α-mediated erk, jnk and p38 activation regulates the ...mek inhibitor suggest a key role of the erk...

IntroductionRhabdomyosarcoma (RMS), the most common paediatric softtissue sarcoma, arises from muscle precursor cells. In RMS, anumber of well known muscle-specific markers are expressedboth in vivo and in vitro (Bouche et al., 1993; Merlino andHelman, 1999; Tonin et al., 1991). RD (rhabdomyosarcomacells), the embryonal RMS tumor cell line, fails to differentiatein spite of the expression of myogenic-specific transcriptionfactors such as myogenin and MyoD. RD cells are believed togain a growth advantage through the action of a mutant N-rasoncogene (Chardin et al., 1985; Kong et al., 1995; Lassar etal., 1989; Olson et al., 1987) and/or mutated tumor suppressorp53 (Germani et al., 1994), which lead to uncontrolledproliferation and secretion of autocrine growth factors andmyogenic inhibitors such as insulin-like growth factors-II(IGF-II) and transforming growth factor-β (TGF-β) (Bouche etal., 2000; De Giovanni et al., 1995; Derynck et al., 1987;Minniti et al., 1994). Nevertheless, the myogenic program can

be rescued by fusing RD cells with fibroblasts, which suggeststhat a positive regulator of myogenic transcription factors isabsent in RD cells but is present in the heterocaryon (Tapscottet al., 1993).

Unlike normal myoblasts, which respond to both theproliferative and differentiative action of insulin and IGFs(Coolican et al., 1997; Florini et al., 1991), RD cells do notdifferentiate in response to IGF. Recent reports have suggestedthat elevated cyclin D1 and CDK activities contribute to theinability of RD cells to arrest growth when cultured in mitogen-deprived medium (Knudsen et al., 1998). Nevertheless, RDcells treated with PKC activators, such as the tumor promoterTPA, progressively acquire a more elongated shape, which isthe typical myogenic morphology, and become unresponsiveto mitogens and undergo growth arrest and myogenicdifferentiation (Aguanno et al., 1990). This suggests thatactivated PKC interferes with the transduction of mitogenicsignals, thereby leaving myogenic transcription factors free to

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We have previously suggested that PKCα has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediatedgrowth arrest and myogenic differentiation in humanembryonal rhabdomyosarcoma cells (RD).

Here, by monitoring the signalling pathways triggeredby TPA, we demonstrate that PKCα mediates these effectsby inducing transient activation of c-Jun N-terminalprotein kinases (JNKs) and sustained activation of both p38kinase and extracellular signal-regulated kinases (ERKs)(all referred to as MAPKs). Activation of MAPKs followingectopic expression of constitutively active PKCα, but not itsdominant-negative form, is also demonstrated.

We investigated the selective contribution of MAPKs togrowth arrest and myogenic differentiation by monitoringthe activation of MAPK pathways, as well as by dissectingMAPK pathways using MEK1/2 inhibitor (U0126), p38inhibitor (SB203580) and JNK and p38 agonist(anisomycin) treatments. Growth-arresting signals aretriggered either by transient and sustained JNK activation(by TPA and anisomycin, respectively) or by preventing

both ERK and JNK activation (U0126) and are maintained,rather than induced, by p38. We therefore suggest a keyrole for JNK in controlling ERK-mediated mitogenicactivity. Notably, sarcomeric myosin expression is inducedby both TPA and U0126 but is abrogated by the p38inhibitor. This finding indicates a pivotal role for p38 incontrolling the myogenic program. Anisomycin persistentlyactivates p38 and JNKs but prevents myosin expressioninduced by TPA. In accordance with this negative role,reactivation of JNKs by anisomycin, in U0126-pre-treatedcells, also prevents myosin expression. This indicates that,unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activationsupports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with theMEK inhibitor suggest a key role of the ERK pathway inregulating myogenic-related morphology in differentiatedRD cells.

Key words: PKCα, MAPKs, RD

Summary

PKCα-mediated ERK, JNK and p38 activationregulates the myogenic program in humanrhabdomyosarcoma cellsAnnunziata Mauro 1,*, Carmela Ciccarelli 1,*, Paola De Cesaris 1, Arianna Scoglio 2, Marina Bouché 2,Mario Molinaro 2, Angelo Aquino 3 and Bianca Maria Zani 1,‡

1Department of Experimental Medicine, University of L’Aquila, Via Vetoio, Coppito II, 67100 L’Aquila, Italy2Department of Histology and Embryology, University of Rome ‘La Sapienza’, Via Scarpa 14, 00161 Rome, Italy3Department of Neuroscience, Section of Pharmacology and Medical Oncology, University of Rome Tor Vergata, Via di Tor Vergata 135, 00133Rome, Italy *These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])

Accepted 28 June 2002Journal of Cell Science 115, 3587-3599 © 2002 The Company of Biologists Ltddoi:10.1242/jcs.00037

Research Article

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initiate muscle differentiation. Thus, knowledge of thedownstream pathways of PKC in RD cells might help thesearch for agonists, other than tumor promoters, capable ofreverting cells back to their non-transformed phenotype.

Growth factor signal transduction pathways involve severalkinases, including those of the MAPK and PI3K/Akt kinasefamilies (Garrington and Johnson, 1999). Within the MAPKfamily, ERKs, JNKs and p38 have been implicated in relayingextracellular signals to the nucleus. MAPK pathways, byregulating transcription factory activity (Hill and Treisman,1995; Treisman, 1996), mediate specific responses, includingproliferation, differentiation, apoptosis and stress (Minden andKarin, 1997; Pang et al., 1995; Racke et al., 1997; Robinsonand Cobb, 1997; Traverse et al., 1992). In a typical pathway,Ras activation initiates a protein kinase cascade that leads toMAPK activation through the intervening protein kinases Rafand MEKs.

Although downstream effectors of MAPKs that elicit specificresponses have yet to be fully identified, recent reports havesuggested that MEF2 is a substrate of activated p38 kinase, atranscriptional target for signalling pathways that controlskeletal myogenesis (Francisco and Eric, 1999; Zhao et al.,1999). In this regard, p38 has been reported to induce muscledifferentiation in both L8 and C2C12 (Cuenda and Cohen,1999; Zetser et al., 1999), whereas PI3K/Akt is thought to beinvolved in differentiation and hypertrophy (Bodine et al., 2001;Jiang et al., 1998; Rommel et al., 2001) of L6 myogenic lines.Moreover, ERKs are activated in C2C12 when myogenesis ispromoted by the removal of mitogens (Gredinger et al., 1998).Recently, a pivotal role for p38 has been documented in thepathological myogenic differentiation of a number of RMSlines, including RD cells transfected with the p38 upstreamkinase MKK6 (Puri et al., 2000). In addition, evidence hasemerged indicating that crosstalk between MAPKs, such as theantagonistic effect of JNKs and p38 in the L6 and C2C12myogenic cell lines, controls myogenesis (Meriane et al., 2000).

