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22, 583-591. Ott, M.-O., Bober, E., Lyons, G., Arnold, H. and Buckingham, M. (1991). Early expression of the myogenic regulatory gene, myf-5, in precursor cells.
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Journal of Cell Science 110, 2771-2779 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JCS9623

The glucocorticoid receptor and AP-1 are involved in a positive regulation of the muscle regulatory gene myf5 in cultured myoblasts Frédéric Auradé1, Curt M. Pfarr2, Catherine Lindon1, Alphonse Garcia1, Michael Primig3, Didier Montarras1,* and Christian Pinset1,* 1Groupe

de Développement Cellulaire, Institut Pasteur, Department of Molecular Biology, 25 rue du Dr Roux, 75724 Paris cedex 15, France 2Department of Cell Biology and Biochemistry, Texas Tech University School of Medicine, 3604 4th Street, Lubbock, Texas 79430, USA 3Unité de Génétique Moléculaire du Développement, Institut Pasteur, Department of Molecular Biology, 25 rue du Dr Roux, 75724 Paris cedex 15, France *Authors for correspondence (e-mail addresses: [email protected]/[email protected])

SUMMARY The muscle regulatory factor, myf5, is involved in the establishment of skeletal muscle precursor cells. Little is known, however, about the control of the expression of the gene encoding this basic helix-loop-helix (bHLH) factor. We have addressed this question in the mouse myogenic cell line, C2, and in a derivative of this cell line where the myf5 gene is the only muscle-specific bHLH factor to be expressed at the myoblast stage. We present evidence that the synthetic glucocorticoid dexamethasone, and the pharmacological agent anisomycin, act synergistically to rapidly up-regulate the levels of myf5 transcript and protein. The glucocorticoid antagonist RU 486 abolishes this synergy, demonstrating the involvement of the glucocorticoid receptor. The expression of a dominant negative mutant of c-jun which interferes with the transactivating properties

of all AP-1 family members also blocks the induction of myf5 by anisomycin and dexamethasone. An activator of protein kinase C (PKCs), 12-O-tetradecanoyl phorbol 13acetate (TPA), abolishes the up-regulation of myf5 gene expression by dexamethasone and anisomycin, and its effect is counteracted by an inhibitor of PKCs, GF 109203X. These results point to the possible involvement of PKCs in the negative control of myf5. Evidence that both positive and negative regulation of myf5 transcripts, described here, does not require the fresh synthesis of transcription factors suggests that myf5 may behave like an immediate early gene.

INTRODUCTION

Downes et al., 1993; Muscat et al., 1994; Münsterberg et al., 1995). The identification of factors which influence expression of the myf5 gene is also hindered by the absence of information on the regulatory sequences at the locus of this gene. Tissue culture approaches have proved fruitful not only for the isolation, but also for the study of the role and the expression of bHLH muscle regulatory factors. Of these, myf5 is expressed predominantly at the myoblast stage (Braun et al., 1989; Montarras et al., 1991) and, unlike MyoD and/or myogenin, is down-regulated at the onset of differentiation (Montarras et al., 1991; Mangiacapra et al., 1992); its expression is not sufficient for autonomous differentiation of myoblasts (Montarras et al., 1996). In this work, we have searched for factors which positively influence expression of the myf5 gene in the mouse myogenic cell line C2 and in a derivative of this cell line. We reasoned that such factors would act as stabilizers of the myoblast phenotype rather than as inducers of differentiation. Our results indicate that dexamethasone and anisomycin synergistically up-regulate myf5 gene expression. The effect of dexamethasone is mediated through the glucocorticoid receptor which functions as a ligand activated transcription factor by binding

Commitment of embryonic cells to the myogenic lineage relies upon muscle regulatory factors of the basic helix-loophelix family. Among these factors, myf5 is the first to be expressed at day 8 post-coitum, in the myotome of the somite of the developing mouse embryo (Ott et al., 1991). In myf5 deficient embryos, initiation of myogenesis is delayed by 2 days (Braun et al., 1992) and a fraction of myogenic precursor cells leaves the myogenic lineage (Tajbakhsh et al., 1996). However, little is known about how myf5 expression is regulated. Grafting experiments in avian embryos have indicated that expression of muscle regulatory factors in the somites could be either positively or negatively influenced by diffusible signaling molecules originating, respectively, from the neural tube or the lateral mesoderm (Münsterberg et al., 1995; Pourquié et al., 1995). Candidate molecules exerting a negative effect include members of the bone morphogenic factor (BMP) family (Pourquié et al., 1996), whereas sonic hedgehog (shh) and members of the wnt family as well as ligands of nuclear receptors (retinoic acid and thyroid hormone) are potential positive regulators (Carnac et al., 1992;

