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Comparative Proteomes of the Proliferating C2C12 Myoblasts and Fully Differentiated Myotubes Reveal the Complexity of the Skeletal Muscle Differentiation Program*

  • Nilesh S. Tannu
    Affiliations
    Departments of Biomedical Engineering, Memphis, TN 38104
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  • Vamshi K. Rao
    Affiliations
    Departments of Biomedical Engineering, Memphis, TN 38104
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  • Ritcha M. Chaudhary
    Affiliations
    Department of Veterans Affairs Medical Center, Memphis, TN 38104
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  • Francesco Giorgianni
    Affiliations
    Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center, Memphis, TN 38104
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  • Abdelwahab E. Saeed
    Affiliations
    Departments of Pharmacology, Memphis, TN 38104
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  • Yong Gao
    Affiliations
    Departments of Pharmacology, Memphis, TN 38104
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  • Rajendra Raghow
    Correspondence
    To whom correspondence should be addressed: University of Tennessee Health Science Center & Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104. Tel.: 901-523-8990 (ext. 7634); Fax: 901-577-7273
    Affiliations
    Departments of Biomedical Engineering, Memphis, TN 38104

    Departments of Pediatrics, Memphis, TN 38104

    Departments of Pharmacology, Memphis, TN 38104

    Department of Veterans Affairs Medical Center, Memphis, TN 38104
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  • Author Footnotes
    The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material.
    * The studies reported here were supported by grants from the Department of Veterans Affairs (DVA), the University of Tennessee Center of Excellence for the Diseases of Connective Tissue, and the National Institutes of Health. R. R. is a Senior Research Career Scientist of the DVA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      When cultured in low serum-containing growth medium, the mouse C2C12 cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C2C12 cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C2C12 myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C2C12 myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.
      The de novo myogenesis from mesoderm-derived committed muscle precursor cells has been studied in the embryos of mouse, chicken, frog, and zebra fish, and in a number of cell and tissue culture models of muscle differentiation. As a result of these studies, key anatomic, genetic, and molecular aspects of this multi-step process have been elucidated (
      • Buckingham M.
      Skeletal muscle formation in vertebrates..
      ,
      • Pownall M.E.
      • Gustafsson M.K.
      • Emerson Jr., C.P.
      Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos..
      ,

      Tannu, N. S., Rao, V. K., and Raghow, R.(2003) Cellular and molecular paradigms of myogenesis, inRecent Research Developments in Molecular Biology (Pandlai, S. G., ed)Vol. 1, pp.73–95, Research Signpost, Trivendrum, India

      ). The final step of myogenesis in vivo entails that the proliferating myoblasts withdraw from cell cycle, elicit a muscle-specific gene expression program and fuse to become multinucleated myotubes.
      The induction of muscle-specific genes during myogenic differentiation is regulated by basic helix-loop-helix (bHLH)
      The abbreviations used are: bHLH, basic helix-loop-helix; MRF, muscle-specific regulatory factor; 2D, two-dimensional; QIT, quadrupole ion trap; GM, growth medium; DMEM, Dulbecco’s modified Eagle’s medium; DM, differentiation medium; MHC, myosin heavy chain; RIPA, radio-immunoprecipitation assay; TBST, TBS-Tween 20; LIMK1, LIM kinase 1; PKA, protein kinase A; MKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; HSP, heat shock protein; CRE, cAMP-responsive element; TIF, transcription intermediary factor; HAT, histone acetyl transferase; FGF, fibroblast growth factor.
      1The abbreviations used are: bHLH, basic helix-loop-helix; MRF, muscle-specific regulatory factor; 2D, two-dimensional; QIT, quadrupole ion trap; GM, growth medium; DMEM, Dulbecco’s modified Eagle’s medium; DM, differentiation medium; MHC, myosin heavy chain; RIPA, radio-immunoprecipitation assay; TBST, TBS-Tween 20; LIMK1, LIM kinase 1; PKA, protein kinase A; MKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; HSP, heat shock protein; CRE, cAMP-responsive element; TIF, transcription intermediary factor; HAT, histone acetyl transferase; FGF, fibroblast growth factor.
      transcription factors such as MyoD, Myf-5, myogenin, and MRF4. The known muscle-specific regulatory factors (MRFs) exhibit distinct but somewhat overlapping spatio-temporal patterns of expression during development of the skeletal muscle. For example, Myf5 is the first of the myogenic bHLH factors to be expressed in the developing embryo followed by MyoD and myogenin. The expression of myogenin and MRF4 occurs later, and apparently the two MRFs directly control transcription of muscle-specific genes prior to the formation of multinucleated myotubes (
      • Charbonnier F.
      • Gaspera B.D.
      • Armand A.S.
      • Van der Laarse W.J.
      • Launay T.
      • Becker C.
      • Gallien C.L.
      • Chanoine C.
      Two myogenin-related genes are differentially expressed in Xenopus laevis myogenesis and differ in their ability to transactivate muscle structural genes..
      ,
      • Smith 2nd, C.K.
      • Janney M.J.
      • Allen R.E.
      Temporal expression of myogenic regulatory genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells..
      ). In contrast, MyoD and Myf-5 are not only expressed in the proliferating myoblasts but may also be needed in an early step of myogenesis (i.e. muscle cell fate specification). Varying degrees of defects in muscle development are caused by loss-of-function mutations in the individual MRF genes (
      • Lassar A.B.
      • Skapek S.X.
      • Novitch B.
      Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal..
      ,
      • Molkentin J.D.
      • Olson E.N.
      Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors..
      ). Consistent with their unique roles, the combined mutations in two MRFs in mice (e.g. MyoD −/−: MRF4 −/−) result in more severe defects in muscle differentiation than those seen in either MyoD −/− or MRF4 −/− mice (
      • Pownall M.E.
      • Gustafsson M.K.
      • Emerson Jr., C.P.
      Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos..
      ). Additionally, MEF2 and regulators of cell cycle such as p21CIP1, p27, and p57KIP2 are also coordinately regulated during muscle differentiation (
      • Buckingham M.
      Skeletal muscle formation in vertebrates..
      ,
      • Pownall M.E.
      • Gustafsson M.K.
      • Emerson Jr., C.P.
      Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos..
      ,

      Tannu, N. S., Rao, V. K., and Raghow, R.(2003) Cellular and molecular paradigms of myogenesis, inRecent Research Developments in Molecular Biology (Pandlai, S. G., ed)Vol. 1, pp.73–95, Research Signpost, Trivendrum, India

      ).
      Mouse C2C12 cells have been used extensively to study the process of myogenic differentiation in culture. The application of DNA microarray technology to the differentiating C2C12 cells has led to the identification of numerous differentially expressed messenger RNAs (
      • Delgado I.
      • Huang X.
      • Jones S.
      • Zhang L.
      • Hatcher R.
      • Gao B.
      • Zhang P.
      Dynamic gene expression during the onset of myoblast differentiation in vitro..
      ,
      • Shen X.
      • Collier J.M.
      • Hlaing M.
      • Zhang L.
      • Delshad E.H.
      • Bristow J.
      • Bernstein H.S.
      Genome-wide examination of myoblast cell cycle withdrawal during differentiation..
      ). Shen et al. (
      • Shen X.
      • Collier J.M.
      • Hlaing M.
      • Zhang L.
      • Delshad E.H.
      • Bristow J.
      • Bernstein H.S.
      Genome-wide examination of myoblast cell cycle withdrawal during differentiation..
      ) reported that the differentially expressed genes of C2C12 cells grown in low serum medium represented regulators of cell cycle (e.g. cyclin D1, p27Kip1, PP2A, and Rb), apoptosis such as DAD1, BAK, Caspase 11, and glycogen synthase kinase-3β, and muscle-specific genes (e.g. MyoD, myogenin, dystroglycan, troponin c, and creatine kinase). These authors showed that expression of Cyclin D1 was readily detected in the proliferating myoblasts while p21WAF1/Cip1 expression increased only when <40% of cells were fused into multinucleated myotubes. In a related study, assessment of global gene expression in the differentiating C2C12 cells up to the stage of myogenin induction showed that <1,500 genes, which could be classified into 12 coordinately regulated group of genes, were significantly altered (
      • Delgado I.
      • Huang X.
      • Jones S.
      • Zhang L.
      • Hatcher R.
      • Gao B.
      • Zhang P.
      Dynamic gene expression during the onset of myoblast differentiation in vitro..
      ). Similar to what was demonstrated by Shen et al. (
      • Shen X.
      • Collier J.M.
      • Hlaing M.
      • Zhang L.
      • Delshad E.H.
      • Bristow J.
      • Bernstein H.S.
      Genome-wide examination of myoblast cell cycle withdrawal during differentiation..
      ), Dalgado and colleagues found that numerous cell cycle signaling-, apoptosis-, cell architecture-, and transcriptional control-specific genes were significantly altered during early phase of myogenic differentiation (
      • Delgado I.
      • Huang X.
      • Jones S.
      • Zhang L.
      • Hatcher R.
      • Gao B.
      • Zhang P.
      Dynamic gene expression during the onset of myoblast differentiation in vitro..
      ).
      Although the genomics-based analyses of myogenesis in C2C12 cells have been highly instructive, we believe that the molecular mechanisms by which proliferating myoblasts leave cell cycle, initiate a program of myogenic gene expression, and become fused into multinucleated myotubes cannot be fully understood from the analysis of the transcriptome alone. This is because the signal transduction pathways mediating the phenotypic conversion of myoblasts into myotubes utilize proteins and the analysis of transcriptome informs us little about the dynamic changes in the rates of translation of various mRNAs or about proteins produced by translation of alternately spliced mRNAs. Therefore, it is desirable to complement the global gene expression analyses with studies examining the proteomes of C2C12 cells undergoing myogenesis in vitro. With a goal to compare the proteomes of the C2C12 cells undergoing differentiation, we analyzed the total cellular, membrane-, and nuclear-enriched proteins from proliferating myoblasts and fully differentiated myocytes by two-dimensional (2D)-PAGE. The differentially regulated protein spots were identified by PDQuest image analysis of the silver nitrate-stained 2D gels followed by MALDI-TOF-MS and LC-ESI-quadrupole ion trap (QIT)-MS/MS. Furthermore, because the status of phosphorylation, a key modification of proteins that regulates numerous signaling cascades, cannot be discerned from analyses of the protein abundance, we also compared the phospho-proteomes of proliferating C2C12 cells and myotubes by using the Pro-Q ® Diamond phospho-protein gel staining. We demonstrate that in addition to many well-known proteins involved with myogenesis, the expression of a number of new proteins capable of regulating inter- and intracellular signaling, cell cycle and apoptosis, cell shape, and transcription is also altered during skeletal muscle differentiation.

