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• W2078443143 abstract "We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGEΔcyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mocktransfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21Waf1, and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGEΔcyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGEΔcyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation. We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGEΔcyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mocktransfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21Waf1, and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGEΔcyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGEΔcyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation. Myogenesis is a multistep process in which the precursors of myofibers, the myoblasts, first proliferate and then differentiate into fusioncompetent cells that finally fuse with each other to form myotubes (1Andres V. Walsh K. J. Cell Biol. 1996; 132: 657-666Crossref PubMed Scopus (503) Google Scholar, 2Arnold H.H. Winter B. Curr. Opin. Genet. Dev. 1998; 8: 539-544Crossref PubMed Scopus (247) Google Scholar, 3Charge S.B. Rudnicki M.A. Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1987) Google Scholar). A similar process occurs in mature skeletal muscles in case of damage; quiescent, mononucleated cells, the satellite cells, that coexist with myofibers, can be activated by a number of extracellular factors to proliferate and then to differentiate as above to repair the damaged myofibers (3Charge S.B. Rudnicki M.A. Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1987) Google Scholar). In both cases, proliferation and differentiation are separate and mutually exclusive events that must occur sequentially in that order. Proliferation arrest is one critical step in the process of embryonic myogenesis and muscle regeneration/repair (3Charge S.B. Rudnicki M.A. Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1987) Google Scholar). Either excessive or defective myoblast and satellite cell proliferation can result in altered skeletal muscle formation because myoblasts cannot activate promyogenic signaling pathways in the former case, and because of the low density and insufficient cell-cell contacts that hamper myoblast fusion into myotubes in the second case. Thus, embryonic myogenesis and the closely related muscle regeneration appear to be the net result of the action of a cohort of factors that balance each others activities in a timely and highly coordinated manner so as to assure a sized extent of muscle tissue formation. These factors will assure an appropriate extent of myoblast and satellite cell proliferation at sites of skeletal muscle formation during embryogenesis and in damaged muscles, respectively, and/or will trigger the myogenic differentiation program in proliferation-arrested myoblasts and satellite cells. Thus, some of these factors stimulate myoblast proliferation and differentiation (e.g. insulin and insulin-like growth factors and their receptors), other factors promote myoblast fusion into myotubes (e.g. BOC, CDO, neogenin and its ligand, netrin), whereas other factors inhibit myoblast proliferation and differentiation (e.g. tumor necrosis factor-α, transforming growth factor-β, and myostatin), and still other factors (e.g. basic fibroblast growth factor, hepatocyte growth factor and, as recently shown, S100B) stimulate myoblast proliferation and/or modulate myoblast differentiation (3Charge S.B. Rudnicki M.A. Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1987) Google Scholar, 4Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar, 5Massague J. Cheifetz S. Endo T. Nadal-Ginard B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8206-8210Crossref PubMed Scopus (405) Google Scholar, 6Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar, 7Maina F. Casagranda F. Audero E. Simeone A. Comoglio P.M. Klein R. Ponzetto C. Cell. 1996; 87: 531-542Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 8Coolican 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, 9Bour B.A. Chakravarti M. West J.M. Abmayr S.M. Genes Dev. 2000; 14: 1498-1511PubMed Google Scholar, 10Ruiz-Gomez M. Coutts N. Price A. Taylor M.V. Bate M. Cell. 2000; 102: 189-198Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 11Langen R.C. Schols A.M. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. FASEB J. 2001; 15: 1169-1180Crossref PubMed Scopus (350) Google Scholar, 12Tortorella L.L. Milasincic D.J. Pilch P.F. J. Biol. Chem. 2001; 276: 13709-13717Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 13McCroskery S. Thomas M. Maxwell L. Sharma M. Kambadur R. J. Cell Biol. 2003; 162: 1135-1347Crossref PubMed Scopus (585) Google Scholar, 14Kang J.S. Yi M.J. Zhang W. Feinleib J.L. Cole F. Krauss R.S. J. Cell Biol. 2004; 167: 493-504Crossref PubMed Scopus (125) Google Scholar, 15Sorci G. Agneletti A.L. Riuzzi F. Marchetti C. Donato R. Mol. Cell. Biol. 2003; 23: 4870-5004Crossref PubMed Scopus (72) Google Scholar). We have reported that amphoterin (HMGB1) 3The abbreviations used are: RAGE, receptor for advanced glycation end products; DM, differentiation medium; DMEM, Dulbecco's modified Eagle's medium; ERK1/2, extracellular signal-regulated kinases 1/2; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GM, growth medium; JNK, c-Jun NH2 terminal protein kinase; MAPK, mitogen-activated protein kinase; RAGEΔcyto, RAGE mutant lacking the cytoplasmic and transducing domain; Rb, retinoblastoma suppressor protein; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; VCAM, vascular cell adhesion molecule; NCAM, neural cell adhesion molecule. and its receptor for advanced glycation end products (RAGE) promote rat L6 myoblast differentiation and myotube formation by up-regulating the expression of the muscle-specific transcription factor, myogenin, via a Rac1-Cdc42-MKK6-p38 MAPK pathway (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar). RAGE is a multiligand receptor belonging to the immunoglobulin superfamily that has been implicated in the regulation of several activities/processes such as protection of neurons against stress and stress-induced neuronal death depending on the nature of the ligand and the duration and intensity of its activation, the inflammatory response, tumorigenesis and, as mentioned above, myoblast terminal differentiation (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar, 17Schmidt A.M. Yan S.D. Yan S.F. Stern D.M. J. Clin. Investig. 2001; 108: 949-955Crossref PubMed Scopus (1058) Google Scholar). Amphoterin (HMGB1), a low molecular weight protein with both intracellular and extracellular regulatory activities (18Muller S. Scaffidi P. Degryse B. Bonaldi T. Ronfani L. Agresti A. Beltrame M. Bianchi M.E. EMBO J. 2001; 20: 4337-4340Crossref PubMed Scopus (377) Google Scholar), is one established RAGE ligand that is normally found in serum and extracellular fluids (17Schmidt A.M. Yan S.D. Yan S.F. Stern D.M. J. Clin. Investig. 2001; 108: 949-955Crossref PubMed Scopus (1058) Google Scholar, 19Huttunen H.J. Rauvala H. J. Intern. Med. 2004; 255: 351-366Crossref PubMed Scopus (151) Google Scholar). Besides stimulating myogenesis in vitro via RAGE activation, amphoterin has been shown to promote skeletal muscle repair when administered to dystrophic mice, via stimulation of measoangioblast homing into the diseased muscle tissue (20Palumbo R. Sampaolesi M. De Marchis F. Tonlorenzi R. Colombetti S. Mondino A. Cossu G. Bianchi M.E. J. Cell Biol. 2004; 164: 441-449Crossref PubMed Scopus (394) Google Scholar), possibly via RAGE binding in the light of our previous data (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar). We found that overexpression of RAGE in L6 myoblasts resulted in a significant extent of myoblast differentiation and fusion into myotube even in growth medium (GM, 10% fetal bovine serum (FBS)) and in an acceleration of differentiation of myoblasts in differentiation medium (DM, 2% FBS), whereas overexpression of a RAGE mutant lacking the cytoplasmic and transducing domain (RAGEΔcyto) resulted in a reduced tendency of myoblasts in DM to differentiate, compared with wild type and mock-transfected myoblasts (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar). Overall, the activation of the MKK6-p38 MAPK signaling pathway for the amphoterin/RAGE pair to promote myoblast differentiation is in line with the notion that p38 MAPK needs to be activated for terminal differentiation to take place (21Cuenda A. Cohen P. J. Biol. Chem. 1999; 274: 4341-4346Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 22Li Y. Jiang B. Ensign W.Y. Vogt P.K. Han J. Cell. Signal. 2000; 12: 751-757Crossref PubMed Scopus (97) Google Scholar, 23Wu Z. Woodring P.J. Bhakta K.S. Tamura K. Wen F. Feramisco J.R. Karin M. Wang J.Y.J. Puri P.L. Mol. Cell. Biol. 2000; 20: 3951-3964Crossref PubMed Scopus (399) Google Scholar, 24Zetser A. Gredinger E. Bengal E. J. Biol. Chem. 1999; 274: 5193-5200Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Specifically, the amphoterin/RAGE pair might be one trigger of the signaling cascade leading to p38 MAPK activation in differentiating myoblasts. In the present study we addressed the question whether RAGE engagement in myoblasts and the resulting p38 MAPK activation might determine changes in myoblast proliferation as well as other processes such as apoptosis, adhesiveness, migration, and invasiveness, which characterize myoblast terminal differentiation and fusion into myotubes (3Charge S.B. Rudnicki M.A. Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1987) Google Scholar). We show here that RAGE activation and signaling in myoblasts result in reduced proliferation, migration, invasiveness, and matrix metalloproteinase (MMP) 1 and 2 activity, and increased apoptosis and adhesiveness, and that these effects rely on amphoterin/RAGE-dependent activation of p38 MAPK. We also show that inoculation into immunocompromised mice of myoblasts overexpressing a signaling-deficient RAGE mutant, but not mock-transfected myoblasts or myoblasts overexpressing full-length RAGE results in tumor formation. Cell Culture Conditions, [3H]Thymidine Incorporation, Transfections, and Apoptosis and Luciferase Assays—Rat L6 myoblasts (clone L6C31) were cultured for 24 h in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin, in a H2O-saturated 5% CO2 atmosphere at 37 °C before decreasing FBS to 2% to induce myoblast differentiation. L6 myoblast clones stably overexpressing RAGE or RAGEΔcyto were selected and characterized as described (15Sorci G. Agneletti A.L. Riuzzi F. Marchetti C. Donato R. Mol. Cell. Biol. 2003; 23: 4870-5004Crossref PubMed Scopus (72) Google Scholar). Experiments were performed using mock-transfected, L6/RAGE and L6/RAGEΔcyto myoblasts in 10 or 2% FBS as indicated in the legends to figures. The anti-amphoterin antibody (BD Pharmingen) used in some experiments was shown to neutralize culture medium amphoterin (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar). For [3H]thymidine incorporation assay, myoblasts (25 × 103 cells/well) were cultivated in 10% FBS for 24 h in 24-multiwell plates, washed with DMEM, serum-starved for 24 h, washed with DMEM, and cultivated in DMEM in the presence of either 10 or 2% FBS for another 24 h in the presence of 1 μCi of [3H]thymidine/ml. Parallel myoblasts treated in the same manner in the absence of [3H]thymidine were incubated with HOECHST 33258 (bisbenzimidazole) as described (25Rao J. Otto W.R. Anal. Biochem. 