Key words: Muscle bHLH, Glucocorticoid, AP-1, Myogenesis

2772 F. Auradé and others to glucocorticoid responsive elements (GREs) present in the promoters of numerous genes (Grove et al., 1980; Geisse et al., 1982). Anisomycin has been shown to activate the expression of c-jun through the stimulation of the stress activated kinases (SAPK), also referred to as the Jun N-terminal kinases (JNK) (Cano et al., 1994, 1996; Kardalinou et al., 1994). Attempts to interfere with these events are presented. MATERIALS AND METHODS Cell culture C2.7 and anti-IGFII C2 cells were previously described (Montarras et al., 1996). All cell culture was performed at 37°C under a humidified atmosphere with 7% CO2. Proliferation medium was a 1:1 (v/v) mixture of MCDB 202 medium and Dulbecco’s modified Eagle’s (DME) medium (both from BICEF), supplemented with 20% (v/v) fetal calf serum (FCS; Jacques BOY, Reims). Initial plating density was between 1.3×103 and 2.5×103 cells/cm2. Cells were harvested 3 days after plating to prepare RNA or protein extracts, except in Fig. 3 where cells were shifted for 24 hours into medium containing 1% FCS, before changing to medium with 10 ng/ml transferrin (Sigma). Dexamethasone (stock solution 10−2 M in 100% EtOH; Sigma) was used at a concentration of 10−7 M, RU 486 (a gift from Dr L. Corcos, INSERM, Rennes) at 10−6 M, anisomycin (Sigma) at 50 ng/ml, puromycin (Sigma) at 10 µg/ml, GF 109203X (a gift from Dr J. Kirilovsky) at 0.8 µM, and 12-O-tetradecanoyl phorbol 13-acetate (TPA; Sigma) at 250 ng/ml. All compounds were added to the culture directly without changing the medium. Plasmids and transient transfection The plasmid used to generate a c-Jun dominant negative protein (FLAG∆169) is described by Ham et al. (1995). It carries a c-jun cDNA deleted in the region encoding the transcriptional transactivation domain (amino acids 1-168). To distinguish the FLAG∆169 from the endogenous c-Jun protein, an 8 amino acid FLAG epitope (recognized by the monoclonal antibody, M2, Eastman-Kodack) is cloned at the amino terminus of the mutant protein. The wild-type c-jun gene is under the control of the RSV promoter. Anti-IGFII cells were plated at 1.6×103 cells/cm2 and transiently transfected 24 hours later by the calcium-phosphate method following the parameters described by Jordan et al. (1996). After 48 hours, cells were fixed and assayed for protein detection by immunofluorescence. Immunofluorescence Cells were grown on plastic tissue culture plates. Between each step, cells were extensively washed with 1× PBS (phosphate buffered saline). All steps were at room temperature. Cells were fixed in 4% paraformaldehyde (w/v) in PBS for 10 minutes, neutralized for 10 minutes in 50 mM NH4Cl in PBS, and permeabilized for 10 minutes in 0.2% Triton in PBS. Incubations with antibodies diluted in PBS containing 0.2% (w/v) gelatin (Merck) were as follows: (1) 1 hour with both a rabbit polyclonal antibody against Myf5 diluted at 1/800 and a mouse monoclonal antibody against c-Jun diluted 1/100 (#J31920; Transduction Laboratories) or a flag epitope (M2) diluted 1/200; (2) 30 minutes with both a polyclonal antibody against mouse immunoglobulins coupled to Texas Red diluted at 1/50 and a polyclonal antibody against rabbit immunoglobulins coupled to biotin (both from Amersham) diluted at 1/200; (3) 20 minutes with streptavidin coupled to fluorescein (Amersham) at 1/100. Cells were mounted in Mowiol (Calbiochem). The myf5 antibody is a polyclonal antibody raised against the N-terminal domain (amino acids 1-72) of the Myf5 protein (M. Primig, manuscript in preparation). It was affinity purified, and did not show any specific reactivity either by western blotting or by immunofluorescence with non-muscle 10T1/2 cells (data not shown). Fluorescence was viewed and recorded by confocal microscopy.

