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• W2016719710 abstract "Activated Raf is a potent inhibitor of skeletal muscle gene transcription and myocyte formation through stimulation of downstream MAPK. However, the molecular targets of elevated MAPK with regard to myogenic repression remain elusive. We examined the effects of activated Raf on myogenin gene expression in avian myoblasts. Overexpression of activated Raf in embryonic chick myoblasts prevented myogenin gene transcription and myocyte differentiation. Treatment with PD98059, an inhibitor of MAPK kinase (MEK), restored myogenin expression but did not reinstate the myogenic program. Using a panel of myogeninpromoter deletion mutants, we were unable to identify a region within the proximal 829-bp promoter that confers responsiveness to MEK. Interestingly, our experiments identified MEF2A as a target of Raf-mediated inhibition in mouse myoblasts but not in avian myogenic cells. Embryonic myoblasts overexpressing activated Raf were unable to drive transcription from a minimal myogenin promoter reporter, containing a single E-box and MEF2 site, to levels comparable with controls. Unlike mouse myoblasts, forced expression of MEF2A did not synergistically enhance transcription from the myogeninpromoter in chick myoblasts, indicating that additional molecular determinants of the block to myogenesis exist. Results of these experiments further exemplify specie differences in the mode of Raf-mediated inhibition of muscle differentiation. Activated Raf is a potent inhibitor of skeletal muscle gene transcription and myocyte formation through stimulation of downstream MAPK. However, the molecular targets of elevated MAPK with regard to myogenic repression remain elusive. We examined the effects of activated Raf on myogenin gene expression in avian myoblasts. Overexpression of activated Raf in embryonic chick myoblasts prevented myogenin gene transcription and myocyte differentiation. Treatment with PD98059, an inhibitor of MAPK kinase (MEK), restored myogenin expression but did not reinstate the myogenic program. Using a panel of myogeninpromoter deletion mutants, we were unable to identify a region within the proximal 829-bp promoter that confers responsiveness to MEK. Interestingly, our experiments identified MEF2A as a target of Raf-mediated inhibition in mouse myoblasts but not in avian myogenic cells. Embryonic myoblasts overexpressing activated Raf were unable to drive transcription from a minimal myogenin promoter reporter, containing a single E-box and MEF2 site, to levels comparable with controls. Unlike mouse myoblasts, forced expression of MEF2A did not synergistically enhance transcription from the myogeninpromoter in chick myoblasts, indicating that additional molecular determinants of the block to myogenesis exist. Results of these experiments further exemplify specie differences in the mode of Raf-mediated inhibition of muscle differentiation. mitogen-activated protein kinase MAPK kinase myogenic regulatory factor myocyte enhancer-binding factor cytomegalovirus reverse transcription-PCR Negative regulation of skeletal myogenesis can be accomplished through growth factor stimulation of several intracellular signaling pathways. The majority of these signaling cascades are associated with the activation of the membrane-localized GTPase, Ras (1Joneson T. Bar-Sagi D. J. Mol. Med. 1997; 75: 587-593Crossref PubMed Scopus (144) Google Scholar). Ras enzymatic activity is a critical component of many ligand-induced responses including those initiated by the myogenesis inhibitory growth factor FGF2 (2Fedorov Y.V. Rosenthal R.S. Olwin B.B. J. Cell Biol. 2001; 152: 1301-1305Crossref PubMed Scopus (21) Google Scholar, 3Weyman C.M. Wolfman A. Endocrinology. 