Myf5 is a member of the muscle regulatory factor family of transcription factors and plays an important role in the determination, development, and differentiation of skeletal muscle. However, factors that regulate the expression and activity of Myf5 itself are not well understood. Recently, a role for the calcium-dependent phosphatase calcineurin was suggested in three distinct pathways in skeletal muscle: differentiation, hypertrophy, and fiber-type determination. We propose that one downstream target of calcineurin and the calcineurin substrate NFAT in skeletal muscle is regulation of Myf5 gene expression. For these studies, we used myotube cultures that contain both multinucleated myotubes and quiescent, mononucleated cells termed ‘reserve’ cells, which share many characteristics with satellite cells. Treatment of such myotube cultures with the calcium ionophore ionomycin results in an ≈4-fold increase in Myf5 mRNA levels, but similar effects are not observed in proliferating myoblast cultures indicating that Myf5 is regulated by different pathways in different cell populations. The increase in Myf5 mRNA levels in myotube cultures requires the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. We used immunohistochemical analyses and fractionation of the cell populations to demonstrate that the calcium regulated expression of Myf5 occurs in the mononucleated reserve cells. We conclude that Myf5 gene expression is regulated by a calcineurin- and NFAT-dependent pathway in the reserve cell population of myotube cultures. These results may provide important insights into the molecular mechanisms responsible for satellite cell activation and/or the renewal of the satellite cell pool following activation and proliferation.
Changes in intracellular calcium have been implicated in regulating diverse processes in skeletal muscle, including differentiation, hypertrophy, and fiber-type determination. In each of these cases, the calcium/calmodulin regulated protein phoshatase calcineurin has been proposed to play a central role in mediating the downstream calcium-dependent signaling (Abbott et al., 1998; Chin et al., 1998; Delling et al., 2000; Dunn et al., 1999; Friday et al., 2000; Musaro et al., 1999; Naya et al., 2000; Semsarian et al., 1999). However, the calcineurin targets that regulate these processes have not been clearly defined.
One potential target of these signals is the muscle regulatory factor (MRF) family of bHLH transcription factors that includes MyoD, Myf5, myogenin, and MRF4 (Megeney and Rudnicki, 1995; Rudnicki and Jaenisch, 1995). The MRFs are themselves members of a larger superfamily of transcription factors that contain a basic region which mediates DNA binding to an E-Box consensus DNA element (Davis et al., 1990), and a helix-loop-helix domain that mediates dimerization. A large number of muscle-specific promoters have been found to contain E-Box elements. Each of the family members was initially described based on its ability to transform 10T1/2 fibroblasts to a muscle phenotype, and their roles have been more clearly defined in the determination and differentiation of skeletal muscle using transgenic and knockout mice (Arnold and Braun, 1996; Arnold and Winter, 1998). In the absence of MyoD and Myf5, myoblasts are not formed in the developing embryo (Rudnicki et al., 1993), while in the absence of myogenin, myoblasts are formed, but differentiation is inhibited (Hasty et al., 1993). MRF4 is believed to play a role in later stages of muscle maturation (Zhang et al., 1995). Clearly, the MRFs play a central role in regulating differentiation of skeletal muscle, one of the proposed calcineurin regulated pathways.
In addition to differentiation, evidence also exists for the participation of the MRFs in the regulation of hypertrophy and fiber-type determination. In several models of hypertrophy including stretch and electrical stimulation, increases in the expression of MRFs occur within muscle fibers, suggesting a role in the growth of existing myofibers (Jacobs-El et al., 1995; Lowe and Alway, 1999). Evidence for a role of MRFs in fiber-type determination is suggested by the fact that MRF expression levels differ between fast and slow muscles (Hughes et al., 1993; Kraus and Pette, 1997). Recently, a transgenic mouse was developed that overexpresses myogenin in post-mitotic fast muscle fibers (Hughes et al., 1999). These mice displayed a shift towards metabolic enzymes characteristic of slow fibers. A role for MyoD in the maintenance of fast fibers has also been proposed (Hughes et al., 1997). As the MRFs play important roles in calcineurin regulated pathways, it is possible that they are directly or indirectly regulated by a calcineurin-dependent pathway.