RNA preparation and northern blotting Whole cell RNA from cultured cells was prepared, electrophoresed and blotted as previously described (Pinset et al., 1988). 10 µg of RNA were analyzed in each case. Homogeneity of loading and transfer of RNA was monitored by ethidium bromide staining of the gels, and by probing filters with a ribosomal protein S26 probe (Vincent et al., 1993). Probes The probe used for myf5 mRNA detection was as described previously (Montarras et al., 1996). The c-jun probe corresponds to the 2.6 kbp EcoRI insert (complete cDNA) of the AH119 plasmid. The c-fos probe corresponds to the 1 kbp NcoI fragment of the cDNA containing the whole coding sequence. The 1.6 kbp junD, 1.8 kbp fosB, 1.5 kbp fra-1, and 2.3 kbp fra-2 probes correspond to the cDNAs excised by EcoRI digestion of, respectively, pUC18, pGEM1, and pKS+ vectors. The GAPDH probe is the 1.4 kbp whole cDNA. The 0.6 kbp BamHI-HindIII S26 probe was described by Vincent et al. (1993). Digested fragments were purified with the Qiaquick extraction kit (QIAGEN). The probes were labelled by random priming in the presence of [α-32P]dCTP and [α-32P]dTTP, each at 3,000 Ci/mmol (Redivue, Amersham). Hybridization was performed at 42°C overnight in 50% formamide, 5× SSC, 10× Denhardt, 200 µg/ml denatured salmon sperm DNA, and 50 mM Hepes, pH 7. In order to quantify the level of mRNA detected, filters were analysed on a Phosphorimager (Molecular Dynamics). The level of ribosomal protein S26 mRNA was used as a standard. Protein preparation and western analysis Cells were harvested and boiled in lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 200 mM NaCl, 1% SDS) supplemented with 1 mM DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, 1 mM benzamidine and 2 mM PMSF (all purchased from Sigma). After protein quantification using the Bradford method (Bradford, 1976), extracts were resolved by 10% SDS-PAGE and were transferred to Hybond C Extra (Amersham) membranes using a trans-blot semi-dry electrophoretic transfer cell (Bio-Rad). Detection of proteins was performed by enhanced chemiluminescence (ECL; Amersham). The dilutions of the rabbit polyclonal antibodies were as follows: anti-cJun, 1/500; anti-c-Fos, 1/2,000 (both previously described in Pfarr et al., 1994); anti-Myf5, 1/500. The antibodies were in PBS containing 0.1% Tween-20 and 5% powdered skimmed milk. The filters were incubated with the first antibody for 1 hour at room temperature with vigourous shaking, followed by secondary incubation with a goat polyclonal antibody against rabbit immunoglobulins labelled with horseradish peroxidase 1/10,000 dilution (Sigma).

RESULTS The observation that the level of myf5 mRNA in myoblasts varies according to culture conditions led us to investigate the sensitivity of myf5 expression to surrounding signals (Montarras et al., 1996). Soluble factors (hormones, pharmocological products) which might promote such variations were therefore tested and, as a result, culture conditions were defined which resulted in positive regulation of the myf5 gene. As illustrated in Fig. 1, RNA blot analysis indicates that dexamethasone treatment in combination with anisomycin promoted an increase in the level of myf5 mRNA both in C2 cells (Fig. 1A), and in a derivative of C2 cells referred to as anti-IGFII C2 myoblasts (Fig. 1B, left panel). Interestingly, these cells, in which IGFII gene expression is abolished by antisense RNA, have also lost expression of the MyoD gene and fail to spontaneously differentiate, requiring addition of IGFs to do so

Regulation of myf5 by anisomycin and dexamethasone 2773

Fig. 1. Effect of dexamethasone and anisomycin treatment on myf5 mRNA accumulation. (A) C2 myoblasts, and (B) anti-IGFII C2 myoblasts. Controls correspond to cells after 3 days of proliferation. Cells were treated as indicated for 2 hours with 10−7 M dexamethasone (Dex.) and 50 ng/ml anisomycin without changing the medium. (B) Right panel, the glucocorticoid antagonist, RU486, was added at 10−6 M, 24 hours prior to dexamethasone and anisomycin treatment. myf5 and GAPDH (A) or S26 (B) were assayed by RNA blot analysis. Histograms show the intensities of myf5 signals (arbitrary units), using S26 signal as a standard.