      EXPERIMENTAL MATERIALS AND METHODS

       Cell Culture—

      C2C12 cells were bought from American Type Culture Collection (ATCC-CRL 1772; Bethesda, MD). Cells were cultured in growth medium (GM; Dulbecco’s modified Eagle’s medium [DMEM] containing 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin) in a humidified incubator at 37 °C with 5% CO2. Cells cultivated in GM were subcultured after they became 70–80% confluent, and the cell passage number was not allowed to exceed 10. To induce differentiation, nearly confluent C2C12 cells were incubated in DMEM containing 2% heat-inactivated horse serum (differentiation medium; DM) for varying lengths of time. The fraction of cells converted into myotubes was assessed by light microscopy of unstained cells or after staining with a monoclonal myosin heavy chain (MHC)-specific primary antibody. The primary antibody was diluted 1:1 with 1% BSA/PBS-Tween 20 followed by secondary reaction with a goat anti-mouse IgG conjugated with FITC. The detailed methods for staining of C2C12 cells with antibody and detection of FITC fluorescence have been outlined previously (
      • Mehra-Chaudhary R.
      • Matsui H.
      • Raghow R.
      Msx3 protein recruits histone deacetylase to down-regulate the Msx1 promoter..
      ).

       Extraction of Proteins—

      Cell monolayers (∼107 cells/15-cm diameter Petri dish) were washed twice with 10 ml of 0.35 m ice-cold sucrose, scraped in 4 ml of 0.35 m ice-cold sucrose, and collected by centrifugation (4,000 rpm for 5 min at 4 °C). Whole-cell proteins were extracted in radio-immunoprecipitation assay (RIPA) buffer [50 mm Tris-HCl at pH 7.5, 150 mm NaCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mm EDTA, and 100-fold diluted fresh cocktails of phosphatase inhibitor I [microcystin LR, cantharidin, and (-)-p-bromotetramisole; catalog no. p2850, Sigma-Aldrich, St. Louis, MO], phosphatase inhibitor II [sodium vanadate, sodium molybdate, sodium tartrate, and imidazole; catalog no. p5726, Sigma-Aldrich], and 40 μl of protease inhibitor mixture (catalog no. 1697498, Roche, Indianapolis, IN). Proteins were precipitated in acetone (final concentration of 80%) at −20 °C overnight, pelleted by centrifugation (14,000 rpm for 20 min at 4 °C), and pellets were air-dried. The protein pellet obtained from cells harvested from a single 15-cm diameter dish was taken up in 200 μl of rehydration buffer [7 m urea, 2 m thiourea, 4% (w/v) CHAPS. The protein solution in rehydration buffer was supplemented with immobilized pH gradient buffer (8 μl/ml), 1 μl (0.5%) of bromphenol blue, and DTT (10 mg/ml)] and kept for 1 h at room temperature. These samples were centrifuged (14,000 rpm for 20 min at 25 °C), and supernatant containing the proteins in complete solution was used for 2D-PAGE.
      We extracted crude membrane fraction according to the published protocol (
      • Simpson R.J.
      • Connolly L.M.
      • Eddes J.S.
      • Pereira J.J.
      • Moritz R.L.
      • Reid E.G.
      Proteomic analysis of the human colon carcinoma cell line (LIM 1215): Development of a membrane protein database..
      ). Cells were suspended in HES buffer [20 mm HEPES, 1 mm EDTA at pH 7.4, and freshly added mixture of protease and phosphatase inhibitors] and broken by freezing and thawing (−80 °C for 30 min) and 30 passes in a Dounce homogenizer (clearance of 0.1016–0.1524 mm). Unlysed cells and nuclei were removed from the cell homogenate by centrifugation (900 × g for 10 min at 4 °C). The crude plasma membranes were recovered as a pellet by centrifuging the post-nuclear supernatant at 100,000 × g for 45 min at 4 °C. Membrane proteins were extracted in RIPA buffer, precipitated by acetone as above and dissolved in re-hydration buffer (
      • Sanchez J.C.
      • Hochstrasser D.
      • Rabilloud T.
      In-gel sample rehydration of immobilized pH gradient..
      ).
      Cells harvested from 15-cm diameter dishes as above were used to isolate nuclei according to the previously reported protocol (
      • Mirkovitch J.
      • Mirault M.E.
      • Laemmli U.K.
      Organization of the higher-order chromatin loop: Specific DNA attachment sites on nuclear scaffold..
      ). Cells were sequentially washed three times in 3 ml of ice-cold isolation buffer [3.75 mm Tris-Cl pH 7.4, 0.05 mm spermine, 0.125 mm spermidine, 0.5 mm EDTA, 1% thiodiglycol, 20 mm KCl] containing protease and phosphatase inhibitors, and centrifuged (900 × g for 5 min at 2 °C) after each wash. The supernatant containing the membrane and cytoplasm was removed after each spin. The crude nuclear pellets were then taken up in 3 ml of ice-cold Triton X-100 lysis buffer [isolation buffer + 0.5% Triton X-100] containing protease and phosphatase inhibitors and washed three times by stepwise resuspension and centrifugation (900 × g for 5 min at 2 °C). Finally, nuclear proteins were extracted in 500 μl of RIPA buffer, precipitated in 80% acetone at −20 °C overnight, and made soluble in 100 μl of rehydration buffer. Concentration of proteins from total cell, membrane, or nuclear extracts was determined by a modified Bradford assay kit (
      • Bradford M.M.
      A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding..
      ) as described by the manufacturer (Pierce, Rockford, IL).

       2D-PAGE—

      One hundred-microgram aliquots of whole-cell, membrane, or nuclear proteins, taken up in 360 μl of rehydration buffer, were electrofocused in Immobiline™ DryStrips (180 × 3×0.5 mm, pH 3–10/4–7 linear) with the IPGphor (Amersham Pharmacia Biotech, Piscataway, NJ) (
      • Bjellqvist B.
      • Ek K.
      • Righetti P.G.
      • Gianazza E.
      • Gorg A.
      • Westermeier R.
      • Postel W.
      Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications..
      ). The IPG strip was rehydrated for 12 h and subjected to sequential IEF at 100 V for 200 V-h, at 500 V for 500 V-h, at 1,000 V for 1,000 V-h, and at 8,000 V for 80,000 V-h. The platform temperature was maintained at 20 °C, and 50-μA current was passed per strip. The IPG strips were equilibrated to reduce the disulfide bonds in a tray containing 3 ml of equilibrating solution per strip [6 m urea, 1.5 m Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS, and 2% (w/v) DTT] with gentle rocking for 10 min. The protein SH groups were blocked by rocking each strip for 10 min in 3 ml of solution containing 6 m urea, 1.5 m Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS, and 2.5% (w/v) iodoacetamide.
      Solubilization of proteins, equilibration of first-dimension strips, and electrophoresis in the second dimension were done in the Protean Dodeca Cell (Bio-Rad, Hercules, CA) apparatus capable of running 12 gels simultaneously. After equilibration, according to the published method (
      • Rabilloud T.
      • Adessi C.
      • Giraudel A.
      • Lunardi J.
      Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients..
      ), the IPG strips were transferred onto vertical 10% SDS-PAGE slab gels (1,800 × 1,800 × 1 mm), using 1% melted agarose as stacking gel. One microliter of the molecular mass marker (Amersham Rainbow marker RPN 800) mixed with 4 μl of running buffer was loaded on 2 mm2 filter paper, which was placed on the acidic end of the IPG strip (
      • Weber K.
      • Osborn M.
      The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis..
      ). Electrophoresis was carried out for 400 min at a steady voltage of 200 V (
      • Sanchez J.C.
      • Hochstrasser D.
      • Rabilloud T.
      In-gel sample rehydration of immobilized pH gradient..
      ).

       Staining of 2D-PAGE and Image Analysis—

      Proteins separated by 2D gels were visualized after staining with Mann’s modified silver staining method, which is compatible with trypsin digestion and MALDI-TOF-MS (
      • Merril C.R.
      • Switzer R.C.
      • Van Keuren M.L.
      Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain..
      ,
      • Shevchenko A.
      • Wilm M.
      • Vorm O.
      • Mann M.
      Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels..
      ). The staining of 2D gels for phospho-proteins was done according to the instructions provided by the manufacturer (Molecular Probes, Eugene, OR). Gels were fixed in 250 ml of 50% methanol and 10% TCA overnight. The fixed gels were then sequentially washed with 250 ml of distilled water for 15 min, incubated with 250 ml of Pro-Q ® Diamond phospho-protein stain for 3 h in the dark and destained with 20% ACN, 50 mm sodium acetate (pH 4) for 3 h. After scanning the images of the gels for phospho-proteins, the gels were silver stained for visualization of the spots for MS analyses. The stained 2D gels were scanned (Hewlett Packard 4470c scanner) and saved as TIFF files using Adobe Photoshop software. The images of the scanned 2D gels were analyzed by PDQuest (version 6.2.1) software (Bio-Rad) (
      • Beresini M.H.
      • Sugarman B.J.
      • Shepard H.M.
      • Epstein L.B.
      Synergistic induction of polypeptides by tumor necrosis factor and interferon-gamma in cells sensitive or resistant to tumor necrosis factor: Assessment by computer based analysis of two-dimensional gels using the PDQUEST system..
      ). To identify valid spots, PDQuest spot detection software was used with appropriate selection of the faintest and the smallest spots and a large representative section of the image containing spots, streaks, and background gradations to make corrections for noise filter. Absorbance of individual protein spots from the replicate gel images was combined to make “master gels” representing proteins from C2C12 cells at different stages of differentiation. The differentially regulated protein spots were identified by quantitative comparisons of master gels. The reproducibility of PDQuest-based quantification of 2D gel images was ensured by three complementary approaches. First, at least three independent sets of synchronized cultures of C2C12 cells were grown in GM or DM to extract myoblast and myotube-specific proteins. All Petri dishes were individually assessed for cell morphology, density of culture, and myotube formation (in DM) on a daily basis to select two to three uniform replicates for each condition of growth. Second, in the initial tests of reproducibility of 2D gels, protein extracts from individual replicate cultures were analyzed by 2D-PAGE. Subsequently, extracts from cells harvested from three to four Petri dishes incubated under identical conditions were mixed and subject to 2D-PAGE to “normalize” dish-to-dish variability. Finally, regardless of the source of the protein extract, either from a single dish or from multiple dishes grown under identical conditions, all samples were run on three to four replicate gels in the Protein Dodeca Cell (Bio-Rad). Unpaired Student’s t test was used to determine if the averages of the myoblast or myotube-specific samples were significantly different using the Microsoft Excel®. Protein spots of interest were subject to MALDI-TOF-MS or LC-ESI-MS/MS (
      • Patterson S.D.
      • Aebersold R.
      Mass spectrometric approaches for the identification of gel-separated proteins..
      ).