We hypothesize that expression of the Myf5 gene is regulated by the calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT). A calcium regulated pathway for controlling Myf5 gene expression has already been proposed. Treatment of skeletal muscle cells in vitro with the physiological peptide arginine-vasopressin induces an increase in cytosolic Ca2+ concentration (Teti et al., 1993), and also results in a strong upregulation of the Myf5 gene (Nervi et al., 1995). However, the downstream mediators of the arginine-vasopressin signal are not known. Regulation of the Myf5 gene is complex, requiring up to 500 kb of genetic sequence to faithfully reproduce the normal pattern of Myf5 expression (Zweigerdt et al., 1997). In addition, genetic elements in distinct locations regulate the expression of Myf5 in different locations during embryonic development (Patapoutian et al., 1993). Few specific DNA elements or transcription factors that bind within the Myf5 promoter have been identified. In one report, an Oct-like binding factor was found to bind to a conserved element within the avian Myf5 promoter, but this element may not be muscle specific, since it was required for expression of a reporter construct in myoblasts as well as fibroblasts (Barth et al., 1998). In the mouse myogenic cell line C2, expression of Myf5 in myoblasts was upregulated by increasing the activity of the glucocorticoid receptor and AP-1 (Aurade et al., 1997). Identification of a calcium-dependent signaling pathway that regulates the expression of Myf5 would help to elucidate the molecular regulation of the Myf5 gene, and may further define the mechanism(s) whereby calcineurin regulates the differentiation, hypertrophy, and fiber type determination of skeletal muscle.
In this report, we show that Myf5 mRNA and protein levels are increased by the calcium ionophore ionomycin in the mononucleated cell fraction of myotube cultures. These mononucleated cells have been termed reserve cells, and were shown to share many characteristics with skeletal muscle satellite cells, including quiescence, self-renewal, and the ability to generate multinucleated myotubes (Yoshida et al., 1998). Ionomycin-induced effects on Myf5 gene expression are not found in proliferating myoblast cultures. The increase in Myf5 mRNA levels is dependent on the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. We conclude that the expression of Myf5 in the reserve cells is regulated by calcineurin and NFAT.
MATERIALS AND METHODS
Antisera, reagents and statistics
Rabbit polyclonal antibodies against Myf5 were purchased from Santa Cruz Biotech., Inc. (Santa Cruz, CA) and obtained as a gift from Dr Didier Montarras. Secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). CSA was a gift of Sandoz (Basil, Switzerland). Ionomycin and PMA were purchased from Sigma Chemical Co. Thapsigargin was obtained from the Alexis Corp. (San Diego, CA). Amphotropic retroviral producer cells (SD-3443) were obtained from the American Type Culture Collection (Rockville, MD). All cell culture reagents were purchased from Life Technologies (Grand Island, NY), except where noted. Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Norcross, GA). Basic fibroblast growth factor (bFGF) was purchased from Promega (Madison, WI). All statistical analyses were performed using a one way analysis of variance and Bonferroni’s pairwise comparisons.
Primary myoblast cultures were prepared from SJL mice and purified to >99% as previously described (Abbott et al., 1998; Rando and Blau, 1994). Growth medium (GM) consisted of Ham’s F10, 20% FBS, 5 ng/ml bFGF, 200 U/ml penicillin G, and 200 μg/ml streptomycin for primary muscle cells, and a similar medium was used for C2C12 muscle cells with the exception of 15% calf serum and 5% FBS in the place of the 20% FBS. Differentiation was induced by changing cells to a low serum, low mitogen differentiation medium (DM: DME, 2% horse serum, 200 U/ml penicillin G, 200 μg/ml streptomycin) for 24-48 hours. Primary cells were differentiated on E-C-L (Upstate Biotechnology, Lake Placid, NY) coated dishes.
Retroviral plasmids, production and infection
The retroviral vectors utilized in these experiments have all been previously described, including CAIN and GFP-VIVIT expression vectors (Friday et al., 2000), an NFAT responsive reporter vector (Abbott et al., 1998; Boss et al., 1998), and an NFATc expression vector (Abbott et al., 2000). Production of infectious retrovirus and infection of primary myoblasts were performed as previously described (Abbott et al., 1998). Experiments using GFP-VIVIT were performed on cells that had been selected for GFP expression by flow cytometry (Friday et al., 2000).