1998; 139: 1794-1800Crossref PubMed Scopus (55) Google Scholar). In addition, sustained Ras activity represents one of the most potent myogenic repressive agents known (4Kong Y. Johnson S.E. Taparowsky E.J. Konieczny S.F. Mol. Cell. Biol. 1995; 15: 5205-5213Crossref PubMed Scopus (65) Google Scholar, 5Olson E.N. Spizz G. Tainsky M.A. Mol. Cell. Biol. 1987; 7: 2104-2111Crossref PubMed Scopus (144) Google Scholar). The means by which activated Ras inhibits skeletal myocyte formation and muscle gene expression remains elusive. However, it is unlikely that repression is mediated by the simple activation of one of the downstream mitogen-activated protein kinases (MAPK),1 such as Jun N-terminal kinase, p38, or ERK1/2 (6Ramocki M.B. White M.A. Konieczny S.F. Taparowsky E.J. J. Biol. Chem. 1998; 273: 17696-17701Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 7Ramocki M.B. Johnson S.E. White M.A. Ashendel C.L. Konieczny S.F. Taparowsky E.J. Mol. Cell. Biol. 1997; 17: 3547-3555Crossref PubMed Scopus (71) Google Scholar, 8Weyman C.M. Wolfman A. Oncogene. 1997; 15: 2521-2528Crossref PubMed Scopus (9) Google Scholar). One of the downstream kinase cascades responsible for transmission of Ras function includes the archetypical signaling axis Raf/MEK/MAPK (9Bar-Sagi D. Mol. Cell. Biol. 2001; 21: 1441-1443Crossref PubMed Scopus (66) Google Scholar). Initiation of this pathway through “mis-expression” of activated Raf leads to repression of the myogenic gene program and inhibition of myocyte formation (10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). The biochemical rationale for suppression of avian skeletal myogenesis by activated alleles of Raf appears to be independent of perturbations in the inherent transcriptional activities of the myogenic regulatory factors (MRFs). However, Raf-initiated morphological transformation of the myoblasts is a direct consequence of increased AP-1 function (11Dorman C.M. Johnson S.E. J. Biol. Chem. 2000; 275: 27481-27487Abstract Full Text Full Text PDF PubMed Google Scholar). The MRFs have long been recognized as master control switches for induction of the skeletal muscle phenotype (12Lassar A. Munsterberg A. Curr. Opin. Cell Biol. 1994; 6: 432-442Crossref PubMed Scopus (143) Google Scholar). Each of the MRFs is required at distinct stages during myogenesis with myogeninassuming a critical role during terminal differentiation and myoblast fusion (13Arnold H.-H. Braun T. J. Dev. Biol. 1996; 40: 345-363PubMed Google Scholar). Mice devoid of myogenin direct a full complement of cells that are located in the appropriate muscle-forming regions (14Hasty P. Bradley A. Morris J.H. Venuti J.M. Edmondson D.G. Olson E.N. Klein W.H. Nature. 1993; 364: 501-506Crossref PubMed Scopus (1016) Google Scholar, 15Venuti J.M. Morris J.H. Vivian J.L. Olson E.N. Klein W.H. J. Cell Biol. 1995; 128: 563-576Crossref PubMed Scopus (222) Google Scholar, 16Vivian J.L. Gan L. Olson E.N. Klein W.H. Dev. Biol. 1999; 208: 44-55Crossref PubMed Scopus (25) Google Scholar). However, the majority of the cells fail to fuse into the multinucleate myocytes capable of contractile protein synthesis. Control of myogenin gene expression requires both E-box-binding factors such as MyoD and myocyte enhancer-binding factor 2C (MEF2C) (17Edmondson D. Cheng T.-C. Cserjesi P. Chakraborty T. Olson E.N. Mol. Cell. Biol. 1992; 12: 3665-3677Crossref PubMed Scopus (256) Google Scholar, 18Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Cell. 1995; 83: 1125-1136Abstract Full Text PDF PubMed Scopus (699) Google Scholar, 19Ridgeway A.G. Wilton S. Skerjanc I.S. J. Biol. Chem. 2000; 275: 41-46Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). cis elements for these two transcription factors reside within the minimal promoter region of themyogenin gene, and deletion of either site in the mouse gene results in a severe reduction in myogenin expression (17Edmondson D. Cheng T.-C. Cserjesi P. Chakraborty T. Olson E.N. Mol. Cell. Biol. 1992; 12: 3665-3677Crossref PubMed Scopus (256) Google Scholar,19Ridgeway A.G. Wilton S. Skerjanc I.S. J. Biol. Chem. 2000; 275: 41-46Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Because myogenin is critical for full differentiation of the myocyte and overexpression of activated kinases often leads to loss of MRF gene expression (20Konieczny S.F. Drobes B.L. Menke S.L. Taparowsky E.J. Oncogene. 