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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Dipartimento di Scienze Biomediche Sperimentali dell'Università di Padova, 2Istituto Interuniversitario di Miologia, and 3Dipartimento di Anatomia e Fisiologia Umana dell'Università di Padova, Padova, Italy
Submitted 6 May 2005 ; accepted in final form 11 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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fatigue; immediate early gene; muscle adaptation; myogenesis
Complementary data seem to suggest a role of Homers in signal transduction of skeletal muscle, and particularly in the regulation of Ca2+ release from SR. Several proteins in the 40- to 47-kDa range referable to CC-Homer have been detected in muscle detergent extracts, as judged by Western blot analysis (31, 33, 39). The data (33) reported the constitutive expression of mRNAs for Homer 1, 2, and 3 in murine skeletal muscles; the complete open reading frame and full-length cDNA clones of both Homer 1a and Homer 1c were obtained from rat skeletal muscle (30). Homer 1 isoforms have been localized by immunofluorescence at the I band of skeletal muscle fibers (29, 36). Moreover, Homers were found to bind to a proline-rich motif of IP3R (35), which is also present in ryanodine receptor (RyR), the predominant Ca2+ release channel of striated muscles (11, 32), and, on the basis of immune precipitation and glutathione S-transferase pull-down experiments, a direct association of Homer 1b/c and RyR1 was postulated (10, 29, 37). Finally, Homer 1 isoforms have been reported to influence in vitro the gating kinetics of RyR1 (although with somewhat conflicting results; see Refs. 10, 15, 37).
In skeletal muscle, the functional role of Homer would be to regulate multimeric complexes involved in signal transduction (33), to act as adapter for RyR (10, 15, 37) and to bind partner for IP3R (29). In particular, the catalytic activity of recombinant Homer 1 isoforms (Homer 1a and Homer 1c) on RyR may provide one of the mechanisms linking Ca2+ signals with dynamic regulation of Homer-mediated protein clustering (10).
The regulatory equilibrium between the constitutive Homers and the ex novo synthesis of their natural dominant competitor IEG Homer could be particularly relevant not only in skeletal muscle resting conditions, but also during pathophysiological conditions, e.g., muscle adaptation, fatigue, regeneration, or hypoxia. It is worth noting that low-voltage electrical stimulation (20) or low-frequency fatigue (9) of skeletal muscle, and experimental models of ischemia (3), all appear to be associated with induction of several IEGs, such as c-fos, c-jun, egr-1, and HSPs. Furthermore, adaptive muscle changes entailing structural rearrangement and morphological remodeling, e.g., development, short-term plasticity, hypertrophy, and regeneration, processes coupled to upregulation of several, distinct IEGs (13, 20), might include a switch in Homer isoform composition. Sarcoplasmic reticulum (SR) biogenesis and triad formation take place in an orderly fashion and are dependent on regulated expression of marker proteins and their selective intracellular targeting (26). A significant temporal relationship between Homer transition and skeletal muscle differentiation might be intriguing. It must be mentioned that a differential Homer expression has been already reported during brain development (31).
In the present study, we undertook a first attempt to determine and characterize stimuli that induce Homer 1a transcription in mammalian striated muscles, the long goal being to unravel mechanisms and intracellular pathways involved in short- and long-term adaptation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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250 g body wt). For marcaine (bupivacaine) injections, fatigue experiments, or transfection experiments, rats were anesthetized with ketamine (1.5 mg/100 g) and euthanized by cervical dislocation. Experimental protocols have been approved by University of Padua Review Boards. Soleus (SOL) a representative slow-twitch skeletal muscle, and extensor digitorum longus (EDL), a representative fast-twitch skeletal muscle, were used.
Antibodies.