RNA was prepared from cells using Trizol Reagent (Life Technologies) following the manufacturer’s protocol. RNA was separated on 1% agarose-formaldehyde gels and transferred to Nytran SPC membranes (Schleicher & Schuell, Keene, NH). Membranes were probed with random-primed cDNA (Rediprime II, Amersham Pharmacia Biotech, Buckinghamshire, UK) labeled with 32P in Rapid-hyb buffer (Amersham Pharmacia Biotech). After high stringency washing, membranes were visualized by autoradiography. Autoradiographs were scanned and quantitated using Scion Image software.
Cells were lysed with RIPA-2 (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) containing protease inhibitors (Mini Complete, Boehringer Mannheim, Indianapolis, IN). Equal amounts of protein (15 μg per lane) (Bradford, 1976) were separated by SDS-PAGE and transferred electrophoretically to a PVDF membrane (Immobilon P, Millipore, Burlington, MA). After non-specific binding was blocked in 5% non-fat milk in TBS for 30 minutes, the membrane was incubated overnight at 4°C in 0.5% non-fat milk in TBS containing a 1:800 dilution of anti-Myf5 (Santa Cruz Biotech., Inc). Blots were washed extensively in TBS containing 0.1% Tween-20 (TBS-T) and then incubated with a donkey anti-rabbit HRP conjugated secondary antibody (1:10,000) in 0.5% non-fat milk in TBS-T. Blots were washed in TBS-T and antibody binding was detected using ECL reagents (Amersham Pharmacia Biotech). To demonstrate relative protein loading membranes were stained with Coomassie Blue (Bio-Rad, Hercules, CA).
Myotube cultures grown in 35 mm dishes were fixed in 3.7% formaldehyde for 10 minutes. After non-specific binding was blocked in TNB buffer (NEN Life Sciences, Boston, MA) for 30 minutes, the cells were incubated overnight at 4°C with a 1:1000 dilution of anti-Myf5 (Lindon et al., 1998) in DMEM containing 10% FBS. Cells were washed with PBS containing 0.1% Tween-20 and then incubated with a biotinylated donkey anti-rabbit secondary antibody for 1 hour in DMEM containing 10% FBS. Antibody binding was detected using the Vectastain Elite Kit (Vector Laboratories, Burligame, CA). Cells were photographed and analyzed using an Axioplan microscope (Carl Zeiss, Thornwood, NY).
Myoblasts containing an NFAT responsive reporter were plated at 4×104 cells per well of 24-well dishes. Cells were infected by two rounds of infection with either control or NFATc retroviruses. After 24 hours, the medium was replaced with DM and the cells were allowed to differentiate for 48 hours. The medium was replaced with the appropriate drug containing medium and incubated for 5-6 hours at 37°C. Cells were washed twice with PBS and 75 μl of Luciferase Cell Culture Lysis Reagent (Promega, Madison, WI) was added to each well. The cell lysates were collected and spun at 12,000 g for 30 seconds. 100 μl of Luciferase Assay Reagent (Promega) was injected into 20 μl of cell lysate and light output was measured after a 5 second delay over a 10 second window using a Turner TD-20e luminometer (Turner Designs, Sunnyvale, CA).
Regulation of Myf5 mRNA levels by a calcineurin-dependent pathway in myotube cultures
The possibility that Myf5 gene expression is regulated by calcium has been suggested by work demonstrating an increase in Myf5 mRNA levels after treatment with arginine-vasopressin, a hormone known to increase cytosolic calcium levels in skeletal muscle cells (Nervi et al., 1995; Teti et al., 1993). To determine if Myf5 mRNA levels could be directly regulated by increasing intracellular calcium, we treated myoblast or myotube cultures with the calcium ionophore ionomycin and determined the effect on the expression of Myf5 mRNA by northern blotting (Fig. 1). In myoblast cultures, ionomycin treatment has no effect on the expression of Myf5 mRNA, but in myotube cultures, ionomycin treatment results in an ≈4-fold increase in Myf5 mRNA levels. To determine if the response of the Myf5 gene to increasing intracellular calcium requires the activity of calcineurin, some cultures were also treated with the calcineurin inhibitor cyclosporine A (CSA). CSA has no effect on Myf5 expression in myoblasts. In myotube cultures, CSA pretreatment blocks the induction of Myf5 mRNA expression following ionomycin treatment. We also find a reduction in the basal expression levels of Myf5 mRNA by ≈30%, but the trend is not statistically significant. We have obtained similar data in human primary muscle cells, C2C12 mouse muscle cells, and primary muscle cells isolated from BALB/C mice (data not shown).
