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• W2056150526 abstract "The Raf/MEK/MAPK signaling module elicits a strong negative impact on skeletal myogenesis that is reflected by a complete loss of muscle gene transcription and differentiation in multinucleated myocytes. Recent evidence indicates that Raf signaling also may contribute to myoblast cell cycle exit and cytoprotection. To further define the mechanisms by which Raf participates in cellular responses, a stable line of myoblasts expressing an estrogen receptor-Raf chimeric protein was created. The cells (23A2RafERDD) demonstrate a strict concentration-dependent increase in chimeric Raf protein synthesis and downstream phosphoMAPK activation. Initiation of low-level Raf activity in these cells augments contractile protein expression and myocyte fusion. By contrast, induction of high level Raf activity in 23A2RafERDD myoblasts inhibits the formation of myocytes and muscle reporter gene expression. Interestingly, treatment of myoblasts with conditioned medium isolated from Raf-repressive cells inhibits all of the aspects of myogenesis. Closer examination indicates that the transforming growth factor-β1 (TGF-β1) gene is up-regulated in Raf-repressive myoblasts. The cells also direct elevated levels of Smad transcriptional activity, suggesting the existence of a TGF-β1 autocrine loop. However, extinguishing the biological activity of TGF-β1 does not restore the myogenic program. Our results provide evidence for the involvement of Raf signal transmission during myocyte formation as well as during inhibition of myogenesis. The Raf/MEK/MAPK signaling module elicits a strong negative impact on skeletal myogenesis that is reflected by a complete loss of muscle gene transcription and differentiation in multinucleated myocytes. Recent evidence indicates that Raf signaling also may contribute to myoblast cell cycle exit and cytoprotection. To further define the mechanisms by which Raf participates in cellular responses, a stable line of myoblasts expressing an estrogen receptor-Raf chimeric protein was created. The cells (23A2RafERDD) demonstrate a strict concentration-dependent increase in chimeric Raf protein synthesis and downstream phosphoMAPK activation. Initiation of low-level Raf activity in these cells augments contractile protein expression and myocyte fusion. By contrast, induction of high level Raf activity in 23A2RafERDD myoblasts inhibits the formation of myocytes and muscle reporter gene expression. Interestingly, treatment of myoblasts with conditioned medium isolated from Raf-repressive cells inhibits all of the aspects of myogenesis. Closer examination indicates that the transforming growth factor-β1 (TGF-β1) gene is up-regulated in Raf-repressive myoblasts. The cells also direct elevated levels of Smad transcriptional activity, suggesting the existence of a TGF-β1 autocrine loop. However, extinguishing the biological activity of TGF-β1 does not restore the myogenic program. Our results provide evidence for the involvement of Raf signal transmission during myocyte formation as well as during inhibition of myogenesis. Myogenesis, the formation of contractile-competent skeletal muscle cells, is tightly regulated by the presence of several critical growth factors in the extracellular environment. As an example, local production of insulin-like growth factor-I (IGF-I) 1The abbreviations used are: IGF, insulin-like growth factor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; AP-1, activator protein 1; MRF, myogenic regulatory factor; PBS, phosphate-buffered saline; TGF-β, transforming growth factor β; tk, thymidine kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; MyHC, myosin heavy chain; DAPI, 4,6-diamidino-2-phenylindole; 4HT, 4-hydroxytamoxifen; RT, reverse transcription; TnI-Luc, troponin I luciferase; STAT, signal transducers and activators of transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FGF, fibroblast growth factor; GDF, growth and differentiation factor. is a strong permissive growth factor for both morphological and biochemical differentiation of myoblasts (1.Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 2.Crown A.L. He X.L. Holly J.M. Lightman S.L. Stewart C.E. J. Endocrinol. 2000; 167: 403-415Crossref PubMed Scopus (47) Google Scholar, 3.Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar, 4.Tureckova J. Wilson E.M. Cappalonga J.L. Rotwein P. J. Biol. Chem. 2001; 276: 39264-39270Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Ectopic IGF-I delivery to skeletal muscle cells increases muscle mass and morphometric measures of strength in birds and mice (5.Barton-Davis E.R. Shoturma D.I. Musaro A. Rosenthal N. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15603-15607Crossref PubMed Scopus (604) Google Scholar, 6.Mitchell P.J. Johnson S.E. Hannon K. Dev. Dyn. 2002; 223: 12-23Crossref PubMed Scopus (26) Google Scholar). On the contrary, several laboratories have documented the repressive effects of transforming growth factor β1 (TGF-β1) and members of the fibroblast growth factor family, chiefly FGF2 (for review see Refs. 7.McLennan I.S. Koishi K. Int. J. Dev. Biol. 2002; 46: 559-567PubMed Google Scholar and 8.Olwin B.B. Hannon K. Kudla A.J. Prog. Growth Factor Res. 1994; 5: 145-158Abstract Full Text PDF PubMed Scopus (42) Google Scholar). The mechanism by which these soluble inhibitory proteins impede skeletal myogenesis involves the activation of specific intracellular signaling cascades. In the case of TGF-β1, a requisite induction of Smad3 protein phosphorylation and nuclear translocation occurs that interferes with muscle gene transcription (9.Liu D. Black B.L. Derynck R. Genes Dev. 2001; 15: 2950-2966Crossref PubMed Scopus (299) Google Scholar). The precise mechanism for FGF2-mediated inhibition of myogenesis is less understood but may involve downstream signaling events that are controlled through G-proteins (10.Fedorov Y.V. Jones N.C. Olwin B.B. Mol. Cell. Biol. 1998; 18: 5780-5787Crossref PubMed Scopus (50) Google Scholar). Overexpression studies and application of specific kinase inhibitors has led to the identification of several essential signaling pathways in skeletal myoblasts. It is firmly established that phosphatidylinositol 3-kinase and p38, a stress-activated mitogen-activated protein kinase (MAPK), both are necessary for the formation of mature myocytes (11.Cuenda A. Cohen P. J. Biol. Chem. 1999; 274: 4341-4346Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 12.Jiang B.H. Zheng J.Z. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14179-14183Crossref PubMed Scopus (117) Google Scholar, 13.Kaliman P. Vinals F. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1996; 271: 19146-19151Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 14.Li Y. Jiang B. Ensign W.Y. Vogt P.K. Han J. Cell Signal. 2000; 12: 751-757Crossref PubMed Scopus (97) Google Scholar, 15.Zetser A. Gredinger E. Bengal E. J. Biol. Chem. 1999; 274: 5193-5200Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Removal of downstream signaling events by the respective kinases through the use of chemical inhibitors abolishes myogenesis. By contrast, initiation of downstream signaling modules by constitutively active Ras, a membrane-localized GTPase that participates in numerous receptor tyrosine kinase-initiated signaling events, results in a severe reduction in muscle gene transcription and myoblast fusion (16.Kong Y. Johnson S.E. Taparowsky E.J. Konieczny S.F. Mol. Cell. Biol. 1995; 15: 5205-5213Crossref PubMed Scopus (65) Google Scholar, 17.Ramocki 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). Activation of Raf, the downstream target of Ras, also abolishes the myogenic program (18.Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar, 19.Perry R.L. Parker M.H. Rudnicki M.A. Mol. Cell. 2001; 8: 291-301Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 20.Samuel D.S. Ewton D.Z. Coolican S.A. Petley T.D. McWade F.J. Florini J.R. Horm. Metab. Res. 1999; 31: 55-64Crossref PubMed Scopus (31) Google Scholar, 21.Winter B. Arnold H.H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar). Interestingly, the means by which Ras and Raf inhibit muscle gene expression does not appear to be directly dependent upon the subsequent phosphorylation and activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Inhibition of MEK/ERK signal transduction does not reinstate the full complement of muscle gene transcription and myoblast fusion to myoblasts expressing constitutively active Ras or Raf (17.Ramocki 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, 22.Dorman C.M. Johnson S.E. J. Biol. Chem. 2000; 275: 27481-27487Abstract Full Text Full Text PDF PubMed Google Scholar). Raf kinase is a serine/threonine kinase whose activation precludes ERK1/2 (MAPK) phosphorylation and a concomitant alteration in gene transcription (23.Herrera R. Sebolt-Leopold J.S. Trends Mol. Med. 2002; 8: S27-S31Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 24.Hindley A. Kolch W. J. Cell Sci. 2002; 115: 1575-1581Crossref PubMed Google Scholar). Signaling through the Raf/MEK/ERK pathway is a common mitogenic response for many cell types. Recently, several groups have reported differential cellular responses as a consequence of Raf/MAPK signal intensity. At lower levels of MAPK activity, epithelial and fibroblasts demonstrate increased proliferative rates (25.Kerkhoff E. Rapp U.R. Cancer Res. 1998; 58: 1636-1640PubMed Google Scholar, 26.Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (419) Google Scholar). By contrast, high levels of Raf/MAPK result in cell cycle arrest and senescence (26.Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (419) Google Scholar, 27.Schulze A. Lehmann K. Jefferies H.B. McMahon M. Downward J. Genes Dev. 2001; 15: 981-994Crossref PubMed Scopus (222) Google Scholar). The Raf-mediated responses are attributed to signaling through MEK/MAPK as well as non-MEK-dependent signaling events. Regulation of Raf activity indicates that the protein physically associates with other kinases, regulatory proteins, and scaffolding proteins (for review see Ref. 28.Chong H. Vikis H.G. Guan K.L. Cell Signal. 2003; 15: 463-469Crossref PubMed Scopus (346) Google Scholar). Indeed, it has become increasingly apparent that Raf kinase can control cellular transcriptional responses through mechanisms that are independent of the archetypical MEK/ERK module. A Raf kinase allele that fails to interact with MEK and cause ERK1/2 phosphorylation retained the ability to activate NFκB-directed transcription and promote neuronal differentiation (29.Pearson 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). These results argue that Raf participates in several downstream signaling cascades. Differential utilization of these pathways may be reflected in the decision of the cell to undergo proliferation, differentiation, apoptosis, and senescence. Because many growth factors exhibit contrasting effects on skeletal myogenesis yet utilize many of the same intracellular signaling pathways, it is likely that signal intensity plays a critical role in the decision to complete terminal differentiation. To this end, myoblasts that express an inducible activated Raf allele were created. Our results indicate that low level Raf activity promotes myogenesis, whereas high level Raf activity inhibits muscle formation. Coincident with repression of differentiation by activated Raf is an increase in TGF-β1 gene expression and Smad-directed transcriptional activity. However, the removal of TGF-β1 from the extracellular environment of the Raf-repressive myoblasts does not reinstate the differentiation program. In summary, Raf signaling modules are both positive and negative mediators of myogenesis that is a direct reflection of signal strength. Cell Culture, Plasmids, and Transfections—23A2RafERDD myoblasts were created by transduction of 23A2 myoblasts with a retrovirus encoding the fusion protein RafERDD (30.Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (323) Google Scholar). RafERDD is comprised of the kinase domain of human c-Raf-1 fused in-frame with the estrogen receptor ligand binding domain. Following infection, the cells were selected in puromycin and clones were isolated by limiting dilution. 