       In-gel Trypsin Digestion—

      The individual protein spots from the 2D gels were excised with pipette tips, minced using a 0.5-ml pestle (Nalge Nunc, Rochester, NY), and destained in 1:1 (v/v) of 30 mm potassium ferricyanide and 100 mm sodium thiosulfate (
      • Gharahdaghi F.
      • Weinberg C.R.
      • Meagher D.A.
      • Imai B.S.
      • Mische S.M.
      Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity..
      ). Gel spots were dehydrated by sonication for 20 min in 100 μl of solution containing 50% (v/v) ACN and 100 mm ammonium bicarbonate until the gel turned opaque white. Twenty micrograms of lyophilized trypsin (883 pmol; Promega, Madison, WI) was reconstituted in 100 μl of 50 mm acetic acid to form the stock solution that was diluted [1:12 (v/v)] in 50 mm ammonium bicarbonate and incubated for 15 min at 37 °C. The gel fragments were dried by vacuum centrifugation for 30 min and incubated overnight with 50 μl of trypsin (16 ng/μl) at 37 °C.
      The supernatant from trypsin digest was transferred to a siliconized microcentrifuge tube. Peptides from the gel pieces were sequentially extracted three times in 50 μl of extraction buffer [60% (v/v) ACN, 5% (v/v) TFA in water]. Each extraction involved 20 min of sonication, followed by centrifugation and removal of the supernatants. The original supernatant and the supernatants from three sequential extractions were combined and dried in a vacuum centrifuge for 3–4 h. The dried peptides were dissolved in 3 μl of 12.5 mg/ml of α-cyano-4-hydroxy-cinnamic acid in 60% (v/v) ACN in water and deposited on paraffin wax-coated stainless-steel MALDI plate (
      • Tannu N.S.
      • Wu J.
      • Rao V.K.
      • Gadgil H.S.
      • Pabst M.J.
      • Gerling I.C.
      • Raghow R.
      Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting..
      ). Alternately, dried peptides were dissolved in 15 μl of 0.1% (v/v) TFA in water and taken up in siliconized microcentrifuge tube for LC-ESI-QIT-MS/MS.

       MALDI-TOF-MS—

      Mass spectrometer analyses were performed using the PerSeptive Biosystems (Framingham, MA) MALDI-TOF Voyager DE™-RP BioSpectrometry™ Workstation operated in the delayed extraction and reflector mode for positive ion detection (
      • Jensen O.N.
      • Podtelejnikov A.
      • Mann M.
      Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps..
      ). The laser wavelength and the repetition rate were 337 nm and 3Hz, respectively. The parameters set were as follows: maximum accelerating voltage, 20,000 V; grid voltage, 57%; mirror voltage ratio, 1.08; guide wire, 0.07% and the extraction delay time of 150 ns. The masses were calibrated internally with the masses of two trypsin auto-digest products: fragment 108–115 ([M+H]+ = 842.509 Da) and fragment 58–77 ([M+H]+ = 2211.104 Da). The results were analyzed with Data Explorer software (Applied Biosystems). The protein identification was carried out using the PeptIdent search engine (us.expasy.org/tools/peptident.html), and the Swiss-Prot/TrEMBL databases were used for the protein search (
      • Henzel W.J.
      • Billeci T.M.
      • Stults J.T.
      • Wong S.C.
      • Grimley C.
      • Watanabe C.
      Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases..
      ). The search parameters used were: pI range of ± 1.0, molecular mass range of ± 40%, mass tolerance of ± 100 ppm, one allowed missed cleavage, cysteine treated with iodoacetamide to form carbamidomethyl-cysteine and methionine as the oxidized form.

       LC-ESI-QIT-MS/MS—

      To remove the residual gel and to desalt the peptides, the mixtures were purified with ZipTipC18 micro-columns (Millipore, Bedford, MA) and eluted into 3 μl of 50% ACN in water. To the eluate, 3 μl of 0.01% hepatofluorobutyric acid was added and the sample mixture was injected onto the column of LC-nanoESI-QIT MS on the LCQDeca instrument (ThermoFinnigan, San Jose, CA). Picofrit™ columns (360-μm outer diameter, 75-μm inner diameter, 15-μm tip inner diameter) from New Objective (Woburn, MA) were used for LC. Samples were analyzed using a gradient program consisting of initial 5-min isocratic elution with 0% B, followed by linear gradient 0–70% B in 50 min, 5-min isocratic elution with 70% B and a linear gradient 70–0% B in 15 min (A = 0.1% formic acid and B = 90% ACN-10% water-0.1% formic acid). Thirty-minute column equilibration time was used between subsequent injections and the injector port was washed with 50 μl of 30% acetone:30% isopropanol:40% water followed by 50 μl of water between injections to prevent carryover of samples. The peptides eluted at 400 nl/min were introduced online into the mass spectrometer and MS and MS/MS spectra were obtained in the data-dependent mode. The instrument cycled through acquisition of a full-scan MS spectrum, followed by five MS/MS scans of the most abundant ions from the MS scans obtained by collision with helium gas. For the MS/MS analysis the collision energy was set to 35% and the dynamic exclusion was 0.5 min.
      The search engine Sequest (LCQDeca software package) was used to analyze the MS/MS data by searching the Swiss-Prot mouse protein sequence database. The protein matches were considered valid if MS/MS data for multiple unique peptides were matched. For single peptide match, the identification was confirmed by manual examination using the following criteria: 1) Sequest cross-correlation (Xcorr) score ≥ 2.0 (doubly charged peptides)/≥ 3.5 (triply charged peptides); 2) a good-quality MS/MS spectrum with the difference in the observed and the theoretical masses of the product ions not more than 0.5 mass unit; 3) continuous stretch of the peptide sequence covered by either the y- or b-ion series; 4) intense y-ions corresponding to a proline residue (if proline was present in the sequence); 5) similar values of observed and theoretical pI and molecular mass of a protein (
      • Giorgianni F.
      • Desiderio D.M.
      • Beranova-Giorgianni S.
      Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry..
      ).

       Western Blot Analysis—

      C2C12 cells were washed twice with warm PBS and incubated for 30 min at 4 °C in RIPA buffer as described above. After removing the insoluble material by centrifugation (14,000 rpm for 5 min at 4 °C), protein concentrations were determined by Bio-Rad DC Protein Assay. Thirty-microgram aliquots of proteins were separated by SDS-PAGE and subject to Western blotting as detailed previously (
      • Shetty S.
      • Takahashi T.
      • Matsui H.
      • Ayengar R.
      • Raghow R.
      Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300)..
      ). Proteins were transferred onto 0.45-μm nitrocellulose membranes. Binding of nonspecific proteins to membranes was blocked by incubating these in the blocking buffer consisting of 5% non-fat milk in TBS plus 0.1% Tween 20 (TBST) for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies diluted in the blocking buffer. Dilution of various antibodies was done according to the recommendation of the manufacturers as follows: anti-MHC (MF20), 1:1,000, anti-cAMP-dependent protein kinase (PKA) α-I and -II regulatory subunit, 1: 200; anti-PKA β-I regulatory subunit, 1:200; anti-actin, 1:200; anti-MyoD, 1:200; anti-MEF-2, 1: 200; anti-LIM kinase 1 (LIMK1), 1:200; anti-phospho serine/threonine protein kinase (pAkt), 1:1000; anti-phospho mitogen-activated protein kinase kinase (MKK) 3/6, 1:1000; anti-phospho P70S6 kinase, 1:1000; anti-phospho extracellular signal-regulated kinase (pERK) 1/2, and 1:1,000 anti-p38 MAP kinase. After three washes with TBST, membranes were incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h. After three more washes with TBST, immunoreactive proteins on the membranes were detected by ECL® reagent and exposure to x-ray film (
      • Shetty S.
      • Takahashi T.
      • Matsui H.
      • Ayengar R.
      • Raghow R.
      Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300)..
      ).

      RESULTS

       Morphology of C2C12 Cells in GM and DM—

      When cultivated in DMEM containing 10% fetal bovine serum, proliferating C2C12 cells grow as mononucleated flattened cells in a monolayer. When confluent cells were incubated in DMEM containing 2% horse serum for 48–72 h, the majority of C2C12 cells assumed elongated morphology and fused to become multinucleated myotubes or myocytes. Such morphological conversion of myoblasts into myotubes accompanies accumulation of muscle-specific proteins, as demonstrated by staining by MHC-specific antibody (Fig. 1A). Changes in the steady-state levels of muscle-specific transcription factors such as MyoD and MEF, and structural proteins (e.g. actin and MHC) could also be readily detected by Western blot analysis of proteins from C2C12 cells grown in DM for various durations (Fig. 1B).
      Figure thumbnail gr1
      Fig. 1A, detection of MHC in C2C12 cells grown either in GM (left) or DM for 72 h (right). A monoclonal antibody against MHC was used as primary antibody that was detected by FITC-conjugated secondary antibody as described in “Materials and Methods.” B, whole-cell lysates prepared from C2C12 cells cultured in GM or cultivated in DM for indicated number of hours were subjected to SDS-PAGE, transferred to membranes, and probed with antibodies for MyoD, MEF, MHC, and actin as detailed in the “Materials and Methods.” The antibody against actin did not discriminate between ubiquitous and muscle-specific actin.