The data using CSA suggest that calcineurin regulates Myf5 gene expression, but CSA may have targets distinct from calcineurin in muscle cells (Lo Russo et al., 1996; Lo Russo et al., 1997). To demonstrate conclusively that the increase in Myf5 mRNA levels observed with ionomycin treatment is calcineurin-dependent, we utilized the physiological calcineurin inhibitor CAIN (Lai et al., 1998). In previous work, we have shown that CAIN effectively inhibits calcineurin activity in skeletal muscle cells (Friday et al., 2000). Myoblasts were infected with either control or CAIN retroviruses and induced to differentiate (Fig. 2). Since calcineurin activity is required for the differentiation of skeletal muscle (Friday et al., 2000), we had to place the cells into differentiation medium prior to CAIN expression. The cells were subsequently treated with either vehicle, ionomycin, or ionomycin plus CSA 24 hours after the initial retroviral infection, a time point at which retroviral encoded proteins are detected, but before maximal expression as determined by visualization of GFP (data not shown). CAIN reduces the expression of Myf5 mRNA in ionomycin treated cultures by ≈35% compared to control cultures. CSA further reduces the levels of Myf5 mRNA to values similar to that of vehicle treated cultures, reflecting the incomplete inhibition of calcineurin by CAIN as a result of the necessity to perform the experiment prior to maximal CAIN expression. Since, both CSA and CAIN inhibit the ionomycin induced increase in Myf5 mRNA levels, we conclude that calcineurin regulates Myf5 gene expression in myotube cultures.
NFAT is a downstream target of calcineurin in the regulation of the Myf5 gene
In T-cells, the major downstream target of calcineurin is the transcription factor NFAT (Rao et al., 1997). Skeletal muscle cells express three isoforms of NFAT (Abbott et al., 1998), but their roles in myogenesis have not been clearly defined. To determine if NFAT is the downstream target of calcineurin in the regulation of the Myf5 gene, we used retroviral mediated gene transfer to overexpress either NFATc or a specific oligopeptide NFAT inhibitor.
To test whether overexpression of NFATc increases NFAT mediated transcription, we used an NFAT responsive luciferase reporter construct. This construct contains multiple copies of an NFAT response element from the IL-2 gene, that requires the calcineurin induced activation of NFAT and the induction of AP-1 activity for transcriptional activation. In this experiment, we used the Ca2+-ATPase inhibitor thapsigargin to activate calcineurin by increasing cytosolic calcium, and the phorbol ester PMA to induce AP-1 activity. As seen in Fig. 3a, treatment with thapsigargin and PMA results in an ≈10-fold increase in luciferase levels in control cells. In cells overexpressing NFATc, the luciferase levels increase by ≈110-fold under similar conditions. An increase in NFAT mediated transcription is also seen in NFATc cells compared to control cells when treated with either PMA or thapsigargin alone. To determine the effect on Myf5 gene expression, myoblasts were infected with either control or NFATc retroviruses. After differentiation, the cells were treated with either vehicle or thapsigargin, and the expression levels of the MRFs were determined by northern blot analyses (Fig. 3b). In accordance with our results using ionomycin, thapsigargin increases the expression of Myf5 mRNA in control cells. After thapsigargin treatment, the levels of Myf5 mRNA are ≈50% less in control cells versus NFATc overexpressing cells (Fig. 3c). The other MRFs, including MyoD and myogenin, are only minimally changed by either NFATc overexpression or thapsigargin treatment when data are normalized to GAPDH levels. We are unable to detect MRF4 in our cultures. These data demonstrate that overexpression of NFATc results in a specific enhancement of the thapsigargin-induced increase in Myf5 mRNA.
