was determined in an ELISA reader (Bio-Rad).
The control without template served as background, which was subtracted to yield the muscle sample absorbances. These were related to the mean of the duplicate reference sample. The amounts of each PCR product were corrected for the different RNA lengths.
Sample Preparation for Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
A modified Laemmli sample buffer containing 100 mM Tris, 20 mM ethylene glycol-bis(Western Blotting
c-Fos protein contents were determined by Western blotting using sarcomericMuscle homogenates (2 µg/lane) were separated on a 10%
SDS-polyacrylamide gel (22). Prestained molecular weight markers were
from Bio-Rad. The proteins were blotted onto polyvinylidene difluoride
(PVDF) membranes (Bio-Rad). Electrophoretic transfer was performed at a
constant voltage of 80 V for 2.5 h in an ice-water bath. Transfer
buffer was 25 mM Tris, 192 mM glycine, 0.02% SDS, and 20% methanol.
After blotting, membranes were washed in TBST and blocked for 2 h at
room temperature with 5% dry milk in TBST. After a brief wash in TBST,
membranes were probed with a monoclonal antibody specific for
c-Fos, diluted 1:100 in TBST, 1% dry milk, and monoclonal antibody against -actinin (Sigma; diluted 1:15,000) at 4°C overnight.
-Actinin was chosen as an internal control (30). After three washes with TBST for 10 min each, membranes were
incubated with peroxidase-coupled polyclonal rabbit anti-mouse immunoglobulin G (IgG) antibody (Amersham ECL kit, diluted 1:10,000) for 2 h at room temperature. Finally, the membranes were washed twice
in TBST and twice in 20 mM Tris (pH 7.6)-137 mM NaCl. Detection was
performed using the Amersham ECL kit according to the manufacturer's instructions. The amounts of c-Fos and
-actinin were determined by two-dimensional densitometric scanning
of X-ray films.
In Situ Hybridization
For in situ hybridization (ISH), 12-µm cryostat sections were cut and thawed onto microscope slides treated with aminoalkylsilane (36) before storage atProbes. PCR products of c-fos (see above) and c-jun (corresponding to nucleotides 338-793 of c-jun sequence) were subcloned into plasmid pCR II using the TA cloning kit (Invitrogen, San Diego, CA), from which the probes were transcribed with T7 RNA polymerase (20). Dependent on the orientation of the insert, either RNA with a sequence complementary to the mRNA was synthesized (antisense probe, used for specific hybridization) or RNA with a sequence identical to the mRNA also was synthesized (sense probe, used as negative control, not shown). In vitro synthesis of digoxigenin-labeled probes was performed as described in the DIG RNA labeling kit (Boehringer).
ISH.
For fixation, the slides with the 12-µm cryostat sections were
transferred from 70°C directly into 4% paraformaldehyde in 1× PBS (137 mM NaCl, 3 mM KCl, and 15 mM sodium or potassium
phosphate, pH 7.4) for 15 min at room temperature and washed for 5 min
in 1× PBS. Digestion with proteinase K (30 µg/ml, Boehringer,
20 min at room temperature) was followed by 5 min in 1× PBS,
another fixation step in 4% paraformaldehyde-1× PBS (5 min),
twice for 1 min in 1× PBS, and twice for 1 min in 2× SSC.
After incubation in 0.25% acetic anhydride-0.1 M TE (10 min) and
subsequently in 0.1 M Tris-glycine (pH 7.0; 30 min), the sections were
dehydrated in a series of graded ethanol steps.
Detection of digoxigenin-labeled probes. The enzymatic detection was done according to the producer's description (Boehringer), using nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate as substrate for alkaline phosphatase. The polyclonal sheep anti-digoxigenin Fab fragment conjugated to alkaline phosphatase (750 U/µl) was used at a dilution of 1:5,000. The color reaction was carried out in the dark with the slides upside down in a tray containing a thin layer of the substrate solution. To get a good signal, it was necessary to run the color reaction for 3-5 days. To avoid the formation of a nonspecific precipitate due to oxidation of the substrate, the slides were covered with a Parafilm sheet to prevent contact with air. Sections were mounted in Kaiser's gelatin (Merck, Darmstadt, Germany).
