Activated Raf Kinase Inhibits Muscle Cell Differentiation Through a MEF2- Dependent Mechanism

Journal of Cell Science 113, 4211-4220 (2000) 4211 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1723

Activated Raf kinase inhibits muscle cell differentiation through a MEF2- dependent mechanism

Barbara Winter* and Hans-Henning Arnold Department of Cell and Molecular Biology, University of Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany *Author for correspondence (e-mail: [email protected])

Accepted 28 September; published on WWW 7 November 2000

SUMMARY

Muscle cell development is dependent on the activity of cell may be involved. Significantly, we observed that elevated type-specific basic-helix-loop-helix transcription factors, Raf kinase activity in L6 muscle cells suppresses the MyoD, Myf-5, myogenin, and MRF4 which collaborate accumulation of MEF2 protein in nuclei, while MEF2 with myocyte enhancer factor 2 proteins to activate muscle- transcription appears unaffected. Moreover, forced specific gene expression. Growth factors and activated Ras expression of MEF2A in 10T1/2 cells rescues MyoD prevent differentiation of myoblasts in culture but the dependent myogenic conversion in the presence of downstream signalling pathways are not well understood. constitutively active Raf kinase and partially restores Here, we demonstrate that active Raf kinase (Raf-BxB) transactivation of a myogenin promoter-dependent completely inhibits myogenic conversion of 10T1/2 cells reporter gene in L6 muscle cells containing activated mediated by Myf-5 and differentiation of L6 myoblasts Raf kinase. From these observations we conclude that as indicated by the absence of myotubes, lack of persistent activation of Raf signalling affects nuclear MEF2 myogenin expression, and markedly reduced expression functions which may explain why myogenin expression and of myosin heavy chain. However, activated Raf inhibits myoblast differentiation are inhibited. transcriptional activation by Myf-5 only partially suggesting that other potential targets of Ras/Raf signalling Key words: Ras, Raf, MEF2, Myogenesis