The following primary antibodies were used for Western blot analysis: polyclonal antibodies specific for either Homer 1b/c, 1a, 2a/b, or 3 (Santa Cruz Biotechnology), monoclonal antibodies for myosin heavy chain (MHC) (MF20; Developmental Studies Hybridoma Bank), and monoclonal antibodies for 
-sarcomeric actin (Sigma). Rabbit anti-goat IgG and goat anti-mouse IgG (both from Sigma) conjugated to alkaline phosphatase were used as secondary antibodies.
cDNA cloning of Homer 1a and cloning of HA-tagged Homer 1a cDNAs. The full-length cDNA referable to Homer 1a (612 bp) was cloned as previously described (30). The nucleotides coding for the nine amino-acid of hemaglutinin (HA) tag were added by PCR at the 5' end; EcoRI-HindIII inserts were cloned into pcDNA3.1 vectors for expression in eukaryotic cells (36).
Fatigue.
A longitudinal opening through skin and fascia was made close to the right knee, and the common peroneal nerve was exposed. The ends of two flexible, multistranded steel wires (model AS 632, Cooner Wire, Chatsworth, CA) with their Teflon insulation removed for the distal 20–25 mm were fixed by sutures on each side of the common peroneal nerve at the level of the knee. The electrodes did not touch the nerve (38). After the electrodes were placed, the opening was sutured, and the proximal ends of insulated wires, exiting through the suture, were connected to a stimulator (Grass S44) through a stimulus isolation unit (Grass). The efficacy of nerve stimulation was checked with the use of supramaximal single stimuli (0.1-ms duration), which usually elicited a strong foot dorsi-flexion due to EDL activation. Muscles were then submitted to a fatigue protocol, consisting in a train of short tetani at low frequency (40 Hz, 300-ms duration, 0.3 s–1) for either 30 or 60 min (12), which reduced force development to 15–20% of the initial value. At the end of the fatigue protocol, both fatigued (right) and control, not fatigued (left) EDL were quickly dissected out, weighted, and frozen in liquid nitrogen.
Bupivacaine-induced necrosis and regeneration of adult rat skeletal muscle. The right soleus muscles were exposed and injected with 0.5 ml of 0.5% bupivacaine, as described previously (22). At different time points (between 0.25 and 21 days postinjection), the muscles were quickly removed and frozen in liquid nitrogen for biochemical analysis.
Electroporation and transfection of soleus muscles. Soleus muscles were exposed and injected with 60 µl of a saline solution containing 50 µg of either Homer 1a or Homer 1a-HA cDNAs. Electroporation was carried out with a BTX ECM 830 square-wave pulse generator using the protocol of Mathiesen (19), with slight modifications (21); pulse stimulation by field electrodes (220 V/linear cm) was applied for six 20-ms pulses at 200-ms intervals. The rats were euthanized 3 days after surgery; the muscles were quickly removed and frozen in liquid nitrogen for biochemical analysis.
Cell cultures. C2C12 myoblasts (MB) were grown in DMEM high glucose (Sigma) supplemented with 10% fetal calf serum (Sigma) (proliferation medium). Cells were seeded on 100-mm-diameter petri dishes. When confluence was reached (day 0), C2C12 myoblasts were induced to differentiate and fuse into multinucleated myotubes once the proliferation medium was changed to DMEM with 2% horse serum (Sigma; differentiation medium).
Homogenates from C2C12 myotubes, from soleus and EDL skeletal muscles, and from cerebrum.
C2C12 MB and myotubes were cultured as described above, harvested in PBS, and lysed with the 
-Gal lysis buffer (-Gal ELISA Kit, Roche) for 30 min at room temperature. After centrifugation at 18,000 g for 15 min, the protein concentration of supernatants was determined by the Bradford assay (Bio-Rad). Supernatants were kept at –20°C until use.
Homogenates of rat skeletal muscles and cerebrum were obtained as follows: frozen tissues were triturated in a mortar, homogenated in 10 volumes of 3% SDS, 1 mM EGTA, boiled for 5 min, and centrifuged at 18,000 g for 15 min to remove debris. Homogenates were kept at –20°C until use. Protein concentration was determined according to Lowry et al. (18).