       Proteome of Total Cell Proteins—

      Total proteins were extracted from proliferating C2C12 myoblasts cultivated in GM and fully differentiated myotubes harvested from cultures incubated for 72 h in DM (80–90% multinucleated myotubes). Four aliquots of 100 μg of protein from each cell type were fractionated on replicate 2D gels (10%, 3–10 linear pH gradient) and images of silver-stained replicates were analyzed by PDQuest to assess their reproducibility. We discovered that <550 protein spots that were detected from either proliferating myoblasts or myotubes on replicate gels had 86–99% reproducibility; this range of reproducibility for replicated 2D patterns is similar to what has been reported previously (
      • Zhan X.
      • Desiderio D.M.
      Differences in the spatial and quantitative reproducibility between two second-dimensional gel electrophoresis systems..
      ). The representative silver-stained 2D images of total cell proteins from C2C12 myoblasts and myocytes fractionated on a 3–10 pH gradient and 10% gels are shown in Fig. 2, A and B, respectively. The PDQuest image analysis of the master images representing myoblasts and myotubes elucidated the putative differentiation-specific protein spots.
      Figure thumbnail gr2
      Fig. 2Representative 2D gels of whole-cell proteins extracted from C2C12 myoblasts (A; top) and myocytes (B; bottom). Protein spots marked by arrows were identified by MALDI-TOF-MS and/or LC-ESI-QIT-MS/MS. The horizontal axis shows the range of pI and the vertical axis shows the position of proteins used as molecular mass markers in kDa.
      When total cell proteins, regardless of whether extracted from myoblasts or myocytes were subject to IEF in a 3–10 pH gradient, distribution of these spots was nonrandom because most spots were clustered in a 4–7 pI range. Therefore in the subsequent experiments we fractionated total cell proteins from the two cell types on a 4–7 pH gradient in the first dimension. Isoelectric focusing of proteins in a 4–7 pH gradient resolved many more spots than those seen in a 3–10 pH gradient 2D gel; 653 and 433 protein spots were resolved from samples of proteins from myoblasts, in a 4–7 versus 3–10 pH gradients, respectively. Similarly, 558 protein spots were observed for myotube-specific proteins subject to IEF on 4–7 pH gradients in the first dimension, whereas about 300 proteins were resolved in a 3–10 pH gradient. A PDQuest-based quantification of the master patterns representing myoblast- and myotube-specific 2D gels revealed numerous protein spots that seemed to have variable expression in the two cell types as judged by their staining intensities (Figs. 2 and 3).
      Figure thumbnail gr3
      Fig. 3Representative 2D gels of total-cell proteins extracted from C2C12 myoblasts (A; top) and myocytes (B; bottom) and resolved in a 4–7 linear pH gradient and 10% SDS-PAGE gels in second dimension. The polypeptide molecular mass scale in kDa is depicted on the y-axis while the x-axis shows the range of pIs. The protein spots marked by arrows were identified by MALDI-TOF-MS and LC-ESI-QIT-MS/MS as detailed in the “Materials and Methods.”

       Proteome of Crude Membrane-enriched Fractions from the Proliferating and Differentiated C2C12 Cells—

      While extracts of whole-cell proteins subjected to IEF on either 3–10 or 4–7 pH gradients in the first dimension resolved numerous proteins, we were aware that these represented only a fraction of the most abundant proteins expressed in C2C12 cells, regardless of their state of differentiation. Therefore, we prepared subcellular fractions from crude plasma membranes and nuclei. Aliquots of membrane-enriched proteins from either C2C12 myoblasts or myotubes were fractionated on three to four replicate pH 4–7 and 10% 2D gels. We should stress here that the purpose of our subcellular fractionation was to enhance the relative abundance of a subset of proteins that may not be detectable in the whole-cell extracts. Thus, our “crude membrane-enriched” fraction is likely to contain additional proteins that are not bona-fide membrane proteins. Furthermore, as has been articulated in detail (
      • Molloy M.P.
      Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients..
      ,
      • Santoni V.
      • Molloy M.
      • Rabilloud T.
      Membrane proteins and proteomics: Un amour impossible?.
      ), analysis of integral membrane proteins by 2D SDS-PAGE poses a number of challenges that include i) their lower solubility in the first dimension, ii) incomplete transfer from the IEF strips to the second-dimensional SDS-PAGE gels, iii) incomplete horizontal resolution due to charge heterogeneity resulting from posttranslational modifications, and iv) relatively low copy number per cell of some membrane proteins (e.g. receptors for hormones). However, these caveats notwithstanding, we achieved our main objective of analyzing many more proteins as a result of subcellular fractionation of C2C12 myoblasts and myotubes than would have been otherwise possible (

      Rao, V. K.(2003)Differential Regulation of Nuclear Proteins in C2C12 Cells Undergoing Myogenesis. M.Sc. thesis, University of Tennessee Health Science Center, Memphis, TN

      ,

      Tannu, N.(2003)Proteomics Based Analysis of C2C12 Cells Undergoing Myogenesis. M.Sc. thesis, University of Tennessee Health Science Center, Memphis, TN

      ).
      Two features of the 2D gel patterns from membrane-enriched proteins shown in Fig. 4 can be readily appreciated visually. First, the overall patterns of membrane-enriched proteins analyzed by 2D gels are less complex compared with the patterns seen with total-cell proteins. Corroborating the visual impression, PDQuest analysis showed that 213 and 144 unique protein spots were detected in the master-images of myoblast- and myocyte-specific membrane protein 2D gels, respectively. Second, a large fraction of the membrane-enriched proteins generated a horizontal string of spots with heterogeneous pIs that likely result from post-translational modifications of proteins. A comparative PDQuest image analysis of master gels representing crude membrane-enriched proteins from myoblasts and myotubes revealed that several proteins were differentially expressed (Fig. 4). We identified a number of these proteins by MALDI-TOF-MS and LC-ESI-QIT-MS/MS. As expected, although the majority of the proteins from crude membrane-enriched fractions were membrane proteins such as IGF-I receptor (spot 39) FGFR-4 (spot 40), jagged 2 (spots 46 and 47), and NADH-ubiqninone oxidoreductase subunit (spot 49), a number of nonmembrane proteins such as baculovirus IAP repeat-containing protein (spot 16), glucosamine-6-phosphate isomerase (spot 62), and LIM domain kinase 1 (spot 11) were also found in this fraction.
      Figure thumbnail gr4
      Fig. 4Representative 2D gels depicting membrane-enriched proteins from C2C12 myoblasts and myocytes. Membrane-enriched proteins extracted from C2C12 myoblasts (A; top) and myotubes (B; bottom) were subjected to IEF in a linear pH 4–7 gradient in the first dimension followed by 10% SDS-PAGE as detailed in “Materials and Methods.” Arrows indicate protein spots that were identified by MS and bioinformatics. The detailed parameters of protein identification are outlined in .

       Proteome of Nuclear Proteins from C2C12 Myotblasts and Myotubes—

      To enhance detection of less-abundant proteins that may be differentially regulated during myogenesis in vitro, we also analyzed proteins from “crude nuclei-enriched” fractions from C2C12 myoblasts and myotubes. We obtained “nuclear-enriched” fractions according to Mirkovitch et al. (
      • Mirkovitch J.
      • Mirault M.E.
      • Laemmli U.K.
      Organization of the higher-order chromatin loop: Specific DNA attachment sites on nuclear scaffold..
      ), as modified to isolate nuclei from C2C12 cells (
      • Tolstonog G.V.
      • Sabasch M.
      • Traub P.
      Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function..
      ). Extensive analysis of nuclear fractions obtained by this approach has revealed that although vast majority of the proteins detected from such preparations represent bona-fide nuclear proteins a large number of cytoplasmic and cytoskeletal proteins (e.g. vimentin cytokeratin and desmin) are also associated with nuclei (
      • Tolstonog G.V.
      • Sabasch M.
      • Traub P.
      Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function..
      ). Therefore, the caveat articulated above with respect to “crude membrane-enriched” fractions should also be applied to the interpretation of our data on the proteome of the “crude nuclear fraction.” In addition to the problem of contamination of nuclei with non-nuclear components, resolution of nuclear proteins on a narrow 4–7 pH gradient 2D gel is incomplete because many nuclear proteins are highly basic.
      Representative 2D images of nuclear proteins from myoblasts and myotubes are shown in Fig. 5. The PDQuest image analysis showed that 413 and 276 nuclei-enriched proteins could be consistently detected from cultures of myoblasts and myotubes, respectively. As we had observed for the membrane-enriched proteins earlier, the 2D patterns of nuclei-associated proteins are also significantly less complex than those of whole-cell proteins. A comparison of the staining intensities of common proteins by PDQuest analysis revealed that several proteins accumulated preferentially either in the nuclei of proliferating myoblasts or fully differentiated myotubes (Fig. 5).
      Figure thumbnail gr5
      Fig. 5Silver-stained 2D gels representing nuclear-enriched protein from C2C12 myoblasts (A; top) and myocytes (B; bottom). Nuclei from proliferating myoblasts or multinucleated myotubes cultured in DM for 72 h were isolated. Proteins were extracted, analyzed by 2D gel electrophoresis, and protein spots marked by arrows were identified by MALDI-TOF-MS and LC-ESI-QIT-MS/MS as outlined in detail in the “Materials and Methods.”

       Quantification of Differentially Expressed Proteins—

      To quantify potential gene products that were differentially regulated during conversion of C2C12 myoblasts into myocytes, we assessed the absorbance of individual protein spots from 2D gels representing total, and membrane-, and nuclear-enriched proteins from each cell type. The staining intensity of each spot was normalized against the sum total of intensities of all detectable spots in the 2D gel; this normalization maneuver of the PDQuest program is designed to correct for the minor differences in protein loading or staining intensity among replicate gels. The values of spot densities were obtained as arbitrary units ranging from 1 × 109 to 1 × 1010 and were divided by a factor of 109. To confirm normalization of spot intensities derived from the PDQuest analysis, we compared the staining intensities of seven protein spots, shown as standard spot protein (SSP) in Fig. 2. This analysis was also used to assess the intrinsic variability of the densitometry-based quantification of protein expression data.
      Quantification of staining intensities of the individual protein spots from 2D gels, as examined by PDQuest-based image analysis, revealed that the vast majority of 2,139 total cell-, membrane-, and nuclei-associated protein spots were qualitatively similar regardless of whether they represented proliferating C2C12 cells or fully differentiated myotubes. Additionally, we observed that the relative staining intensities of numerous protein spots showed only low (10–20%) to moderate (20–50%) change associated with the state of cellular differentiation (

      Rao, V. K.(2003)Differential Regulation of Nuclear Proteins in C2C12 Cells Undergoing Myogenesis. M.Sc. thesis, University of Tennessee Health Science Center, Memphis, TN

      ,

      Tannu, N.(2003)Proteomics Based Analysis of C2C12 Cells Undergoing Myogenesis. M.Sc. thesis, University of Tennessee Health Science Center, Memphis, TN

