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• W1993415211 abstract "To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype. To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype. Skeletal muscle represents an important site of action for the IGFs, 1The abbreviations used are: IGF, insulin-like growth factor; Erk, extracellular signal-regulated protein kinase; IRS, insulin receptor substrate; PTB, phosphotyrosine binding; SH2, Src homology 2; CH1, -2, collagen homology 1 and 2; MEK, mitogen-activated and extracellular signal-regulated protein kinase kinase; PD98059, an inhibitor of activation of MEK by Raf; MEM, minimum essential medium; BCS, bovine calf serum; BSA, bovine serum albumin; PY99, phosphotyrosine antibody; Akt, protein kinase B; MAP, mitogen-activated protein; FITC, fluorescein isothiocyanate; Grb2, growth factor receptor binding protein-2; Sos, son of sevenless; EGF, epidermal growth factor; TBS, Tris-buffered saline. because specific high affinity IGF-I receptors are expressed in this tissue (1Beguinot F. Kahn C.R. Moses A.C. Smith R.J. J. Biol. Chem. 1985; 260: 15892-15898Abstract Full Text PDF PubMed Google Scholar, 2Shimizu M. Webster C. Morgan D.O. Blau H.M. Roth R.A. Am. J. Physiol. 1986; 251: E611-E615PubMed Google Scholar), and both IGF-I and IGF-II stimulate growth and differentiation of skeletal muscle cells (3Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar). Muscle cells also synthesize and secrete IGF-I, IGF-II, and IGF-binding proteins, providing a cell model for integrated autocrine and paracrine control of mitogenic and metabolic actions by the IGFs. IGF signaling in skeletal muscle involves activation of specific cell surface receptors, containing a tyrosine kinase domain within their cytoplasmic portion and undergoing autophosphorylation on specific tyrosine residues upon ligand binding. Receptor autophosphorylation triggers tyrosine phosphorylation of intracellular substrate proteins, including IRS-1, IRS-2, Crk-II, and the Shc proteins (4LeRoith D. Werner H. Beitner-Johnson D. Roberts Jr., C.T. Endocr. Rev. 1995; 16: 143-163Crossref PubMed Scopus (1251) Google Scholar, 5LeRoith D. Endocrinology. 2000; 141: 1287-1288Crossref PubMed Scopus (98) Google Scholar). The Shc proteins are widely expressed signaling mediators that are tyrosine-phosphorylated by multiple receptor or receptor-associated tyrosine kinases and are capable of stimulating the Ras/MAP kinase pathway after binding to the Grb2 adaptor (6Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1010) Google Scholar, 7Pronk G.J. de Vries-Smits A.M. Buday L. Downward J. Maassen J.A. Medema R.H. Bos J.L. Mol. Cell. Biol. 1994; 14: 1575-1581Crossref PubMed Google Scholar). The Shc proteins originate by alternative use of three distinct translation starting points on a longer transcript (p42Shc, p52Shc, and p66Shc) and of two translation starting points on a shorter transcript (p46Shc and p52Shc); the two mRNA transcripts are generated by alternative splicing from a single gene and are regulated by distinct promoters (8Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar, 9Migliaccio E. Mele S. Salcini A.E. Pelicci G. Lai K.M. Superti-Furga G. Pawson T. Di Fiore P.P. Lanfrancone L. Pelicci P.G. EMBO J. 1997; 16: 706-716Crossref PubMed Scopus (362) Google Scholar, 10Ventura A. Luzi L. Pacini S. Baldari C.T. Pelicci P.G. J. Biol. Chem. 2002; 277: 22370-22376Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). As a consequence, p66Shc contains the entire sequence of p46Shc and p52Shc, with one phosphotyrosine binding (PTB) domain, one src homology 2 (SH2) domain, and one collagen homology (CH1) region. In addition, p66Shc has a second CH2 domain of 110 amino acids, located at the NH2-terminal portion of the molecule, that is not present in p46Shc or p52Shc. Recent reports have established a specific role for the p66Shc isoform. p46Shc and p52Shc are found in every cell type with similar reciprocal relationship, whereas p66Shc expression varies from cell type to cell type and is also lacking in some cell types (8Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar). Evidence for divergent regulation of p66Shcversus p46/p52Shc has emerged from studies that demonstrated specific serine phosphorylation of p66Shc through an MEK-mediated pathway in response to insulin (11Kao A.W. Waters S.B. Okada S. Pessin J.E. Endocrinology. 