The involvement of both PKC-dependent and PKC-independent pathways of Raf/MEK activation in response to anagonist has been reported as a possible mechanism of MAPKactivation (Qiu and Leslie, 1994; Schonwasser et al., 1998). Theinvolvement of PKCs in Raf activation has been mainly studiedin cells that activate ERKs in response to TPA (El Shemerly etal., 1997; Kaneki et al., 1999; Miranti et al., 1999; Racke et al.,1997). In fact, Raf and MEK phosphorylation have beenobserved in cells co-transfected with PKCα and β, whichprovides strong evidence for the involvement of PKCs in theregulation of these pathways (Marquardt et al., 1994; Sozeri etal., 1992). Furthermore, MEK-1 activation is mediated byconventional, novel and atypical PKC isoforms in a Raf-dependent and -independent manner (Schonwasser et al., 1998).

In this study, we report that activation of the myogenicprogram of RD cells is dependent on PKCα-mediated ERK,JNK and p38 activation. The use of MAPK inhibitors andagonists allowed us to dissect TPA-mediated kinase activationand thereby correlate a cell response with specific kinasepathways. The results obtained show an anti-mitogenic role foractivated JNKs and a role for activated p38 in the expressionof muscle-specific genes. Moreover, ERK activation may beinvolved in the maintenance of the RD myogenic-relatedmorphology.

Materials and MethodsCell cultures and treatmentsThe human rabdomyosarcoma cell line (ATCC, Rockville MD) wascultured as previously described (Germani et al., 1994). One day afterplating, cells were treated with 10–7, 10–8 and 10–9 M TPA to producea dose-response curve, and 10–7 M TPA was established as the dosethat was effective for both MAPK activation and myogenicdifferentiation. For inhibitory studies, 10 µM U0126 (Promega) and2.5 µM SB203580 (Calbiochem) were added 5 minutes and 1 hour,respectively, before treatments were started. These concentrations areeffective in dose-response experiments. For PKC inhibition, 60 nMRo320432 (Calbiochem) was added 6 hours before any treatment wasstarted. 10 ng/ml of anisomycin were added for 30 minutes and 5 days,1 hour prior to TPA treatment. This concentration is effective for JNKand p38 activation without altering phospho-ERKs in a dose-responseexperiment.

For growth analysis, RD cells were harvested in trypsin-EDTA andcounted in a hemocytometer chamber.

Subcellular fractionation, SDS-PAGE and immunoblottingTotal extracts were prepared by scraping cells in 2% SDS containing2 mM phenyl methyl sulphonyl fluoride (PMSF), 10 µg/ml antipain,leupeptin and trypsin inhibitor, 10 mM sodium fluoride and 1 mMsodium orthovanadate and sonicating them for 30 seconds. Nuclearand membrane fractions were obtained by lysing cells in 10 mM Tris-HCl pH 7.5 containing protease and phosphatase inhibitors,homogenized by 20 strokes in a Dounce homogenizer andcentrifuging them at 900 g. The nuclei-containing pellets wereresuspended in 2% SDS containing proteases and phosphatasesinhibitors. The 900 g supernatants were centrifuged at 100,000 g tosediment the enriched membrane fractions. An aliquot of total lysatesand nuclear and membrane fractions were used to evaluate the amountof proteins (Lowry et al., 1951). Equal amounts of total lysates ormembrane and nuclear fractions (100 µg) were separated by 10%SDS-PAGE (Laemmli, 1970) and transferred to a nitrocellulosemembrane (Hybond C Extra Amersham) following standardprocedures (Towbin et al., 1979). 8% SDS-PAGE was used for myosindetection. Immunoblottings were performed with the followingantibodies: 1:1000 of anti-phospho-ERKs, anti-phospho-JNKs andanti-phospho-p38 (New England Biolabs, NEB), all of whichrecognise the phosphorylated/activated forms of these kinases; 1:250antibodies which recognize the total ERKs, JNKs and p38 (SantaCruz); 1:10 MF20 (supernatant of hybridoma kindly provided by D.Fischman, Cornell University); 1:1000 anti-PKCα and β1(Transduction Laboratory); 1:500 anti-PKCγ as previously described(Aquino et al., 1990); 1:500 anti-PCNA and 1:250 anti-hemagglutinin(Santa Cruz). Peroxidase-conjugated anti-mouse (1:3000) or anti-rabbit IgG (1:1500) purchased from Amersham or from Santa Cruzwas used for ECL (Amersham) detection.

As the anti-phospho-MAPK antibodies detect each kinase onlywhen dually phosphorylated, an increase in phosphorylation is anindex of their activation. Densitometric analysis of bands, relative toboth total and phosphorylated proteins, provided quantification(phospho-MAPK:total-MAPK) of TPA-induced ERK, JNK and p38activation expressed as a fold increase over the control value, whichwas arbitrarily set at 1.

Immunofluorescence and FACS analysisFor the immunofluorescence study, untreated and treated cells werefixed with 4% paraformaldehyde (SIGMA) for 15 minutes at roomtemperature, washed three times, permeabilized in 0.2% Triton X-100in PBS for 20 minutes and saturated for 1 hour with 1% of BSA(immunoglobulin free, SIGMA) in PBS. Undiluted MF20 wasincubated for 1 hour and immunocomplexes were detected using

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3589PKCα-mediated MAPK activation during myogenesis

(1:50) FITC-conjugated rabbit anti-mouse IgG (Zymed). For FACSanalysis, untreated and treated cells were washed, and the pellets wereresuspended in 50% FBS in PBS. Cells were fixed overnight afterthree parts of 70% cold ethanol had been added. Fixed cells werewashed twice and resuspended in PBS additioned with 50 µg/ml ofpropidium iodide and 100 U/ml of DNAse-free RNAse (SIGMA).Cell cycle analysis of cells stained with propidium iodide wasperformed using a Coulter Epics XL flow cytometer (BeckmanCoulter).