(Montarras et al., 1996). Since MyoD has previously been proposed to interfere with myf5 expression (Peterson et al., 1990; Rudnicki et al., 1992; Montarras et al., 1996), we chose to work with this cell line, which, of the myogenic bHLH factors, only expresses myf5 at the myoblast stage. The analysis by northern blot shows that anisomycin at 50 ng/ml and dexamethasone at 10−7 M act synergistically to upregulate the expression of the myf5 gene (Fig. 1B, left panel): whereas separate treatments had no (dexamethasone) or little (2-fold, anisomycin) effect, the addition of both led to a strong (12-fold) and rapid elevation of the level of myf5 RNA which peaked at 2 hours. This high level of myf5 mRNA remains detectable for up to 24 hours (not shown). The expression of ribosomal protein S26, and of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts, which are not altered by these treatments, was used to monitor RNA loading. The halflife of the myf5 transcript which is close to 1 hour in proliferating myoblasts (Carnac et al., 1993, and our observations) was not affected by dexamethasone and anisomycin (data not shown). It is interesting to note that prolonged treatment with dexamethasone and anisomycin did not promote autonomous differentiation of either anti-IGFII C2 myoblasts or parental C2 myoblasts (data not shown). Fig. 2 illustrates the effect of dexamethasone and anisomycin on the accumulation of the Myf5 protein. Indirect immunofluorescence experiments were performed using a specific polyclonal antibody directed against the aminoterminal domain of the Myf5 protein. In untreated proliferative anti-IGFII myoblasts, the nuclei displayed varying levels of protein accumulation, with most cells staining weakly, and a few cells staining strongly. In contrast, the nuclei of treated cells displayed homogeneous intense staining.

Dexamethasone is a synthetic analogue of the glucocorticoid hormone cortisol. We have thus further investigated whether the effect of dexamethasone is mediated by the glucocorticoid receptor (GR), a nuclear receptor which, when activated by ligand-binding, functions as a transcription factor (Grove et al., 1980; Geisse et al., 1982). The drug RU 486 is a synthetic antiglucocorticosteroid hormone which binds to the GR with the same affinity as dexamethasone (Kd=3.10−9 M), but which reduces the nuclear transfer capacity of the ligand-receptor complex, as well as binding to the glucocorticoid response element (GRE) (Jung-Testas and Baulieu, 1983; Bourgeois et al., 1984). To antagonize GR function, anti-IGFII C2 cells were exposed for 24 hours to RU 486. These cells were then incubated in the presence of 10−7 M dexamethasone and 50 ng/ml anisomycin for 2 hours. As shown in Fig. 1 (right panel), RU 486 treatment abolished the effect of dexamethasone on myf5 expression (compare with dexamethasone-anisomycin treatment, Fig. 1B, left panel), bringing the level of myf5 induction down to that obtained with anisomycin treatment alone. This result indicates that the effect of dexamethasone upon myf5 gene expression is mediated by glucocorticoid receptor activation. Anisomycin at 50 ng/ml is known to be a potent and selective signalling agonist for the activation of immediate early (I.E.) gene induction (Cano et al., 1994). This process, called superinduction, is responsible for a strong and sustained induction of the I.E. genes c-fos and c-jun. We investigated the expression of both the jun and fos gene families (c-jun, junB, junD, c-fos, fosB, fra-1 and fra-2) by RNA blot analysis under conditions of anisomycin and dexamethasone treatment (Fig. 3). JunB transcripts were not detected in the proliferating antiIGFII C2 myoblasts and remained undetectable even after

2774 F. Auradé and others

Fig. 2. Immunocytolocalization of the Myf5 protein. Proliferating anti-IGFII C2 myoblasts were treated for 4 hours with 10−7 M dexamethasone and 50 ng/ml anisomycin (Dex. + anisomycin) without medium change. Control cells were untreated. The images correspond to the sum of the optical sections obtained by confocal microscopy. Bar, 10 µm.