Immunocytochemistry
For immunocytochemistry, 8- to 10-µm cryostat sections were first cut and then thawed onto microscope slides treated with aminoalkylsilane (20). Sections were incubated overnight with 50 µl of first antibody [polyclonal anti-c-Fos (AB-2) and polyclonal anti-c-Jun (Ab-2), both obtained from Oncogene Science], diluted 1:1,000 and 1:300, respectively, in PBS-5% dry milk, covered with coverslips, and incubated at 4°C in a moisture chamber for 24 h. Sections were then washed six times for 10 min each in PBS. Incubations with 50 µl of biotinylated anti-rabbit IgG (Vectastain ABC kit, Vector Laboratories, Burlingame, CA), diluted 1:200 in PBS-5% dry milk, were at room temperature for 30 min with coverslips. Sections were again washed three times for 10 min each in PBS. The biotinylated secondary antibody was detected using the avidin-biotin complex system (Vectastain ABC kit) according to the manufacturer's instructions and incubation at room temperature for 30 min. Color reaction was carried out using the Fast 3,3'-diaminobenzidine tablet set from Sigma with 0.03% NiCl2 for enhancement. It was stopped after ~4 min by thoroughly washing with running tap water for 10 min. Sections were then dehydrated in a series of graded ethanol steps and two times in xylene for 3-4 min each. They were then embedded in Roti-Histokitt (Roth, Germany).Histochemistry
Histochemical staining of myofibrillar adenosinetriphosphatase (ATPase) was carried out as previously described (20).Statistics
The Student's paired t-test was used in comparisons between groups. P < 0.05 was considered significant. All data in text and figures are presented as means ± SE. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR Quantification
The concentrations of all RNAs of the fos/jun family were determined by reverse-transcription PCR. Table 1 shows that the concentrations of most but not all RNAs of the fos/jun family were transiently increased after a single exercise bout. The inductions were in most cases only temporary. The highest induction was observed for the fos members with a c-fos mRNA increase of over 20-fold. fosB was not detectable before exercise, but the induction must have been greater than sixfold compared with the low 3-h level. fosBdel increased 27-fold and fra-1 9-fold; fra-2 was the only fos member whose concentration did not change within the first 3 h after exercise. The jun family mRNAs showed less induction: about threefold in the case of c-jun and junB. junD increased only marginally (1.5-fold).
|
By use of a "special" primer design (see METHODS), we were able to identify and quantify fosBdel mRNA, a spliced version of fosB mRNA that generates a truncated form of FosB [also called fosB-S (25), delta fosB (26), fosB2 (35), and fosB/SF (1)]. This truncated form has not been described previously for humans. The deletion comprises a 144-bp stretch in the 3'-coding region and results in a shift of the reading frame and a stop codon immediately after the deleted part. It is believed that the deletion of the 3'-end leads to a FosB variant lacking the transactivation domain that may have inhibitory functions by competing with other Fos family members for binding to Jun members (26).
ISH
The induction of c-fos and c-jun was evident in individual muscle fibers as demonstrated by the visualization of these mRNAs by ISH on cryostat sections. Figure 1 shows that no c-fos mRNA signal above background was detectable before exercise. At 4 min after exercise, however, clear signals were evident, localized in a "halo" of stain in the cytoplasm around a fiber's nuclei as well as in the nuclei themselves (see also Fig. 3). Similar results were obtained in biopsies taken 0.5 h after the end of exercise (not shown).
|
The same was true for c-jun mRNA (Fig. 2): virtually no signal before exercise and a clear signal in a large portion of nuclei in biopsies taken 4 min after exercise. Note that the limit of detection is higher in the ISH experiments than in the PCR (e.g., preexercise levels could not be detected with ISH). Therefore the difference in the strength of induction between c-fos and c-jun mRNAs is not as obvious as in the bulk quantification with PCR.
|
Figure 3 indicates that at least some of the nuclei are likely to express both c-fos and c-jun mRNAs at detectable levels after the run. It shows the same area on two successive 10-µm cryostat sections. Arrowheads point to corresponding locations positive for c-fos as well as c-jun mRNAs, most probably indicating the location of nuclei expressing both transcripts.