INTRODUCTION MEF2 binding site which is important for correct spatio- temporal expression of the myogenin gene during mouse The formation of skeletal muscle occurs in a series of development and MEF2 appears to maintain sustained high coordinated events during which pluripotent mesodermal cells level expression of myogenin in myocytes (Black et al., 1998; first give rise to proliferating myoblasts which subsequently Hasty et al., 1993; Yee and Rigby, 1993). withdraw from the cell cycle and differentiate into In order to execute terminal differentiation myoblasts must multinucleated myotubes that activate the muscle-specific gene withdraw from the cell cycle which is facilitated by the absence expression program. The progression of cells through this of growth promoting factors in their environment (Lassar and developmental program is associated with the activation of four Munsterberg, 1996). In fact, high serum concentrations in cell type-specific basic-helix-loop-helix (bHLH) transcription culture or the presence of specific growth factors, such as factors, referred to as Myf-5, MyoD, myogenin and MRF4 (for fibroblastic growth factors (FGFs) or members of the review see, Arnold and Winter, 1998). When overexpressed in transforming growth factor (TGF) family suppress myoblast non-muscle cells, each of these proteins is capable of differentiation (Brennan et al., 1991; Li et al., 1992). Activated converting cells to the muscle phenotype in vitro but targeted Ras, a crucial component of the intracellular signalling cascade gene disruptions in the mouse indicate that Myf-5 and MyoD downstream of many growth factor receptors has also been have overlapping roles in early myogenic cell determination, shown to profoundly inhibit muscle cell differentiation (Kong distinct from myogenin and possibly MRF4 which are et al., 1995; Konieczny et al., 1989; Lassar et al., 1989; necessary for terminal differentiation. For transcriptional Sternberg et al., 1989; Vaidya et al., 1991). Deregulation of activation of muscle-specific target genes the myogenic growth control and transformation generally interferes with regulatory factors (MRFs) collaborate with members of the myogenic differentiation as indicated by the severe repression myocyte enhancer factor 2 (MEF2) family of proteins in two of myogenesis by many activated oncogenes including kinases possible ways (Olson et al., 1995). They either interact directly and transcription factors (Alema and Tato, 1994). The with their cognate DNA binding site, present in many muscle- monomeric GTPase Ras functions as a crucial molecular specific enhancers and promoters, or they form complexes with switch in multiple intracellular signalling pathways including MRFs as essential cofactors (Molkentin and Olson, 1996; those controlling cell proliferation and transformation (Hill and Molkentin et al., 1995). The myogenin promoter contains a Treisman, 1995; Sun et al., 1994; Avruch et al., 1994; White 4212 B. Winter and H.-H. Arnold et al., 1995; Herkowitz, 1995). The mechanism by which Dulbecco’s modified Eagle’s medium (DMEM) supplemented with activated Ras inhibits myoblast differentiation is not well 10% fetal bovine serum (GM). For differentiation 10T1/2 cells were understood, although it seems reasonable to assume that the shifted to DMEM with 3% horse serum and L6 cells to DMEM classical Raf/MEK/MAPK cascade may play a role in specific containing 5% horse serum, 1% glucose, and 0.1% insulin (DM). protein phosphorylation events that result in altered activities Transient transfections were performed using calcium phosphate of transcription factors. Possible phosphorylation targets in precipitation as described previously (Winter et al., 1997). 18 hours after transfection cells were transferred to DM and harvested 48 hours myoblasts include the MRFs, however, no MAPK dependent later. CAT and β-galactosidase activities were determined in cell modifications have yet been found. Indeed, transactivation extracts according to standard procedures (Gorman, 1985). When functions of MyoD and MRF4 are retained in muscle cells used, the MEK inhibitor PD09859 and the MEK/ERK inhibitor which constitutively express activated Ras (Kong et al., 1995). U0126 were added to the medium 12 hours after plating and the This suggests that inhibition of myogenesis by activated Ras medium was changed every 24 hours with fresh drug. All transfections cannot be exclusively ascribed to repression of MRF functions were standardized by cotransfection of 5 µg of RSV-LacZ plasmid. and other potential effectors may be important. The reporter plasmid 15-500 CAT, contains the promoter and The roles of downstream intermediates in the Ras initiated enhancer of the mouse myosin light chain1/3 gene linked to CAT signalling pathways are also controversial. According to (Rosenthal et al., 1990). Expression vectors pEMSV Myf-5, pEMSV Gredinger et al. ERK activity is upregulated in differentiating MyoD, and Myf-5 mut lacking amino acids 156 to 169 have been described previously (Winter et al., 1992; Winter et al., 1997). PE/OZ C2 muscle cells and overexpression of activated MEK or CAT containing oligomerized MEF2 binding sites in front of a activated Raf1 stimulates transcriptional activity of MyoD and minimal TK promoter as well as MyoLacZ, which contains the LacZ leads to myotube formation in the presence of high serum gene driven by the muscle-specific myogenin promoter (Molkentin et concentrations suggesting that the MAPK pathway positively al., 1995) were used as reporters for transactivation with pMSV regulates MyoD activity (Gredinger et al., 1998). Rho, another MEF2A (Buchberger and Arnold, 1999). Ras pT24 (Kanda et al., downstream effector of Ras, also seems to be required for 1993), and Raf-BxB or RAF-BxB-ERTM (Kerkhoff and Rapp, 1997), differentiation of C2C12 myoblasts, as a dominant negative were used as expression constructs for constitutive activated Ras and form of Rho prevents differentiation (Takano et al., 1998). Raf, respectively. RAF-BxB-ERTM expression was induced by 100 nM or 200 nM 4-hydroxytamoxifen in the culture medium. L6 Raf- Thus, neither MEK nor Rho appear to mediate the inhibitory TM effect of activated Ras on muscle cell differentiation but rather BxB-ER cell lines were generated by stable transfection and selection for G418 resistent clones. seem to play a positive role for myogenesis (Ramocki et al., 1997). Using overexpression of a MAPK phosphatase to inhibit Myogenic cell differentiation and immunohistochemistry p42 ERK2 activity in C2C12 muscle cells Bennett and Tonks Differentiation of L6 muscle cells and myogenic conversion of 10T1/2 made the contrasting observation that inactivation of MAPK cells by Myf-5 or MyoD (alone or in combination with MEF2A, Ras activity relieves the inhibitory effects of mitogens and results pT24, Raf-BxB, or Raf-BxB-ERTM) was assessed by immunostaining in muscle-specific gene expression, although MAPK may be for myosin heavy chain using monoclonal MF20 antibody (Bader et required later for myotube fusion (Bennett and Tonks, 1997). al., 1982) as described previously (Winter et al., 1997). MEF2 protein Thus, MAPK effects on muscle cells have been reported as in cells was identified with rabbit polyclonal MEF2 antiserum (Santa being mitogenic and inhibitory for differentiation or promoting Cruz) used in a 1:200 dilution and either fluorescein (FITC)- myogenic differentiation. conjugated donkey anti-rabbit second antibody (dianova) or peroxidase-coupled secondary antibody (Vectastain). 4,6-diamidino- Considering the widely disputed and confusing roles of the 2-phenylindole (DAPI, 0.1 µg/ml) was used to stain nuclei. MAPK pathway in muscle cell differentiation, in particular the apparently contrasting effects of activated Ras and the Raf-1 immunoprecipitation kinase cascade assay downstream components of the cascade, MEK and MAPK, we Subconfluent cultures of L6 wild-type cells or L6Raf-BxB-ERTM cell decided to investigate the effects of activated Raf on muscle- lines 19, 34, and 35 were cultured for 1 hour in DM with or without specific regulatory factors. We demonstrate that activated Raf tamoxifen and protein extracts from whole cells were prepared inhibits Myf-5 mediated myogenic conversion of 10T1/2 cells according to Eckner et al. (Eckner et al., 1994). Raf was and inducible Raf activity in L6 muscle cells prevents immunoprecipitated from extracts (500 µg of total protein) using anti- myogenin expression and differentiation. Furthermore we human Raf-1 antibody (Upstate Biotechnology) and G-agarose beads. show that MEF2 protein fails to accumulate in nuclei of cells Raf kinase activity was determined by serial activation of MEK and MAP kinase2 and phosphorylation of MBP according to the which contain elevated Raf activity. The inhibitory effect of protocol provided by the manufacturer (Upstate Biotechnology). activated Raf on muscle conversion of 10T1/2 cells can be Phosphorylated MBP was separated on SDS-PAGE and detected on alleviated by overexpression of MEF2A suggesting that this X-ray film. transcription factor may be a critical target of the Ras/Raf cascade. As MEF2 is required for the transcription of RT-PCR analysis in stably transfected cells myogenin which is essential for differentiation, the lack of Total RNA was extracted from L6 and L6Raf-BxB-ERTM cell lines MEF2 protein in the nucleus of cells containing activated Raf using the RNA clean kit (AGS) and reverse transcribed (5 µg of total may explain their failure to activate the differentiation RNA) with AMV reverse transcriptase. RT-PCR reactions were program. performed with Tli-Taq polymerase as suggested by the manufacturer (Promega). Oligonucleotides used in the PCR reactions were as follows: Raf-BxB-ERTM 5′ (5′-ccagctaattgacacattgc-3′) and 3′ (5′- tcatcgaagcttcactgaag-3′); myogenin 5′ (5′-caaccaggaggagcgcgatctccg- MATERIALS AND METHODS 3′) and 3′ (5′-aggcggctgtgggagttgcattcact-3′); Myf-5 5′ (5′-tgtatcccc- tcaccagaggat-3′) and 3′ (5′-ggctgtaatagttctccacctgtt-3′); MEF2 5′ Cell cultures, transfections and plasmids (5′-ccctttcgtccggcgaaggtc-3′) and 3′ (5′-catgccgccatctgccctcag-3′); C3H10T1/2 fibroblasts and rat L6 muscle cells were grown in 5′ribosomal L6 protein (RPa7) 5′ (5′-gaagctcatctatgagaaggc-3′) and 3′ Activated Raf kinase inhibits muscle cell differentiation 4213