TransfectionOne day after plating (3.5×105 cells/ml), cells were transientlytransfected with 0.5 µg of constitutively active PKCα-coding vector(A25E) or a dominant-negative version of PKCα (K368R), kindlyprovided by Dr Baier (University of Innsbruck) (Baier-Bitterlich etal., 1996) or with 0.5 µg of constitutively active MKK2 (KW71A),kindly provided by Dr Ahn (Howard Hughes Medical Institute)(Mansour et al., 1996). In the present paper, MKK2 is named MEK2,according to the current nomenclature (Cohen, 1997). For the in vivoc-Jun and Elk1 trans-reporting system (Stratagene), cells weretransfected with the following plasmids: (i) 0.1 µg of pFA-c-Jun orpFA-Elk1 activator plasmids, which express proteins that consist ofthe DNA-binding domain of yeast GAL4 and the activation domainsof c-Jun or Elk1; (ii) 1 µg of pFR-Luc plasmid, which contains asynthetic promoter with five tandem repeats of the GAL4-bindingsites controlling expression of the luciferase gene. In this assay thefusion activators, which are phosphorylated and activated by JNKsand ERKs, transactivate the luciferase promoter. Thus, the extent ofluciferase activity reflects the activation of a specific kinase and thecorresponding signal transduction pathway. Lipofectamine Plusreagent was used as the transfectant according to the manufacturer’sinstructions (GIBCO BRL). One day after transfection, cells weretreated with TPA, or left untreated, for 1 day. Total lysates fromtransfected cells were processed either for SDS-PAGE andimmunoblotting or assayed for luciferase activity according to themanufacturer’s instructions (Promega).

ResultsTPA activates ERKs, JNKs and p38To assess the effect of TPA on ERK, JNK and p38 (MAPKs)activation, time-course experiments were performed bytreating RD cells with 10–7 M TPA, the concentration that wefound to be effective upon early activation of ERKs, JNKs andp38 and late accumulation of sarcomeric myosin (Aguanno etal., 1990) (data not shown). Immunoblots of total cell lysateswere performed with antibodies against phospho-activeMAPKs and total MAPKs.

Analysis of MAPK activation (Fig. 1) shows, in control RDcells (C0), a high level of phospho-ERKs and phospho-p38 buta low level of phospho-JNKs. In culture, a drastic decrease inphospho-ERKs and phospho-JNKs and a significant increasein phospho-p38 occur over time (C2-C5, Fig. 1). Short TPAtreatment (30 minutes to 7 hours) induces a rapid and sustainedincrease in both phospho-ERKs (2.3) and p38 (2.4) incomparison with the levels of control cells, whereas theincrement in phospho-JNK/p46, though more marked (8.6), isless persistent.

After prolonged treatments (2-5 days), neither basal (C2, C5)nor TPA-induced phospho-JNKs are detectable, whereas adecrease in ERK phosphorylation of control cells causes moreevident phospho-ERK stimulation (3.2-4.3), which is in contrastto the decrease in p38 stimulation following an increase in the

basal phosphorylation level in control cells. Moreover, TPAdoes not alter the expression level of total kinases and the foldincreases in ERK, JNK and p38 phosphorylation provide aquantification of kinase activation (see Materials and Methods).The ongoing activation of MAPKs is also demonstrated by amarked increase in the phospho-ATF2 and phospho-c-jun, thedownstream targets of MAPKs (data not shown). These resultsdemonstrate that TPA induces the concomitant activation ofthree distinct MAPK cascades.

PKCα-dependent MAPK kinase inductionWe previously suggested that the effects of TPA on RD celldifferentiation may be caused by the activation of PKCα ratherthan other isoforms, (γ and β1) (Bouche et al., 1995). In thispaper, we demonstrate that there is a significant and rapid(30 minutes) TPA-induced selective PKCα membranetranslocation that is not accompanied by either any expressionof PKCγ or an increase in PKCβ1 membrane translocation(Fig. 2A).

Fig. 1.TPA induces phosphorylation/activation of ERKs, JNKs andp38. The time course of ERK, JNK and p38 phosphorylations in RDcells, either untreated (C0, C2d and C5d) or treated with 10–7 M TPAfor different times (30 minutes to 5 days). Immunoblots of totallysate were performed using antibodies against phospho-active formsof MAPKs. Each blot was re-probed with antibodies that recognizetotal proteins. Densitometric analysis of bands, relative to both totaland phosphorylated proteins, provided quantification (phospho-MAPK:total-MAPK) of TPA-induced ERK, JNK and p38 activationexpressed as a fold increase over the control value arbitrarily set at1.The data shown are representative of three independentexperiments.

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PKCα-induced activation of ERKs, JNKs and p38 was thenstudied in transiently transfected RD cells with a constitutivelyactive mutant form of PKCα (A25E) and with a control vector(CMV) by analysing the phosphorylated forms of ERKs, JNKsand p38 after immunoblottings of total lysates 24 hours aftertransfection. The results in Fig. 2B show that the increase inPKCα expression (two-fold) in A25E-transfected cells over thelevel of control vector CMV-transfected cells induces asignificant increase in ERK phosphorylation (1.7-fold) and aneven more marked increase in the levels of phosphorylatedJNKs (2.2-fold for p46 and 7.3-fold for p54) and p38 (7.3-fold). It is noteworthy that a marked phosphorylation ofJNK2/p54 is present in PKCα-transfected cells. Moreover, theextent of kinase activation in PKCα-transfected cells isconsistent with that observed after 30 minutes to 7 hours ofTPA treatment (Fig. 1).

To further demonstrate the role of PKCα in MAPKactivation, we tested the effect of both the wild-type (WTPKCα) and dominant-negative mutant of PKCα ( DN PKCα)by using a c-Jun or Elk1 trans-reporting system designed forthe assessment of in vivo MAPK activation (see Materials andMethods). In these experiments, RD cells were co-transfectedwith WT PKCα or DN PKCα with activator plasmid (pFA-c-Jun or pFA-Elk1) and a reporter plasmid pFR-Luc and wereleft untreated or were treated with TPA. The results in Fig. 2Cshow that Elk1- and c-Jun-driven luciferase activity(respectively, Elk-Luc and Jun-Luc) are induced by TPA inboth control vector- (CMV+TPA) (4.7- and 2.1-foldrespectively) and PKCα-transfected cells (WT PKCα + TPA)(4.2- and 3-fold respectively). By contrast, the ectopicexpression of the DN-mutant form of PKCα decreases TPA-induced luciferase activity (DN PKCα +TPA) by about 61%for Elk-Luc and 38% for Jun-Luc. Taken together, these resultsindicate that activation of PKCα by TPA mediates MAPKpathway activation.