SDS-PAGE, Western blot analysis, and quantitative densitometry. SDS-PAGE on 12.5% gels, transfer to nitrocellulose, and Western blot analysis were carried out essentially as previously described (22).
Homers and myofibrillar proteins were quantified by densitometry of immunoblots of homogenates from fatigued EDL, bupivacaine-treated soleus, transfected soleus, and corresponding controlateral muscle, as well as from C2C12 MB and myotubes. Densitometric analysis was performed with Image for Windows software (version Beta 4.0.2; Scion, Frederick, MD; www.scioncorp.com). Each value for the Homer isoforms, MHC, or -actin in regenerating muscles is given as ratio to the corresponding controlateral sample, with the exception of Homer 3; in the latter case, absolute optical density (OD) values are provided because Homer 3 was undetectable in control muscles. Because morphological and histochemical recovery is attained at 3–4 wk after bupivacaine injection (see Ref. 5 for a review) and because Homers and myofibrillar proteins had returned to control levels by postinjection day 21, we assessed the time course change by testing the differences between each time point and the value at postinjection day 21 (see also Statistical analysis).
In C2C12 MB/myotubes, the value for Homer 1b/c/d, 1a, and 3 is given relative to that obtained just before the induction of differentiation (day 0 value). Changes in Homer expression were assessed by comparing each experimental value (between day 1 and day 8) to that obtained in proliferating myoblasts (see also Statistical analysis).
RNase protection assay. Antisense RNA probes were produced using T7 polymerase (Roche Molecular Biochemicals) on linearized Homer 1a and Homer 1c in the presence of biotinylated ribonucleotides. RNasePAs were carried out using the RPA III kit (Ambion) hybridizing 10–50 µg of total RNA from each tissue to 600 pg of biotinylated RNA probe. After RNase digestion, the protected RNA probe was run on 5% polyacrylamide/8 M urea denaturing gels and then electrotransferred on positively charged nylon membranes (Amersham). The biotinylated RNA probes were detected by using the BrightStar BioDetect kit (Ambion). Undigested RNA probes were also run on the same gel as size markers. The Homer 1a probe was made up of 247 nucleotides and the expected size of the Homer 1a protected fragment is 179 nucleotides; the same probe is also able to protect a Homer 1b/c fragment of 146 bases. Quantitation was carried out by densitometric analysis performed with an imaging densitometer (model G5-670, Bio-Rad). The OD value of Homer 1a fragment was normalized to that of Homer 1b/c. The time course of changes of Homer transcripts was determined by comparison of each value to that obtained from control muscles (see Statistical analysis).
Statistical analysis. One-way ANOVA was used to evaluate the changes over time for both relevant proteins and mRNA. All values are means ± SE. Post hoc comparison were performed with Fisher's protected least-significant difference test. Dunnett's test was applied to compare each time point value with either the postinjection day 21 value in the case of soleus regeneration, or the MB value in C2C12 differentiation, or the control value in RNasePA. Correlations between changes in Homer isoforms and MHC content were assessed by linear regression analysis. Differences were considered statistically significant at the 0.05 level of confidence.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Skeletal muscle fatigue: expression of Homer 1 isoforms in EDL, a fast-twitch skeletal muscle of the rat. Transcription and transduction of Homer 1 isoforms in skeletal muscle might be influenced by fatigue: thus we measured the content of Homer 1a and of long Homer 1 isoforms, both at mRNA and protein levels, in EDL fatigued by repeated tetani applied for either 30 or 60 min, as detailed in MATERIALS AND METHODS.
RNasePA analysis (not shown) indicated that there was no difference between fatigued and controlateral, control muscles, with respect to both Homer 1b/c and Homer 1a.