23A2 and 23A2RafERDD myoblasts were cultured on gelatinized tissue culture grade plasticware in Dulbecco's modified Eagle medium containing 15% fetal bovine serum (BioWhittaker), 1% penicillin-streptomycin, and 0.5% Geneticin (Invitrogen). Differentiation was induced in confluent cultures by continuous culture in Dulbecco's modified Eagle medium supplemented with 2% horse serum, 1% penicillin-streptomycin, and 0.5% Geneticin. For the measurement of muscle-specific reporter gene activity, myogenic cells cultured in 6-well tissue plasticware were transiently transfected with 1 μg of troponin I luciferase (TnI-Luc), the minimal E-box reporter plasmid, 4Rtk-Luc, or a multimerized AP-1 binding site reporter (AP-1-Luc) and 50 ng of pRL-tk, a Renilla luciferase plasmid as a monitor of transfection efficiency, using standard calcium phosphate methods (16.Kong Y. Johnson S.E. Taparowsky E.J. Konieczny S.F. Mol. Cell. Biol. 1995; 15: 5205-5213Crossref PubMed Scopus (65) Google Scholar, 22.Dorman C.M. Johnson S.E. J. Biol. Chem. 2000; 275: 27481-27487Abstract Full Text Full Text PDF PubMed Google Scholar). The cells were maintained in differentiation media for 48 h in the presence or absence of varying concentrations of 4-hydroxytamoxifen (4HT, Sigma) prior to lysis and measurement of luciferase activities (Dual-luciferase, Promega, Madison, WI). Inhibition of MEK activity was achieved by supplementation of differentiation medium with 20 μm PD98059. The amount of corrected luciferase activity generated by 23A2 or 23A2RafERDD myoblasts treated with vehicle was set to 100%. Each experiment was replicated a minimum of three times. For conditioned medium experiments, the culture medium was removed from myoblasts treated for 48 h with Me2SO or varying amounts of 4HT. The medium was centrifuged to remove debris and brought to a final concentration of 2% horse serum. The resulting medium was applied to confluent 23A2 myoblasts, and the cells were maintained for an additional 48 h prior to fixation. Heat inactivation of the conditioned medium was performed as described previously (31.Weyman C.M. Wolfman A. Oncogene. 1997; 15: 2521-2528Crossref PubMed Scopus (9) Google Scholar). RNA Isolation, Northern Blots, and RT-PCR—Total RNA was isolated with STAT60-denaturing solution per the manufacturer's recommendations (Tel-Test, Friendswood, TX). Twenty micrograms of total RNA were separated through 1% agarose gels containing 2% formaldehyde, transferred to nitrocellulose membrane, and irreversibly cross-linked to the medium by UV. The blots were hybridized with 32P-labeled cDNA probes generated by random hexamer priming (DecaPrime, Ambion, Houston, TX). The probes corresponded to regions contained within the TGF-β1 and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene-coding regions. Northern blot analysis was performed using Ultra-Hyb (Ambion) at 42 °C overnight according to the manufacturer's recommendations. Blots were washed with 2× SSC, 0.1% SDS once at room temperature and twice with 1× SSC, 0.1% SDS at 42 °C. Blots were exposed to phosphorimaging screens for the visualization and quantification of message levels. For the semi-quantitative assessment of mRNA levels, 1 μg of total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase. Amplification of GAPDH and TGF-β1 was performed using Taq polymerase (Fisher Scientific, Pittsburgh, PA), gene-specific primers, and thermocycle conditions of 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 90 s for a total of 35 cycles. To ensure that quantification was accomplished in the linear range of amplification, an aliquot was removed from each reaction after 25, 30, and 35 cycles. GDF-8 was amplified as described previously (32.Rios R. Carneiro I. Arce V.M. Devesa J. Biochem. Biophys. Res. Commun. 