A. B.

Fig. 1. Raf-BxB inhibits Myf-5 induced 2 300 100 myogenesis in 10T1/2 cells. (A) C3H10T1/2 cells were transiently transfected with 5 µg pMSVa2 (control vector), pMSV Myf-5, or pMSV Myf-5 together with a 3-fold excess of 200 activated Ras pT24 and Raf-BxB expression vector. Following shift to DM for 48 hours, 50 cells were fixed and immunostained for MHC 100 % CAT activity expression as described in Materials and Methods. 10 representative microscope fields from each experimental group were scored for cells per cm MMF20 positive myofiber formation using the MHC specific MF20 antibody. The average number of Myf-5 - 1 x 1 x 1 x Myf-5 1 x 1 x 1 x 2 myofibers per cm is indicated. (B) C3H10T1/2 Ras pT24 - - 3 x - Raf-BxB - 2 x 3 x cells were transiently cotransfected with pMSV Raf-BxB - - - 3 x 1 x 1 x Myf-5, increasing amounts of Raf-BxB and a 15-500 1 x CAT reporter which is driven by the muscle- specific MLC promoter/enhancer (15-500 CAT). After 48 hours in DM, cell extracts were C. D. prepared, normalized for protein content, and assayed for CAT activity. CAT values are 100 100 expressed relative to that of Myf-5 alone set at 100%. Each column represents the mean of at least three independent transfections. (C) Transient transfections of 10T1/2 cells with pMSV Myf-5mut which lacks the only putative MAPK phosphorylation site (aa 156 to 160) 50 50 within the Myf-5 protein, together with 15-500 % CAT activity % CAT activity CAT reporter and increasing amounts of Raf- BxB expression vector. CAT activities were normalized to that of Myf-5mut which was set to 100%. (D) C3H10T1/2 cells were transiently transfected with pMSV MEF2A, increasing Myf-5mut 1 x 1 x 1 x MEF2A 1 x 1 x 1 x amounts of Raf-BxB, and a CAT reporter which Raf-BxB - 2 x 3 x Raf-BxB - 2 x 3 x contains oligomerized MEF2 binding sites in front of a TK minimal promoter (PE/OZ CAT). 15-500 CAT 1 x 1 x 1 x PE/OZCAT 1 x 1 x 1 x