|
From Figs. 1 and 2, it is also evident that the signals of c-fos and c-jun were not detectable in all muscle fibers. Figure 4 shows a comparison of c-fos and c-jun expression with a fiber type staining on consecutive cryosections. It is evident that c-fos and c-jun were expressed in the same muscle fibers and often the same nuclei, which is more explicitly shown in Fig. 3, and, in addition, that the expression of c-fos and c-jun was not restricted to a particular fiber type. There are "positive" fibers [fibers 1 (type I), 3 (type IIa), and 5 (type IIb)] and "negative" fibers [fibers 2 (type I), 4 (type IIa), and 6 (type IIb)]. The expression of c-fos and c-jun was "patchy," confined to specific regions across the cross-sectional area (marked with a star in Fig. 4). There, most nuclei appeared to express the mRNAs at high levels. Other regions showed low or no expression (marked with a circle in Fig. 4), labeling only sparse nuclei at low levels. Although it is sometimes difficult to judge whether a particular muscle fiber is stained, the existence of all three muscle fiber types in both regions of high and low staining underlines the fact that the expression of c-fos and c-jun mRNAs was not restricted to a particular muscle fiber type.
|
c-Fos Protein Levels
The induction of c-fos was also detectable at the protein level. Before exercise, no specific signal could be detected on cryostat sections stained with a monoclonal antibody against c-Fos (Fig. 5). However, although weaker than the signal obtained with ISH, a clearly positive staining of nuclei was seen in sections of biopsies taken at 4 or 30 min after the exercise bout. Again, not all muscle fibers were expressing c-Fos protein at detectable levels. Figure 6 shows a comparison of ISH with an immunocytochemistry staining of adjacent sections of the same biopsy, demonstrating that, in general, c-Fos protein appeared in those fibers also expressing higher levels of c-fos mRNA.
|
|
The total amount of c-Fos protein in biopsies was determined by Western blotting of protein extracts (Fig. 7). c-Fos protein was found to be expressed before exercise and increased slightly but significantly 2.4-fold 4 min after exercise and 14-fold after 30 min and was not significantly different from the preexercise level by 3 h after the end of the run.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
mRNA Levels of fos and jun Genes
In the present study, we have focused on the expression of the mRNAs from all members of the fos and jun transcription factor gene families after a single running bout of 30 min.The observed early and transient induction of fos and jun genes (Table 1) is typical for IEGs. It does not require de novo protein synthesis and may at least in part be explained by the low stability of the transcripts (reviewed in Ref. 2). In the course of the present study, we found, at the mRNA level, pronounced induction of the fos members except fra-2 during or immediately after exercise. The jun mRNAs were less affected but still showed significant upregulation. The fact that the IEG induction was also observed 30 min after exercise demonstrates that an influence of the biopsy sampling intervention can be excluded, since this was the only biopsy that was taken from the contralateral leg. Recent studies of IEG induction in animal models of phenotypic transformation of skeletal muscle differ substantially from the results presented here. Michel et al. (23) found c-fos mRNA (and egr-1 mRNA) to have increased only after 4 h in chronically stimulated rabbit tibialis anterior muscle. c-jun mRNA induction was even later. In accordance with our data, the concentration of junD mRNA did not change. Goldspink et al. (14), after electrical stimulation of rabbit extensor digitorum longus muscle, also found c-fos and c-jun to have increased after 4-6 h. Interestingly, the combination of stretch and stimulation led to a biphasic induction of both mRNAs with peaks after ~1 h and later between 4 and 6 h. Both mRNAs seemed to be upregulated to similar magnitudes. Qualitatively equivalent results were found after the same treatment in rabbit latissimus dorsi muscle by the same authors (28). In an ensuing study (11), they found c-jun mRNA to be increased to a larger extent than c-fos mRNA when reapplying stretch but lower c-jun mRNA peaks than c-fos after a 1-h continuous stretch. These results are in contrast to our findings, in which fos and jun genes are upregulated immediately after exercise and fos genes are more affected than jun genes.
The study of Whitelaw and Hesketh (34) also showed that the induction
of IEGs may be quite specific to a given stimulus. Hypertrophy of rat
soleus and plantaris muscles by tenotomy or clenbuterol administration
(-adrenoreceptor agonist) showed no changes in the expression of
c-fos mRNA, whereas another IEG, c-myc, was rapidly increased. After
denervation of the sciatic nerve in mice, only
c-fos mRNA was temporarily
induced after 1.5 h, whereas
c-jun and
junB mRNA levels increased after
only 24 h (5). junD mRNA, as in
the present experiments, did not change, hence it seems to be quite
insensitive to such stimuli in general (3).
The observed differences between the human experiment and animal models of exercise may be the result of species-specific signal transduction pathways. A more likely explanation, however, is that the physiological response of human endurance running differs substantially from that of animal models (7), leading to distinct early genetic events.