Western blot analysis (Fig. 1) was carried out with antibodies specific for either Homer 1b/c or Homer 1a. The apparent molecular weight of the immunostained proteins (47 and 27 kDa, respectively) was consistent with the calculated mass of Homer 1b/c and Homer 1a, respectively (4, 17). Antibodies for Homer 1b/c yielded single, unique bands, being unable to discriminate between long forms of Homer 1; however, because mRNA coding for Homer 1d (an additional alternative splicing variant of Homer 1) is specifically expressed in skeletal muscle (28), we cannot exclude that the 47-kDa-labeled polypeptide might also include Homer 1d. Therefore, we will refer to the immunoreactive Homer 1 long forms as Homer 1b/c/d hereafter. Western blot analysis showed that, relative to controlateral, control muscles, Homer 1a and Homer 1b/c/d were virtually unchanged in fatigued EDL after tetani applied for either 60 min (Fig. 1) or 30 min (not shown). Average densitometric data, derived from four distinct experiments for each group, were not significantly different (see Fig. 1).
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Soleus muscles of the rat were transiently transfected by electroporation with cDNAs coding for either recombinant Homer 1a or epitope-tagged (HA)-Homer 1a: the very same 27-kDa protein band was recognized by both anti-Homer 1a and anti-HA antibodies in homogenates from Homer 1a-HA transfected soleus (Fig. 2, lane 2). Notably, in the nontransfected, controlateral soleus, a slightly less intense band was evidenced by anti-Homer 1a antibodies (Fig. 2, lane 3), whereas no labeling was observed after incubation with anti-HA antibodies (Fig. 2, lane 1).
Finally, the comparison between homogenates of Homer 1a-transfected soleus and of controlateral muscle (Fig. 2, lanes 5 and 4) showed that the immunoreactive 27-kDa band is increased, as expected, in transgenic muscles. Taken together, these data demonstrated that anti-Homer 1a antibodies specifically recognize the "short" Homer 1 isoform and that Homer 1a appears to be "constitutively" expressed in skeletal muscle.
Homer 1a and Homer 1b/c/d protein levels in regenerating soleus skeletal muscle. We next asked whether there are changes in either Homer 1a or Homer 1b/c/d associated to skeletal muscle regeneration. The bupivacaine-induced muscle degeneration/regeneration model was implemented in the soleus of adult rat and the content of Homer 1 isoforms was monitored by Western blot analysis on muscle homogenates obtained from 0.25 to 21 days postinjection.
Relative levels of both isoforms were different over time but they displayed distinct expression patterns (Fig. 3). With regard to Homer 1a, a nonsignificant decrease in protein level was observed at 6-, 12-, and 24-h postinjection. Homer 1a expression then significantly increased and attained levels that were two- to threefold higher than those detected in control muscles between 6 and 10 days (see Fig. 3, inset, lane 1 vs. lane 2). Homer 1a decreased at postinjection day 15 and returned to control levels at 21 days postinjection.
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14% at 6-h postinjection, and gradually increased until 21 days postinjection, when control values were fully attained. At postinjection day 10, Homer 1b/c/d levels were only slightly lower than in control (see Fig. 3, inset, lane 3 vs. lane 4). Up to 3 days postinjection, necrosis is the prevalent phenomenon so that reduction of protein levels relative to control is plausible. Later and up to postinjection day 15, when regeneration is in full-blown, relative differences between Homer 1a and Homer 1b/c/d became conspicuous.
RNasePA in regenerating soleus skeletal muscle. Differences in protein content might be due to either increased transcription, reduced turnover of mRNA and protein, or both. Some clues to this effect were sought by RNase protection assay. Plot of Fig. 4 shows the Homer 1a-to-Homer 1b/c ratio as a function of postinjection time. Compared with control (0 on the x-axis), the ratio was significantly twofold higher up to 1 day postinjection, and virtually back to baseline by postinjection day 3. Because the Homer 1b/c/d protein content increased steadily and monotonically (cfr. Fig. 3), if we assume that the relative mRNA had increased accordingly, it would appear as though transcription for Homer 1a was upregulated after bupivacaine injection, i.e., when activation of satellite cells occurs.