      ). In contrast, a small subset of polypeptides that were abundant in the extracts of proliferating C2C12 cells such as a LIM domain-containing protein (spot 7), insulin-like growth factor I receptor (spot 39), jagged 2 (spots 46 and 47) were not seen in myotube-specific 2D gels, while other proteins such as MKK3 (spots 9) or a baculovirus IAP-repeat containing protein-4 (spot 16) were detected only in the 2D gels of the fully differentiated myotubes (

      Tannu, N. S., Rao, V. K., and Raghow, R.(2003) Cellular and molecular paradigms of myogenesis, inRecent Research Developments in Molecular Biology (Pandlai, S. G., ed)Vol. 1, pp.73–95, Research Signpost, Trivendrum, India

      ). As shown in Fig. 6, although the regulation of about 70 proteins fell between the “all or none” extremes their steady-state levels were significantly different between myoblasts and myotubes (p < 0.05 by unpaired Student’s t test). The extent of differential expression of such proteins ranged from as low as 1.32-fold enhancement noted for calsenilin (spot 36) to a 42-fold increase in the steady-state levels of the microtubule-associated, APC-binding protein EB1 (spot 17). We reasoned that these highly regulated proteins were mechanistically related to the process of myogenic differentiation of C2C12 cells and, therefore, systematically established their identities by peptide mass fingerprinting and amino acid sequencing as outlined below.
      Figure thumbnail gr6
      Fig. 6Quantification of proteins assessed to be differentially expressed during the conversion of C2C12 myoblasts into MHC-positive multinucleated myotubes. All protein spots were quantified by scanning of the silver-stained images by PDQuest image analysis software. The absorbance in arbitrary units in a given spot was normalized and all absorbance values were divided by a factor of 109. Relative levels of proteins involved in the re-organization of cytoskeleton and signaling (A), intracellular processing/folding and membrane-enriched molecules (B), and nuclear and miscellaneous proteins (C) in proliferating C2C12 cells (black bars) and fully differentiated myotubes (white bars) are presented. The numbers denoting various proteins on the x-axis are the same as shown in and .

       Phospho-proteomes of C2C12 Myoblasts and Myocytes—

      To determine changes in protein phosphorylations in C2C12 undergoing myogenesis, we stained the 2D gels of whole-cell protein extract with the Pro-Q ® Diamond phospho-protein gel stain. The representative gels of whole-cell proteins of myoblasts and myocytes fractionated in 4–7 pH range and stained for phospho-proteins are shown in the Fig. 7. PDQuest-based comparisons of the gels stained for phospho-proteins showed that 108 and 122 putative phospho-protein spots were resolved in the myoblasts and myotubes, respectively. Thus, only about 15–20% spots of the total cellular proteins stained by silver nitrate were stained by Pro-Q ® Diamond stain, regardless of whether cells were actively proliferating or fully post-mitotic. A PDQuest-based image analysis was done to quantify the staining intensities of the putative phospho-proteins and is represented in the Fig. 8. The absorbance in arbitrary units in a given spot was normalized, and all the absorbance values were divided by a factor of 104. The targeted phospho-protein spots were identified by MALDI-TOF-MS (Table I). Although the specificity of the Pro-Q ® Diamond for staining phospho-proteins has been rigorously established (
      • Martin K.
      • Steinberg T.H.
      • Cooley L.A.
      • Gee K.R.
      • Beechem J.M.
      • Patton W.F.
      Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye..
      ,
      • Schulenberg B.
      • Aggeler R.
      • Beechem J.M.
      • Capaldi R.A.
      • Patton W.F.
      Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye..
      ,
      • Steinberg T.H.
      • Agnew B.J.
      • Gee K.R.
      • Leung W.Y.
      • Goodman T.
      • Schulenberg B.
      • Hendrickson J.
      • Beechem J.M.
      • Haugland R.P.
      • Patton W.F.
      Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology..
      ), we wish to inject a note of caution in the interpretation of these data. The identification of a spot, stained with Pro-Q ® Diamond, as a phospho-protein by MS may be seriously biased if an abundant protein (which may or may not be a phospho-protein) co-migrates with this particular spot. Therefore, the MS-mediated identification of a spot as a phospho-protein must be independently validated by other methods (i.e. by precursor ion scanning for phosphate ion or neutral loss by MS technique). We have not independently verified whether every spot assigned to the “phospho-proteome” of C2C12 cells, as judged by Pro-Q ® Diamond staining, is indeed a bona-fide phospho-protein. However, it is worth noting that out of 26 differentially regulated “phospho-proteins,” at least half (spots 25, 83, 84, 89, 91, 92, 95, 96, 97, 100, 101, 104, and 105) are known phospho-proteins as evident from the published literature. In addition, we have obtained sequencing information on three additional spots by LC-ESI-QIT-MS/MS to confirm their phospho-protein nature (R. Raghow, unpublished observation). In light of this technical concern, the assignment of the remaining polypeptides as phospho-proteins may only be considered tentative.
      Figure thumbnail gr7
      Fig. 7Representative 2D gels of phospho-proteins from actively growing myoblasts (A; top) and fully differentiated myotubes (B; bottom). The protein separation was achieved in a 4–7 linear pH gradient in the first dimension and by 10% SDS-PAGE in the second dimension. Gels were stained using Pro-Q ® Diamond reagent and quantified by PDQuest image analysis as described in the “Materials and Methods.” The identities of the protein spots indicated by arrows were established by MALDI-TOF-MS and are outlined in .
      Figure thumbnail gr8
      Fig. 8Quantification of the phospho-proteins detected in myoblasts (black bars) and myocytes (white bars). The data were obtained by PDQuest analysis of phospho-proteins detected by Pro-Q ® Diamond staining of 2D gels representing whole-cell proteins from myoblasts (black bars) and myocytes (white bars). The relative intensities of the phospho-proteins in myoblasts and myocytes are the normalized values in arbitrary units in a given spot as outlined in the “Materials and Methods.”
      Table IThe proteins identified by MALDI-TOF-MS and LC-ESI-QIT-MS/MS from C2C12 myoblasts and myocytes
      Spot no.ProteinAccession no.ParametersMALDI-MS/LC-ESI-QIT-MS/MS*Regulation
      MrpIPeptidesCoverage (%)
      Cytoskeletal proteins
      1Actin, cytoplasmic 1 (β)P02570417365.291952.4NS
      Actin, cytoplasmic 2 (γ)P02571417365.31934.2
      A-X actinQ61276416935.21726.9
      *Actin, skeletal muscle 2 (α)P53482419765.2353.6*
      2Actin, cytoplasmic 2 (γ)P02571417365.31517.1NS
      3*F-actin capping protein α-2 subunitP47754329675.5771*
      4*Myosin heavy chain, fast skeletal muscleP025652228165.680.7*
      5*Myosin light chain 1, skeletal muscleP05977205944.9885.6*
      6Tropomyosin β chainP58774328364.66621.1
      17Microtubule-associated protein RP/EB family member 1 (APC-EB1)Q15691299995.02419
      8Calponin, acidic isoform (Calponin 3)Q15417364145.69423.7
      70Myosin light chain 1, atria/fetal isoformP09541210274.97847.4
      Signaling proteins
      9Dual specificity MAP kinase kinase 3 (MKK3)P46734393186.2622
      10cAMP-dependent protein kinase type II-alpha regulatory chainP13861453874.96522.6
      11LIM domain kinase 1 (LIMK1)P53668727006.461217.8
      14Serine/threonine protein kinase Akt-2Q60823557415.98714
      15Sestrin 1 (p53-regulated protein PA26)P58006566325.64416.3
      16Baculoviral IAP repeat-containing protein 4Q60989560795.6849.3
      7LIM domain-containing protein 1Q9BQQ9UDUD414.1
      72LIM and SH3 domain protein 1 (Lasp-1)Q61792299946.61831.6
      18*75-kDa glucose-regulated proteinP38647735285.9147*NS
      19*Growth arrest-specific protein 1 (GAS 1)Q01721356945.683.1*
      71Follistatin-related protein 1Q62356324615.41413.9
      82Serine/threonine protein phosphatase 2AP13353356085.3426.5
      Protein processing/folding
      20Protein disulfide isomeraseP09103571434.752133.7
      2178-kDa glucose-regulated proteinP20029724225.013247.3
      22*78-kDa glucose-regulated proteinP20029724225.0148.5*
      23*78-kDa glucose-regulated proteinP20029724225.0134.2*
      2460-kDa heat shock protein, mitochondrialP19226609555.35923.4NS
      25*Heat shock protein HSP90-βP11499831944.971826.3/50.4*
      26EndoplasminP08113924754.723036.4NS
      28*Ubiquitin carboxyl-terminal hydrolase isozymes L5Q9WUP7376165.243.3*
      29*Prefoldin subunit 2O705911653368.3*
      29*Phosducin-like protein (PHLP)Q9DBX2344064.793.4*
      30Peptidyl-prolyl cis-trans isomerase EQ9QZH3331615.4421.1
      73Chain 1: Plasminogen activator inhibitorP22777428486.03619.8
      7626S Proteasome regulatory subunitQ03527491845.87614.8
      Membrane-associated proteins
      31Chain 1: AgrinP253042086455.01711.6
      32DR β-chain antigen binding domainQ30788UDUD548.2
      33Annexin IP04083385836.6832.5
      34Annexin IVP55260357435.32517.9
      35Annexin IVP97429358585.43532.8
      36Calsenilin (DRE-antagonist modulator)Q9QXT8294305.46520.7
      37Chain 1: Frizzled 4Q9QZH0564306.558.4
      38Ephrin type-B receptor 6 precursorO08644623006.03613.9
      39Insulin-like growth factor I receptorQ60751715305.01710.3
      40Fibroblast growth factor receptor 4P22455855006.3645.8
      40Integrin β-2P11835826966.8645.6
      43*Integrin α-V precursorP434061152775.461.4*
      46Chain 1: JAGGED 2Q9QYE51320505.381019.3
      47Chain 1: JAGGED 2Q9QYE51320505.38824.3
      48Peripheral plasma membrane protein CASKO705891047106.161315.5
      49NADH-ubiquinone oxidoreductase 49-kDa subunitP17694491745.95614
      50*Guanine nucleotide-binding protein β subunit 4P29387373545.592.9*
      51Guanine nucleotide-binding protein G(I)/G(S)/G(T) β subunit 3Q61011372405.4148.8NS
      52Guanine nucleotide-binding protein β subunit like protein 12.3 (receptor of activated protein kinase C1)P25388350767.6410.7NS
      53Granulocyte-macrophage colony-stimulating factor (GM-CSF)Q00941387758.02512.8NS
      12Paired box protein Pax-7P4723932911UD529.3
      Nuclear proteins
      45*Myocyte-specific enhancer factor 2A (MEF2A)Q60929537246.433*
      54Ribonucleoprotein F (RNP F)Q9Z2X1457305.31622.7
      55TBP-associated factor (TAF) 28kDQ99JX1233335.16419.9
      56Exosome complex exonuclease RRP42 (EC 3.1.13.-)Q9D0M0316655.07419.6
      57Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)P09867221327.8432.3
      43*Transcription intermediary factor 1-βQ62318888475.5211.5*
      58*Similar to eukaryotic translation initiation factor 4A, isoform 1Q9BRK6395485.63.2*
      60Zinc finger MYND domain-containing protein 10Q99ML0506326.1610.5
      61Centrosomal protein 2Q9BV73365775.7953
      74Histone acetyl transferase type B subunit 2Q60973477904.89612.9
      75M-phase inducer phosphatase 3P48967500466.38412.1
      Miscellaneous proteins
      45*40S ribosomal protein SA (34/67-kDa laminin receptor)P14206327194.7423.7*
      45*Vacuolar ATP synthase subunit EP50518265889.25.3*
      45*Secreted protein acidic and rich in cysteine (SPARC)P07214344504.7714*
      4*α enolase (2-phospho-d-glycerate hydro-lyase)P17182471406.3664.1*
      62Glucosamine-6-phosphate isomerase (glucosamine-6-phosphate deaminase) (Oscillin)O088958325506521.1
      63ATP synthase β chain, mitochondrialP56480517004.991238.7NS
      64Chain 1: CalreticulinP14211463004.33621.8
      65Adenylate kinase isoenzyme 5Q9Y6K8221005.38426.8
      66NADPH-dependent carbonyl reductase 3O75828307005.82421NS
      67Immunoglobulin-binding protein 1P78318392205.26522.4
      68Immunoglobulin-binding protein 1P78318392205.26419.8
      77Chain 1:ATP synthase β chainP56480517494.991747.2
      78Aldo-keto reductase family 1 member C13Q8VC28370576.67527.6
      79Guanine deaminaseQ9R111510135.36412.3
      80Chain 1: protein C21ORF63 homologP58659440375.98420.2
      81Protein-glutamine glutamyl transferase EQ08189249735.54724.7
      Phospho-proteins
      25Heat shock protein HSP90-β (tumor-specific transplantation 84-kDa antigen)P11499T83.194.971620
      83VimentinP2015253.555.06815.9
      84A disintegrin and metalloproteinase domain-containing protein 17Q9Z0F8-273.885.75610.8
      85Zinc finger protein 305O4330970.226.2858.3
      86Quinone oxidoreductase-like 1 (QOH-1) (ζ-crystallin homolog)O9582538.665.49524.1
      87Annexin A5 (Annexin V) (Lipocortin V)P4803635.754.83418.2
      88NIF3-like protein 1Q9EQ8038.825.91927
      89Transcription factor E2-αP1580630.236.25411.7
      90CDK-activating kinase assembly factor MAT1 (CDK7/cyclin H assembly factor) (p36) (p35)P5194935.845.6448.4
      91Mitogen-activated protein kinase 4 (MAP kinase isoform p63)P3115262.626.0548.3
      92Lamin AP4867874.216.54812.3
      93Lamin AP4867874.216.54919.4
      94TNF receptor-associated factor 5P7019164.147.71712.4
      95Mitogen-activated protein kinase kinase kinase 7 (MKK7)Q6207364.226.2369.7
      96Ribosomal protein S6 kinase α 3 (pp90RSK2)P1865483.696.41712.6
      97Splice isoform 3 of Rho guanine nucleotide exchange factor 1Q61210-389.295.7898.1
      98Crooked neck-like protein 1Q9CQC178.76.6247.3
      99POU domain, class 5, transcription factor 1P2026338.216.05411.4
      100Protein kinase C-α binding proteinQ6208346.525.3359.4
      101Ribosomal protein S6 kinase α 3P1865483.696.4167.4
      102Splice isoform 2 of tropomyosin α 3 chainP21107-229.024.75722.2
      103Annexin VP4803635.754.83416.3
      104Inhibitor of nuclear factor κ-B kinase α subunitQ6068066.87.23512.7
      105B-Raf proto-oncogene serine/threonine-protein kinaseP28028UDUD48.2
      106G2/mitotic-specific cyclin B1P2486048.057.148.6