(5′-aagacgaaggagctgcagaac-3′). For each primer pair the appropriate 5 under these conditions was analyzed in 10T1/2 cells on the numbers of PCR cycles were predetermined to ensure that the MRF dependent reporter construct 15-500 CAT which contains reactions were still in the linear range. Generally 25 cycles were the muscle-speciﬁc MLC1/3 enhancer to drive transcription. suitable. One third of each reaction was separated on agarose gels. This reporter was readily activated by Myf-5 but cotransfection RPa7 was used to calibrate the reaction input. of Raf-BxB resulted in a concentration dependent inhibition of reporter activity (Fig. 1B). However, even high concentrations of Raf-BxB expression plasmid, sufficient to completely block RESULTS myogenic conversion of 10T1/2 cells, only partially inhibited transactivation by Myf-5. Similar results were obtained in Activated Raf inhibits Myf-5 mediated myogenic transactivation experiments with MyoD (data not shown). conversion of 10T1/2 cells Sequence inspection of Myf-5 protein revealed the presence of It has been established that constitutively active Ras inhibits a putative phosphorylation site for MAP kinase in the C- myogenesis (Kong et al., 1995). In order to determine the effect terminal half (consensus sequence PTSPT; aa156-160). To test of the immediate downstream component of Ras within the the potential importance of this consensus motif for the MAPK pathway, an expression plasmid for the constitutively inhibition by activated Raf a Myf-5 deletion mutant lacking activated Raf mutant Raf-BxB was cotransfected with the this site was analyzed in a transient transactivation experiment expression vector for Myf-5 into 10T1/2 cells which were in 10T1/2 cells. As shown in Fig. 1C, the Myf-5 deletion then scored for myogenic conversion by the appearance of mutant activated the 15-500 CAT reporter and was inhibited by multinucleated myotubes and the expression of sarcomeric Raf-BxB in the same way as wild-type Myf-5 suggesting that myosin heavy chain (MHC). While transiently expressed Myf- direct phosphorylation of this site by MAP kinase is probably 5 efficiently activated MHC expression in 10T1/2 cells, not implicated in the Raf effect. Taken together these coexpression of Raf-BxB completely prevented MHC observations suggested to us that activated Raf may have activation similar to activated Ras pT24 which was used as additional targets to interfere with muscle cell differentiation. positive control (Fig. 1A). Transcriptional activation by Myf- As members of the MEF2 family of transcription factors are 4214 B. Winter and H.-H. Arnold

Fig. 2. Active Raf kinase inhibits differentiation of rat L6 myoblasts. (A) Three stably transfected rat L6 myoblasts containing Raf-BxB- ERTM (L6Raf-BxB-ERTM 19, 34, and 35) and wild-type L6 cells were cultivated for 24 hours in DM or DM containing tamoxifen at the indicated concentrations. Myofiber formation was assessed by immunostaining with MF20 monoclonal antibody to detect MHC expressing cells. While tamoxifen does not affect differentiation in L6 cells, induction of Raf-BxB kinase by tamoxifen prevents differentiation. (B) Raf kinase activity was determined in extracts of each cell line by the Raf-1 immunoprecipitation kinase cascade assay (Raf – MEK – MAPK) as described in Materials and Methods. Kinase activity was estimated by the phosphorylation of myelin basic protein (MBP). The control represents phosphorylation background obtained in cell extracts without immunoprecipitation of Raf-1. also important for muscle-specific gene expression, the (Kerkhoff and Rapp, 1997). L6 cells were chosen, because they effect of Raf-BxB on MEF2A mediated transactivation was express Myf-5 and no MyoD and thus closely resemble the examined in transient transfections of 10T1/2 cells. In contrast situation in the 10T1/2 cell conversion experiments. In contrast to Myf-5 and MyoD, transactivation of the MEF2-specific to wild-type L6 cells which readily formed multinucleated reporter PE/OZ CAT by MEF2A was not inhibited by Raf-BxB myotubes expressing myosin heavy chain after 4 days in indicating that activated Raf exerts its inhibitory effect differentiation medium (DM) with or without tamoxifen, three specifically and does not block transcriptional activation in randomly selected stable L6Raf-BxB-ERTM cell lines, 19, 34, general (Fig. 1D). Thus, activated Raf blocks the Myf-5 and 35, formed drastically fewer or failed to form myotubes induced muscle phenotype in 10T1/2 cells similar to activated and did not express MHC in the presence of 100 or 200nM Ras but it seems unlikely that Myf-5 is a direct phosphorylation tamoxifen in DM but differentiated like wild-type L6 cells in target of the MAPK cascade and reduced Myf-5 activity is the absence of tamoxifen (Fig. 2A). Mononucleated spindle probably not solely responsible for the observed inhibition of shaped cells which stained positively for MHC were myogenesis. occassionally observed in clone L6Raf-BxB-ERTM 35 in the presence of 200 nM tamoxifen but could be further reduced by Raf signalling prevents differentiation of L6 300 nM tamoxifen (data not shown). Raf kinase activity in the myoblasts inducible L6Raf-BxB-ERTM cell lines was determined by the To evaluate the effect of activated Raf in muscle cells we Raf-1 immunoprecipitation kinase cascade assay as described generated stable L6 cell lines containing Raf-BxB-ERTM in Materials and Methods. Low level Raf kinase activity which plasmid which encodes the Raf-BxB-estrogen receptor (ERTM) was unaffected by the addition of tamoxifen was found in L6 fusion protein that allows regulated activity of Raf kinase in muscle cells, whereas in L6Raf-BxB-ERTM clones Raf kinase cells by the addition of 4-hydroxytamoxifen to the medium activity was increased at least two- to threefold within one hour Activated Raf kinase inhibits muscle cell differentiation 4215