1989; 4: 473-481PubMed Google Scholar, 21Lassar A.B. Thayer M.J. Overell R.W. Weintraub H. Cell. 1989; 58: 659-667Abstract Full Text PDF PubMed Scopus (209) Google Scholar), we reasoned that Raf signaling pathways may inhibit skeletal myogenesis by disruption of myogeninexpression and function. In this report, we find that activated Raf suppresses myogenin expression in avian myoblasts and that this repression is achieved by MEK activation. One of the downstream inhibitory targets of Raf signaling is the transcription factor MEF2. Overexpression of MEF2A reverses the negative effects of activated Raf on myogenin reporter gene expression in mouse myoblasts. However, additional transcriptional intermediates are involved in full repression of myogenin gene expression by Raf kinase as MEF2C overexpression is unable to fully overcome the detrimental effects of the Raf/MEK/MAPK signaling module. Expression plasmids coding for Myc-tagged activated human Raf (pCS2+MT shuttle Raf BXB (CMV-Raf BXB), pCS2+MT shuttle Raf CAAX (CMV-Raf CAAX)) have been described (10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). RCAS(A)-Raf BXB and RCAS(A)-Raf CAAXare replication-competent retroviruses containing the coding sequence for activated alleles of human Raf (10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). Mammalian expression plasmids encoding activated MEK1 (pBABE MEK E217/E221) or dominant inhibitory MEK1 (pBABE MEK-A221) were a generous gift from Dr. M. H. Cobb (University of Texas-Southwestern, Dallas, TX). myogeninpromoter deletions were constructed by PCR of genomic chick DNA. In brief, genomic DNA isolated from embryonic day-10 myocytes was amplified with Pfu DNA polymerase (Stratagene, Loyola, CA), and the primers 5′-CGAGGTCGACGGTATCGATAAG (5′-primer) and 5′-GGGAGGACGTGTGCGCGGCTC (3′-primer), which span the region from 829 nucleotides upstream of the transcriptional start site (+1) of themyogenin gene to the translational start site, respectively (22Malik S. Huang C.-F. Schmidt J. Eur. J. Biochem. 1995; 230: 88-96Crossref PubMed Scopus (25) Google Scholar). The fidelity of the amplified DNA fragment (−829/+40) was confirmed by automated sequencing (Davis Sequencing, Davis, CA). Themyogenin promoter fragment was inserted into the vector pGL2-basic (Promega, Madison, WI) to create −829mgn-Luc. 5′-deletion mutants of −829mgn-Luc were created by PCR using Pfu DNA polymerase, the 3′-primer (see above), and the following 5′-primers: −829, 5′-CCCTCGAGGTCGACGGTATC; −668, 5′-CTCCGCTGGAATCTGGCCCAG; −545, 5′-GAAGGGTGAAAACCCATCCC; −470, 5′-GAACTGTTCCTCTCAGGGCTG; and −228, 5′-AGGGCAGCTCCCACCATGCC. An Xba restriction site was included at the beginning of the 5′-primer, and a BglII restriction site was included on the 3′-primer to allow for directional cloning into pGL2. The fidelity of the reporters was verified by automated DNA sequencing. Myoblasts were isolated from the hind limbs of embryonic day-10 (ED10) chicks (SPAFAS, Preston, CT) as described previously (10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). The cells were seeded onto gelatin-coated tissueware and cultured in growth media composed of Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 2% chicken serum, 1% penicillin-streptomycin, and 20 μg/ml Geneticin (Invitrogen). 18 h post-plating, the myoblasts (2 × 105) were transiently transfected by calcium phosphate precipitate formation with 0.5 μg of CMV-Raf BXB or CMV-Raf CAAX; 0.5 μg of CMV-LacZ or 0.1 μg of pRL-CMV; and 1.0 μg of −829mgn-Luc or an equimolar amount of the respectivemyogenin promoter deletion reporter plasmids. The cells were incubated with the DNA precipitates for 4 h followed by a brief osmotic shock (20% glycerol in Dulbecco's modified Eagle's medium). The cultures were maintained in growth media for an additional 48 h prior to lysis and measurement of luciferase and β-galactosidase orRenilla luciferase activities. The luciferase values were normalized to β-galactosidase or Renilla to correct for transfection efficiencies. Statistical significance between the treatments was determined by analysis of variance. Chick ED10 myoblasts (1 × 106) were transduced with RCAS, RCAS-Raf CAAX, and RCAS-Raf BXB and treated for 48 h with 20 μm PD98059 (New England Biolabs, Beverly MA) or an equivalent amount of Me2SO. The cells were washed with phosphate-buffered saline and lysed with 5 ml of RNA STAT-60 (Tel-Test, Friendswood, TX). The lysates were processed according to the manufacturer's directions, and the total RNA pellet was dissolved in sterile water, quantified, and stored frozen at −80° C until needed. One microgram of total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) in a reaction volume of 20 μl. Two microliters of the