1997; 138: 2474-2480Crossref PubMed Scopus (51) Google Scholar). Knock-out experiments, in which the p66Shc gene has been selectively inactivated in mice, suggest an important role of this protein in the regulation of cellular responses to oxidative stress, apoptosis, and life span (12Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1477) Google Scholar, 13Napoli C. Martin-Padura I. de Nigris F. Giorgio M. Mansueto G. Somma P. Condorelli M. Sica G. De Rosa G. Pelicci P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2112-2116Crossref PubMed Scopus (332) Google Scholar). However, the intracellular signaling pathways mediating the unique actions of p66Shc remain to be established. The mitogenic and survival signals evoked by the IGFs have been extensively studied in multiple cell types, including skeletal muscle cells. Activation of the MEK/Erk pathway in response to the IGFs appears to mediate myoblast growth and differentiation (14Coolican 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, 15Sarbassov D.D. Jones L.G. Peterson C.A. Mol. Endocrinol. 1997; 11: 2038-2047Crossref PubMed Scopus (71) Google Scholar). IGF-I stimulation of DNA synthesis is blocked by the MEK inhibitor PD98059 (16Milasincic D.J. Calera M.R. Farmer S.R. Pilch P.F. Mol. Cell. Biol. 1996; 16: 5964-5973Crossref PubMed Scopus (102) Google Scholar), and myotubes do not survive or differentiate when the MEK/Erk pathway is similarly blocked by PD98059 (17Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar). Furthermore, augmentation of IGF-I-stimulated cell proliferation in dexamethasone-treated L6 myoblasts is associated with increased Shc and decreased IRS-1 tyrosine phosphorylation (18Giorgino F. Smith R.J. J. Clin. Invest. 1995; 96: 1473-1483Crossref PubMed Scopus (57) Google Scholar, 19Giorgino F. Pedrini M.T. Matera L. Smith R.J. J. Biol. Chem. 1997; 272: 7455-7463Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), suggesting that under conditions of glucocorticoid excess the mitogenic response of muscle cells to the IGFs can be modulated by altering the activity of the Shc/Erk pathway. The specific contribution of the p66Shc isoform to IGF-I action on skeletal muscle cells has not been investigated. In this study, we show that selective reduction of p66Shc in L6 skeletal muscle cells results in up-regulation of the MEK/Erk pathway, leading to increased Erk-1/2 phosphorylation and nuclear localization in the absence of IGF-I stimulation. This is associated with a dramatic perturbation of the actin cytoskeleton, leading to abnormal cell shape, growth, and differentiation of the L6 myoblasts. The abnormalities in both signaling reactions and organization of the actin cytoskeleton are corrected by the MEK inhibitor PD98059, suggesting that in skeletal muscle cells the p66Shc isoform may physiologically exert an inhibitory role on MEK/Erk, which is necessary for full responsiveness of this signaling pathway to the IGFs and maintenance of normal cell morphology. Cell Culture—L6 rat skeletal muscle myoblasts were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% donor calf bovine serum (BCS) (both from Invitrogen), 2 mm l-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, and non-essential amino acids in a 5% CO2 atmosphere at 37 °C. Myoblasts were differentiated into myotubes with 2% horse serum (Invitrogen), 2 nm triiodothyronine (Sigma), and 20 nm insulin. For IGF-I studies, cells were incubated in complete medium containing 0.5% bovine serum albumin (BSA) without calf bovine serum for 16 h, and then stimulated with 100 nm IGF-I (GRO PEP, Adelaide, Australia) for the indicated times or left untreated. To block the MEK/Erk signaling pathway, cells were incubated with 20 μm PD98059 (Calbiochem, Merck KGaA) for the indicated times. Antibodies—A monoclonal phosphotyrosine antibody (PY99) and polyclonal IGF-I R β-subunit (C-20) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal MEK-1/2, phospho-MEK-1/2 (Ser-217/Ser-221), Akt, phospho-Akt (Ser-473), and phospho-p42/44 MAP kinase (Erk-1/2) (Thr-202/Tyr-204) antibodies were from Cell Signaling Technology (Beverly, MA). Polyclonal and monoclonal Shc antibodies were from BD Transduction Laboratories (Lexington, KY). A