Fig. 3. Activated Raf inhibits transcriptional activation of myogenin in differentiation-deficient L6Raf- BxB-ERTM cells. Total RNA from the indicated cell lines cultured either in DM with (+) or without (−) tamoxifen for 24 hours was used for RT-PCR to analyze transcript accumulation of Raf-BxB-ERTM, Myf-5, MEF2, and myogenin. 0 time indicates addition of tamoxifen. PCR for ribosomal protein L6 mRNA was used as input control. after the addition of tamoxifen to the culture medium (Fig. 2B). expression has been reported to be upregulated during We also determined the accumulation of myogenin, Myf-5, and differentiation and in response to myogenin (Ridgeway et al., MEF2 transcripts in wild-type L6 cells and in L6Raf-BxB- 2000). The second possibility, however, appeared less likely, ERTM clones grown for 24 hours in DM with or without because accumulation of MEF2 transcripts was not inhibited tamoxifen. Semiquantitative RT-PCR revealed that tamoxifen by activated Raf. completely suppressed myogenin expression in L6Raf-BxB- ERTM cells but had no effect on the accumulation of Myf-5 or Overexpression of MEF2A rescues the Raf kinase- MEF2 mRNA (Fig. 3). Tamoxifen had also no effect on the mediated differentiation defect transcription of these genes in wild-type L6 cells. As expected, To investigate whether overexpression of MEF2 may alleviate expression levels of BxB-ERTM mRNA determined as internal the differentiation block caused by activated Raf 10T1/2 cells control were found to be essentially unaltered by the culture were transiently transfected with Myf-5 (data not shown) conditions. These results indicate that elevated and sustained or MyoD together with constitutively active Raf-BxB Raf kinase activity strongly and specifically inhibits myogenin expression plasmid and increasing concentrations of MEF2A expression in muscle cells consistent with their failure to expression vector. Cells were scored for myogenic conversion differentiate. 48 hours after transfection using MF20 antibody to detect MHC expressing cells. As shown in Fig. 5, the inhibition of MEF2 protein is markedly reduced in nuclei of MyoD induced myogenic conversion of 10T1/2 cells by Raf- differentiation defective L6Raf-BxB-ERTM cells BxB was completely abolished by overexpressed MEF2A. The myogenin promoter contains E-boxes for binding of the Under these conditions MEF2A accumulated in the nucleus, myogenic bHLH factors and a consensus binding site for possibly because high concentrations of MEF2A protein MEF2 proteins (Buchberger et al., 1994; Molkentin et al., obtained by forced expression overwhelmed the system 1995). It has been shown that both types of transcription factors thereby escaping the inhibition by Raf. To demonstrate that are necessary for correct activation of the myogenin gene MEF2A can also rescue muscle-specific gene expression in (Molkentin et al., 1995). As myogenin transcription is blocked L6 cells reporter plasmid MyoLacZ, containing the myogenin in L6Raf-BxB-ERTM cells expressing activated Raf we wanted promoter driving LacZ, was transiently transfected into L6 to know, if one or both of these transcription factors may be Raf-BxB-ERTM cells cultured with or without tamoxifen lacking. Although RT-PCR had indicated that Myf-5 and (Fig. 6). While MyoLacZ was readily activated in L6 Raf- MEF2 transcripts accumulate normally in these cells, we BxB-ERTM at low Raf kinase activity (minus tamoxifen), it reasoned that protein levels may be altered. While antibody was completely inhibited by increased kinase activity (plus staining for Myf-5 protein demonstrated comparable levels in tamoxifen). This inhibition could be rescued at least in part L6 wild-type and L6Raf-BxB-ERTM cells (data not shown), an by cotransfection of MEF2A expression vector but not by antibody recognizing MEF2A and less efficiently MEF2C Myf-5. Taken together, these results suggest that activated revealed a dramatic reduction of MEF2 protein in nuclei of Raf interferes with nuclear function of MEF2 in muscle cells L6Raf-BxB-ERTM cells grown for 24 hours in tamoxifen and this effect can be overcome by high concentrations of containing DM (Fig. 4A). This inhibitory effect was reversible MEF2 protein. as removal of tamoxifen from the cultures resulted in efficient That endogeneous MEF2 activity is indeed modulated by cell differentiation and nuclear accumulation of MEF2 protein Raf kinase activity was further investigated in L6 Raf-BxB- (Fig. 4B). Thus, activated Raf seems to reduce MEF2 protein ERTM using the MEF2-specific reporter construct PE-OZ CAT levels in muscle cell nuclei and this may be the reason why (Molkentin et al., 1995) which contains multimerized MEF2 myogenin is not expressed. Alternatively, the lack of nuclear binding sites to direct CAT expression. As shown in Fig. 7, this MEF2A may be the result of the differentiation block, as MEF2 reporter gene was readily activated in cells growing in the 4216 B. Winter and H.-H. Arnold