first strand reaction was amplified with Taq (Promega, Madison, WI) and the following primer sets: myogenin (F-GCAGCCTCAACCAGGAGGA, R-CTCCTCTGGGAACGTCACG) and glyceraldehyde-3-phosphate dehydrogenase (F-AGTCATCCCTGAGCTGAATG, R-AGGATCAAGTCCACAACACG). The amplification products were separated through ethidium bromide-containing agarose gels, visualized, and photographed. ED10 myoblasts were transduced with RCAS, RCAS-Raf CAAX, or RCAS-Raf BXB retroviruses as described previously (10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). For the inhibition of MEK activity, the cultures were incubated with growth media supplemented with 20 μmPD98059 (Cell Signaling, Beverly, MA). Extracts of nuclear proteins were prepared, and total protein content was determined by Bradford assay (Bio-Rad). Ten micrograms of nuclear proteins were electrophoretically separated through 12% denaturing gels and transferred to nitrocellulose. The blots were incubated for 60 min in 10 mm Tris, pH 8.0, 150 mm NaCl, and 0.1% Tween 20 (TBST) containing 5% nonfat dry milk. Subsequently, the blots were incubated for 1 h at room temperature with anti-myogenin (1:3,000, a kind gift from Dr. Bruce Paterson, National Institutes of Health). The blots were washed extensively with TBST prior to incubation for 1 h with anti-rabbit peroxidase-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA). The blots were further washed with TBST, and the myogenin immunoreactive complexes were visualized by chemiluminescence (ECL, AmershamBiosciences). Myoblasts (2 × 105) transiently transfected with 5 μg of pBABE MEK-E217/E221, pBABE MEK-A221, CMV-Raf BXB, or CMV-Raf CAAX were analyzed in a similar fashion. Briefly, the cells were lysed with SDS-PAGE sample buffer 3 days after transfection. Total cellular proteins were separated through 12% polyacrylamide gels, transferred to nitrocellulose, and analyzed using anti-Myc (1:5,000; 9E10 ascites, Developmental Hybridoma Bank, University of Iowa, Iowa City, IA) for the detection of Raf proteins and anti-MEK1 (1:500; C-18, Santa Cruz Biotechnology, Santa Cruz, CA) for the detection of MEK-A221 proteins. Visualization of immunocomplexes was achieved using the appropriate peroxidase-conjugated secondary antibody and chemiluminescence. Early work demonstrated that constitutive Ras activity leads to a loss of MRF expression in myocytes, which may contribute to the inhibitory effects of the kinase (21Lassar A.B. Thayer M.J. Overell R.W. Weintraub H. Cell. 1989; 58: 659-667Abstract Full Text PDF PubMed Scopus (209) Google Scholar). Because Raf signaling is a product of Ras action and Raf is an inhibitor of myogenesis, we examined the relative amounts of myogenin protein in Raf-transformed chick myoblasts (10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). In brief, equal amounts of nuclear proteins were analyzed by Western blot using a polyclonal myogenin antibody. Chemiluminescent detection revealed equivalent amounts of myogenin in RCAS- and RCAS-Raf BXB-transduced myoblasts (Fig. 1 A). By contrast, the level of myogenin protein synthesized by RCAS-Raf CAAX-transformed myogenic cells was significantly reduced. To determine the effects of MEK inactivation on myogenin production, RCAS-, RCAS-Raf BXB-, and RCAS-Raf CAAX-transduced cells were treated with 20 μm PD98059 and analyzed by Western blot. PD98059 is a chemical agent with preferential specificity for MEK that acts to suppress activation of this kinase, leading to an inhibition of MAPK activity (23Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3248) Google Scholar, 24Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3924) Google Scholar, 25Wang X. Studzinski G.P. Exp. Cell Res. 2001; 268: 294-300Crossref PubMed Scopus (38) Google Scholar). Results demonstrate that the relative levels of myogenin protein synthesized by RCAS- and RCAS-Raf BXB-transduced myoblasts are unaffected by the MEK inhibitor. However, treatment of RCAS-Raf CAAX cells with the chemical agent restored myogenin protein expression. The re-establishment of myogenin expression is not a reflection of altered protein translation. Semi-quantitative RT-PCR for myogenin mRNA expression in the Raf-transformed cells is analogous to the Western blot findings. RCAS- and RCAS-Raf BXB-transduced cells transcribe