       MALDI-TOF-MS and LC-ESI-QIT-MS/MS Identification of Proteins—

      Table I represents a list of proteins extracted from 2D gels (Figs. 2–6) of C2C12 myoblasts and myocytes and identified by MALDI-TOF-MS and/or LC-ESI-QIT-MS/MS. Although majority of the proteins extracted from 2D gels were identified by MALDI-TOF-MS, the identity of some proteins was also corroborated by peptide sequencing using LC-ESI-QIT-MS/MS. Thus, heat shock protein β-90 (HSP-90β) was identified by MALDI-TOF-MS initially; the identity of HSP-90β was confirmed by amino acid sequence analysis of its tryptic peptides by LC-ESI-QIT-MS/MS. The combined sequence coverage by MALDI-TOF and LC-ESI-QIT MS unequivocally established the identity of HSP-90β. The list of 106 proteins and phospho-proteins identified by 2D-PAGE and MS and bioinformatics methods is presented in Table I. We have attempted to organize the differentially expressed proteins into functional categories, fully realizing that such a classification is somewhat arbitrary because a number of proteins could be assigned to more than one functional category.

       Validation of Selected Proteomics Data by Western Blotting—

      The list of putatively regulated proteins depicted in Table I is a snapshot of proteins from proliferating myoblasts versus quiescent, multinucleated myotubes. Therefore, we assessed temporal changes in expression of a subset of proteins C2C12 myoblasts incubated in the DM for 6, 12, 24, 48, 72, 96, and 120 h. Cellular proteins were then fractionated by denaturing SDS-PAGE and analyzed by Western blotting. In addition to proteins known to be associated with skeletal muscle differentiation such as MyoD, MEF, MHC, and actin (Fig. 1B), we analyzed changed in the levels of LIMK1, PKA, AKT, p70S6 kinase, and various members of the MAP kinase family in C2C12 cells undergoing myogenesis in DM (N. Tannu, data not shown). The ∝-I, ∝-II, and β-I subunits of PKA were expressed constitutively in C2C12 cells regardless of their growth in GM or DM. The ∝-II subunit of PKA showed a steady decrease in expression up to 48 h, after which it showed enhanced accumulation up to the 120 h time point. The expression of LIMK1 was steady in the initial 12 h in DM but declined progressively as myoblasts exited cell cycle and became differentiated into bona fide multi-nucleated myotubes (72–120 h). The overall pattern of sequential expression of LIMK1 is consistent with the PDQuest and MS data. The phosphorylated form of Akt (pAkt) also showed steady expression up to 48 h after serum withdrawal but was up-regulated in C2C12 cells incubated in DM for 72–120 h. The apparent discrepancy in the expression Akt2 as assessed from PDQuest-based analysis and Western blot analysis may be due to failure of our antibody to distinguish between Akt1 and Akt2 (N. Tannu, data not shown). The pattern of p38 MAP kinase expression did not change during growth in DM. The levels of pMKK3/6 were dynamically regulated. The steady-state levels of MKK 3/6 and phosphorylated MKK7 went up when C2C12 cells were transferred to DM for 72 h. The p70S6 kinase was abundantly expressed in proliferating C2C12 myoblasts but dramatically declined after the cultures were transferred to DM for 12 h or longer.

      DISCUSSION

      We compared whole-cell, crude membrane-, and nuclear-enriched proteins from C2C12 myoblasts and myotubes by a noncandidate, large-scale method of proteomics that involved fractionation of proteins by 2D-PAGE, PDQuest image analysis of silver-stained gels, and identification of target proteins by MS. We should note, however, that assignment of some proteins as “membrane” or “nuclear” may be an artifact of our subcellular fractionation method. This caveat notwithstanding, such crude fractionation enabled us to quantify 20–30% more proteins than would have been otherwise possible from the analyses of the whole-cell proteins without subcellular fractionation (

      Rao, V. K.(2003)Differential Regulation of Nuclear Proteins in C2C12 Cells Undergoing Myogenesis. M.Sc. thesis, University of Tennessee Health Science Center, Memphis, TN

      ,

      Tannu, N.(2003)Proteomics Based Analysis of C2C12 Cells Undergoing Myogenesis. M.Sc. thesis, University of Tennessee Health Science Center, Memphis, TN