To determine if NFAT activity is required for regulation of the Myf5 gene by calcineurin, we infected cells with either a control retrovirus or a retrovirus expressing the oligopeptide NFAT inhibitor GFP-VIVIT (Aramburu et al., 1999). We have shown previously that this construct blocks ≈95% of the NFAT mediated transcription in primary muscle cells (Friday et al., 2000). The cells were differentiated and then treated with either vehicle or ionomycin. To identify whether pathways distinct from NFAT also participate in the regulation of the Myf5 gene by calcineurin, some cultures were also treated with CSA. Myf5 mRNA levels were determined by northern blot analyses (Fig. 4). In cultures containing GFP-VIVIT, the level of Myf5 mRNA in vehicle and ionomycin treated cultures was not statistically different, suggesting that NFAT is the sole downstream effector of calcineurin.
Calcineurin regulates Myf5 expression in mononuclear cells of myotube cultures
Myf5 mRNA levels do not always correspond to the levels of the Myf5 protein (Kitzmann et al., 1998; Lindon et al., 1998). The mRNA level is relatively unchanged throughout the cell-cycle, but protein levels vary dramatically. To determine if the increase in Myf5 mRNA levels due to increasing intracellular calcium also increases Myf5 protein levels, we treated myotube cultures with either vehicle, ionomycin, or ionomycin plus CSA (Fig. 5a). Immunoblot analyses were performed using an antibody against Myf5. Ionomycin treatment increases Myf5 protein levels, and the increase is blocked by the addition of CSA.
Myotube cultures do not represent a homogeneous population of cells. In addition to the multinucleated, differentiated cells, myotube cultures contain mononucleated, non-dividing cells that have been referred to as reserve cells (Yoshida et al., 1998; Kitzmann et al., 1998). These cells remain undifferentiated, and upon the readdition of serum, can reenter the cell cycle. In one report, reserve cells expressed high levels of Myf5 (Kitzmann et al., 1998). To determine the cell type in myotube cultures that accounts for the ionomycin-induced increase in Myf5 protein levels, we performed immunohistochemical analyses. Consistent with the previous reports, Myf5 protein is primarily expressed in mononucleated cells in myotube cultures (Fig. 5b). Occasionally, Myf5-positive nuclei are present in myotubes (data not shown). The number of Myf5 positive nuclei was counted in 15 random fields and the data were plotted as either the total number of Myf5 positive nuclei or the percentage of Myf5 positive nuclei in mononucleated cells (Fig. 5c). Ionomycin treatment does not change the number of Myf5 positive nuclei. In vehicle treated cells, ≈96% of the Myf5 positive nuclei were in mononucleated cells, and the ratio is unchanged with ionomycin treatment. Since a change is not observed in either the number or type of cells that express Myf5 following ionomycin treatment, these data suggest that calcineurin regulates Myf5 expression in the cells that are already expressing Myf5, the reserve cell population.
C2C12 myotube cultures can be separated into a mononuclear enriched cell fraction containing reserve cells and a multinucleated enriched cell fraction containing myotubes by a brief exposure to trypsin (Kitzmann et al., 1998). Biochemically, these cell fractions can be identified based on their differential expression of the MRFs, the reserve cells express Myf5 and the myotubes express MyoD and myogenin. We utilized the limited trypsinization technique to determine the cell fraction responsible for the ionomycin-induced increase in Myf5 mRNA levels (Fig. 6).
C2C12 cells were induced to differentiate for 48 hours and then cultures were treated with either vehicle or ionomycin. After a brief trypsinization, the mononucleated and multinucleated enriched cell fractions were analyzed by northern blotting for expression of Myf5, MyoD and myogenin mRNA. We observe a reciprocal expression pattern of Myf5 versus MyoD and myogenin in the two cell fractions. Myf5 mRNA levels are higher in the mononucleated cell fraction compared to the multinucleated fraction in both vehicle and ionomycin treated cultures. MyoD and Myogenin are preferentially expressed in the multinucleated cell fraction. There is no change in the expression level of myogenin with ionomycin treatment, whereas a slight decrease in MyoD expression is observed with ionomycin treatment. These results are consistent with the hypothesis that the increase in Myf5 mRNA following ionomycin treatment occurs primarily in the mononucleated cell fraction, but since the two fractions are not pure, we can not rule out the possibility that Myf5 gene expression is also activated in myotubes by ionomycin. Based on the data obtained using both immunohistochemistry and limited trypsinization, we conclude that calcineurin regulates Myf5 expression in the reserve cell population of myotube cultures.