abundant amounts ofmyogenin message, whereas RCAS-Raf CAAX-transduced myoblasts direct a fraction of the amount (Fig. 1 B). myogenin expression is affected by MEK activity, and re-establishment of gene expression to levels comparable with controls is achieved by inhibition of MEK. However, the restoration of myogenin does not remove the Raf-imposed block to myoblast differentiation (10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). To better understand the means by which MEK contributes to Raf-induced repression ofmyogenin, a panel of myogenin promoter deletions was constructed. The largest myogenin promoter fragment was amplified from chicken genomic DNA with primers designed from the published myogenin promoter sequence (22Malik S. Huang C.-F. Schmidt J. Eur. J. Biochem. 1995; 230: 88-96Crossref PubMed Scopus (25) Google Scholar). The 829-bpmyogenin promoter region was analyzed by TFSEARCH, a web-based search engine for the identification of putativecis elements, and the results were used to develop the deletion mutants. As shown in Fig. 2, several DNA binding sites exist within the proximal promoter that are known to be affected by MAPK activity including Ras-response element-binding proteins, cAMP response element-binding proteins, E-boxes, and MEF2 binding sites (26Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1668) Google Scholar, 27Thiagalingam A. de Bustros A. Borges M. Jasti R. Diamond L. Mabry M. Ball D.W. Baylin S.B. Nelkin B.D. Mol. Cell. Biol. 1996; 16: 5335-5345Crossref PubMed Scopus (121) Google Scholar). ED10 chick myoblasts were transiently transfected with −829mgn-Luc, −668mgn-Luc, −545mgn-Luc, −470mgn-Luc, −228mgn-Luc, or pGL2-basic, CMV-β-galactosidase, and CMV or CMV-Raf CAAX. After 48 h the cells were lysed and analyzed for luciferase and β-galactosidase activity. As shown in Fig.3, all of the myogeninpromoters are capable of supporting strong luciferase expression. The smallest promoter fragment, −228mgn-Luc, drives reporter gene expression to levels comparable with the largest myogeninpromoter reporter (−829mgn-Luc), indicating that all of the elements necessary for transcription are present in the proximal 228 base pairs. Additionally, constitutive expression of activated Raf dramatically inhibits each of the myogenin promoters to levels approximating 70% of control cells lacking Raf CAAXexpression. No differences were found between the various reporters with regard to the extent of inhibition. Therefore, the ciselements affected by Raf signaling likely are contained within the minimal promoter reporter, −228mgn-Luc.Figure 3Activated Raf inhibits myogeninpromoter reporter activity. Chick myoblasts (2 × 105) were transfected with 1 μg of −829mgn-Luc or an equimolar amount of 5′-deletion reporter, 0.5 μg of CMV-LacZ, 0.5 μg of CMV, or CMV-Raf CAAX. After 48 h cells were lysed, and luciferase and β-gal activities were measured. Normalized luciferase activity produced by pGL2 was set equal to one. Means and S.E. are from three independent experiments.View Large Image Figure ViewerDownload (PPT) To identify the region of the myogenin promoter that responds to MEK signaling, the panel of myogenin promoter reporters was tested in Raf-expressing myoblasts treated with the MEK inhibitor PD98059. Embryonic day-10 chick myoblasts were transiently transfected with equimolar amounts of −829mgn-Luc or −228mgn-Luc, pRL-renilla, and CMV or CMV-Raf CAAX. After 48 h, the cells were lysed, and luciferase and Renilla luciferase activities were measured. As shown in Fig. 4 A, both the full-length (−829mgn-Luc) and minimal (−228mgn-Luc) promoter reporters efficiently activate luciferase expression in the absence of Raf signaling, and the levels of luciferase are reduced significantly in the presence of activated Raf. Moreover, treatment of Raf CAAX myoblasts with 20 μm PD98059 did not restore −829mgn-Luc or −228mgn-Luc transcriptional activities to levels comparable with those found in control myoblasts. Similar findings were found for the remaining deletion mutants (data not shown). Because the chemical inhibitor increases basalmyogenin promoter activity, ED10 myoblasts were transiently transfected with either the full-length reporter or the minimal promoter and expression plasmids encoding Raf CAAX and MEK-A221, a