      ). PDQuest image analysis of the most abundant 2,139 proteins revealed that vast majority of these most likely represented gene products relegated to structural and/or housekeeping functions and apparently did not undergo major regulation. In contrast, expression of 75 polypeptides was consistently altered as mononucleated, proliferating C2C12 myoblast cells exited cell cycle and became MHC-positive, post-mitotic multi-nucleated myotubes. In addition, we identified 26 phospho-proteins that underwent differential expression during myogenic differentiation of C2C12 cells. Included among the differentially regulated proteins were mediators of inter- and intracellular signaling, cell shape, protein folding and stability, cell proliferation and apoptosis, and putative regulators of transcriptional and post-transcriptional modes of muscle-specific gene expression. We should note that although most of the differentially expressed proteins seen here are already known to be either directly or indirectly involved in myogenesis, a number of gene products (e.g. HSP90, transcription intermediary factor 1β (TIF1β) and IKB kinase α subunit) with unprecedented involvement in skeletal muscle differentiation were also uncovered by our experiments. Furthermore, although we found the proteomics-based approach to be valuable in generating new information, we were surprised that some well-known mediators of muscle differentiation such as myogenin and cell-cycle regulator p21 were not readily detected in our analysis. The most plausible explanation for this discrepancy is that a given spot on a 2D gel may consist of more than one protein and analysis of such spots by MALDI-TOF generates complex mixture of peptide fingerprints. Therefore, by choosing only the best-matched protein and ignoring additional less perfect peptide fingerprint matches, we are likely to miss minor proteins obscured by the more abundant ones.
      Initiation of differentiation in C2C12 cells following their growth in DM leads to profound changes in the transduction of inter- and intra-cellular signals, locomotion, and cell shape that precede the fusion of myoblasts into multinucleated muscle cells and depends on the reorganization of their cytoskeleton and plasma membranes (
      • Schmidt A.
      • Hall M.N.
      Signaling to the actin cytoskeleton..
      ). Temporal changes in the steady-state levels of a number of key protein kinases known to regulate cell architecture, proliferation, and apoptosis seen in our study are consistent with this scenario. Coordinate regulation of p70sk6 and pERK in cells incubated in DM suggests involvement of these kinases in the cell-cycle regulation and imposition of quiescence before induction of the myogenic differentiation program. Similarly, the levels of pAKT/PKB and p38 were also regulated during this process. The precise regulatory relationship among the various kinases remains to be determined. Furthermore, changes in the steady-state levels of α- and β-actin, calponin, CapZ, APC-EB1, and tropomyosin and myosin light chain during myogenesis in vitro underscore the role of cell shape changes during this process.
      The C2C12 myotubes accumulated the ubiquitous and muscle-specific actins and some proteins known to associate with actin. Tropomyosin binds to actin and in association with troponin regulates calcium-dependent contraction of muscle. Anti-CapZ antibodies or expression of dominant-negative CapZ delay myofibril assembly and the formation of striations in the muscle cell (
      • Schafer D.A.
      • Hug C.
      • Cooper J.A.
      Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments..
      ). Phosphatidylinositol-4,5-bisphosphate dissociates CapZ from the barbed ends of actin filaments in vitro (
      • Schafer D.A.
      • Jennings P.B.
      • Cooper J.A.
      Dynamics of capping protein and actin assembly in vitro: Uncapping barbed ends by polyphosphoinositides..
      ). Thus, the observed up-regulation of both CapZ and EB1 in the myotubes underscores a role of actin- and microtubule-based cytoskeleton network in differentiating muscle.
      Cell-cell and cell-extracellular membrane interactions facilitate myotube formation and the enhanced levels of basement membrane proteoglycan agrin, the metricellular protein SPARC, and αV and β2 integrin in the C2C12 myotubes are consistent with a role of extracellular membrane in myogenesis. Agrin facilitates clustering of acetylcholine receptors and acetylcholinesterase at neuromuscular junction (
      • Campanelli J.T.
      • Hoch W.
      • Rupp F.
      • Kreiner T.
      • Scheller R.H.
      Agrin mediates cell contact-induced acetylcholine receptor clustering..
      ). An enhanced expression of agrin in C2C12 myotubes reflects their intrinsic potential to form neuromuscular synapse. Three members of the calcium- and phospholipid-binding family of proteins, the annexins, were up-regulated in C2C12 myotubes. Annexin-I is known to promote membrane fusion and exocytosis, and elevated levels of annexin-I reflect its role in the fusion of C2C12 cells needed to form multinucleated myotubes.
      The levels of two LIM domain-containing proteins and LIMK1 were significantly altered in C2C12 cells in DM. While the LIM- and SH3-domain containing protein (Lasp-1) preferentially accumulated in the myotubes, LIM-domain protein1 and the LIMK1 were both down-regulated. Lasp-1, a cAMP-dependent actin-binding protein (
      • Chew C.S.
      • Parente Jr., J.A.
      • Zhou C.
      • Baranco E.
      • Chen X.
      Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell..
      ,
      • Schreiber V.
      • Moog-Lutz C.
      • Regnier C.H.
      • Chenard M.P.
      • Boeuf H.
      • Vonesch J.L.
      • Tomasetto C.
      • Rio M.C.
      Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions..
      ) may be causally associated with the changes in cell architecture. LIMK1 phosphorylates cofilin and abolishes its ability to de-polymerize actin. Apparently, LIMK1 initially rises in cells incubated in DM but declines progressively as myoblasts exit cell cycle and become myotubes (
      • Geneste O.
      • Copeland J.W.
      • Treisman R.
      LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics..
      ,
      • Pawlak G.
      • Helfman D.M.
      MEK mediates v-Src-induced disruption of the actin cytoskeleton via inactivation of the Rho-ROCK-LIM kinase pathway..
      ). Because the mature muscle cells do not express LIMK1, we propose that its steady decline during myogenic differentiation of C2C12 cells facilitates this process. Three members of the LIM-only proteins, FHL1, FHL2, and FHL3, are expressed in skeletal and heart muscle (
      • Chu P.H.
      • Ruiz-Lozano P.
      • Zhou Q.
      • Cai C.
      • Chen J.
      Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system..
      ). Exogenous expression of FHL2 has been shown to enhance the rate of myotube formation in C2C12 cells (
      • Martin B.
      • Schneider R.
      • Janetzky S.
      • Waibler Z.
      • Pandur P.
      • Kuhl M.
      • Behrens J.
      • von der Mark K.
      • Starzinski-Powitz A.
      • Wixler V.
      The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts..
      ). In light of these observations, we can only speculate whether changes in LIM-domains-containing protein 1 and LIMK1 are mechanistically related to the process of myogenic differentiation.
      The steady-state levels of two signaling kinases, MKK3 and PKA, were reciprocally altered during in vitro conversion of C2C12 cells into myotubes. The MKK3, a dual-specificity MAP kinase, together with MKK4, MKK6, and MKK7, is an upstream activator of p38 MAP kinase involved in muscle differentiation (
      • Derijard B.
      • Raingeaud J.
      • Barrett T.
      • Wu I.H.
      • Han J.
      • Ulevitch R.J.
      • Davis R.J.
      Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms..
      ). Expression of dominant-negative MKK3 severely inhibited the synthesis of MyoD, troponin T, p21 and p27, and myotube formation in C2C12 cells (
      • Cabane C.
      • Englaro W.
      • Yeow K.
      • Ragno M.
      • Derijard B.
      Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway..
      ). Thus, preferential accumulation of MKK3 in the C2C12 myotubes corroborates previous studies indicating that MAP kinase cascade plays a critical role in skeletal muscle differentiation.
      Coordinated control of intracellular cAMP, PKA, phosphorylation of cAMP-responsive element (CRE) binding protein and transcription of CRE-driven promoters during myogenesis has also been documented (
      • Siow N.L.
      • Choi R.C.
      • Cheng A.W.
      • Jiang J.X.
      • Wan D.C.
      • Zhu S.Q.
      • Tsim K.W.
      A cyclic AMP-dependent pathway regulates the expression of acetylcholinesterase during myogenic differentiation of C2C12 cells..
      ). Thus, the reduced level of type II-α regulatory subunit of PKA seen in C2C12 myotubes is consistent with published data showing PKA to be a negative regulator of muscle differentiation.
      We found that the serine/threonine protein kinase Akt2/PKB preferentially accumulated in C2C12 myotubes. A positive feedback loop between Akt2 and MyoD has been demonstrated previously. Akt2 promoter possesses multiple MyoD binding sites and is activated by MyoD; in turn, Akt2 facilitates the actions of MyoD and enhances muscle specific gene expression (
      • Kaneko S.
      • Feldman R.I.
      • Yu L.
      • Wu Z.
      • Gritsko T.
      • Shelley S.A.
      • Nicosia S.V.
      • Nobori T.
      • Cheng J.Q.
      Positive feedback regulation between Akt2 and MyoD during muscle differentiation. Cloning of Akt2 promoter..
      ,
      • Vandromme M.
      • Rochat A.
      • Meier R.
      • Carnac G.
      • Besser D.
      • Hemmings B.A.
      • Fernandez A.
      • Lamb N.J.
      Protein kinase B beta/Akt2 plays a specific role in muscle differentiation..
      ). It was reported recently that Akt binds to HSP90 and if Akt-HSP90 complex formation was inhibited, it led to de-phosphorylation and inactivation of Akt (
      • Sato S.
      • Fujita N.
      • Tsuruo T.
      Modulation of Akt kinase activity by binding to Hsp90..
      ). The de-phosphorytaion of free Akt was catalyzed by phosphatase 2A (PP2A), a hetero-trimer composed of structural (A), phosphatase regulatory (B), and catalytic (C) subunits (
      • Mumby M.C.
      • Walter G.
      Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth..
      ). The B subunit of PP2A, represented by α, β, and γ isoforms, is abundant in muscle. Based on previously published observations (
      • Kaneko S.
      • Feldman R.I.
      • Yu L.
      • Wu Z.
      • Gritsko T.
      • Shelley S.A.
      • Nicosia S.V.
      • Nobori T.
      • Cheng J.Q.
      Positive feedback regulation between Akt2 and MyoD during muscle differentiation. Cloning of Akt2 promoter..
      ,
      • Vandromme M.
      • Rochat A.
      • Meier R.
      • Carnac G.
      • Besser D.
      • Hemmings B.A.
      • Fernandez A.
      • Lamb N.J.
      Protein kinase B beta/Akt2 plays a specific role in muscle differentiation..
      ,
      • Sato S.
      • Fujita N.
      • Tsuruo T.
      Modulation of Akt kinase activity by binding to Hsp90..
      ), the reduced levels of HSP90 and a concomitant increase in PP2A protein seen here are likely to be mechanistically related to the process of muscle differentiation. We should also note that DNA microarray hybridization studies revealed that expression of mRNA-encoding PP2A was highly enhanced in C2C12 myoblasts (
      • Delgado I.
      • Huang X.
      • Jones S.
      • Zhang L.
      • Hatcher R.
      • Gao B.
      • Zhang P.
      Dynamic gene expression during the onset of myoblast differentiation in vitro..
      ).
      Chromatin remodeling and regulation of transcription of muscle-specific genes are mechanistically related to the process of differentiation. The proliferating C2C12 cells reprogram their genome to induce muscle-specific gene expression prior to myotube formation. It is not surprising therefore that the steady-state levels of several nuclear proteins including Pax-7, MEF2A, TAF28, the transcription intermediary factor 1β (TIF1β), histone acetyl transferase (HAT), HnRNP A1, and RNP-F were altered in the differentiating C2C12 cells.
      The Pax-3 and Pax-7 genes are expressed in the segmental plate and somites and play a central role in the development of muscle (
      • Ziman M.R.
      • Kay P.H.
      Differential expression of four alternate Pax7 paired box transcripts is influenced by organ- and strain-specific factors in adult mice..
      ). The muscles of Pax-7−/− mice lacked satellite cells completely (
      • Seale P.
      • Sabourin L.A.
      • Girgis-Gabardo A.
      • Mansouri A.
      • Gruss P.
      • Rudnicki M.A.
      Pax7 is required for the specification of myogenic satellite cells..
      ). Activated satellite cells expressing Pax-7 are involved in postnatal growth and repair of skeletal muscle fibers. Because expression of Pax-7 in fully differentiated muscle is negligible, the observed down-regulation of Pax-7 in C2C12 cells in DM is compatible with its putative role in the process of myogenesis.
      The MADS-domain containing factor MEF2A is involved in muscle-specific and growth factor-related transcription (
      • Pownall M.E.
      • Gustafsson M.K.
      • Emerson Jr., C.P.
      Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos..
      ,