untreated

superinduction (not shown). Two hours of anisomycin treatment resulted in strong activation of c-jun, junD, c-fos, fra1 and fra-2 and weaker activation of fosB (not shown), compared to the steady state levels of these transcripts in proliferating anti-IGFII C2 cells (Fig. 3, first and second lanes). However, none of these transcripts showed the same pattern of

Fig. 3. Northern blot analysis of the expression of Jun/Fos family members in anti-IGFII C2 cells. After 3 days of proliferation, cells were shifted to a medium supplemented with 1% FCS for 24 hours, then to a medium containing 10 µg/ml transferrin (control), anisomycin, or anisomycin and dexamethasone, at, respectively, 50 ng/ml and 10−7 M for 2 hours. The low level of myf5 mRNA in control cells is due to the down-regulation of the transcript under these culture conditions. For each transcript, blots and exposure times were the same, despite some background variations. S26 mRNA detection shows the homogeneity of RNA loading.

Dex. + anisomycin induction as myf5 once treated with dexamethasone and anisomycin (third lane). Addition of dexamethasone antagonizes the effect of anisomycin on fosB (not shown), c-fos, fra-1, fra2 and junD induction. In contrast, synthesis of c-jun mRNA, strikingly elevated upon superinduction, is not affected by dexamethasone treatment. Thus, among the seven transcripts of the Jun/Fos family genes that we have analysed, only c-jun mRNA accumulates upon anisomycin treatment and is not downregulated after addition of dexamethasone. Given these findings, we evaluated the role of c-jun in the induction of myf5. We tested the effect of interfering with cjun function by transiently transfecting anti-IGFII C2 cells with an expression vector encoding a dominant negative version of the mouse c-Jun protein (FLAG∆169) (Ham et al., 1995), which is missing the amino-terminal transactivation domain (amino acids 1-168). This deletion abolishes the ability of the protein to activate transcription without affecting its capacity to dimerize and to bind DNA. Anti-IGFII C2 cells were transiently transfected with the FLAG∆169 construct or a wild-type c-jun expressing vector. Transfected cells were treated with 50 ng/ml anisomycin and 10−7 M dexamethasone for 4 hours and analysed by immunofluorescence for the presence of Myf5 and FLAG∆169 or cJun proteins (Fig. 4). Analysis by confocal microscopy showed that the level of Myf5 in untransfected cells is strongly induced by the combination of anisomycin and dexamethasone (green staining), as previously observed in Fig. 2. In contrast, overexpression of the FLAG∆169 mutant Jun protein (red staining) prevented accumulation of high levels of Myf5 protein (Fig. 4A, compare left and right panels). In contrast up-regulation of Myf5 was not impaired upon overexpression of a wild-type version of c-Jun (Fig. 4B). The analysis of three independent experiments indicated that 68% of the FLAG∆169 positive nuclei expressed undetectable levels of Myf5 protein, whereas, in surrounding untransfected cells, cells transfected with a wild-type c-jun construct (Fig. 4B) or with a NLS-β-galactosidase expression vector as an unrelated nuclear epitope (not shown), only about 5% of nuclei were Myf5 negative. These results suggest an involvement of AP-1 family members in the induction of myf5 by anisomycin and dexamethasone.

Regulation of myf5 by anisomycin and dexamethasone 2775

Fig. 4. A c-Jun dominant negative protein antagonizes myf5 induction. Proliferating anti-IGFII C2 cells were transiently transfected with a plasmid encoding a flagged c-jun mutant protein Flag∆169 (A) or a wild-type c-Jun protein (B), and treated 48 hours after transfection with dexamethasone and anisomycin as in Fig. 1. Red staining: Flag∆169 mutant protein (A, right panel) or wild-type c-Jun (B, right panel). Green staining: Myf5 protein. The images correspond to the sum of the optical sections by confocal microscopy. For this reason, co-localization of red and green stainings for Myf5 and wild-type c-Jun protein detection appear in yellow. Bar, 10 µm.