Evidence that PKCα is an upstream effector of MAPKactivation was provided by the use of PKC inhibitor Ro320432

at a concentration (60 nM), which selectively inhibits thePKCα and β isoforms (Wilkinson et al., 1993). To analyseMAPK phosphorylation, RD cells were pre-treated withRo320432 for 6 hours and were then left untreated or weretreated with TPA for 30 minutes. Immunoblotting analysisshows that Ro320432 does not affect the level ofphosphorylated MAPKs in untreated cells but completelyprevents TPA-induced phosphorylation of ERKs, JNKs andp38 (Fig. 3A).

Likewise, to assess PKCα dependence on growth arrest andmyogenic differentiation, growth curve (0-6 days) and latesarcomeric myosin heavy chain (MHC) (6 days) accumulationwere analysed in RD cells pre-treated with Ro320432 and thentreated with TPA. The results, shown in Fig. 3B, clearlydemonstrate that PKC inhibition prevents TPA-mediatedgrowth arrest (Ro+TPA) without affecting the level of controlcell proliferation (Ro). Fig. 3C shows that Ro320432 inhibitsthe TPA-induced sarcomeric MHC accumulation. It isnoteworthy that, since PKCβ activation is not altered by TPAtreatment, the results obtained by using Ro320432 can beascribed to the selective inhibition of PKCα. Taken together,these results demonstrate that PKCα is an upstream effector ofboth TPA-induced MAPK activation and myogenic phenotypeexpression in RD cells.

MEK1/2 inhibition induces downregulation of both ERKand JNK pathways and correlates with morphologicalchangesSustained ERK activation after prolonged treatment (2-5 days)with TPA may involve ERKs in the induction of late biologicalresponses to TPA (Bennett and Tonks, 1997). We investigatedthe MEK1/2 inhibitor, U0126, which was used effectively toinvestigate the role of ERKs in regulating cellular responsesowing to the fact that it inhibits the MEK/ERK pathway bypreventing the activation of MEK1/2 and by blocking activatedMEK1/2 (Favata et al., 1998). We initially determined whether


Fig. 2. PKCα-dependent MAPK kinase phosphorylation.(A) Immunoblots of total brain extract and of cytosoland membrane fractions prepared from control and RDcells treated with TPA for 30 minutes. (B) Immunoblotsof total lysate from RD cells transfected with controlvector (CMV) and with constitutively active PKCα-expressing vector (A25E) using the same antibodies asthose described in the legend for Fig. 1 and with specificantibodies that recognize PKCα, β1 and γ. The filter wasnormalized with an antibody specific to p54/JNK2. Thedata shown are representative of four independent experiments. The increases (fold over the CMV-transfected cells) in phosphorylation levelsare indicated for each sample. (C) Luciferase assay for detection of activated c-Jun and Elk1 (see Materials and Methods). RD cells co-transfected with activator plasmid GAL4-jun or GAL4-Elk1 and reporter plasmid GAL4-luc together with empty vector (CMV), WT PKCα orDN PKCα (dominant-negative version, K368) were left untreated or treated with TPA for 24 hours 1 day after transfection.


the simple inhibition of ERKs leads to an alteration in the basaland TPA-induced phosphorylation levels of p38 and JNKs. Atime-course experiment was performed with 10 µM U0126, theminimal concentration required to obtain maximalinhibition of ERK phosphorylation in a dose-responseexperiment (data not shown).

Thus, the pattern of MAPK activation was analysedafter immunoblotting of RD cells (Fig. 4A) pre-treatedwith 10 µM U0126 and left untreated (U) or treated withTPA for 30 minutes, 2 and 5 days (U+TPA). Within 30minutes, both in the presence and in the absence of TPA,a marked reduction in ERK and, unexpectedly, JNKphosphorylation is observed in U0126-treated cells (Fig.4A). By contrast, p38 phosphorylation is insteadincreased after 30 minutes of U0126 treatment. After 2

and 5 days of treatment, the U0126-mediated inhibition ofERK phosphorylation is still present, both in the absence andpresence of TPA, although to a lesser extent, whereas JNKphosphorylation is undetectable in both treated and untreatedcells. After 2 and 5 days, phospho-p38 does not significantlychange in U0126-treated cells, either with or without TPA, butdoes increase in control cells (C; Fig. 4A) as already seen inthe experiment shown in Fig. 1. It is noteworthy that p38phosphorylation is rapidly (30 minutes) stimulated by U0126,which suggests that, upon MEK inhibition, either p38 is theonly active MAPK or the activation of p38 is inverselycorrelated with the inactive state of ERKs or JNKs.

To rule out the possibility of a time-dependent inactivationof U0126 in the culture medium, we tested the 30 minute and5 day conditioned U0126-containing media for their ability toinhibit ERK phosphorylation within 30 minutes of incubationof parallel RD cultures. Immunoblotting analysis shows thatboth these conditioned media retain their capacity to preventERK phosphorylation (Fig. 4B).

Furthermore, prolonged U0126 treatment (3-6 days) inducesdrastic morphological changes in both untreated and TPA-

Fig. 3.The PKC inhibitor abrogates MAPK activation, growth arrestand myosin expression. (A) Immunoblots of total lysates from RDcells, either untreated (C) or treated with TPA for 30 minutes, pre-treated (6 hours) with 60 nM PKC inhibitor Ro320432 (Ro) in thepresence and absence of TPA, using the antibodies described in thelegend for Fig. 1. (B) Growth curve of RD cells either untreated (C)or treated with TPA for different times (0, 1, 3 and 6 days, TPA) andafter pre-treatment with the PKC inhibitor (Ro, Ro + TPA). Eachvalue represents the mean±s.e.m. of three samples. (C) Immunoblotsof total lysate from cells, either untreated (C) or treated with TPA for6 days (TPA) and pre-treated with Ro320432, in the absence orpresence of TPA, (Ro, Ro + TPA) using anti-myosin heavy chain(MHC) antibody. The data shown are representative of threeindependent experiments.