      Tannu, N. S., Rao, V. K., and Raghow, R.(2003) Cellular and molecular paradigms of myogenesis, inRecent Research Developments in Molecular Biology (Pandlai, S. G., ed)Vol. 1, pp.73–95, Research Signpost, Trivendrum, India

      ,
      • Molkentin J.D.
      • Olson E.N.
      Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors..
      ). MEF2A forms homodimers and also associates with other bHLH proteins that bind to the promoters/enhancers of muscle-specific genes to modulate their transcription. The MEF2 proteins also interact with HAT-containing co-activators such as p300/CBP, and some members of the MAP kinase family (
      • Han J.
      • Jiang Y.
      • Li Z.
      • Kravchenko V.V.
      • Ulevitch R.J.
      Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation..
      ,
      • Sartorelli V.
      • Huang J.
      • Hamamori Y.
      • Kedes L.
      Molecular mechanisms of myogenic coactivation by p300: Direct interaction with the activation domain of MyoD and with the MADS box of MEF2C..
      ). Altered regulation of TIF1β that binds to the heterochromatin protein 1 (HP1) has been shown to occur during the differentiation of F9 cells (
      • Cammas F.
      • Oulad-Abdelghani M.
      • Vonesch J.L.
      • Huss-Garcia Y.
      • Chambon P.
      • Losson R.
      Cell differentiation induces TIF1beta association with centromeric heterochromatin via an HP1 interaction..
      ). Because treatment of C2C12 cells with a specific inhibitor of HAT or expression of dominant-negative CBP drastically reduced their ability to form myotubes (
      • Polesskaya A.
      • Naguibneva I.
      • Fritsch L.
      • Duquet A.
      • Ait-Si-Ali S.
      • Robin P.
      • Vervisch A.
      • Pritchard L.L.
      • Cole P.
      • Harel-Bellan A.
      CBP/p300 and muscle differentiation: No HAT, no muscle..
      ), altered levels of MEF2A, TIF1β, and HAT seen here underscore a central role for chromatin remodeling in myogenesis.
      A number of proteins known to regulate cell proliferation and apoptosis such as Gas1, BIRC4, follistatin-related protein, IGF-I, and FGF receptors were also altered in differentiating C2C12 cells. Both programmed cell death and myogenic differentiation share a need for dynamic assembly and disassembly of actin-based cytoskeleton. Caspases are the primary regulators of programmed cell death (
      • Enari M.
      • Hug H.
      • Nagata S.
      Involvement of an ICE-like protease in Fas-mediated apoptosis..
      ). BIRC4 inhibits caspase-3, -7, and -9 (
      • Deveraux Q.L.
      • Takahashi R.
      • Salvesen G.S.
      • Reed J.C.
      X-linked IAP is a direct inhibitor of cell-death proteases..
      ). The caspase3 −/− mice elicited severe muscle deficit, and abolition of caspase-3 in C2C12 cells dramatically decreased myotube formation (
      • Fernando P.
      • Kelly J.F.
      • Balazsi K.
      • Slack R.S.
      • Megeney L.A.
      Caspase 3 activity is required for skeletal muscle differentiation..
      ). The exact role of BIRC4 in promoting myogenesis in C2C12 remains to be established.
      The IGF-I receptor was down-regulated in C2C12 myocytes. IGF-I apparently has time-dependent actions during myogenesis; initially it promotes C2C12 myoblast proliferation and inhibits cell differentiation while subsequently IGF-I induces cell-cycle exit and promotes differentiation. Regulated expression of myogenin in response to IGF-I is thought to mediate both early and late actions of this growth factor (
      • Adi S.
      • Bin-Abbas B.
      • Wu N.Y.
      • Rosenthal S.M.
      Early stimulation and late inhibition of extracellular signal-regulated kinase 1/2 phosphorylation by IGF-I: A potential mechanism mediating the switch in IGF-I action on skeletal muscle cell differentiation..
      ,
      • Foulstone E.J.
      • Meadows K.A.
      • Holly J.M.
      • Stewart C.E.
      Insulin-like growth factors (IGF-I and IGF-II) inhibit C2 skeletal myoblast differentiation and enhance TNF alpha-induced apoptosis..
      ).
      We found that the receptor for FGF-4 was down-regulated during the differentiation of C2C12 myoblasts. FGFs stimulate myoblast proliferation and inhibit their differentiation into myotubes. Coordinated regulation of FGFs and their cognate receptors (FGFRs) in murine and chicken myoblasts undergoing differentiation has been reported (
      • Itoh N.
      • Mima T.
      • Mikawa T.
      Loss of fibroblast growth factor receptors is necessary for terminal differentiation of embryonic limb muscle..
      ,
      • Moore J.W.
      • Dionne C.
      • Jaye M.
      • Swain J.L.
      The mRNAs encoding acidic FGF, basic FGF and FGF receptor are coordinately downregulated during myogenic differentiation..
      ,
      • Templeton T.J.
      • Hauschka S.D.
      FGF-mediated aspects of skeletal muscle growth and differentiation are controlled by a high affinity receptor, FGFR1..
      ). Enforced expression of FGFR1 increased myoblast proliferation and delayed differentiation, while expression of a truncated FGFR1 had the opposite effect (
      • Scata K.A.
      • Bernard D.W.
      • Fox J.
      • Swain J.L.
      FGF receptor availability regulates skeletal myogenesis..
      ). A seminal role of FGFR4 signaling in the regulation of Myf5, MyoD, and MHC genes and muscle differentiation was recently demonstrated (
      • Marics I.
      • Padilla F.
      • Guillemot J.F.
      • Scaal M.
      • Marcelle C.
      FGFR4 signaling is a necessary step in limb muscle differentiation..
      ).
      Extracellular proteolysis is associated with myogenesis and uPA −/− mice have decreased ability for muscle regeneration (
      • Carmeliet P.
      • Schoonjans L.
      • Kieckens L.
      • Ream B.
      • Degen J.
      • Bronson R.
      • De Vos R.
      • van den Oord J.J.
      • Collen D.
      • Mulligan R.C.
      Physiological consequences of loss of plasminogen activator gene function in mice..
      ). Thus, an increased accumulation of the plasmin activator inhibitor seen in C2C12 myocytes corroborates the central role of uPA in myogenesis.
      Notch 1 expression is associated with immature myoblasts, while cells expressing the Notch ligands, delta1 and serrate2, are more advanced in myogenesis. Interactions between Notch and its ligands are thought to sustain myoblasts in a proliferative state (
      • Conboy I.M.
      • Conboy M.J.
      • Smythe G.M.
      • Rando T.A.
      Notch-mediated restoration of regenerative potential to aged muscle..
      ,
      • Conboy I.M.
      • Rando T.A.
      The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis..
      ). Because the levels of Jagged-2, a trans-membrane Notch ligand, declined in the multinucleated C2C12 myocytes, we posit that homothalic Notch1-Jagged signaling may modulate myotube formation.
      Intracellular Ca2+ concentration regulates contraction-relaxation, cell motility, cell cycle progression, and apoptosis (
      • Clapham D.E.
      Calcium signaling..
      ), and lowering of Ca2+ inhibits myogenic differentiation (
      • Shainberg A.
      • Yagil G.
      • Yaffe D.
      Control of myogenesis in vitro by Ca2+ concentration in nutritional medium..
      ). Calreticulin (Crt), located in the endoplasmic reticulum, is thought to regulate Ca2+ homeostasis of muscle cells (
      • Li J.
      • Puceat M.
      • Perez-Terzic C.
      • Mery A.
      • Nakamura K.
      • Michalak M.
      • Krause K.H.
      • Jaconi M.E.
      Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis..
      ,
      • Tharin S.
      • Hamel P.A.
      • Conway E.M.
      • Michalak M.
      • Opas M.
      Regulation of calcium binding proteins calreticulin and calsequestrin during differentiation in the myogenic cell line L6..
      ). It was reported that although the Crt −/− embryonic stem cells differentiated into cardiac myocytes, they had severely reduced expression of ventricular myosin light chain 2. The accumulation of MEF2-C into the nuclei was also impaired in Crt −/− cardiac cells. A direct link between the observed up-regulation of Crt and myotube formation needs to be experimentally established.
      The G-protein mediated GTP and GDP exchange is involved in regulating numerous physiological reactions. We noted that the levels of guanine deaminase that converts guanine to xanthine and ammonia, and also removes the guanine base from guanine containing metabolites, were significantly enhanced in C2C12 myotubes. Guanine deaminase may regulate the pools of intracellular GTP and guanylate, both as a housekeeping function and in response to differentiation signals.
      DNA microarray-based analyses of about 12,000 genes revealed that ∼12% transcripts were differentially regulated in differentiating C2C12 cells (
      • Delgado I.
      • Huang X.
      • Jones S.
      • Zhang L.
      • Hatcher R.
      • Gao B.
      • Zhang P.
      Dynamic gene expression during the onset of myoblast differentiation in vitro..
      ,
      • Shen X.
      • Collier J.M.
      • Hlaing M.
      • Zhang L.
      • Delshad E.H.
      • Bristow J.
      • Bernstein H.S.
      Genome-wide examination of myoblast cell cycle withdrawal during differentiation..
      ). Our proteomics-based investigation has revealed that from more than 2,000 proteins analyzed here about 6% were differentially regulated. The apparent gap between the genomics and proteomics data may be reconciled if we consider the underlying differences between the two techniques. First, the analysis of gene expression by DNA microarrays represents an order of magnitude greater coverage of the genome, essentially 100%. Second, a greater sensitivity of the nucleic acid probes used for hybridization to DNA microarrays enables one to detect relatively small differences in the relative rates of steady-state accumulation of mRNAs. With these caveats notwithstanding, we conclude that the comparative analysis of protein expression from proliferating C2C12 cells and fully differentiated myotubes has yielded interesting and interpretable data that shed important light on the process of skeletal myogenesis.

      Acknowledgments

      We are grateful to Dominic Desiderio for allowing us ready access to the mass spectrometry facility of the Stout Neuroscience laboratory and to Jacquelyn Fountain for providing competent secretarial assistance.

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