2001; 280: 561-566Crossref PubMed Scopus (128) Google Scholar). Amplicons were separated through 2% agarose gels containing ethidium bromide, visualized under UV, and photographed. Western Blot and Scanning Densitometry—23A2RafERDD myoblasts were differentiated in the presence or absence of 4HT. After 48 h, the cells were lysed in 4× SDS-PAGE sample buffer and an aliquot was removed for protein quantification. Equal amounts of protein were electrophoretically separated through denaturing gels and transferred to nitrocellulose membrane. The blots were incubated with 5% nonfat dry milk in TBST (10 mm Tris, pH 8.0, 150 mm NaCl, 0.1% Tween 20) to remove nonspecific binding sites. Primary antibodies were diluted in blocking buffer, and the blots were incubated overnight at 4 °C with shaking. Antibodies and dilutions included the following: anti-myogenin (F5D ascites, Developmental Hybridoma Bank, University of Iowa, 1:5000); anti-myosin heavy chain (MF20 hybridoma supernatant, Developmental Hybridoma Bank, 1:5); anti-ERK1/2 and anti-phosphoERK1/2 (Cell Signaling); and anti-estrogen receptor (Santa Cruz Biotechnology, Santa Cruz, CA, 1:300). After extensive washing with TBST, the blots were reacted with the appropriate peroxidase secondary antibody for 45 min at room temperature. Visualization of protein bands was accomplished by chemiluminescence and autoradiography. Multiple exposures to x-ray film were used to ensure that the linear range of densitometry was maintained. Autoradiograms were scanned on a Storm 860 phosphorimaging system (Molecular Dynamics, Amersham Biosciences). Immunocytochemistry—23A2 and 23A2RafERDD myocytes were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. The cells were washed with PBS, and nonspecific antigen sites were blocked by incubation with 5% horse serum in PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cultures were incubated with anti-myosin heavy chain (MF20) for 60 min at room temperature. After exhaustive washing with PBS, the cells were reacted with donkey anti-mouse fluorescein isothiocyanate (Vector Laboratories, Burlingame, CA, 1:200) for 45 min at room temperature. Cultures were washed with PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Immunofluorescent detection was accomplished using a Nikon TE200 inverted phase microscope equipped with epifluorescence. Representative photomicrographs were captured to slide film and assembled in Adobe Photoshop. Creation and Characterization of an Inducible Raf Myogenic Cell Line—Previous work has clearly demonstrated that overexpression of activated alleles of Raf are inhibitory to myocyte formation and muscle gene expression (17.Ramocki 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, 18.Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar, 21.Winter B. Arnold H.H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar). More importantly, MAPK-dependent repression appears to be a function of Raf signaling intensity in avian myoblasts (18.Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar). To extend these observations, an activated Raf kinase that is responsive to the estrogen analog, 4-hydroxytamoxifen (4HT), was stably expressed in 23A2 mouse myoblasts (23A2RafERDD). Confluent cultures of 23A2RafERDD myoblasts were differentiated in the presence or absence of increasing concentrations of 4HT. After 48 h, the cells were lysed for Western analysis of chimeric Raf (α-estrogen receptor), activated MAPK (α-phosphoMAPK), and total MAPK (α-MAPK) expression. As shown in Fig. 1, a dose-dependent increase in the relative amount of RafERDD protein is found with amounts 4HT from 0.25 nm to 2.5 μm (lanes 2–6). Coincident with the increase in chimeric Raf protein is an elevation in the amounts of phosphorylated MAPK. Phosphorylated ERK2 is preferentially activated at 4HT concentrations as low as 0.25 nm, whereas activated ERK1 is first apparent at 4HT concentrations of 25 nm or greater. No differences in the amounts of total MAPK (ERK1/2) were evident. Activation of ERK1/2 signal transmission leads to an increase in AP-1-directed transcriptional activity in many cell types including skeletal myoblasts. To ensure that the Raf/ERK pathway was functional in 23A2RafERDD myoblasts, semiconfluent cells were transiently transfected with a multimerized AP-1 reporter plasmid and pRL-tk, a plasmid encoding Renilla luciferase as a marker of transfection efficiency. Subsequently, the cells were treated with increasing amounts of 4HT for 48 h prior to lysis and measurement of luciferase activities. Control 23A2 myoblasts were treated in an analogous fashion. AP-1-Luc reporter activity was normalized to Renilla luciferase activity. Results