The importance of calcium in regulating the molecular events surrounding the physiological changes that occur during differentiation, hypertrophy, and fiber-type determination in skeletal muscle cells has been shown recently by a number of different groups (Chin et al., 1998; Delling et al., 2000; Dunn et al., 1999; Friday et al., 2000; Musaro et al., 1999; Naya et al., 2000; Semsarian et al., 1999; Wu et al., 2000). The MRFs have also been proposed to play important roles in these same pathways (Hasty et al., 1993; Hughes et al., 1999; Hughes et al., 1997; Hughes et al., 1993; Jacobs-El et al., 1995; Kraus and Pette, 1997; Lowe and Alway, 1999; Rudnicki et al., 1993), but a link between calcineurin-dependent signaling and MRF expression or activity has not been previously proposed.
In this report, we show that Myf5 gene expression is regulated by calcineurin and NFAT in the reserve cell population of myotube cultures. The concept of reserve cells was proposed by two different groups based on the identification of quiescent, mononuclear cells in differentiated myotube cultures that could reenter the cell cycle upon the readdition of mitogenic serum (Kitzmann et al., 1998; Yoshida et al., 1998). Unlike proliferating myoblasts which express both Myf5 and MyoD, the reserve cells express only Myf5. Ectopic expression of MyoD forces cells into the differentiation pathway and eliminates the formation of reserve cells (Yoshida et al., 1998). Reserve cells do not express p21 (Yoshida et al., 1998), a marker of terminal differentiation, proliferating cell nuclear antigen (Yoshida et al., 1998) or cyclin A (Kitzmann et al., 1998), markers of cell proliferation, suggesting that reserve cells are in a quiescent state. Reactivation of reserve cells with growth medium generates progeny that form myotubes and reserve cells upon the removal of serum. The characteristics of quiescence, self-renewal and generation of myotubes by reserve cells are shared with the resident satellite cells of muscle fibers that are responsible for the growth and regeneration of skeletal muscle (Schultz and McCormick, 1994; Yablonka-Reuveni, 1995). Data obtained from MyoD null mice provide in vivo support for the reserve cell hypothesis, in that satellite cells expressing only Myf5 display a propensity for self-renewal rather than proceeding down the differentiation pathway (Megeney et al., 1996). Based on the characteristics shared by satellite cells and reserve cells, the reserve cells may represent an in vitro model for satellite cells. Since Myf5 is one of the first genes to be induced following satellite cell activation (Cooper et al., 1999; Cornelison and Wold, 1997), our results demonstrating that a calcium-dependent signaling pathway activates Myf5 expression may provide important insights into the molecular mechanisms responsible for satellite cell activation. Additionally, our results may help elucidate the mechanism whereby activated satellite cells generate progeny that replenish the satellite cell pool. Since satellite cells do not express either MyoD or Myf5, the reserve cells may represent an intermediary step between the proliferating muscle cell and the reformation of a quiescent satellite cell.
Regulation of Myf5 gene expression by calcineurin and NFAT represents a distinct calcineurin-dependent pathway from those previously proposed based on several observations. First, the reserve cells represent a distinct population of cells based on their MRF expression profile, as compared to either proliferating myoblasts or terminally differentiated myotubes and myofibers, the cell types in which calcineurin-dependent pathways were previously described. Second, we have proposed that calcineurin initiates the myogenic program in proliferating myoblast cultures (Friday et al., 2000), a process that normally results in the down-regulation of the Myf5 gene, as seen in the difference in Myf5 mRNA expression between myoblasts and myotubes in Fig. 1a. However, in the reserve cell population, activation of calcineurin results in an increase in Myf5 expression. Finally, the calcineurin-dependent pathway that initiates differentiation is NFAT-independent (Friday et al., 2000), whereas the regulation of Myf5 expression in reserve cells is NFAT-dependent.