monoclonal MAP kinase (Erk-1/2) antibody was from Zymed Laboratories (San Francisco, CA). A monoclonal vinculin antibody was from Sigma-Aldrich. Transfection Studies—To generate L6 myoblasts stably expressing reduced levels of the p66Shc isoform, a 287-bp fragment of the cDNA encoding the p52/p46Shc proteins (from nucleotide 55 to nucleotide 342), indicated as as1, was generated by amplification using the pMJ/Shc plasmid (kindly provided by Dr. J. Schlessinger, New York, NY) as a template. A 326-nucleotide cDNA fragment corresponding to the NH2-terminal 109 amino acids unique to p66Shc, indicated as as2, was generated by PCR amplification using the rat p66Shc cDNA (kindly provided by Dr. J. E. Pessin, New York, NY). To generate stable transfectants, the cDNAs of interest were cloned in antisense orientation into the mammalian expression vector pCR3.1 (Invitrogen), containing a G418 resistance gene, under control of the cytomegalovirus promoter. The as1- and as2-containing plasmids were transfected into L6 myoblasts by liposome-mediated gene transfer using LipofectAMINE™ (Invitrogen), and stable transfectants, indicated as L6/Shcas1 and L6/Shcas2, were selected by their ability to grow in neomycin-containing medium. Multiple stable clones of L6 cells hypoexpressing Shc were obtained within 4 weeks. Plasmid integration into the cell genome was confirmed by PCR amplification of genomic DNA with forward and reverse oligonucleotide primers corresponding to the pCR3.1 (5′-TAA TAC GAC TCA CTA TAG GG-3′) and Shc (5′-CTG GCA CAG TGG CCC TGT CCA TCC-3′) sequences, respectively. The amount of Shc protein expressed in transfected L6 myoblasts was determined by immunoblotting total cell lysates with Shc antibodies, as previously described (18Giorgino F. Smith R.J. J. Clin. Invest. 1995; 96: 1473-1483Crossref PubMed Scopus (57) Google Scholar). Immunoprecipitation and Immunoblotting—For immunoprecipitation studies, L6 skeletal muscle cells were washed twice with Ca2+/Mg2+-free PBS and then scraped in ice-cold lysis buffer containing 50 mm HEPES (pH 7.5), 150 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10% glycerol, 10 mm sodium pyrophosphate, 10 mm sodium fluoride, 2 mm EDTA, 2 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 2 mm sodium orthovanadate, and 1% Nonidet P-40. Cell lysates were then centrifuged at 12,000 × g for 10 min, and the resulting supernatant was collected and assayed for protein concentration using the Bradford dye binding assay kit with BSA as a standard. Equal amounts of cellular proteins (500 μg) were subjected to immunoprecipitation with the indicated antibodies overnight at 4 °C. The resulting immune complexes were adsorbed onto 70 μl of protein A-Sepharose beads (Amersham Biosciences) for 2 h at 4 °C, washed three times with lysis buffer, and then eluted with Laemmli buffer for 5 min at 100 °C. For immunoblotting studies, equal amounts of cellular proteins were resolved by electrophoresis on 7% or 10% SDS-polyacrylamide gels, as appropriate, directly or following immunoprecipitation with the specific antibodies, as indicated. The resolved proteins were electrophoretically transferred to nitrocellulose membranes (Hybond-ECL, Amersham Biosciences) using a transfer buffer containing 192 mm glycine, 20% (v/v) methanol, and 0.02% SDS. To reduce nonspecific binding, the membranes were incubated in TNA buffer (10 mm Tris-HCl, pH 7.8, 0.9% NaCl, 0.01% sodium azide) supplemented with 5% BSA and 0.05% Nonidet P-40 for 2 h at 37 °C, or in phosphate-buffered saline (PBS) supplemented with 3% nonfat dry milk for 2 h at room temperature, as appropriate, and then incubated overnight at 4 °C with the indicated antibodies. The proteins were visualized by enhanced chemiluminescence (ECL) using horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG (Amersham Biosciences) and quantified by densitometric analysis using Optilab® image analysis software (Graftek SA, Mirmande, France). Immunofluorescence Analyses—To visualize the actin cytoskeleton, L6 cells were grown on coverslips in complete medium for the indicated times, then fixed with 4% paraformaldehyde and permeabilized at -20 °C with 100% methanol. Fixed cells were incubated with fluorescein isothiocyanate-conjugated (FITC) phalloidin (purchased from Sigma-Aldrich) for 30 min and