Although it is not yet completely understood how the different Fos and Jun combinations affect AP-1 activity in vivo (2, 21), their differential upregulation, distinct from some of the animal models, opens the possibility that a different stimulus (e.g., strength training) might elicit a different pattern of response. This tenet needs to be tested experimentally.
Our data indicate that, at least for c-fos, the increased mRNA is efficiently translated into protein (Fig. 7). For c-jun, comparisons of PCR results and Western blots could not be made, since all the antibodies tested were not sufficiently sensitive. We have also found good correlations of c-fos mRNA in ISH and its corresponding protein in immunocytochemistry sections (Fig. 6). It has been suggested that due to the short half-life of c-Fos protein (t1/2 ~2 h) and c-fos mRNA (t1/2 ~10-15 min), the amount of protein reasonably well reflects the activity of the gene (31). In rare cases, we have found exceptions to this concept in which sparse fibers showed distinct ISH signals but no protein staining. We assume that these fibers have just begun to express c-fos mRNA and have not yet accumulated sufficient amounts of its protein.
Intramuscular Expression of c-fos and c-jun
In our study, both c-fos and c-jun mRNAs were induced in the muscle fibers of several regions in a biopsy, whereas others showed little or no induction (Fig. 4). In addition, as indicated in Fig. 3, c-fos and c-jun mRNAs can be increased in the same nuclei. The fact that this IEG response is observed in some but not all fibers argues for the notion that this is due to a specific stress experienced by this fiber subset.Not only the magnitude and time course but also the intramuscular expression pattern of the fos and jun genes differed from that of animal models. In contrast to the impact of running on human vastus lateralis, electrical stimulation of rabbit latissimus dorsi resulted in a pronounced induction of c-Fos only in interstitial cells and not in myonuclei (28). Interestingly, passive stretch of the same muscle did result in increased c-Fos staining of myonuclei. No regional differences across the muscles were reported. These results have tentatively been associated with the increase in the capillary density in response to electrostimulation as well as cell proliferation and hypertrophy after stretch (28).
In our study, it was not possible to unequivocally identify the origin of the positive nuclei, especially in the ISH, for which the morphology of the sections is somewhat degraded by the proteinase K digest and the swelling of the nuclei in the hybridization step (20). The majority of the positive nuclei can be identified as myonuclei, but it cannot be ruled out that fibroblasts, infiltrating macrophages, satellite cells or other nuclei also contribute to the observed response.
Thus, although the adaptive response of skeletal muscle to endurance training in humans has been considered comparable to the effects after electrical stimulation of animal muscles, the specificity, magnitude, time course, and cell type specificity of fos and jun responses are distinct.
fos and jun Genes as Markers for Fiber Recruitment?
The patchy intramuscular distribution pattern of c-fos and c-jun mRNAs and protein after exercise raises the question of the stimuli for their induction. Our initial hypothesis that these oncogenes may serve as markers of muscle fiber recruitment is refuted. It is well accepted that motor units are recruited in a hierarchical manner, type I fibers being first, followed by type IIa and IIb fibers (reviewed in Ref. 9). The exercise intensity used in our study is expected to involve primarily type I and type IIa fibers (10, 17, 32); type IIb fibers should not have been activated significantly. The patchy and not fiber type-specific signal of c-fos and c-jun mRNAs in our study does not seem to be compatible with this concept. A similar regional and patchy expression has recently also been found in stretched and stimulated rat tibialis anterior muscle for myogenic regulatory factor-4 (21) and for qmf-1, the chicken homologue of MyoD, after stretch-induced injury (15). No explanation as to the nature of these findings was offered. The observation that passive stretch but not electrostimulation of rabbit latissimus dorsi muscle induces c-fos in muscle fiber nuclei (28) indicates that our observed increase in oncogene expression may be related to regional differences in muscle tension. Because our exercise protocol consisted of treadmill running, a substantial eccentric load was imposed on vastus lateralis. If eccentric exercise were indeed responsible for the patchy expression pattern, using a model of concentric exercise of similar intensity and duration should result in a different pattern (or absence) of an AP-1 response. At the moment, however, we cannot exclude that other, yet unidentified stimuli are responsible for this unexpected pattern of IEG expression in adult skeletal muscle. Additionally, further insights into the nature of the signals related to IEG induction might be gained through a comparison with trained subjects undergoing a single exercise bout. ![]() |
ACKNOWLEDGEMENTS |
---|
This study could not have been performed without the devoted collaboration of the five subjects. We greatly appreciated the support provided by the Institute of Sports Science in Magglingen, Switzerland, especially by Toni Held and Fredy Grossenbacher. We are also grateful to Barbara Krieger for providing photographic material and to Valentin Djonov, Annette Dräger, and Rolf Jaggi for help in setting up the immunocytochemistry. Jean-Marc Burgunder and Kai Rösler provided the sections of dystrophic muscles.