We then tested whether the activation of a conventional AP1 complex (c-Jun/c-Fos) by the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) could enhance the effect of dexamethasone and anisomycin (Fig. 5). Proliferating antiIGFII C2 cells were treated for 30 minutes or for 2 hours with dexamethasone and anisomycin with or without 250 ng/ml TPA. Stimulation of myf5 expression by dexamethasone and anisomycin (Fig. 5A) was strongly repressed by TPA treatment, despite the hyperinduction of c-fos and c-jun mRNA. We have also observed that TPA alone eliminates the basal level of myf5 expression (not shown). Western blot analysis, performed on extracts from cells treated as in Fig. 5A, confirmed that TPA provoked a marked increase of c-Fos protein levels and, to a lesser extent, of c-Jun, together with the inhibition of Myf5 protein accumulation (Fig. 5B). Moreover, addition to the cells of either fresh serum or TGFβ (both inducers of c-fos expression) also antagonizes the enhancement of myf5 expression (not shown). In order to see whether the inhibitory effect of TPA required de novo protein synthesis, i.e. whether the effect of TPA was mediated by c-Fos, protein synthesis was inhibited by puromycin which, in contrast to anisomycin, does not activate stress activated protein kinases (Edwards and Mahadevan, 1992). The result presented in Fig. 6A shows unambiguously that: (1) inhibition of myf5 by TPA occurs in the absence of protein synthesis (4th and 5th lanes); (2) accumulation of myf5 mRNA upon dexamethasone and anisomycin treatment, similarly, does not require de novo protein synthesis (2nd and 3rd lanes). TPA has been widely documented to mobilize a group of phospholipid-dependant kinases belonging to the protein kinase C (PKC) family (see Jaken, 1996, for review). We used an inhibitor of the PKC family, GF 109203X, to investigate the involvement of these kinases. Fig. 6B shows that GF 109203X

Fig. 5. TPA treatment impairs myf5 mRNA and protein accumulation. (A) myf5, c-fos, c-jun and S26 transcripts were assayed by northern analysis. Dexamethasone at 10−7 M (Dex.) and anisomycin at 50 ng/ml were added, without change of medium, to proliferative anti-IGFII C2 cells in the presence or absence of TPA (250 ng/ml). Treatments were for 30 minutes and 2 hours. Control represents untreated cells. S26 shows the homogeneity of RNA loading. (B) Myf5, c-Fos and c-Jun proteins were detected by western analysis on samples recovered after 2 hours treatment performed as in A.

2776 F. Auradé and others 1) is unlikely to be due to transcript stabilization, particularly given the half-life of the transcript which is about one hour in non-treated cells, and in cells treated with dexamethasone and anisomycin (Carnac et al., 1993, and our observations). Thus, the increase is likely to be due to a stimulation of the rate of transcription of the myf5 gene. In addition, we have shown that the GR is implicated in the mechanism leading to elevated levels of myf5. Since the regulation of gene transcription via the GR has been well documented (see Cato and Wade, 1996, for review), we favor the idea that the observed increase in myf5 mRNA is due to direct transcriptional activation. Although there are precedents for the transcriptional regulation of the myogenic bHLH genes by ligand activated transcription factors, thyroid hormone, and retinoic acid receptors (Carnac et al., 1992, 1993; Albagli-Curiel et al., 1993; Downes et al., 1993; Muscat et al., 1994), the implication of the glucocorticoid hormone, dexamethasone, in the induction of myf5 gene transcription is the first positive hormonal control of myf5 gene expression to be reported to date. Fig. 6. TPA represses myf5 induction independently of protein synthesis through a mechanism involving PKC activation. myf5 and S26 transcripts were assayed by northern analysis. Proliferative antiIGFII C2 cells were pre-treated 30 minutes with TPA (250 ng/ml) before addition of dexamethasone (Dex., 10−7 M) and anisomycin (Aniso., 50 ng/ml) for 2 hours. Control represents untreated cells, S26 detection was used to assess homogeneous RNA loading. (A) Puromycin (Puro., 10 µg/ml) was added to the cells where indicated 15 minutes before TPA. (B) Cells were treated for 15 minutes prior to TPA addition with the PKC inhibitor GF 109203X (GF, 0.8 µM).