demonstrate a dose-dependent increase in AP-1-Luc activity with increasing amounts of 4HT (Fig. 2). At concentrations of 4HT greater than 250 nm, a decline in AP-1 luciferase activity was evident. Moreover, the response is directly attributed to the induction of RafER activity as 23A2 myoblasts failed to initiate AP-1-transcriptional activity upon treatment with 4HT. These results indicate that the Raf/ERK signaling axis is intact and functional in the myogenic cells. As final confirmation of the integrity of the Raf signaling system in 23A2RafERDD myoblasts, confluent cultures of cells were treated for 48 h in differentiation medium containing vehicle only (Me2SO) or 1 μm 4HT. Subsequently, cells were fixed and immunostained for myosin heavy chain (α-MyHC). Control 23A2RafERDD myocytes treated with vehicle readily differentiate into large multinucleated myofibers that express copious amounts of the contractile protein, MyHC (Fig. 3). By contrast, activation of the Raf/ERK signaling pathway leads to a severe reduction in both myofiber number and MyHC protein expression. These results directly reflect published reports of the effects of activated Raf on skeletal myogenesis. Thus, 23A2RafERDD myoblasts represent a myogenic cell line that retains its muscle features in the absence of Raf activity and is differentiation-defective in the presence of Raf signal transmission. Contrasting Effects of Raf Signal Transduction on Skeletal Myoblasts—To understand the effects of signal intensity of myogenesis, 23A2RafERDD myoblasts were differentiated in the presence of increasing amount of 4HT. After 48 h, the cells were lysed and equal amounts of total cellular proteins were analyzed by Western blot for the expression of muscle proteins (anti-MyHC, anti-myogenin). Scanning densitometry was performed on the chemiluminescent autoradiograms. Muscle protein expression was normalized to total ERK2 expression, an internal marker for protein loading, transfer, and detection efficiency. In the absence of 4HT, 23A2RafERDD myoblasts differentiate into myocytes that express both MyHC and myogenin (Fig. 4). Unexpectedly, low level induction of Raf activity using concentrations of 4HT (0.25 or 2.5 nm) causes an increase in the amount of contractile and regulatory protein expression. Supplementation of differentiation medium with 25 nm 4HT or greater causes the predicted loss of MyHC and myogenin protein expression. The biphasic response of 23A2RafERDD myoblasts to increasing 4HT concentrations is further reflected in muscle-specific reporter gene activity. 23A2 and 23A2RafERDD myoblasts were transiently transfected with TnI-Luc and pRL-tk. After 48 h in differentiation medium supplemented with 4HT, the cells were lysed and luciferase activities were measured. TnI luciferase enzymatic activity did not vary as a function of 4HT treatment in control 23A2 myoblasts, indicating that the estrogen analog does not significantly alter basal muscle gene expression (Fig. 5). The levels of Raf activity directed in response to 25 nm 4HT or greater are sufficient for a reduction in TnI-Luc activity. However, low level Raf activity (0.25–2.5 nm 4HT) directs only a slight increase in muscle reporter gene activity. It is likely that a larger portion of the TnI regulatory region is necessary to obtain a substantial increase in reporter gene transcription. Our results clearly demonstrate that Raf has contrasting effects on muscle formation that are intensity-dependent.Fig. 5Raf activation alters two distinct phases of myogenesis. 23A2 and 23A2RafERDD myoblasts transiently transfected with TnI-Luc and pRL-tk were cultured in differentiation medium supplemented with increasing concentrations of 4HT. TnI-Luc was normalized to Renilla luciferase activity. The amount of reporter activity directed by 23A2 myocytes in the absence of 4HT was set to 100%. Low level Raf activity causes a slight increase in muscle reporter gene activity, whereas high level Raf activity inhibits myogenesis. Data represent the mean ± S.E. of at least three independent experiments. RLU, relative light units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previously, we have documented the existence of MEK-independent signaling in response to activated Raf (18.Dorman C.M. Johnson S.E. Oncogene. 