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Expression of Homer 2a/b in skeletal muscle is still controversial (31, 39), whereas it is undoubtedly detectable in cerebellum extracts (31, 39). In preliminary Western blot experiments, the reactivity to Homer 2a/b antibodies of homogenates from untreated soleus and cerebellum was investigated: both homogenates displayed a doublet (42 and 47 kDa), referable to the two long forms of Homer 2, 2a, and 2b, respectively (31), the latter being predominant in soleus (not shown). Soleus muscle homogenates from regenerating and controlateral samples were then analyzed by being immunoblotted with antibodies specific for Homer 2a/b. Homer 2b appeared to be the predominant isoform in regenerating and untreated soleus (Inset of Fig. 5A, lanes 1 and 2, respectively); Homer 2a was indeed only barely detectable and for this reason only the expression pattern of Homer 2b has been described. Relative levels of Homer 2b were reduced by 80% up to 3 days postinjection, then gradually increased and returned to control values 10 days postinjection (Fig. 5A) with a pattern formally similar to that of Homer 1b/c/d.
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47 kDa (Fig. 5B, inset, lane 4). Homer 3 content dramatically increased at postinjection day 3 and remained elevated until postinjection day 6, the increase being 8-fold compared with postinjection day 21 (Fig. 5B). Expression likewise decreased steeply, i.e., very low at postinjection day 10 (Fig. 5B, inset, lane 5) and almost undetectable at postinjection day 21.
Variation of -actin and MHC protein content in soleus during the degeneration/regeneration process.
Initial changes in Homer proteins levels may be merely, or at least in part, because of muscle necrosis induced by bupivacaine. Evidence for the extent of muscle necrosis was gained by measuring the relative content of two structural muscle-specific proteins by immunoblot analysis (Fig. 6A). Both -actin and MHC showed a dramatic reduction between 6 h and 3 days postinjection, then rapidly increased by postinjection day 6 and returned to control values between 15 and 21 days postinjection. These findings might be taken as evidence that the maximum decrease in muscle mass takes place before/on postinjection day 3, and that the increase of both Homer 1/b/c/d and Homer 2b is related to muscle mass increase later than postinjection day 3: in both cases, plots of MHC vs. Homer 1b/c/d (Fig. 6B) and of MHC vs. Homer 2b (Fig. 6C) and linear regression analysis show a positive relationship with R = 0.97 and R = 0.95, respectively. No significant relationship was found between Homer 1a and MHC (R = 0.02, data not shown), i.e., the pattern of Homer 1a is unrelated to muscle mass increase. On the other hand, regression analysis applied to Homer 3 vs MHC (Fig. 6D) shows a negative relationship with R = 0.92, i.e., Homer 3 linearly decreased as muscles mass approached steady-state conditions at postinjection day 21.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Moreover, we provide evidence for the transient and rapidly decaying expression of Homer 3 during regeneration and observe a time course different from that of Homer 1a; thus the role of the Homer family proteins in regeneration appears to be pleiomorphic.
Expression of Homer 1 in skeletal muscle. After bupivacaine treatment, from postinjection day 3 to postinjection day 21, Homer 1b/c/d displayed an expression pattern superimposable to that of myofibrillar proteins (Figs. 3 and 6, A and B) and of creatine phosphokinase, a myogenic marker of myotube formation, monitored under similar experimental conditions (8). Not only myogenic cells (satellite cells, myoblasts) and myotubes but also regenerating muscle fibers appear to express long isoforms of Homer 1. Homer 1/b/c/d never overshoots expression levels of control, controlateral samples, and, thus its increase appears to parallel restoration of muscle mass. A similar pattern was displayed also by C2C12 myoblasts and myotubes: thus long isoforms of Homer 1 have undoubtedly a myogenic origin.
The paradigm for Homer 1a transcription, as hypothesized by Brakeman et al. (4) in neural tissue has become less stringent because Homer 1a does not only appear to behave as an IEG product, but it also appears to be constitutively expressed: Homer 1a protein was detected in control brain lysates (31) and in quiescent cultured hyppocampal neurones (17); moreover, Homer 1a was detected in cultured cortical neurones upon inhibition of proteasome activity (1).