at 0.8 µM abolishes the inhibition mediated by TPA, and allows full myf5 transcript up-regulation by dexamethasone and anisomycin. This implies that some members of the protein kinase C family are involved in the negative control of myf5 expression. Our results indicate that both positive and negative regulation of myf5 gene expression occurs in the absence of de novo protein synthesis, and thus is strictly dependent on transduction events. DISCUSSION In the present paper, we describe culture conditions which result in a positive regulation of the level of myf5 mRNA and protein. The addition of the synthetic glucocorticoid dexamethasone and the pharmacological agent anisomycin leads in 2 hours to a 12-fold elevation of the steady state level of myf5 mRNA in muscle cells, without requiring fresh protein synthesis. Dexamethasone or anisomycin alone have little effect on myf5 mRNA and protein levels, demonstrating clearly that these two compounds act in synergy to positively regulate expression of the myf5 gene. Furthermore, our results implicate the action of the glucocorticoid receptor (GR) and the involvement of members of the AP-1 family in the regulation of the myf5 gene. Among the members of the jun and fos family, we propose that it is c-jun which acts, cooperatively with the glucocorticoid receptor, to enhance myf5 expression. myf5 mRNA accumulation is enhanced upon dexamethasone and anisomycin treatment A 12-fold induction of the myf5 mRNA within two hours (Fig.

c-jun plays a critical role in the positive control of myf5 Mahadevan and co-workers have demonstrated that anisomycin activates the c-Jun protein through activation of SAPK and stimulation of c-jun, and to a lesser extent of c-fos, transcription (Cano et al., 1994). Although anisomycin is known to inhibit protein synthesis at concentrations higher than 70 ng/ml, its ability to stimulate intracellular signaling is maintained at sub-inhibitory concentrations (Mahadevan and Edwards, 1991). Thus, for this study we used 50 ng/ml in order not to affect protein synthesis. However, we observe that lower concentrations of anisomycin (as low as to 1 ng/ml) in conjunction with dexamethasone still promote an induction of myf5 mRNA (F. Aurade, D. Montarras and C. Pinset, unpublished observation). Among the members of the AP-1 family, c-jun is the only gene which is strongly induced by treatment with both anisomycin and dexamethasone in anti-IGFII C2 cells. Anisomycin alone up-regulates c-fos and the fos family members fosB, fra-1 and fra-2 (Fig. 3), but addition of dexamethasone strongly attenuates this effect (Karagianni and Tsawdaroglou, 1994). Similarly, anisomycin induction of junD is also attenuated by dexamethasone (Fig. 3). Expression of a dominant negative c-Jun mutant protein in transient transfection assays resulted in strong inhibition of the dexamethasone-anisomycin effect (Fig. 4). The mutant protein (FLAG∆169) is deleted in the amino-terminal domain which encompasses the transactivation domain of c-Jun. This mutant can dimerize with other members of the Jun and Fos family and bind to DNA, but prevents transcriptional activation of AP1 dependent target genes, probably by occupation of AP-1 binding sites in place of functional Jun/Jun or Jun/Fos dimers (see Brown et al., 1994, for a discussion of the mechanism of inhibition). The results of transfection with this c-Jun mutant protein demonstrate that AP-1 family members are implicated in the anisomycin-dexamethasone induction of myf5. Positive regulation of myf5 by dexamethasone and anisomycin occurs in the absence of de novo protein synthesis (Fig. 6A). We have established that the c-Jun protein is present in cells at the time of induction while the c-Fos protein is absent (Fig. 5B). Thus, while c-fos is excluded from the positive regulation of myf5,