1999; 18: 5167-5176Crossref PubMed Scopus (41) Google Scholar, 33.Johnson S.E. Dorman C.M. Bolanowski S.A. J. Biol. Chem. 2002; 277: 28742-28748Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). To determine the role of MEK in the RafER-imposed block to myogenesis, confluent 23A2RafERDD myoblasts were transiently transfected with TnI-Luc and pRL-tk. The myoblasts were treated for 48 h with increasing amounts of 4HT and 20 μm PD98059 or vehicle only. Cell lysates were collected, and luciferase activities were measured. The amount of muscle reporter gene activity directed in the absence of 4HT and the chemical MEK inhibitor was set to 1. As expected, the selected concentrations of 4HT caused a reduction in the amount of TnI-Luc reporter activation (Fig. 6). Suppression of MEK function restored muscle gene transcription as evidenced by an increase in TnI-Luc activity to levels comparable to controls. However, strong stimulation of Raf signaling initiated by 2.5 μm 4HT was refractile to the effects of the MEK inhibitor. Western analysis of replica samples demonstrates that treatment with PD98059 reinstates myosin heavy chain expression to levels <10% of controls (data not shown). These results duplicate previous observations using constitutive Raf alleles of various signaling intensities (21.Winter B. Arnold H.H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar, 33.Johnson S.E. Dorman C.M. Bolanowski S.A. J. Biol. Chem. 2002; 277: 28742-28748Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Raf Inhibition of Myogenesis Involves Secretion of a Soluble Factor—It has been reported that the inability of myoblasts to differentiate in the presence of activated Raf is a product of sequestration of MEF2 in the cytoplasm (21.Winter B. Arnold H.H. J. Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar). However, forced expression of the transcription factor in avian myoblasts does not reinstate the muscle gene program, arguing that additional factors are involved (33.Johnson S.E. Dorman C.M. Bolanowski S.A. J. Biol. Chem. 2002; 277: 28742-28748Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Moreover, rhabdomyosarcoma cells secrete bioactive TGF-β1 that acts in an autocrine manner to suppress differentiation (34.Bouche M. Canipari R. Melchionna R. Willems D. Senni M.I. Molinaro M. FASEB J. 2000; 14: 1147-1158Crossref PubMed Scopus (44) Google Scholar). To further characterize the differentiation-defective phenotype of myoblasts directing extreme levels of Raf/ERK signal, conditioned medium was collected from 23A2RafERDD myoblasts that were treated with Me2SO, 2.5 nm 4HT, or 2.5 μm 4HT in differentiation medium. Cell debris was removed by centrifugation, and the supernatants were supplemented with horse serum to a final concentration of 2%. An aliquot of conditioned medium isolated from the 2.5 μm of treatment group was heat-inactivated prior to supplementation with horse serum (31.Weyman C.M. Wolfman A. Oncogene. 1997; 15: 2521-2528Crossref PubMed Scopus (9) Google Scholar). Confluent cultures of 23A2 myoblasts were allowed to differentiate for 48 h in the conditioned medium followed by fixation and immunostaining for MyHC. Control cultures were treated with Me2SO, 2.5 nm 4HT, or 2.5 μm 4HT and analyzed as described. Conditioned medium from 23A2RafERDD myoblasts differentiated in the presence or absence of 2.5 nm 4HT did not alter the ability of myoblasts to fuse into MyHC-positive myofibers (Fig. 7). By contrast, medium harvested from 23A2RafERDD myoblasts treated with 2.5 μm 4HT dramatically inhibited myocyte formati" @default.
• W2056150526 created "2016-06-24" @default.
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• W2056150526 date "2004-01-01" @default.
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• W2056150526 title "Transforming Growth Factor β1 Is Up-regulated by Activated Raf in Skeletal Myoblasts but Does Not Contribute to the Differentiation-defective Phenotype" @default.
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