Fig. 4. Activated Raf signalling reduces accumulation of MEF2 protein in nuclei of myoblasts. (A) L6 Raf-BxB-ERTM 19 cells and wild-type L6 cells were cultivated for 24 hours in GM and then shifted to DM containing tamoxifen. Accumulation of MEF2 protein was determined by immunohistochemical staining with MEF2 antibody and nuclei were stained with DAPI as described in Materials and Methods. Note that nuclei in proliferating myoblasts have marginal MEF2 content, while differentiated myotubes in wild-type L6 accumulate nuclear MEF2. In contrast, L6Raf-BxB-ER cells with induced kinase activity fail to accumulate MEF2 protein in nuclei. (B) Wild-type L6 cells and L6 Raf- BxB-ER cells were grown in DM containing tamoxifen for 24 hours (a) and then shifted to DM without tamoxifen for 24 (b), 48 (c), and 120 (d) hours. Cultures were fixed at indicated time points and stained with αMEF2 antibody. Note that the differentiation block exerted by activated Raf kinase activity is reversible, as L6 Raf-BxB-ER cells differentiate in DM without tamoxifen like L6 wild-type cells. absence of tamoxifen but lacked activation in cells with high DISCUSSION Raf kinase activity in the presence of TM. Mitogenic signals or growth factors and activated Ras Nuclear accumulation of MEF2 is not directly protein as one of the canonical downstream components of controlled by MAP kinases but its inhibition by Raf intracellular signalling pathways inhibit muscle cell depends on MAP kinase activity differentiation by largely unknown mechanisms (Kong et al., To begin to investigate whether inhibition of nuclear MEF2 1995). In particular, the final targets of the signal cascades, accumulation by Raf kinase involves the canonical downstream presumably transcription factors in the cell nucleus have not signaling pathway of MAP kinases, we employed the specific been identified. In this study we demonstrate that sustained Raf inhibitors, PD 098059 and U 0126 which block MEK and kinase activity prevents differentiation of myoblasts and MEK plus ERK activity, respectively. In L6 Raf-BxB-ERTM abolishes the ability of Myf-5 and MyoD to convert 10T1/2 cells nuclear MEF2 was detected by antibody staining in the fibroblasts to muscle cells. Under these conditions the capacity presence of either inhibitor similar to untreated L6 control cells of Myf-5 and MyoD to transactivate E-box dependent target over a time course of 48 hours. This suggests that MEF2 genes is only partially impaired. Moreover, we find no evidence translocation to the nucleus does not depend on the MAPK that Myf-5 may constitute a direct phosphorylation target for pathway (Fig. 8). Interestingly, application of PD 098059 or U MAPK. Taken together these observations suggest that 0126 to L6 Raf-BxB-ERTM cells grown in the presence of TM additional crucial regulators of myogenesis may be affected by which normally prevents nuclear accumulation of MEF2, the Ras/Raf cascade. A hallmark of differentiating muscle resulted in extensively MEF2 positive nuclei and enhanced cells is the expression of myogenin which under normal differentiation. This observation suggests that MAP kinase circumstances is required for differentiation (Olson et al., activity is required for the inhibition exerted by Raf, although 1995). In accordance with this notion the differentiation- MAPK is not necessary for normal MEF2 translocation to the defective myoblasts expressing activated Raf lack myogenin nucleus. Whether MAP kinase directly phosphorylates MEF2 expression. As myogenin gene activation depends on both, under these conditions needs further investigations. Myf-5 or MyoD and members of the MEF2 family of Activated Raf kinase inhibits muscle cell differentiation 4217