kinase-defective MEK. After 48 h the cells were lysed, and luciferase activity was measured. The amount of luciferase activity was corrected for transfection efficiency by normalizing for β-galactosidase activity. Myoblasts expressing activated Raf directed low levels of −829mgn-Luc and −228mgn-Luc reporter activity as expected (Fig. 4 B). Cells cotransfected with CMV-Raf CAAX and pBABE-MEK-A221 failed to direct transcription from −829mgn-Luc to levels comparable with myoblasts transfected with reporter only. In an analogous fashion, the dominant inhibitory MEK-A221 protein was unable to restore full activity to the −228mgn-Luc reporter in the presence of activated Raf. The inability of the MEK inhibitors to restore myogeninpromoter function in the presence of Raf CAAX is not a product of insufficient MEK-A221 protein expression as determined by Western blot (Fig. 4 C). In brief, myoblasts were transfected with the expression plasmids coding for MEK-A221 and Raf CAAX. After 48 h, the cells were lysed with SDS-PAGE buffer, and equal amounts of protein were electrophoretically separated and transferred to nitrocellulose. The blots were probed with anti-MEK1. Chemiluminescent detection revealed that abundant amounts of MEK-A221 were produced in the presence of Raf CAAX. Thus, reestablishment of myogenin gene expression by inhibition of MEK activity occurs independently of the cis-DNA elements located in the 829-bp promoter region. Recently it was reported that an inducible Raf BXB inhibits L6 myoblasts from terminal differentiation, and the block to myogenesis is a consequence of MEF2 localization to the cytoplasm (28Winter B. Arnold H.-H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar). Raf CAAX inhibits the minimalmyogenin promoter (−228mgn-Luc), and the region contains a single MEF2 DNA binding site (Fig. 2). To examine the effects of activated Raf on MEF2 and E-box function, each of the sites was mutated in the minimal promoter reporter to create −228mMEF2mgn-Luc and −228mEmgn-Luc, respectively. The promoter reporters were transfected into ED10 myoblasts with pRL-CMV, CMV, or CMV-Raf CAAX. After 48 h the cells were harvested, and luciferase activities were measured. As shown in Fig. 5, both the wild-type −228mgn-Luc and −228mEmgn-Luc direct abundant levels of luciferase activity in the absence of Raf signals. Mutation of the single MEF2 site within the context of the minimal promoter completely abolishes transcriptional activity as reported previously (22Malik S. Huang C.-F. Schmidt J. Eur. J. Biochem. 1995; 230: 88-96Crossref PubMed Scopus (25) Google Scholar). Cotransfection of CMV-Raf CAAX significantly repressed activation of both wild-type and the E-box-deficient promoter reporters. Moreover, cotransfection of pMT2-MEF2A with −228mgn-Luc or −228mEmgn-Luc significantly enhanced the level of reporter activity compared with controls. Unexpectedly, cotransfection of the ED10 myoblasts with expression plasmids coding for Raf CAAX and MEF2A did not restore the levels of luciferase activity to levels comparable with control muscle cells transfected with −228mgn-Luc or −228mEmgn-Luc plus MEF2A. In addition, coexpression of activated Raf with MEF2 did not result in sequestration of the transcription factor in the cytoplasm (Fig. 6). These results are attributable to differences between primary avian muscle cells and C3H10T1/2 myoblasts as we were able to duplicate the results of Winter and Arnold (28Winter B. Arnold H.-H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar). Fibroblasts were transfected with expression plasmids encoding avian MyoD (CMD), activated Raf, or MEF2A and −228mgn-Luc, −228mEmgn-Luc, or −228mMEF2mgn-Luc. After 48 h in differentiation-permissive media the cells were harvested, and reporter activities were measured. Activated Raf suppressed transcription from the wild-type reporter (Fig. 7). The transcriptional block was effectively removed by coexpression of MEF2 with Raf CAAX as reported previously (28Winter B. Arnold H.