Within this framework, it is not surprising that we found the constitutive expression of Homer 1a both in fast-twitch and in slow-twitch skeletal muscles of the rat. The present finding fits well with our own previous finding concerning the presence of mRNA coding for either Homer 1a or Homer 1c in skeletal muscle (30). We did not assess quantitatively the relative level of Homer 1a vs. Homer 1b/c/d in each muscle type, but it is clear that Homer 1b/c/d was expressed in comparable amounts, whereas Homer 1a was more represented in slow-twitch muscles (about twofold).
Thus, in skeletal muscle, as in other tissues, Homer 1a might play additional roles besides the classic one, i.e., competitive antagonist of Homer 1b/c: it might have a role of its own in cell regulation.
During muscle regeneration and after postinjection day 3, Homer 1a increased markedly compared with controlateral samples and remained 2.5-fold higher until postinjection day 10. Therefore the increase of Homer 1a is not positively correlated to muscle mass increase and could be the result of several concomitant processes affecting transcription as well as protein stability and turnover: 1) satellite cells are known to be activated and to start proliferation on postinjection days 4–5 (14), whereas at postinjection day 10, myotubes and regenerating fibers constitute the vast majority of the cell population; 2) in C2C12 cell cultures, Homer 1a expression increases as proliferation and differentiation occur (Fig. 7); and 3) proteasome activity increases between 3 and 7 days during muscle regeneration but then rapidly returns to control levels (7). In this respect, it is noteworthy that only Homer 1a, but not Homer 1b/c, -2, or -3, has been reported to be degraded by proteasomes and that this mechanism is responsible for the rapid turnover rate of Homer 1a protein observed both in cultured neurons and upon expression in heterologous cell lines (1). If Homer 1a escapes proteasome-mediated degradation by some yet unknown mechanism, protein accumulation ensues (1). Indeed, treatment with phorbol esters led to a moderate upregulation and increased synaptic localization of Homer 1a, even in the absence of transcriptional activity (1). Moreover, upon blockade of proteasomes, Homer 1a mRNA levels rose markedly in primary neuronal cell cultures (24).
Even if the possible contribution of inflammatory cells cannot be ruled out in the initial phases, Homer 1a accumulation in regenerating soleus is likely associated to proliferation of satellite cells (day 3 onward) as well as to differentiation of myotubes (day 10 onward); interestingly, postnatal brain development, up to 5 wk, has been reported to be associated to increase of mRNA coding for Homer 1a (4).
The long-lasting Homer 1a accumulation might be due not only to increase of mRNA levels (Fig. 4) but also to decreased protein turnover should proteasome activity be reduced. Compared with upregulation of Homer 1a in hippocampus by seizure-induced neuronal activation, i.e., mRNA peaks around 1 h and protein at 4 h (4), our findings are quite different, mRNA peaks 12–24 h, protein accumulates after day 3 up to day 7.
Thus, there appears to be two pools of Homer 1a, the constitutive pool, and the inducible pool, that might be referable to the IEG one and include transient expression during muscle differentiation. Interestingly, in hippocampal cultures, Okabe et al. (23) argued that the composition of Homer-mediated clustering of protein complexes is highly dynamic and postulated that Homers rely on two regulatory systems for their distribution and interaction with other proteins: 1) a local, protein synthesis-independent signaling cascade that rapidly redistributes Homers within dendritic compartments, and 2) an inducible, Homer 1a-dependent system, linked to stimulus-transcription coupling, that regulates the global state of signaling complex formation with a slower, yet long-lasting, time course.