1992; 207: 186-192Crossref PubMed Scopus (207) Google Scholar) to normalize incorporated [3H]thymidine to DNA content. For cell number measurements, myoblasts were cultivated in 10% FBS for 24 h in 96-multiwell plates at a density of 4 × 103 cells/well and then in DMEM in the presence of either 10 or 2% FBS for 1-7 days. Cell density was measured by a tetrazolium-based (MTT) colorimetric assay. To analyze the cell cycle and measure apoptosis, myoblasts were seeded onto 35-mm plastic dishes (18 × 104 cells/dish) in 10% FBS for 24 h, washed with DMEM, and cultivated for 24 or 48 h in DMEM in the presence of either 10 or 2% FBS. Cells were stained with propidium iodide in hypotonic buffer and subjected to fluorescence-activated cell sorting (FACS) analysis as described (26Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4426) Google Scholar). This procedure allows the determination of the percentage of apoptotic (hypodiploid) nuclei as well as that of normal (diploid) nuclei in the same cell population irrespective of the cell volume. In experiments performed in the presence of the p38 MAPK inhibitor, SB203580 (Calbiochem) (2 μm, final concentration), control cells received an equal volume of vehicle (dimethyl sulfoxide). FACS analysis was also employed to measure the mean cell volume as described (27Ahmadzadeh M. Hussain S.F. Faber D.L. J. Immunol. 1999; 163: 3053-3063PubMed Google Scholar). Transient transfections were carried out using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. Briefly, myoblasts cultured in 10% FBS without antibiotics were transfected with the reporter gene p21Waf1-luc, reporter gene cyclin D1-luc, or empty vector. After 6 h, cells were cultivated in 10 or 2% FBS, as indicated in the figure legends. After another 24 h cells were harvested to measure luciferase activity. p21Waf1 and cyclin D1 promoter transcriptional activities were normalized for transfection efficiency by cotransfecting cells with a cDNA encoding green fluorescent protein. The percentage of green fluorescent protein-positive cells (20 to 25%) was determined by FACS analysis. Western Blot Analyses—To detect phosphorylated and total extracellular signal-regulated kinase (ERK) 1/2, phosphorylated and total c-Jun NH2-terminal protein kinase (JNK), phosphorylated and total retinoblastoma suppressor protein Rb, tubulin, caspase-3, Bcl-2, integrin β1, VCAM, NCAM, and caveolin-3 in myoblast extracts by Western blotting, myoblasts were cultivated as detailed in the legend of pertinent figures, washed twice with PBS, and solubilized with 2.5% SDS, 10 mm Tris-HCl, pH 7.4, 0.1 m dithiothreitol, 0.1 mm tosylsulfonyl polyclonal chloromethyl ketone protease inhibitor (Roche). The following antibodies were used: polyclonal antibody specific to phosphorylated (Thr202/Tyr204) ERK1/2 (1:1,000, New England BioLabs), polyclonal anti-ERK1/2 antibody (1:5,000, Sigma), polyclonal anti-phosphorylated (Ser807/Ser811) Rb antibody (1:1,000, Cell Signaling Technology), polyclonal anti-Rb antibody (1:1,000, Cell Signaling Technology), monoclonal anti-α-tubulin antibody (1:10,000; Sigma), polyclonal anticaspase-3 antibody (1:1,000; Cell Signaling Technology), polyclonal anti-Bcl-2 antibody (1:1,000; BD Pharmingen), monoclonal anti-integrin β1 antibody (1:2,000; BD Transduction Laboratories), monoclonal anti-VCAM antibody (1:2,000; BD Pharmingen), monoclonal anti-NCAM antibody (1:2,000; Sigma), and monoclonal anti-caveolin-3 antibody (1:2,000; BD Transduction Laboratories). The immune reaction was developed by enhanced chemiluminescence (ECL) (SuperSignal West Pico, Pierce). Adhesion, Migration, and Invasiveness Assays, and F-actin Staining—For adhesion experiments, mock-transfected, L6/RAGE, and L6/RAGEΔ cyto myoblasts (50 × 103 cells in 0.1 ml of DMEM containing 10% FBS) were seeded into each well, incubated for 3 h, and further processed as described (28Tietze L. Borntraeger J. Klosterhalfen B. Amo-Takyi B. Handt S. Gunther K. Merkelbach-Bruse S. Exp. Mol. Pathol. 