subsequently washed three times with PBS. Coverslips were mounted on glass slides with Gel mount (Biomeda, Foster City, CA). Fluorescence signals were detected by conventional epifluorescence microscopy (Leica DMLB microscope with a Sensicam 12 Bitled charge-coupled device camera, Bansheim, Germany), and all images were captured at the same magnification. To examine vinculin-containing focal adhesions, L6 cells grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Incubation with primary antibody against vinculin (1:400) was conducted at room temperature for 16 h, followed by incubation with secondary Alexa488 Fluor anti-mouse goat antibody (1:500, Molecular Probes, Eugene, OR) for 1 h and washing three times with PBS. Cells were finally stained with TO-PRO-3 (1:10,000, Molecular Probes) to visualize nuclei, and coverslips were mounted using Gel Mount (Biomeda). Images were acquired on a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems, Heerbrugg, Switzerland), and all images were taken at the same magnification. To study the intracellular localization of Erk-1/2, L6 cells were grown on glass slides, arrested at 50% confluence in serum-free medium for 24 h, and then incubated in the absence or presence of 100 nm IGF-I for 30 min. To assess total Erk-1/2, control and IGF-I-treated cells were fixed with methanol/acetone (70:30, v/v) for 10 min at -20 °C. After a 10-min rehydration with multiple PBS washes at 25 °C, the cells were blocked with PBS/10% BCS for 45 min and then incubated with the monoclonal MAP kinase antibody in PBS/10% BCS (1:100) for 60 min at 25 °C. Cells were then washed three times with PBS and incubated with FITC-conjugated anti-mouse antibodies in PBS/10% BCS (1:100) for 60 min at 25 °C in the dark. To evaluate the localization of phospho-Erk-1/2, control and IGF-I-treated cells were fixed with 3% paraformaldehyde for 20 min and permeabilized with 100% methanol for 5 min at -20 °C. After a 10-min rehydration with multiple PBS washes at 25 °C, cells were blocked with blocking buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% Triton X-100 (Tris-buffered saline 1×, TBST) supplemented with 5.5% BCS for 45 min, and then incubated with the polyclonal phospho-p44/p42 MAP kinase (Thr-202/Tyr-204) antibody in Tris-buffered saline 1× (TBS) containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl/3% BSA (1:100) for 60 min at 25 °C. Cells were then washed three times with TBST and incubated with FITC-conjugated anti-rabbit antibodies in TBST/3% BSA (1:100) for 60 min at 25 °C in the dark. Finally, cells were washed three times with PBS, mounted under glass coverslips with CITIFLUOR medium, examined under epifluorescent illumination with excitation-emission filters for FITC by epifluorescence microscopy (Leica DMRXA microscope), and photographed with an Olympus inverted microscope equipped with phase-contrast and UV illumination through an FITC filter. Thymidine Incorporation—Thymidine incorporation was carried out as previously described (18Giorgino F. Smith R.J. J. Clin. Invest. 1995; 96: 1473-1483Crossref PubMed Scopus (57) Google Scholar). Briefly, L6 cells were grown in 35-mm multiwell plates to ∼50% confluence in MEM with 10% BCS. Following incubation in serum-free MEM for 16 h, L6 myoblasts were incubated with or without 100 nm IGF-I for 16 h at 37 °C. The cells were then incubated for 1 h at 37 °C in fresh MEM containing 0.2% BSA, 25 mm HEPES (pH 7.6), and 1 μCi/ml [3H]thymidine. The medium was removed, and the cells were washed twice with ice-cold PBS, left in 10% trichloroacetic acid for 30 min on ice, and washed twice with ice-cold 10% trichloroacetic acid. The cells were then solubilized in 0.1 n NaOH for 30 min at 37 °C, and the amount of 3H was quantitated by liquid scintillation counting. For each condition, experiments were carried out in triplicate. Statistical Analyses—All data are expressed as mean ± S.E. Data are expressed as percentage of control or basal control values, as appropriate. Statistical analyses were performed by unpaired Student's t tests. Generation of L6 Skeletal Muscle Myoblasts with Reduced p66Shc Protein Levels—To determine the specific contribution of the p66Shc isoform to IGF-I action and signaling in skeletal muscle cells, the cellular levels of this IGF-I receptor substrate were selectively