3600 100 DM 3300 90 DM+TM

3000 80

70 2700 60 2400 50 2100 40 1800 MyoLacZ positive cells (% of control) 30 1500

20 1200

MF20 positive cells per field 10 900 MyoLacZ + + + 600 Myf5 + 300 MEF2 A +

Fig. 6. Overexpression of MEF2A rescues the Raf kinase-mediated MyoD 1 x 1x 1 1x 1 x 1 x 1 x - differentiation defect in L6Raf-BxB-ERTM cells. L6Raf-BxB-ERTM x MEF2 - 7.5x - 3x 5 x 7.5x 7.5x cells were transiently transfected with the expression vector pMSV Raf-BxB - 2.5x 2.5x 2.5x 2.5x - Myf-5 or pMSV MEF2A together with the muscle-specific reporter gene MyoLacZ driven by the myogenin promoter. Following shift to Fig. 5. Overexpression of MEF2A overcomes the differentiation DM (control) or DM plus tamoxifen (DM+TM) cells were fixed and block by Raf-BxB in MyoD induced 10T1/2 cells. 10T1/2 cells were analyzed by LacZ staining. MyoLacZ positive cells are expressed transiently transfected with the expression vectors for MyoD and relative to that of the control (DM-TM) set at 100%. MEF2A, and inhibitory amounts of Raf-BxB vector. Myogenic conversion of 10T1/2 cells was determined by antibody staining with negative MEF2A mutant which prevents differentiation in MF20 after 48 hours in DM. MyoD together with MEF2A results in muscle cell cultures (Ornatsky et al., 1997). That MEF2 efficient activation of MHC expression and myotube formation proteins positively collaborate with myogenic bHLH which is strongly inhibited by Raf-BxB. Cotransfected MEF2A expression vector rescues muscle cell differentiation in a transcription factors has been demonstrated by enhanced concentration-dependent manner. Columns indicate mean numbers of conversion of 10T1/2 cells when both proteins were MF20 positive cells per microscopic field. Ten independent fields overexpressed (Black et al., 1998; Molkentin et al., 1995). were counted for each transfection. Moreover, there is ample evidence that MEF2 proteins form complexes with MRFs and constitute essential cofactors for transcription factors (Molkentin et al., 1995), we examined transcriptional activation (Molkentin et al., 1995). So their presence in myoblasts with constitutively active Raf. undoubtedly, MEF2 proteins in concert with MRFs are key Transcription of Myf-5 and MEF2 were essentially unaffected reguators of muscle-specific gene expression and constitute by Raf signalling, at least over a time course of 24 hours, strategic targets for cellular signalling pathways that control however, we failed to detect MEF2 protein in the nucleus, growth and differentiation. while Myf-5 protein was present. That the lack of MEF2 MEF2 proteins have been implicated as targets for several protein is probably causally related to the inhibitory Raf effect signal transduction pathways including MAP kinases, such as can be concluded from the fact that differentiation can be p38 MAPK (Zetser et al., 1999) and BMK1 (Han et al., 1997; rescued by overexpression of MEF2A. Thus, activated Raf Kato et al., 1997). Both kinases are upregulated during signalling seems to interfere with normal MEF2 function by a myogenic differentiation of C2C12 cells and enhance MEF2C posttranscriptional mechanism. Whether MEF2 protein under or MEF2A dependent reporter gene expression, possibly these conditions is rapidly degraded or not translocated to the through direct phosphorylation (Zetser et al., 1999). Recent nucleus has yet to be investigated. One also cannot exclude studies demonstrate that MEF2A, but not MEF2B or MEF2D, completely the possibilty that a Raf-induced conformational is a substrate for p38 and phosphorylation of MEF2A in the change of MEF2 or complex formation with other proteins may heterodimer complex with MEF2D augments MEF2- prevent its detection by the used antibody. dependent gene expression (Zhao et al., 1999). Moreover, it In Drosophila mutants lacking mef2 all muscle cell lineages was shown recently that signal independent activation of p38 fail to differentiate underlining its crucial function in kinase drives rhabdomyosarcoma cells into differentiation myogenesis (Ranganayakulu et al., 1995). The importance of (Puri et al., 2000). So far, no evidence has been presented that MEF2A, the predominant isoform expressed at the onset of MEF2A or MEF2C are directly phosphorylated by mitogen myogenesis in vertebrates (Ornatsky and McDermott, 1996), activated protein kinases ERK1 or ERK2 (Ornatsky et al., has also been demonstrated by overexpression of a dominant- 1999) and our data with Erk inhibitors support the idea that 4218 B. Winter and H.-H. Arnold

50 MAPK are not involved in nuclear accumulation of MEF2. A testable hypothesis could be that activated Raf may antagonize 45 posttranscriptional modiﬁcation and function of MEF2 protein by p38 MAPK, possibly through downregulation of p38 kinase 40 activity via MAPK. Crosstalk between distinct signalling pathways is not without precedence. We are currently 35 investigating whether activated p38 signalling affects the TM 30 diffentiation behaviour of our Raf-BxB-ER cell lines. In agreement with our results in L6 muscle cells Ramocki 25 et al., described the inhibition of MyoD function by activated Raf signalling in C2C12 cells (Ramocki et al., 1997), whereas

% CAT activity 20 Gredinger et al. in the same cell line observed induced ERK activity at the onset of terminal differentiation and suggested 15 that the MAPK cascade enhances the transcriptional activity of MyoD (Gredinger et al., 1998). More recently, Dorman and 10 Johnson reported inhibition of differentiation by constitutively active Raf via the Raf/MEK/MAPK axis in primary chick 5 myocytes (Dorman and Johnson, 1999). The reasons for these controversal results are unclear but are likely to lie in different PE/OZ CAT + + + + experimental conditions. In rat pheochromocytoma PC12 cells TM - ++--+ -+ which serve as a model for neuronal differentiation the duration L6 WT L6BxB-ER 19 L6BxB-ER 34 L6BxB-ER 35 and strength of MAPK activation determine growth versus differentiation promoting signals (Marshall, 1995). In these Fig. 7. Active Raf kinase inhibits transcriptional activation of a cells sustained ERK activity results in differentiation, whereas MEF2 speciﬁc CAT reporter in L6 Raf-BxB-ERTM cells. Three TM short-lived activation leads to proliferation. In the context of stable L6 Raf-BxB-ER cell lines (L6 BxB-ER 19, 34, and 35) and the above mentioned studies and our own it is interesting to wild-type L6 cells were transiently transfected with the PE/OZ CAT reporter construct which contains oligomerized MEF2 binding sites note that different Raf mutants were used in various studies to in front of a TK minimal promoter. After 48 hours in DM (−TM) or activate the pathway and this may result in different levels DM plus tamoxifen (+TM), cell extracts were prepared, normalized of activity. Furthermore, it seems important whether MyoD for protein content, and assayed for CAT activity (%). Each column function is measured in cells grown in high serum which represents the mean of at least three independent transfections. is growth promoting or in low serum which allows