Fig. 4.Effect of U0126 on ERK, JNK and p38phosphorylations. (A) Immunoblots of total lysates fromuntreated cells (C), cells treated with TPA (TPA) and 10 µMU0126 in the absence (U) or presence of TPA (U + TPA) for30 minutes, 2 and 5 days, using anti-phospho-active ERK andJNK antibodies. For normalization, filters were re-probed withantibodies that recognize total proteins. (B) Immunoblots oftotal lysate from cells incubated for 30 minutes with controlmedium (C30 min, C5d) or with U0126-containingconditioned medium derived from cells treated with U0126 for30 minutes (U 30 min) and 5 days (U 5d), using anti-phospho-active ERK antibody. The data shown are representative offour independent experiments.

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treated cells, appear as round-shaped (Fig. 5), although no celldetachment is observed even after 15 days of treatment. Theseresults show that inhibition, by U0126, of the MEK/ERKpathway in RD cells parallels inhibition of JNK phosphorylationand is accompanied by a drastic morphological change.

Interestingly, the U0126-mediated JNK downregulationsuggests that MEK1/2 are upstream activator kinases of JNKs,just as U937 cells have been reported to be (Franklin and Kraft,1995). To verify this hypothesis, RD cells were transfectedwith constitutively active HA-tagged-MEK2 or empty vector;subsequent immunoblotting analysis of total lysates shows asignificant increase in the level of both phospho-JNK andphospho-ERK in MEK2-transfected cells (MEK2) whencompared with control cells (CMV) (Fig. 6A).

To assess the in vivo activation of the JNK pathway, parallelRD cell cultures were co-transfected with either constitutivelyactive HA-tagged-MEK2 (CA MEK2) or control vector(CMV) and with both pFA-c-Jun activator plasmid and areporter plasmid pFR-Luc. A dramatic increase in luciferaseactivity is detected in MEK2-transfected cells when comparedwith control vector-transfected cells (Fig. 6B).

Taken together, these results demonstrate that, in RD cells,MEK2 is an upstream activator kinase of JNK.

Selective JNK activation as well as ERK and JNKmodulation induce growth arrestThe concomitant deletion of MAPK activation, growth arrestand myogenic-specific marker expression induced by the PKCinhibitor (Fig. 3) led us to investigate whether ERK, JNK andp38 pathways play distinct roles in these effects by using aselective MAPK agonist or inhibitor. To study possible changesin growth potential of RD cells, we used anisomycin, which isreported to act as a true signaling agonist of JNKs and p38

(Hazzalin et al., 1998), U0126, shown here to inhibit ERKs andJNKs (Fig. 4), and SB203580, which inhibits p38.

We first performed a time-course experiment to investigatewhether long-lasting anisomycin treatment induces persistentJNK activation without altering cell viability. Ten ng/ml ofanisomycin is the dose that induces maximal JNK activationand is ineffective on ERKs either in the presence or absence ofTPA (data not shown) (Cano et al., 1994). For the time-courseexperiment, cells were left untreated or were treated, fordifferent periods of time, with 10 ng/ml of anisomycin, added1 hour before TPA treatment. Immunoblots of total lysate showthat anisomycin persistently stimulates (30 minutes-5 days)phosphorylation of p38 and JNKs, though it does this to alesser extent at 5 days (Fig. 7A). Moreover, 3 day anisomycinpre-treatment does not impair the activation of myosinexpression during chase in the presence of TPA (Fig. 7B) evenat doses as high as 50 ng/ml. Notably, anisomycin-pre-treatedcells synthesize more myosin than control cells during chasein the presence of TPA (Fig. 7B). These results rule out thepossibility that prolonged anisomycin treatment affects cellviability.


Fig. 5.Effects of U0126 on the morphology of RD cells. Phasecontrast morphology of RD cells either untreated (C) or treated withTPA for 6 days in the absence (TPA) and in the presence of U0126(U + TPA).

Fig. 6. MEK2-dependent JNK activation. (A) Immunoblots of RDcells transfected with the constitutively active form of HA-taggedMEK2 (CA-MEK2) or with the empty vector (CMV) usingantibodies that recognize phospho-active ERKs and hemagglutinin(HA). (B) A luciferase assay for detection of activated JNKs (seeMaterials and Methods); luciferase activity (units/plates) was assayedin total lysates from RD cells co-transfected with a constitutivelyactive form of MEK2 (CA-MEK2) or with empty vector (CMV) andboth with activator plasmid GAL4-Jun and reporter plasmid GAL4-luc. The data shown are representative of two independentexperiments.


We therefore investigated whether cell proliferation isaffected by prolonged JNK or p38 activation by treating cellswith anisomycin, in the presence of the p38 inhibitorSB203580, to exclude the contribution of p38. 2.5 µM ofSB203580 is the minimal concentration that does not affecteither ERK and JNK activation or, as recently reported,PKB/Akt (Lali et al., 2000) (data not shown). Cells weretreated and the number of cells was counted after 2 to 6 daysof treatment. Fig. 8A shows that anisomycin induces drasticgrowth arrest, which is not modified by SB203580. Similarly,TPA, which concomitantly activates ERKs, JNKs and p38, alsoinduces growth arrest, which is not modified by SB203580even after 4 days of treatment (Fig. 8B). However, a 30%growth increase, after 6 days of treatment, occurs inSB203580-treated cells with or without TPA. These results ledus to hypothesize that the growth arrest pathway is triggeredby highly activated JNKs, rather than p38, to counterbalancethe mitogenic effects of a threshold level of active ERKs.

To verify this hypothesis, we used U0126, which, bydownregulating ERK and JNK pathways (Fig. 4A), drasticallyalters the critical MAPK pathways balance, which is, in turn,likely to be the cause of the transduction of mitogenic signals.Cells were treated with U0126 for different periods of time,and the number of cells was counted after 2 to 6 days of culture.Fig. 8C shows that U0126 induces drastic growth arrest, whichis not reversed by p38 inhibition for up to 4 days of culture,whereas a 38% growth recovery occurs between days 4 and 6of SB203580 treatment.