-H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar). Interestingly, CMD was unable to activate transcription from −228mMEF2mgn-Luc, which contains a viable E-box. MEF2A readily activated −228mEmgn-Luc, and the levels of activity were enhanced significantly by coexpression with activated Raf. Thus, activated Raf inhibits avian myogenesis through a mechanism that is not entirely reflected in disrupted MEF2 function.Figure 6MEF2A localizes to the nucleus in the presence of Raf CAAX. Chick myoblasts were transiently transfected with pMT2-MEF2A (A) or pMT2-MEF2A and CMV-Raf CAAX (B). Cells were fixed and immunostained for MEF2A protein expression. Arrows indicate MEF2-containing nuclei. Representative photomicrographs at 400× are shown.View Large Image Figure ViewerDownload (PPT)Figure 7MEF2A restores myogeninpromoter reporter activity to Raf-expressing myoblasts.C3H10T1/2 fibroblasts were transfected with 1 μg of −228mgn-Luc reporter plasmids, 0.1 μg of pRL-CMV, 0.5 μg of CMV-CMD, and 0.5 μg of pMT2-MEF2A, CMV-Raf CAAX, or both plasmids. Luciferase activities were normalized to Renilla activity. −228mEmgn-Luc and −228mMEFmgn-Luc lack a functional E-box and MEF2 site, respectively.View Large Image Figure ViewerDownload (PPT) Constitutive activation of components of the Ras/Raf/MEK/MAPK signaling axis consistently has demonstrated a strong repressive effect on skeletal myogenesis (4Kong Y. Johnson S.E. Taparowsky E.J. Konieczny S.F. Mol. Cell. Biol. 1995; 15: 5205-5213Crossref PubMed Scopus (65) Google Scholar, 10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar, 20Konieczny S.F. Drobes B.L. Menke S.L. Taparowsky E.J. Oncogene. 1989; 4: 473-481PubMed Google Scholar, 28Winter B. Arnold H.-H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar, 29Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (303) Google Scholar). However, the mechanism by which chronic activity of the archetypical signaling module inhibits muscle gene expression is less well understood. Members of the myogenic regulatory factors retain inherent DNA binding and transcriptional activation capacities, thus suggesting that the molecular means for the repression of gene function is levied at alternate transcriptional mediators (4Kong Y. Johnson S.E. Taparowsky E.J. Konieczny S.F. Mol. Cell. Biol. 1995; 15: 5205-5213Crossref PubMed Scopus (65) Google Scholar, 10Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). Our study demonstrates that loss ofmyogenin gene expression accompanies elevated Raf kinase signaling in a MEK-dependent manner. Constitutive expression of Raf CAAX, a full-length membrane-localized Raf protein, dramatically reduces the number of myogenin mRNA transcripts and subsequent protein synthesis. Surprisingly, RCAS-Raf BXB-transduced myoblasts transcribe abundant amounts of myogenin mRNA, yet the cells remain differentiation-defective. Moreover, restoration of myogenin gene transcription by treatment of the RCAS-Raf CAAX myocytes with PD98059 does not reverse the negative effects of the kinase on morphological or biochemical differentiation. From these results, we conclude that loss of myogenin expression is not the molecular target of activated Raf that leads to repression of the muscle differentiation program. The predominant signaling cascade induced by Raf is the sequential activation of MEK and MAPK (1Joneson T. Bar-Sagi D. J. Mol. Med. 1997; 75: 587-593Crossref PubMed Scopus (144) Google Scholar). However, Raf kinase interacts with additional kinase intermediates that may serve to amplify or instigate alternate, MEK-independent signaling (30Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 31Morrison D.K. Cutler Jr., R.E. Curr. Opin. Genet. Dev. 1997; 9: 174-179Google Scholar, 32Pearson G. Bumeister R. Henry D.O. Cobb M.H. White M.A. J. Biol. Chem. 2000; 275: 37303-37306Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). A direct physical association occurs between Raf and MEKK1, a dual-specific kinase that signals primarily through downstream Jun N-terminal kinase with minimal activation of MAPK (30Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Protein complexes comprised of MEKK1, MEK, and MAPK also can be found, suggesting that the kinase may serve as a scaffold protein for the Raf/MEK/MAPK module. Moreover, a mutated Raf protein that fails to interact with MEK and induce MAPK activity retains its ability to transactivate NFκB reporter genes, further supporting the growing body of evidence that Raf represents a signaling bifurcation point (32Pearson G. Bumeister R. Henry D.O. Cobb