With regard to the putative role of Homer 1 during muscle differentiation, a tentative although speculative role might be derived from models put forward in nonmuscle cells. It is known that Homer 1 isoforms control plasma membrane (PM) targeting of mGluR5 in neurons: Homer 1b caused intracellular retention of the receptor, whereas Homer 1a reversed the Homer 1b-mediated effect, triggering PM localization of the receptor (2). Moreover, it has been demonstrated that Homer 1b inhibits PM localization of mGluR5 in heterologous cells, causing mGluR5 to be retained in the endoplasmic reticulum. In contrast, mGluR5 alone or mGluR5 coexpressed with Homer 1a successfully travels through the secretory pathway to PM (27). These data are consistent with a model, in which the activity-regulated increased expression of Homer 1a disrupts the binding of Homer 1b to mGluR and leads to increased trafficking of mGluR to PM. Transient upregulation of Homer 1a during muscle differentiation might be involved in protein redistribution related to SR biogenesis and/or formation of peripheral junctions and triads.
Change of Homer 3 expression during regeneration. Interestingly, no constitutive expression of Homer 3 was detected in control soleus muscle. This finding is in qualitative agreement with previous findings by Soloviev et al. (33), who reported that mRNA for Homer 3 is the least represented among Homer transcripts in skeletal muscle of the adult mouse, and by Xiao et al. (39) and by Shiraishi et al. (31) who reported no expression of Homer 3, as judged by Western blot analysis of skeletal muscles of the adult rat and mouse, respectively. On the other hand, during regeneration, there was a conspicuous and sustained expression of Homer 3 between postinjection day 3 and day 6. Compared with Homer 1a upregulation, Homer 3 buildup started earlier and its decay was inversely related to increase of muscle mass (see Figs. 5B, and 6, A and D).
Consistently, in C2C12 cell cultures, Homer 3 expression was already at plateau when Homer 1a started its ascending phase (Fig. 7). Taken together, these data indicate that Homer 3 might be characteristic of satellite cell activation and of the following early phases of myoblast proliferation (6). Thus upregulation of Homer 3 and that of Homer 1a are clearly distinct phenomena. The reasons for such a different behavior are unknown.
Interestingly, transient expression of Homer 3 has been already observed during postnatal development of the brain (31). In cultured T cells, on serum stimulation, Homer 3 has been reported (16) to be overexpressed and to translocate to the nucleus, thus regulating T cell activation via interaction with specific transcription factors. It is uncertain whether Homer 3 is indeed an IEG in skeletal muscle.
Expression pattern of Homer 2b during regeneration. Expression pattern was similar to that of Homer 1b/c/d, and, thus, Homer 2 could be expressed by myogenic cells during regeneration and as a constitutive isoform in adult muscle. Our observations are interesting in relation to previous findings by Pattison et al. (25), who reported downregulation of Homer 2 mRNA in disuse atrophy, an adaptive phenomenon characterized by reduced number and size of muscle fibers. Thus increase of Homer 2b during regeneration might be associated to mass recovery (cfr. Fig. 6C) and be also instrumental to transition toward hypertrophy.
Final remarks. The relevant biological question stemming from the present work is whether Homer (either 1a and/or 3) transcription is causally involved in differentiation and regeneration of skeletal muscle, or is an epiphenomenon associated to activation of specific myogenic programs. The distinct expression pattern displayed by Homer 1a and Homer 1b/c/d, as well as the temporal dissociation of upregulation transiently shown by Homer 1a and Homer 3, would indicate a role for both Homer 1a and Homer 3 in muscle differentiation and regeneration, whereas Homer 1b/c/d and Homer 2b appear to be part of the muscle growth program. If Homer 1a and Homer 3 were to play a role in differentiation and regeneration, as judged by data obtained in C2C12 cell cultures, then Homer 3 might be mainly involved in early phases of myogenesis up to myoblast differentiation and Homer 1a in later phases of myogenesis, i.e., formation and maturation of myotubes.
Negative results obtained in fatigue experiments do not rule out a role for Homer 1a; in fact, other patterns of fatigue might do so and additional investigations are in order.
Finally, a variety of functional studies (10, 15, 37) would have no relevance at all, under resting conditions, if Homer 1a were an IEG only. The constitutive expression of Homer 1a, as shown here, corroborates the putative role of Homer 1a in skeletal muscle, i.e., Homers may play a role in signal transduction of skeletal muscle, in particular regulation of Ca2+ release from SR.
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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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