reduced by expressing specific Shc antisense RNA sequences. For this purpose, undifferentiated L6 skeletal muscle cells were stably transfected with two independent Shc antisense sequences, designated as1 and as2. as1 represents a 287-nucleotide cDNA fragment corresponding to both the p46/p52Shc and p66Shc mRNA transcripts, whereas as2 represents a 326-nucleotide cDNA fragment specific to the NH2-terminal 109 amino acids unique to the p66Shc mRNA transcript (Fig. 1A). Independent clones of L6 cells showing decreased expression of p66Shc were identified by immunoblotting with anti-Shc antibodies and selected for further analyses. Both as1 and as2 induced a marked and selective reduction in p66Shc protein levels in multiple clones of L6 myoblasts (Fig. 1, B and C). as1 decreased the p66Shc protein ∼90% compared with untransfected wild-type L6 myoblasts (p < 0.05) or L6/Neo myoblasts transfected with the empty pCR3.1 vector. By contrast, the protein levels of the other Shc isoforms, i.e. p52Shc and p46Shc, were not significantly different in L6/Shcas1, L6/Neo, and untransfected L6 myoblasts (Fig. 1B). A 65% reduction in p66Shc was obtained following overexpression of the as2 cDNA construct (p < 0.05), which also did not affect p52Shc or p46Shc protein levels (Fig. 1C). Therefore, L6 skeletal muscle cell lines with marked reduction of p66Shc were established by stable transfection of as1 or as2. The selective inhibition of p66Shc expression in the absence of changes in expression levels of the other Shc isoforms may be potentially explained by different sensitivity to antisense-mediated inhibition of protein translation of the two mRNA transcripts encoding the Shc proteins (8Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar, 9Migliaccio E. Mele S. Salcini A.E. Pelicci G. Lai K.M. Superti-Furga G. Pawson T. Di Fiore P.P. Lanfrancone L. Pelicci P.G. EMBO J. 1997; 16: 706-716Crossref PubMed Scopus (362) Google Scholar, 10Ventura A. Luzi L. Pacini S. Baldari C.T. Pelicci P.G. J. Biol. Chem. 2002; 277: 22370-22376Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Therefore, both as1 and as2 interfere with the mRNA transcript encoding p66Shc, whereas the mRNA transcript encoding p46/52Shc may be resistant to interference by as1 (Fig. 1A). Erk-1/2 Signaling in L6/Shcas Myoblasts—The Shc proteins regulate cellular responses through activation of the Grb2/Sos/Ras signaling cascade, leading to phosphorylation and activation of the MAP kinase family members Erk-1 and Erk-2. To verify whether selective reduction of the p66Shc protein levels could modify this signaling pathway, we analyzed basal and IGF-I-induced Erk-1/2 phosphorylation in L6/Shcas1 and L6/Shcas2 myoblasts with decreased p66Shc protein levels. Activation of Erk-1 and Erk-2 was evaluated by immunoblotting with phospho-Erk-1/2 (Thr-202/Tyr-204) antibodies in two independent clones of L6/Shcas1 myoblasts (C6 and D28) and two independent clones of L6/Shcas2 myoblasts (E15 and E21). In the L6/Shcas1 clones, basal Erk-1 and Erk-2 phosphorylation was increased 411 and 360% of control, respectively (p < 0.05) (Fig. 2A). By contrast, basal phosphorylation of Erk-1 and Erk-2 was similar in L6 myoblasts transfected with the empty vector (L6/Neo, clones N1 and N5) and in untransfected wild-type L6 myoblasts (respectively, p = 0.32 and p = 0.97), indicating that plasmid transfection and clonal selection in neomycin-containing medium did not affect the level of Erk phosphorylation. No change in total Erk-1 or Erk-2 protein content was evident in wild-type L6, L6/Neo, and L6/Shcas1 myoblasts (Fig. 2A). Increased phosphorylation of Erk isoforms in the basal state was also evident in two independent clones of L6/Shcas2 myoblasts compared with control (Fig. 2B; p < 0.05), although this change was of lower magnitude (i.e. 280 and 200% increases of Erk-1 and Erk-2 phosphorylation, respectively, in clones E15 and E21 versus control) as compared with that seen in L6/Shcas1 myoblasts. Total protein content of Erk-1 and Erk-2 was slightly and not significantly increased in L6/Shcas2 compared with control myoblasts (Fig. 2B). Therefore, stable overexpression of either as1 or as2 in L6 myoblasts results in decreased p66Shc content and increased phosphorylation of Erk-1 and Erk-2 not due to changes in total Erk