1999; 66: 131-139Crossref PubMed Scopus (21) Google Scholar, 29Aumailley M. Mann K. von der Mark H. Timpl R. Exp. Cell Res. 1989; 181: 463-474Crossref PubMed Scopus (212) Google Scholar). The supernatant with non-adherent cells was removed by two washes with warmed culture medium. Attached cells were fixed with 30% methanol/ethanol for 15 min at room temperature, stained with 0.1% crystal violet (Sigma) in PBS, extensively washed with distilled water, and dried at room temperature. The dye was resuspended with 50 μl of 0.2% Triton X-100/well, and color yield was measured using an enzyme-linked immunosorbent assay reader at 590 nm. For migration assay, we used Boyden chambers (pore size, 8 μm) (BD Biosciences). Individual myoblast clones (5 × 104 cells in 0.5 ml of DMEM) were placed in the upper chamber, and 0.75 ml of DMEM containing 10% FBS was placed in the lower chamber. After 20 h in culture, cells on the upper side of the filters were removed with cottontipped swabs, and the filters were fixed in methanol for 2 min and stained with 0.05% crystal violet in PBS for 15 min. Cells on the underside of the filters were viewed and counted under a microscope. Each clone was plated in triplicate in each experiment. For the invasiveness assay, conditions were as described for migration assay except that biocoat Matrigel invasion chambers (pore size, 8 μm) (BD Biosciences) were used. For F-actin staining, myoblasts were seeded onto 13-mm glass coverslips in plastic multiwell dishes (7.5 × 104 cells/dish) in DMEM containing 10% FBS for 24 h and washed in PBS. Cells were then fixed for 10 min in 3.7% paraformaldehyde in PBS, extensively washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 2 min, washed again, and incubated with fluorescein-labeled phalloidin (Sigma) (1:250 in PBS) for 1 h in a humid chamber at room temperature. After three washes in PBS, the cells were mounted in 80% glycerol, containing 0.02% NaN3 and p-phenylenediamine (1 mg/ml) in PBS to prevent fluorescence fading and viewed on a Leica DM Rb fluorescence microscope equipped with a digital camera. Gelatin Zymography—Mock-transfected, L6/RAGE, and L6/RAGEΔ cyto myoblasts were cultivated in a 150-cm2 flask in DMEM containing 10% FBS, after which the supernatant was collected, centrifuged to remove detached cells, and concentrated. The protein concentration of the supernatants was determined using the Bio-Rad protein microassay system with bovine serum albumin as a standard. Samples were stored at -70 °C until use. Aliquots (10 μg of total protein per sample) were electrophoresed at constant voltage on a 10% polyacrylamide gel containing 2 mg/ml of gelatin. The gels was rinsed three times for 15 min in 2.5% Triton X-100 to remove SDS and renature the proteins and then incubated in MMP activation buffer (50 mm Tris-HCl, pH 7.5, with 5 mm CaCl2) for 24 h at 37 °C with constant shaking. Gels were stained overnight in 0.5% Coomassie Blue R-250 and destained for 1 h in 40% methanol, 10% acetic acid. Proteinase activity was quantified by densitometric scanning. In Vivo Tumor Growth—For tumor growth in vivo, female (NOD/SCID) mice weighing ∼ 20 g were inoculated subcutaneously with 5 × 106 L6/wt, L6/RAGEΔcyto, or L6/RAGE myoblasts and monitored for ∼3.5 months. The mice were sacrificed by cervical dislocation. Consent was obtained by the Ethics Committee of the University of Perugia. Tumor masses were excised and weighed, and tumor volume was calculated by the equation: tumor volume = x2y/2, where x and y correspond to the width and thickness of the tissue, respectively. Tumors were then fixed with 4% paraformaldehyde in PBS (2 days at 4 °C), extensively washed in PBS, and paraffin-embedded. Sections were