![]() |
FOOTNOTES |
---|
The study was supported by grants from the Swiss National Science foundation (Grant 3100-042449.94/1), the Research Council of the Swiss Commission for Sports, and the Swiss Society for the Promotion of Research on Muscle Diseases.
Address for reprint requests: A. Puntschart, Dept. of Anatomy, University of Bern, Bühlstr. 26, CH-3012 Bern, Switzerland.
Received 5 May 1997; accepted in final form 12 September 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abu-Shakra, S. R.,
A. J. Cole,
and
D. B. Drachman.
Nerve stimulation and denervation induce differential patterns of immediate early gene messenger RNA expression in skeletal muscle.
Mol. Brain Res.
18:
216-220,
1993.[Medline]
2.
Angel, P.,
and
M. Karin.
The role of jun, fos and the AP-1 complex in cell-proliferation and transformation.
Biochim. Biophys. Acta
1072:
129-157,
1991[Medline].
3.
Berger, I.,
and
Y. Shaul.
The human junD gene is positively and selectively autoregulated.
DNA Cell Biol.
13:
249-255,
1994[Medline].
4.
Bergström, J. Muscle electrolytes in man.
Scand. J. Clin. Lab. Invest.
14, Suppl. 68: 1-110, 1962.
5.
Bessereau, J.-L.,
B. Fontaine,
and
J.-P. Changeux.
Denervation of mouse skeletal muscle differentially affects the expression of the jun and fos protooncogenes.
New Biologist
2:
375-383,
1990[Medline].
6.
Booth, F. W.,
and
K. M. Baldwin.
Muscle plasticity: energy demand and supply processes.
In: Handbook of Physiology. Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Am. Physiol. Soc., 1996, sect. 12, chapt. 24, p. 1075.
7.
Booth, F. W.,
and
D. Thomason.
Molecular and cellular adaptation of muscle in response to exercise: perspective of various models.
Physiol. Rev.
71:
541-585,
1991
8.
Chomczinski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
9.
Cope, T. C,
and
M. J. Pinter.
The size principle: still working after all these years.
News Physiol. Sci.
10:
280-286,
1995.
10.
Costill, D. L.,
P. D. Gollnick,
E. D. Jansson,
B. Saltin,
and
E. M. Stein.
Glycogen depletion pattern in human muscle fibers during distance running.
Acta Physiol. Scand.
89:
374-383,
1973[Medline].
11.
Dawes, N. J.,
V. M. Cox,
K. S. Park,
H. Nga,
and
D. F. Goldspink.
The induction of c-fos and c-jun in the stretched latissimus dorsi muscle of the rabbit: responses to duration, degree and re-application of the stretch stimulus.
Exp. Physiol.
81:
329-339,
1996[Abstract].
12.
Dobrazanski, P.,
T. Noguchi,
K. Kovary,
C. A. Rizzo,
P. S. Lazo,
and
R. Bravo.
Both products of the fosB gene, FosB and its short form, FosB/SF, are transcriptional activators in fibroblasts.
Mol. Cell Biol.
11:
5470-5478,
1991[Medline].
13.
Eppley, Z. A.,
J. Kim,
and
B. Russell.
A myogenic regulatory gene, qmf-1, is expressed by adult myonuclei after injury.
Am. J. Physiol.
265 (Cell Physiol. 34):
C397-C405,
1993
14.
Goldspink, D. F.,
V. M. Cox,
S. K. Smith,
L. A. Eaves,
N. J. Osbaldeston,
D. M. Lee,
and
D. Mantle.
Muscle growth in response to mechanical stimuli.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E288-E297,
1995
15.
Held, T., R. Kummer, and B. Marti. Estimation of endurance
capacity on the basis of a lactate based, submaximal two level field
test (Abstract). Proc. Swiss Congr. Sports
Med. p. 3, 1996.
16.
Hennemann, E.,
G. Somjen,
and
D. O. Carpenter.
Functional significance of cell size in spinal motoneurons.
J. Neurophysiol.
28:
560-580,
1965
17.
Henriksson, J.,
and
R. C. Hickner.
Training-induced adaptations in skeletal muscle.