protein content. L6/Shcas1 myoblasts were used in subsequent studies, because they showed greater decrease in p66Shc protein levels and more prominent increase in Erk-1/2 phosphorylation as compared with L6/Shcas2 myoblasts. To assess the responsiveness of Erk-1/2 phosphorylation to IGF-I stimulation, control and L6/Shcas myoblasts were treated with 100 nm IGF-I for various times and subjected to immunoblotting with phospho-Erk-1/2 antibodies. In control L6/Neo cells, IGF-I markedly increased phosphorylation of both Erk isoforms, which was evident after 3 min of stimulation, peaked at 5 min, and remained sustained up to 30 min (Fig. 2C). By contrast, Erk-1/2 phosphorylation was elevated in the basal state in L6/Shcas myoblasts and showed no further increase during IGF-I stimulation (Fig. 2C). Activation of Erk-1/2 following phosphorylation on Thr-202/Tyr-204 results in translocation of the phosphorylated kinases from the cytoplasm, where they are normally retained presumably via a cytoplasmic anchoring complex, to the nucleus (20Lenormand P. Sardet C. Pages G. L'Allemain G. Brunet A. Pouyssegur J. J. Cell Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (585) Google Scholar). To investigate the intracellular localization of activated Erk-1/2 in myoblasts with reduced p66Shc levels, L6/Shcas cells were studied by immunofluorescence using antibodies to total or phosphorylated Erk-1/2 and compared with control. In control L6 myoblasts, Erk-1/2 appeared uniformly distributed in the cell cytoplasm under basal conditions. IGF-I stimulation induced an increase in the Erk-1/2 signal in the cell nucleus and perinuclear region (Fig. 3A). Increased nuclear fluorescence in IGF-I-treated cells was also observed using antibodies to phosphorylated Erk-1/2 (Fig. 3B), indicating IGF-I-dependent nuclear translocation of the phosphorylated form of Erk-1/2. As compared with control cells, L6/Shcas myoblasts showed a greater amount of Erk-1/2 in their nucleus already in the basal state, which did not augment upon IGF-I stimulation (Fig. 3A). The nuclear Erk-1/2 in the unstimulated L6/Shcas myoblasts was constitutively phosphorylated, as demonstrated by immunofluorescence with phospho-Erk-1/2 antibodies (Fig. 3B). Thus, L6/Shcas myoblasts show constitutive activation and nuclear translocation of Erk-1/2 proteins in the absence of IGF-I stimulation. IGF-I exerts a dual effect on proliferation and differentiation of skeletal muscle cells, which relies upon an intact Erk-1/2 responsiveness to this growth factor (16Milasincic D.J. Calera M.R. Farmer S.R. Pilch P.F. Mol. Cell. Biol. 1996; 16: 5964-5973Crossref PubMed Scopus (102) Google Scholar, 17Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar, 21Adi S. Bin-Abbas B. Wu N.Y. Rosenthal S.M. Endocrinology. 2002; 143: 511-516Crossref PubMed Scopus (54) Google Scholar). Because Erk activation was altered in L6/Shcas compared with control L6 myoblasts, IGF-I effects on DNA synthesis and cell differentiation were next determined. In untransfected L6 and L6/Neo myoblasts IGF-I stimulation resulted in a 3-fold increase in DNA synthesis, measured by determining the rates of [3H]thymidine incorporation into DNA (p < 0.05 versus basal) (Fig. 4A). By contrast, in L6/Shcas myoblasts the IGF-I effect on DNA synthesis was modest and statistically not significant (Fig. 4A). Differentiation into myotubes, which is largely mediated by IGFs secreted in an autocrine manner (3Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar), was also impaired in the L6/Shcas myoblasts. Both wild-type L6 and L6/Neo myoblasts became elongated and fused to form the characteristic multinucleated myotubes when grown in the differentiation medium (Fig. 4B). By contrast, L6/Shcas myoblasts showed elongation and some degree of alignment, but did not develop into myotubes (Fig. 4B). IGF-I Receptor Signaling in L6/Shcas Myoblasts—The constitutive activation of Erk in the absence of IGF-I stimulation in myoblasts with reduced p66Shc levels could potentially result from increased activity of steps in the IGF-I signaling cascade upstream of Erk. To explore this possibility, tyrosine phosphorylation and t" @default.
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• W1993415211 date "2004-10-01" @default.
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