stained with hematoxylin/eosin, and histopathology was performed by an independent pathologist. Statistical Analysis—The data were subjected to analysis of variance with SNK post-hoc analysis using a statistical software package (GraphPad Prism version 4.00, GraphPad Software, San Diego, CA). Statistical significance was assumed when p < 0.05. RAGE Activation in Myoblasts Reduces Proliferation and Stimulates Apoptosis: Role of Amphoterin and p38 MAPK—L6/RAGEΔcyto myoblasts incorporated more [3H]thymidine and L6/RAGE myoblasts incorporated less [3H]thymidine than did L6/mock myoblasts in both GM and DM (Fig. 1A). By MTT assay, a larger number of L6/RAGEΔcyto myoblasts and a smaller number of L6/RAGE myoblasts were obtained at any day of cultivation in both GM (Fig. 1B) and DM (Fig. 1C) between days 1 and 7, compared with L6/mock myoblasts. Moreover, after 1 and 2 days of cultivation in both GM (Fig. 1D) and DM (Fig. 1E) by FACS analysis a larger fraction of L6/RAGEΔcyto myoblasts and a smaller fraction of L6/RAGE myoblasts were in the S and G2/M phases of the cell cycle (with a low percentage of L6/RAGE myoblasts in G2/M phase at 48 h, pointing to the inability of these cells to complete cell division), and a smaller fraction of L6/RAGEΔcyto myoblasts and a larger fraction of L6/RAGE myoblasts were in the G0/G1 phase, compared with L6/mock myoblasts. Collectively, these data suggested that RAGE might transduce an antiproliferative signal in myoblasts. Neutralization of culture medium amphoterin with an anti-amphoterin antibody (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar) resulted in an increased [3H]thymidine incorporation by L6/RAGE and L6/mock myoblasts compared with their respective controls, and no effects in the case of L6/RAGEΔcyto myoblasts (Fig. 1A). Notably, on administration of anti-amphoterin antibody the levels of [3H]thymidine incorporation were similar in the three L6 clones under study. Neutralization of the culture medium amphoterin with an anti-amphoterin antibody also reduced the fractions of L6/mock and L6/RAGE myoblasts in the G0/G1 phase and increased those in S and G2/M phases, whereas without an effect on L6/RAGEΔcyto myoblasts (Fig. 1F). Thus, amphoterin appeared to exert a regulatory role on myoblast proliferation, promoting proliferation arrest via RAGE engagement and stimulation of RAGE transducing activity. This effect of amphoterin would add to the reported promyogenic activity of the protein via activation of a RAGE-Cdc42-Rac-MKK6-p38 MAPK pathway (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar). In addition to reduced proliferation, L6/RAGE myoblasts exhibited a larger extent of apoptosis than did L6/mock myoblasts which in turn showed a larger extent of apoptosis than did L6/RAGEΔcyto myoblasts in both GM and DM, with larger percentages in DM than in GM as expected, after 1 day of cultivation (Fig. 2A). Similar results were obtained after 2 days of cultivation (Fig. 2A). Also, neutralization of culture medium amphoterin reduced apoptosis in both L6/mock and L6/RAGE myoblasts, whereas without effect in the case of L6/RAGEΔcyto myoblasts (Fig. 2B), suggesting that amphoterin was the RAGE ligand involved in RAGE-dependent stimulation of myoblast apoptosis. RAGE-dependent activation of p38 MAPK (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar) was responsible for RAGE-mediated effects on myoblast proliferation and apoptosis. In fact, treatment with the p38 MAPK inhibitor, SB203580, reduced the fraction of L6/RAGE and L6/mock myoblasts in the S and G2/M phases of the cell cycle and decreased the fraction of these cells in the G0/G1 phase, without affecting L6/RAGEΔcyto myoblasts (Fig. 2C), and decreased apoptosis in L6/RAGE, L6/RAGEΔcyto, and L6/mock myoblasts (Fig. 2D), probably because of lack of inhibition of Raf activity under these conditions (30Lee J. Hong F. Kwon S. Kim S.S. Kim D.O. Kang H.S. Lee S.J. Ha J. Kim S.S. Biochem. Biophys. Res. Commun. 