Regulation of myf5 by anisomycin and dexamethasone 2777 c-jun is a likely candidate, although we have not examined the presence of other AP-1 family members at the protein level. RNA blot analysis (Fig. 3) indicates that fra-1 and junD transcripts are present in the cells prior to induction by dexamethasone and anisomycin. The positive implication of AP-1 family members in myf5 expression contrasts with the inhibitory effect of c-Jun and cFos on MyoD (Lassar et al., 1989; Bengal et al., 1992), and suggests that AP-1 factors may contribute to the reciprocal regulation between myf5 and MyoD (Peterson et al., 1990; Rudnicki et al., 1992; Montarras et al., 1996). Negative regulation of myf5 gene expression occurs through activation of PKCs The phorbol ester TPA strongly antagonizes the induction of myf5 (Fig. 5A and B). Whereas TPA produced a large and sustained induction of c-fos mRNA leading to a high level of the c-Fos protein (Fig. 5B), the inhibition of protein synthesis did not alter the negative effect observed on myf5 gene expression (Fig. 6A). Moreover, since both c-fos mRNA and protein are undetectable in control cells, it is unlikely that the c-fos gene product is implicated in the inhibition of myf5 induction upon TPA treatment. Since the effect of TPA is antagonized by the inhibitor of PKCs, GF 109203X, it is likely that inhibition of myf5 induction by TPA requires activation of members of the PKC family (Fig. 6B). PKCs were shown previously to impair the activity of the muscle regulatory proteins MyoD, myogenin and MRF4 (Li et al., 1992a,b; Hardy et al., 1993; for review, see Lassar et al., 1994). Our results indicate that the negative action of PKCs may also occur, in the case of myf5, at the transcriptional level. Possible mechanism for the synergistic effect of dexamethasone and anisomycin upon myf5 expression Our results suggest that expression of the myf5 gene may be positively controlled by two types of transcription factors: members of the AP-1 family and the glucocorticoid receptor activated by dexamethasone. The synergy between AP-1 members and the glucocorticoid receptor is reminiscent of the regulation of eucaryotic genes coding for proliferin, α-fetoprotein and collagenase 1. GR and AP-1 bind either to a partially overlapping promoter site (composite AP-1/glucocorticoid response element) in the case of mouse proliferin and α-fetoprotein genes (Diamond et al., 1990; Zhang et al., 1991), or to a classical AP-1 site in the promoter sequence of the human collagenase-1 gene (Teurich and Angel, 1995). While GR inhibits c-Jun/c-Fos transactivation activity, GR enhances the activity of c-Jun homodimers. Although we have excluded c-Fos from the negative regulation of myf5 gene expression, this type of mechanism illustrates possible interactions between activated GR and alternative AP-1 complexes for the regulation of several eucaryotic genes. Another possibility, which does not exclude the one above, is that the synergy of action of dexamethasone and anisomycin could arise from an increased accessibility of the myf5 promoter to transcription factors due to conformational changes in chromatin domains mediated by one or both of these compounds (see Zaret and Yamamoto, 1984; Barratt et al., 1994, for discussion). Identification and characterisation of

the myf5 regulatory elements will be necessary in order to clarify these issues. Dexamethasone and anisomycin promote neither myf5 gene expression nor myogenesis in the pluripotent embryonic cell line C3H10T1/2 (data not shown). Since this cell line expresses both c-jun and the glucocorticoid receptor, this result demonstrates that the up-regulation of myf5 expression mediated by anisomycin and dexamethasone treatment relies on transcriptional enhancement rather than on transcriptional activation of a silent gene. What happens in vivo? The widespread expression of c-jun during development in proliferating tissues and its high levels of expression in certain differentiating tissues, including skeletal muscle, has led to the idea that it may be involved both in the proliferative response of early embryonic cells to growth factors and in the differentiation of specific cells types during embryogenesis (Wilkinson et al., 1989). No muscle abnormality was described in either c-jun or GR deficient mice (Johnson et al., 1993; Cole et al., 1995). Determining whether muscle development is affected in these mice, however, may not be straightforward. The muscle phenotype of myf5 deficient embryos is relatively subtle: in these embryos, initiation of myogenesis, which normally occurs at 8 days post-coitum (Ott et al., 1991), is delayed by 2 days, and the appearance of myoblasts relies on MyoD expression (Braun et al., 1992; Rudnicki et al., 1993). c-jun expression is also associated with muscle regeneration and denervation (Bessereau et al., 1990). Interestingly, myf5 is also mobilised in muscle regeneration; its expression is associated with the reentry of satellite cells into the cell cycle (Smith et al., 1994). It remains now to determine when and to what extent the myf5 regulatory mechanisms we have uncovered contribute to myogenesis. It is also possible that the properties of myf5 as an immediate early gene may be important in regeneration following the stress resulting from injury. We thank Dr J. Ham for the cJun∆169 construct, Dr F. Mechta for junD, RSV-wild-type c-jun and AH119 plasmids, Dr J. Kirilovsky for the gift of the GF 109203X inhibitor, R. Hellio for confocal microscopy, and G. Antolini for manuscript preparation. This work was supported by grants from the Pasteur Institute, the Centre National de la Recherche Scientifique and the Association Française contre les Myopathies. F.A. was the recipient of a fellowship from the Association Française contre les Myopathies, C.L. benefited from a grant from the Société des Amis des Sciences, M.P. was supported by a Schrödinger fellowship of the FFW.

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