Based on our results, we present the model outlined in Fig. 7 for calcineurin mediated signaling during differentiation and generation of reserve cells in skeletal muscle cultures. Proliferating myoblasts express both Myf5 and MyoD mRNA. Upon the removal of mitogenic stimuli, myoblasts enter one of two distinct pathways. In the first pathway, calcineurin acts on an as yet unknown substrate to initiate the differentiation pathway into multinucleated myotubes that express MyoD, but not Myf5 (Friday et al., 2000). In a second pathway, myoblasts withdraw from the cell cycle and become quiescent reserve cells that express Myf5, but not MyoD. Increasing intracellular calcium in the reserve cells activates calcineurin and NFAT resulting in an increase in Myf5 mRNA.
Our data demonstrate that the expression of Myf5 in post-natal skeletal muscle cells is regulated by different pathways under differing culture conditions. We find no evidence that the expression of Myf5 in proliferating myoblasts is regulated by calcineurin or NFAT. The high level of Myf5 mRNA expression observed in myoblasts is not decreased by CSA treatment, nor does pharmacological activation of calcineurin and NFAT with ionomycin result in an increase in Myf5 expression. One explanation for these results is that even though ionomycin robustly activates NFAT in various cell types, specific NFAT isoforms in skeletal muscle do not always undergo nuclear translocation. We have previously shown that only the NFAT4 isoform can undergo nuclear translocation in myoblast cultures in response to calcium (Abbott et al., 1998), even though other NFAT isoforms are present. In differentiating cell cultures, both the NFATc and NFATp isoforms can translocate to the nucleus in response to calcium, suggesting that it is these isoforms that regulate Myf5 gene expression in reserve cells. Our results using overexpresssion of NFATc support this model. An alternative explanation is that NFAT requires a coactivator that is not present in proliferating cells. In T-cells, the major partner for NFAT mediated gene transcription is AP-1 (Rao et al., 1997), but in skeletal muscle cells alternate NFAT binding partners have been proposed, including GATA-2 (Musaro et al., 1999) and MEF2 (Chin et al., 1998; Wu et al., 2000). The identity and importance of NFAT binding partners in the regulation of the Myf5 gene are not known.
The transcriptional regulation of the Myf5 gene is complex, requiring more than 500 kb of genomic sequence to faithfully recapitulate Myf5 expression during development (Zweigerdt et al., 1997). Of particular importance is the finding that distinct genomic elements regulate Myf5 expression in different regions of the embryo (Patapoutian et al., 1993; Yoon et al., 1997). The enhancer elements that regulate Myf5 expression in post-natal cells have not been identified, but based on the results in embryonic development, it would not be surprising to find that a genomic region regulated Myf5 gene expression in post-natal muscle cells that was distinct from those regions that regulate embryonic expression. Further, different enhancer elements may regulate Myf5 gene expression in myoblasts versus reserve cells.
The regulation of Myf5 gene expression by calcineurin is mediated by the transcription factor NFAT, suggesting that NFAT binding sites should be present in the enhancer elements regulating Myf5 expression in reserve cells. However, due to the complex nature of the Myf5 gene and its regulation, it will be difficult to find the specific elements amidst the 500 kb of DNA required for proper gene regulation (Zweigerdt et al., 1997). A cursory examination of the human Myf5 promoter sequence revealed several putative NFAT binding sites, but these sites do not activate transcription in response to calcium in myotube cultures using a luciferase reporter gene (data not shown), suggesting that some other region is responsible for NFAT mediated regulation. Future work in identifying the specific regions of DNA that regulate Myf5 in post-natal cells will allow us to renew a search for NFAT binding sites in Myf5 enhancers. In summary, we have shown that Myf5 gene expression is regulated by a calcineurin and NFAT-dependent pathway in the reserve cell population of myotube cultures. Future work will aim to define the relationship between the reserve cells and satellite cells, and to characterize the role of calcineurin and NFAT in the activation and/or renewal of the satellite cell pool.
We thank Jonathan Gephart for technical assistance, Dr Didier Montarras for the Myf5 antibody, Dr Anjana Rao for the GFP-VIVIT cDNA, and Dr Jeffery Molkentin for help with the CAIN construct. Supported by grants to GKP from the National Institutes of Health (DE11987, AR-43410 and DE13040).