Moreover, in order to investigate whether the decrease in cell

numbers (Fig. 8A-C) observed during the various treatmentswas a result of withdrawal from the cell cycle, we analysed thenuclear distribution of proliferating cell nuclear antigen(PCNA), which is known to be downregulated in growth

Fig. 7.Effects of anisomycin on JNK and p38 phosphorylations.(A) Immunoblots of total lysates of control (C) and TPA-treated cellsboth in the absence (TPA) and in the presence of 10 ng/mlanisomycin (AN, AN + TPA) for 30 minutes and 5 days usingantibodies specific for phospho-active JNKs and p38. Fornormalization, filters were re-probed with antibodies that recognizetotal protein. (B) Immunoblots, using anti-sarcomeric MHCantibody, of total cells lysates from RD cells untreated (C) or treatedwith different doses of anisomycin (5, 10, 50 ng/ml, AN) for 3 daysand chased for a further 3 days in the presence and in the absence ofTPA. The data shown are representative of three differentexperiments.

Fig. 8.Effects of SB203580, U0126 and anisomycin on growthpotential. Growth curves of RD cells untreated (C) or pre-treated for1 hour with SB203580 (SB) and (A) treated with anisomycin (AN),(B) treated with TPA or (C) with U0126 for 2, 4 and 6 days. Eachvalue represents the mean±s.e.m. of three samples. (D) Immunoblotsof nuclear extracts from RD cells, either untreated (C) or treated withTPA, anisomycin and U0126 using the anti-PCNA antibody. Thedata shown are representative of three independent experiments.

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arresting cells (Mercer et al., 1991). The distribution of RDcells in the cell cycle by flow cytometric analysis was alsoanalysed. Immunoblots of nuclear extracts from untreated cellsand from cells treated with TPA, U0126 and anisomycin for 3days (Fig. 8D) shows a consistent reduction in nuclear PCNAin TPA-treated cells, whereas nuclear PCNA in U0126- andanisomycin-treated cells is undetectable. Furthermore, theresults of the flow cytometric analysis point to G1 arrest inTPA-, anisomycin- and U0126-treated cells between days 1and 4 of treatment (Table 1).

Taken together, these results indicate that JNK activation orcomplete ERK and JNK downregulation are sufficient toinduce growth arrest, whereas p38 activity is likely tocontribute to maintaining a steady state in RD cells alreadytending towards myogenic differentiation.

Opposite roles of p38 and sustained JNK activation inmyogenic differentiationThe differentiated phenotype of myogenic lines, including RD,has recently been ascribed to selective p38 activation (Puri etal., 2000; Wu et al., 2000a). In this study, since activated p38was detected in the absence (U0126) or in the presence ofdifferent levels of activated ERKs and JNKs (TPA) or in thepresence of highly and persistently activated JNKs(anisomycin), we investigated whether activated p38 isnecessary and sufficient to induce myogenic phenotypeexpression independently of other activated MAPKs. For thispurpose, analysis of sarcomeric MHC expression wasperformed by immunoblotting of lysates (Fig. 9A) and byimmunofluorescence (Fig. 9B) in RD cells treated with TPA,U0126, in the presence and in the absence of SB203580, andwith TPA in the presence of anisomycin for 6 days. As shownin Fig. 9A, both U0126-treated and TPA-treated cellsaccumulate more sarcomeric MHC (U) than controlproliferating RD cells (C); moreover, U0126 potentiates theeffect of TPA on MHC expression (U+TPA). By contrast, bothSB203580 and anisomycin inhibit TPA-mediatedaccumulation of sarcomeric MHC (SB+TPA, AN+TPA), andSB203580 also inhibits U0126-mediated MHC accumulation(U+SB).

Immunofluorescence analysis confirmed the results of theimmunoblotting and shows that filamentous fluorescence ispresent in TPA-treated cells, whereas unassembled diffusedmyosin staining is observed in U0126-treated round cells both

in the presence and in the absence of TPA (Fig. 9B). It isnoteworthy that myosin accumulation in U0126-treated cellsrules out any toxic effect of this inhibitor, which suggests thatU0126-treated cells are metabolically active.

These data indicate that p38 plays a pivotal role in theactivation of the myogenic program and that persistentlyactivated JNK might antagonise the differentiating effects ofactivated p38. To further verify the latter hypothesis, we treatedU0126-pre-treated RD cells, in which both JNKs and ERKs arecompletely downregulated, with anisomycin for 30 minutes, 3and 5 days, to reactivate JNK in the absence of activated ERKs.We then analysed the myosin expression and the level ofphospho-active-JNKs by immunoblotting experiments. Theresults in Fig. 10 show that TPA- and U0126-mediated myosinexpression is strongly inhibited by anisomycin at day 3 as wellas at day 5. Interestingly, U0126 induces MHC accumulationearlier (3 days) than TPA treatment does, whereas anisomycin


Fig. 9.Effects of SB203580, U0126 and anisomycin on myosinexpression. (A) Immunoblot, using anti-MHC antibody, of totallysates from RD cells, untreated (C) or treated with U0126 in theabsence (U) or presence of SB203580 (SB+U), with TPA in theabsence (TPA) or in the presence of U0126 (U+TPA), SB203580(SB+TPA) and anisomycin (AN+TPA) for 6 days.(B) Immunofluorescence microscopy of cells treated as indicatedabove. The data shown in A and B are representative, respectively, offour and two independent experiments.

Table 1. Cell cycle analysis of control and growth arrestedRD cells

Sample G1 S G2/M

C1d 45.7 38.3 15.7TPA 1d 47.6 30.7 21.8AN 1d 43.2 31.9 24.5U0126 1d 80.4 8.4 9.9C 4d 48.2 30.5 20.9TPA 4d 58.5 26 15.8AN 4d 62.8 13.3 19.5U0126 4d 87.6 5.5 4.3

FACS analysis of RD cells either untreated and treated with TPA, U0126and anisomycin. The values represent the percentage of the total population ofcells analysed in G1, S, G2/M.


alone (AN) does not induce MHC expression in spite ofsustained p38 activation (Fig. 7). As expected, besidesactivating p38, anisomycin potently and persistently (from 30minutes to 5 days) re-activates JNK in U0126-pre-treated cellsat all times of treatment (Fig. 10).

These results suggest that JNKs counteract the p38-inducedmyogenic program, thereby indicating that p38 activation isrequired but is not sufficient to induce the expression ofmyogenic markers when sustained activation of JNKs occurs.

DiscussionGrowth arrest and tissue-specific gene expression are distinctstages in myogenic differentiation that are likely to beregulated by distinct signaling pathways. We have identifiedthe ERK, JNK and p38 cascades as targets of the signaltransduction pathway induced by PKCα activation in RD cells.