M.H. White M.A. J. Biol. Chem. 2000; 275: 37303-37306Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Our previous work indicates that the membrane-localized form of constitutively active Raf signals through downstream kinases that are MEK-independent (11Dorman C.M. Johnson S.E. J. Biol. Chem. 2000; 275: 27481-27487Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we report that MEK is present in embryonic chick myoblasts transduced with RCAS-Raf CAAX and that the chemical inhibitor PD98059 suppresses the functional activity of MEK in these cells. Thus, Raf CAAXdisrupts transcription from the myogenin gene in a MEK-dependent manner. We chose to look for MEK-responsivecis elements within the promoter of myogenin in an effort to identify targets of Raf/MEK/MAPK signaling that contribute to repression of myogenesis. Serial deletions of the largest promoter fragment did not reveal the presence of elements that are solely regulated by Raf/MEK actions. Interestingly, overexpression of a kinase-defective MEK (MEK-A221), disruption of the Raf/MEK interface (RKIP-N60), or treatment with PD98059 did not restore full transcriptional activity to −228mgn-Luc in the presence of Raf CAAX. These findings illustrate that the MEK-responsivecis element lies outside of the boundaries of themyogenin promoter regions examined. Recently, MEF2 was identified as a critical myogenic regulator whose function was disrupted as a consequence of activated Raf signaling (28Winter B. Arnold H.-H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar). L6 myoblasts overexpressing activated Raf fail to differentiate into mature myocytes partially because of the cytoplasmic localization of MEF2. Forced expression of MEF2 resulted in restoration of myoblast fusion and muscle gene transcription. In a similar manner, we found that activated Raf suppresses transcription from the minimalmyogenin promoter in C3H10T1/2 myoblasts, and the block to reporter gene activation is overcome by compulsory MEF2A expression. Thus, MEF2 likely is a target of Raf/MEK/MAPK signaling that lends to the inhibitory effects of the kinase in mouse myogenic cells. However, additional transcription factors also are altered in response to activated Raf signaling that contribute to the block to avian myogenesis. Embryonic chick myoblasts transfected with the minimal promoter (−228mgn-Luc) and expression plasmids coding for activated Raf and MEF2A did not direct reporter gene activity levels comparable with those found in avian myoblasts expressing MEF2 alone. Although MEF2 does partially restore transcription from the myogeninpromoter in the presence of Raf kinase, the levels fail to reach those directed by the synergistic actions of endogenous MRF and MEF2. Moreover, immunostaining for MEF2 failed to demonstrate a localization of the factor to the cytoplasm in the presence of constitutive Raf activity. These results argue that additional inhibitory mechanisms are initiated by activated Raf in avian myoblasts that contribute to the block to myocyte formation. The role of the MEK/MAPK signaling with regard to myogenic repression remains controversial. Recently, it was reported that a nuclear-localized, activated MEK can inhibit skeletal muscle gene transcription (33Perry R.L.S. Parker M.H. Rudnicki M.A. Mol. Cell. 2001; 8: 291-301Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Moreover, the repressive actions of MEK involved a physical association of the kinase with the transcriptional activation domain of MyoD and were independent of the basic helix-loop-helix and chromatin-remodeling domains of the MRF. A direct interaction of the MRFs and MEK may partially explain the repressive effects of Raf that are not attributable to disrupted MEF2 function. Under moderate levels of Raf activity (Raf BXB), myogenesis is inhibited by sequestration of MEF2 in the cytoplasm in a MAPK-dependent manner. Extreme levels of Raf activity (Raf CAAX) prevent muscle gene transcription by mechanisms that involve MAPK-mediated MEF2 translocation to the cytoplasm and MEK-MRF complex formation and repression. The validity of these inferences remains to be experimentally tested. We thank Dr. Bruce Paterson for provision of the anti-myogenin antibody." @default.
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