2002; 298: 765-771Crossref PubMed Scopus (66) Google Scholar). RAGE-dependent regulation of myoblast apoptosis was further investigated by analyzing the levels of activated caspase-3 and the antiapoptotic factor, Bcl-2. We found that L6/RAGEΔcyto myoblasts in GM and DM exhibited a smaller extent and L6/RAGE myoblasts exhibited a larger extent of caspase-3 activation, compared with L6/mock myoblasts (Fig. 2E). Also, L6/RAGEΔcyto myoblasts exhibited higher levels and L6/RAGE myoblasts exhibited lower levels of Bcl-2, compared with L6/mock myoblasts (Fig. 2E), suggesting that RAGE signaling might negatively regulate Bcl-2 expression in myoblasts. Collectively, these data suggested that, besides promoting myoblast differentiation (16Sorci G. Riuzzi F. Arcuri C. Giambanco I. Donato R. Mol. Cell. Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar), RAGE activation by amphoterin might cause proliferation arrest and promote apoptosis in myoblasts via stimulation of p38 MAPK. RAGE Activation in Myoblasts Increases p21Waf1 Induction and Reduces Cyclin D1 Induction and Rb Phosphorylation—Because increased levels of the proliferation inhibitor p21Waf1 and decreased levels of cyclin D1 and extents of Rb phosphorylation accompany myoblast proliferation arrest and differentiation (24Zetser A. Gredinger E. Bengal E. J. Biol. Chem. 1999; 274: 5193-5200Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 31Missero C. Calautti E. Eckner R. Chin J. Tsai L.H. Livingston D.M. Dotto G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5451-5455Crossref PubMed Scopus (328) Google Scholar, 32Rao S.S. Kohtz D.S. J. Biol. Chem. 1995; 270: 4093-4100Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 33Porrello A. Cerone M.A. Coen S. Gurtner A. Fontemaggi G. Cimino L. Piaggio G. Sacchi A. Soddu S. J. Cell Biol. 2000; 151: 1295-1304Crossref PubMed Scopus (98) Google Scholar), we next analyzed the role of the amphoterin/RAGE pair in p21Waf1 and cyclin D1 induction and extent of Rb phosphorylation. In both GM and DM, L6/RAGEΔ cyto myoblasts exhibited a smaller induction of p21Waf1, a larger induction of cyclin D1, and higher levels of phosphorylated Rb, whereas the opposite was observed in L6/RAGE myoblasts, compared with L6/mock myoblasts (Fig. 3, A-C). Neutralization of culture medium amphoterin reduced the levels of p21Waf1 induction in L6/RAGE and L6/mock myoblasts to nearly those detected in L6/RAGEΔcyto myoblasts (Fig. 3A). Similarly, neutralization of culture medium amphoterin resulted in an increase in cyclin D1 induction (Fig. 3B) and the extent of Rb phosphorylation (Fig. 3D) in L6/RAGE and L6/mock myoblasts to the levels observed in L6/RAGEΔ cyto myoblasts. These data suggested that induction of p21Waf1 and cyclin D1 and the extent of Rb phosphorylation in myoblasts is under the control of the amphoterin/RAGE pair signaling. Thus, we concluded that the amphoterin/RAGE pair might transduce antiproliferative signals in myoblasts via up-regulation of p21Waf1 induction, down-regulation of cyclin D1 induction, and reduction of Rb phosphorylation. RAGE Activation in Myoblasts Results in ERK1/2 and JNK Inactivation—We have previously shown that the amphoterin/RAGE pair stimulates myogenic different" @default.
• W2078443143 created "2016-06-24" @default.
• W2078443143 creator A5016331365 @default.
• W2078443143 creator A5054695573 @default.
• W2078443143 creator A5065739247 @default.
• W2078443143 date "2006-03-01" @default.
• W2078443143 modified "2023-09-28" @default.
• W2078443143 title "The Amphoterin (HMGB1)/Receptor for Advanced Glycation End Products (RAGE) Pair Modulates Myoblast Proliferation, Apoptosis, Adhesiveness, Migration, and Invasiveness" @default.
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