TPA-mediated PKC-α activation induces MAPKpathwaysAnalysis of MAPK pathways in proliferating RD cells revealsa high level of activated ERKs and a low level of activatedJNKs. These findings, which are in keeping with data in theliterature, suggest that activated ERKs correlate withproliferation, whereas activated JNKs correlate with cellularresponses to stress (Iordanov et al., 1998; Rosette and Karin,1996). Surprisingly, though myogenic phenotype is induced byp38 activation (Puri et al., 2000; Wu et al., 2000a), theconsistent increase in phospho-p38 in cultured control RD cellsfails to induce myogenesis, suggesting that p38 downstreampathways are inhibited. TPA, which promotes the myogenesisprocess in RD cells, induces a rapid and sustained increase inphospho-active ERKs and p38 and a transient activation inJNKs. Concomitant activation of the three MAPKs is not acommon event in other myogenic cell lines or in other celltypes, although we have previously observed similar results inan inflammatory-like response induced by TNF-α treatment inSertoli cells (De Cesaris et al., 1998; De Cesaris et al., 1999).It has also been reported that TPA-induced macrophagicdifferentiation of U937 leukemic cells requires ERK, JNK andp38 activation (Franklin and Kraft, 1997).

In this study, we demonstrate that PKCα is an upstreamkinase of MAPK cascades, which regulate growth arrest andmyogenic differentiation. Notably, the PKC inhibitorRo320432 prevents MAPK phosphorylation as well as growtharrest and myogenic differentiation. The involvement of PKCαin the activation of the three MAPK pathways is strongly

supported by transient transfection experiments. In fact,ectopic expression of the constitutively active form of PKCα,but not its dominant-negative form, induces ERK, JNK and p38activation. In conclusion, these data suggest that PKCα is anessential activator of the three MAPK cascades which, in turn,play relevant roles in growth arrest and myogenicdifferentiation in RD cells (Fig. 11).

JNKs activation is controlled by MEK2 and is involved ingrowth arrestAlthough it has been demonstrated that normal andpathological myogenesis is dependent on p38 activation, thepathways inducing growth arrest, which enable myogenictumor cells to activate myogenic-specific programs, requirefurther investigation.

The use of MAPK inhibitors allowed us to dissect the TPA-mediated kinase activation and, hence, to show that ERKs andJNKs are involved in the regulation of withdrawal from the cellcycle and that p38 is required for the initiation of the myogenicdifferentiation program. The MEK1/2 inhibitor, U0126, whichdrastically and persistently inhibits ERK and, unexpectedly,JNK activation, induces rapid (1 day) and drastic growth arrest.This is demonstrated by a decreased growth potential (2-6days), an increased number of G1-arrested cells as shown byFACS analysis, as well as by a drastic decrease in nuclearPCNA. These effects are not modified after prolonged U0126treatment, although the extent of ERK inhibition decreasedslightly between days 2 and 5, and this decrease is not due tothe instability of the drug in the culture medium. Moreover, theinhibition of JNK activation is not a result of a non-specificeffect of U0126, since cells transfected with a constitutivelyactive form of MEK2 express a much higher level of activatedJNKs than control cells transfected with empty vector do.

Besides preventing ERK and JNK activation, U0126 alsoinduces an increase in activated p38; thus, growth inhibitionmay be due either to the downregulation of ERKs and/or JNKsor to the activation of p38. By using the p38 inhibitorSB203580 together with the MEK inhibitor, we demonstratethat inhibition of p38 can only revert growth arrest afterprolonged incubation times (6 days). Similarly, in TPA-treatedcells, in which the three MAPKs are activated, p38 inhibitiondoes not reverse growth arrest before 6 days. These data pointto a role of p38 in the maintenance, rather than in the induction,of growth arrest.

Since there is no specific JNK inhibitor with which to gainan insight into the role of JNKs in growth arrest, we usedanisomycin, a potent JNK and p38 agonist. The fact that

Fig. 10. Effect of anisomycin-mediated JNK re-activation onMHC accumulation. Immunoblots of total lysates from RD cellsuntreated (C) and treated with anisomycin (AN), with TPA andU0126 in the absence (TPA, U) and in the presence ofanisomycin (AN + TPA, AN + U) for 30 minutes, 3 and 5 days.Filters were probed with antibodies recognizing phospho-activeJNKs and MHC. The data are representative of three independentexperiments.

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anisomycin does not affect ERK activity permits us to studythe induction of strong JNK activation in the absence of ERKmodulation. We found that persistent activation of JNKs inanisomycin-treated RD cells induces growth arrest even in thepresence of the p38 inhibitor, as shown by the reduction ingrowth potential and in nuclear PCNA, as well as by anincreased number of G1-arrested cells.

All these data indicate that a critical level of activated ERKssustains deregulated growth of RD cells (control cells) and thata high level of activated JNKs is required to counteract theproliferative action of ERKs (TPA-treated cells). When theERK pathway is abrogated (U0126-treated cells), the JNKpathway is concomitantly downregulated, and growth arrestoccurs following the lack of proliferative action of ERKs. Weconclude that growth arrest of RD cells comes from twoalternative pathways triggered either by downregulation ofERKs or by activation of JNKs, which thus confer a growtharresting function on JNKs (Fig. 11).

Crosstalk between MAPKs modulates myogenic-relatedmorphology and differentiation Interestingly, the MEK inhibitor induces a round as opposed tothe typical spindle-shaped morphology, without detachment orapoptotic events; this suggests that activated ERK or JNK mayplay a role in maintaining myogenic-related morphology. It hasrecently been reported that in the C2C12 myogenic cell line,MAPK-specific phosphatase (MKP-1) overexpressiondownregulates ERK activity sufficiently to rescue myogenic-specific gene expression but prevents mature myotubeformation. These results point to a role for activated ERKsduring early and late myogenic differentiation (Bennett andTonks, 1997). Furthermore, the role of activated ERKs in latemyogenesis has been confirmed by other studies in which theMEK inhibitor PD98059 prevents the myoblast fusion processwithout affecting the expression of muscle-specific genes inC2C12 cells (Gredinger et al., 1998). By comparing the datafrom those studies with our results, which show that the MEKinhibitor prevents the acquisition of a myogenic-relatedmorphology, we suggest that a critical level of activated ERKs,which probably affects cytoskeleton or sarcomeric

organization, may be responsible for the morphologicalphenotype of RD cells. Indeed, the recovery of ERKphosphorylation, during U0126 treatments, may reflect arequirement of the ERK pathway during the late myogenicprocess. However, we cannot rule out the possibility that theacquisition of a myogenic-related morphology requiresfunctional interaction between the three MAPKs.