Fig. 8. Nuclear accumulation of MEF2 is not controlled by MAP kinases but its Raf-mediated inhibition depends on MAPK activity. Wild-type L6 cells and L6Raf-BxB-ERTM 35 cells were cultivated for 24 hours, 48 hours, and 72 hours in DM or DM plus tamoxifen (DM+TM) in the presence or absence of the specific inhibitors PD 098059 (50 µM) and U 0126 (10 µM) which block MEK and MEK plus ERK activity, respectively. Accumulation of MEF2 protein was determined by immunohistochemical staining with MEF2A antibody at 24 hours and 48 hours in culture. Myofiber formation was assessed at 72 hours by immunostaining wth MF20 monoclonal antibody to detect MHC expressing cells as outlined in the lower panel of the figure. While nuclear accumulation of MEF2 is not affected by either MEK inhibitor, application of PD 098059 or U 0126 to L6 Raf-BxB-ERTM cells grown in the presence of tamoxifen resulted in extensively MEF2 positve nuclei and enhanced differentiation. Activated Raf kinase inhibits muscle cell differentiation 4219 differentiation. In fact, there is evidence that the MAPK Dorman, C. M. and Johnson, S. E. (1999). Activated Raf inhibits avian cascade functions differently at early versus late stages of myogenesis through a MAPK-dependent mechanism. Oncogene 18, 5167- muscle differentiation (Bennett and Tonks, 1997). While 5176. Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCaprio, J. A., MAPK activity at an early stage inhibits differentiation, it is Lawrence, J. B. and Livingston, D. M. (1994). Molecular cloning and required for myotube fusion but not for MHC expression at a functional analysis of the adenovirus E1A-associated 300-kD protein (p300) later stage. It is worthwhile mentioning, however, that we have reveals a protein with properties of a transcriptional adaptor. Genes Dev. 3, not observed serum-dependent differences of Myf-5 or MyoD 869-884. Gorman, C. (1985). High efficiency gene transfer into mammalian cells. DNA activity in the presence of Raf-BxB. Support for the inhibitory Cloning II (ed. D. M. Glover), pp. 143-190. Oxford: IRL Press. effect of MAPK signalling on muscle cell differentiation Gredinger, E., Gerber, A. N., Tamir, Y., Tapscott, S. J. and Bengal, E. comes from the observation that p42 ERK2 activity is (1998). Mitogen-activated protein kinase pathway is involved in the specifically downregulated by the MAPK phosphatase-1 differentiation of muscle cells. J. Biol. Chem. 273, 10436-10444. (MKP-1) at the onset of differentiation and overexpression of Han, J., Jiang, Y., Li, Z., Kravchenko, V. V. and Ulevitch, R. J. (1997). Activation of the transcription factor MEF2C by the MAP kinase p38 in MKP-1 antagonizes the inhibitory effect of mitogens (Bennett inflammation. Nature 386, 296-299. and Tonks, 1997). The idea, however, that activated ERK Hasty, P., Bradley, A., Morris, J. H., Edmondson, D. G., Venuti, J. M. and simply prevents myoblasts to exit from the cell cycle is not in Olson, E. N. (1993). Muscle deficiency and neonatal death in mice with a full agreement with published data showing that constitutive targeted mutation in the myogenin gene. Nature 364, 501-506. Raf activity does not result in cycling myonuclei (Dorman and Herkowitz, I. (1995). MAP kinase pathway in yeast: for mating and more. Cell 80, 187-197. Johnson, 1999). The same study demonstrates that Raf-BxB Hill, C. S. and Treisman, R. (1995). Transcriptional regulation by does not lead to apoptosis in myocytes. Thus, despite all this extracellular signals: mechanisms and specificity. Cell 80, 199-211. information it remains enigmatic by which mechanisms the Kanda, H., Lee, G. H., Nomura, K., Ohtake, K. and Kitagawa, T. (1993). activated Ras/Raf pathway interferes with muscle cell Malignant transformation of the mouse liver epithel cell line by transfection of an activated c-H-ras gene with a point mutation at codon 12. differentiation and how and to which extent the individual Carcinogenesis 14, 1061-1063. kinase cascades contribute to the decision of proliferation Kato, Y., Kravchenko, V. V., Tapping, R. I., Han, J., Ulevitch, R. J. versus differentiation. Our study provides evidence for the first and Lee, J. D. (1997). BMK1/ERK5 regulates serum-induced early time that the transcription factor MEF2A constitutes an gene expression through transcription factor MEF2C. EMBO J. 16, 7054- important target for integrating the signals initiated by Ras and 7066. Kerkhoff, E. and Rapp, U. R. (1997). Induction of cell proliferation in Raf. Lack of nuclear MEF2 may help to explain why muscle- quiescent NIH 3T3 cells by oncogenic c-Raf-1. Mol. Cell. 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