In: Oxford Textbook of Sports Medicine, edited by M. Harries,
C. Williams,
W. D. Stanish,
and L. J. Nicheli. Oxford, UK: Oxford Univ. Press, 1996, p. 27-45.
18.
Hoffmann, G. E.,
M. S. Smith,
and
J. G. Verbalis.
c-fos and related immediate early gene products as markers of activity in neuroendocrine systems.
Front. Neuroendocrinol.
14:
173-213,
1993[Medline].
19.
Jacobs-El, J.,
M.-Y. Zhou,
and
B. Russell.
MRF-4, myf-5, and myogenin mRNAs in the adaptive responses of mature rat muscle.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1045-C1052,
1995
20.
Jostarndt, K.,
A. Puntschart,
H. Hoppeler,
and
R. Billeter.
Fiber type-specific expression of essential (akali) myosin light chains in human skeletal muscles.
J. Histochem. Cytochem.
44:
1141-1152,
1996
21.
Karin, M.
The regulation of AP-1 activity by mitogen-activated protein kinases.
J. Biol. Chem.
270:
16483-16486,
1995
22.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
23.
Michel, J. B.,
G. A. Ordway,
J. A. Richardson,
and
R. S. Williams.
Biphasic induction of immediate early gene expression accompanies activity-dependent angiogenesis and myofiber remodeling of rabbit skeletal muscle.
J. Clin. Invest.
94:
277-285,
1994[Medline].
24.
Morgan, J. I.,
and
T. Curran.
Immediate-early genes: ten years on.
Trends Neurosci.
18:
66-67,
1995[Medline].
25.
Mumberg, D.,
F. C. Lucibello,
M. Schürmann,
and
R. Müller.
Alternative splicing of fosB transcripts results in differentially expressed mRNAs encoding functionally antagonistic proteins.
Genes Dev.
5:
1212-1223,
1991[Abstract].
26.
Nakabeppu, Y.,
and
D. Nathans.
A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity.
Cell
64:
751-759,
1991[Medline].
27.
Neufer, P. D.,
G. A. Ordway,
G. A. Hand,
J. M. Shelton,
J. A. Richardson,
I. J. Benjamin,
and
R. S. Williams.
Continuous contractile activity induces fiber type-specific expression of HSP70 on skeletal muscle.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1828-C1837,
1996
28.
Osbaldeston, N. J.,
D. M. Lee,
V. M. Cox,
J. E. Hesketh,
J. F. J. Morison,
G. E. Blair,
and
D. F. Goldspink.
The temporal and cellular expression of c-fos and c-jun in mechanically stimulated rabbit latissimus dorsi muscle.
Biochem. J.
308:
465-471,
1995[Medline].
29.
Puntschart, A.,
H. Claassen,
K. Jostarndt,
H. Hoppeler,
and
R. Billeter.
mRNAs of enzymes involved in energy metabolism and mtDNA are increased in endurance-trained athletes.
Am. J. Physiol.
269 (Cell Physiol. 38):
C619-C625,
1995[Abstract].
30.
Puntschart, A.,
M. Vogt,
H. R. Widmer,
H. Hoppeler,
and
R. Billeter.
Hsp70 expression in human skeletal muscle after exercise.
Acta Physiol. Scand.
157:
411-417,
1996[Medline].
31.
Schmidt, J.
Depolarization-transcription coupling in excitable cells.
Rev. Physiol. Biochem. Pharmacol.
127:
251-279,
1995.
32.
Vøllestad, N. K.,
and
P. C. S. Blom.
Effect of varying exercise intensity on glycogen depletion in human muscle fibers.
Acta Physiol Scand.
125:
395-405,
1985[Medline].
33.
Weis, J.
Jun, Fos, MyoD1, and myogenin proteins are increased in skeletal muscle fiber nuclei after denervation.
Acta Neuropathol.
87:
63-70,
1994[Medline].
34.
Whitelaw, P. F.,
and
J. E. Hesketh.
Expression of c-myc and c-fos in rat skeletal muscle. Evidence for increased level of c-myc mRNA during hypertophy.
Biochem. J.
281:
143-147,
1992[Medline].
35.
Yen, J.,
R. M. Wisdom,
I. Tratner,
and
I. M. Verma.
An alternative spliced form of FosB is a negative regulator of transcriptional activation and transformation by Fos proteins.
Proc. Natl. Acad. Sci. USA
88:
5077-5081,
1991[Abstract].