Surprisingly, myosin expression is detected in MEK-inhibitor-treated cells in spite of their round morphology. Thefinding that myogenic differentiation of RD cells occurs bothafter TPA-mediated ERK and JNK activation and after theirdownregulation, by the MEK inhibitor, seems contradictory. Itis noteworthy that both treatments induce an increase in p38activation. Similarly, p38 activation, following ERKdownregulation by the MEK inhibitor PD98059, has alreadybeen reported in non-myogenic cell lines, but has been foundto be associated with apoptosis (Berra et al., 1998).Furthermore, growth arrest, an obligatory step fordifferentiation as well as for the myogenic process, isdramatically impaired in RD cells, probably because of thepredominance of ERK-mediated mitogenic signals. Thus, anexplanation for our contradictory data comes from the findingthat, in RD cells, the attainment of the myogenic process canoccur either when the ERK pathway is abrogated or when it isantagonised by JNKs. Growth arrest is necessary but notsufficient to induce the myogenic phenotype, although boththese events can be dissociated. In fact, the p38 inhibitorSB203580 prevents myosin accumulation in both growtharrested TPA- and U0126-treated cells. Moreover, this findingclearly demonstrates that p38 is responsible for promoting themyogenic program, which is in agreement with data in theliterature, thereby showing that p38 mediates myogenic-specific gene expression (Wu et al., 2000a).

Interestingly, no myosin expression is found in cells treatedwith TPA and anisomycin, the latter inducing long-lived p38and JNK activation. The failure of TPA to induce myogenicdifferentiation in RD cells in the presence of anisomycin maybe because of persistent JNK activation, which can inhibit thep38-mediated myogenic pathway (Fig. 11). JNKs activate c-Jun, which may antagonise MyoD function by impairingmyogenic differentiation (Bengal et al., 1992). In addition, a


Fig. 11. A model of PKCα-mediated signal transductionpathways in differentiating RD cells. A schematicpresentation of PKCα-mediated MAPKs cascades induced byTPA and the proposed roles of ERKs, JNKs and p38 in theregulation of growth arrest and myogenic differentiation inRD cells (solid arrows). The MAPK pathways targeted by theagonist, as well as inhibitors and a negative regulator ofJNK’s effect on p38, are indicated by dashed and dotted lines.PKCα-mediated MAPK activation induces growth arrest andmyogenic differentiation. When ERK and JNK aredownregulated (encircled), by U0126, growth arrest and p38-mediated myogenic differentiation occur. Inhibition of thep38 pathway, by SB203580, prevents myogenicdifferentiation. Activation of both p38 and JNKs, byanisomycin, induces growth arrest but prevents myogenicprocess owing to a persistent and highly activated JNK,which negatively regulates the p38 pathway.


role for JNKs has recently been proposed in the loss of Myf5nuclear localization, an event which occurs early in themyogenic process (Meriane et al., 2000). It is noteworthy thatduring anisomycin removal, cells progressively re-acquire theability to activate the TPA-mediated myogenic program, whichsuggests that the removal of the JNK agonist permitsmyogenic-specific gene expression. In addition, RD cells pre-treated with a high anisomycin concentration (50 ng/ml)synthesized, during chase, more myosin than cells treated withlower doses. This result supports the finding that growthinhibition, caused by persistently active JNK, althoughinhibitory for myogenic-specific gene expression, does notirreversibly impair the myogenic process but allows RD cellsto be more responsive to differentiation signals induced byTPA. Moreover, since transient JNK activation, following TPAtreatment, does not impair myosin expression, it could bepostulated that transient activation of JNKs does not interferewith the p38-induced myogenic program whereas persistentactivation does. The pathway that induces myogenic markersin RD cells might, therefore, be dependent on a balancebetween JNK and p38 activities. In agreement with this, theU0126-induced myosin expression is also drastically inhibitedby anisomycin treatment, which persistently re-activates JNKseven after long incubation times, thus supporting the inhibitoryrole of JNK on the myogenic gene expression program.

The authors of one recent noteworthy paper found that p38plays a role in both growth arrest and myogenic phenotypeexpression in RD cell lines stably transfected with the p38upstream kinase MKK6 (Puri et al., 2000). The partialdiscrepancy between those results and ours, concerning therole of p38 in growth arrest, may depend on the differentexperimental approaches used to induce myogenicdifferentiation in RD cells. The forced expression of aconstitutively active isoform of MKK6 kinase, when used tostudy downstream kinase activation, may impair the temporalsequence of responses related to kinase modulatory events. Bycontrast, in this study, treatment with agonists that activateendogenous kinase cascades expanded the temporal scale ofthe differentiation process, thereby allowing a more detailedcharacterization of events.

ConclusionsIt may be hypothesized that RD cells, unlike untransformedsatellite cells, which are their normal precursors, develop thetransformed phenotype because of their failure to respond tophysiological differentiative signals, for example, IGFs(Coolican et al., 1997). Differentiative signals inducing thesatellite cell myogenic program have been reported to bemediated by the MAPK p38, which is involved in the inductionof myogenic-specific marker expression (Wu et al., 2000b).

Interestingly, here we demonstrate that the coordinatedactivation of ERKs, JNKs and p38, all playing distinct roles ingrowth arrest and expression of myogenic-specific markers in RDcells, is controlled by activated PKCα. Knowledge of pathwaysthat induce growth arrest in RD cells provides importantinformation concerning the role of PKCα in the balance betweenproliferation and differentiation in the pathological myogenicprocess. Moreover, our data point both to a crucial role for MAPKactivation length and to a drastic change in the scenario ofactivated kinases that induce RD cells to attain the growth arrest

state alone or to move on towards myogenic differentiation. Webelieve that it may be possible to exploit these results for futurestudies of novel therapeutic approaches.

We are grateful to S. Adamo for his helpful discussion and C.Giacinti for her skillful collaboration. We are particularly indebted toA. Floridi for his generous help and support in the course of this work.We also thank Lewis Baker for reviewing the English in themanuscript. This work was supported by grants from MURST 40%,AIRC, Telethon (D107) and ASI.

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α-mediated erk, jnk and p38 activation regulates the ...mek inhibitor suggest a key role of the erk...

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