identification of secreted proteins during skeletal muscle ... · identification of secreted...

Identification of Secreted Proteins during Skeletal Muscle

Development

X’avia C. Y. Chan,†,‡ John C. McDermott,*,†, ‡ and K. W. Michael Siu*,‡,§

Department of Biology, Centre for Research in Mass Spectrometry, and Department of Chemistry,York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3

Received September 3, 2006

The differentiation program of skeletal muscle cells is exquisitely sensitive to secreted proteins. Wedeveloped a strategy to maximize the discovery of secreted proteins, using mass spectrometry-basedproteomics, from cultured muscle cells, C2C12, grown in a serum-free medium. This strategy led tothe identification of 80 nonredundant proteins, of which 27 were secretory proteins that were identifiedwith a minimum of two tryptic peptides. A number of the identified secretory proteins are involved inextracellular matrix remodeling, cellular proliferation, migration, and signaling. A putative network ofproteins involving matrix metalloproteinase 2, SPARC, and cystatin C that all interact with TGFâ signalinghas been postulated to contribute toward a functional role in the myogenic differentiation program.

Keywords: Conditioned media (CM) • One-dimensional gel electrophoresis (1D-SDS-PAGE) • Matrix-assisted laserdesorption/ionization quadrupole time-of-fight mass spectrometry (MALDI-QqTOF MS) • Extracelluar matrix (ECM)• Matrix metalloproteinase-2 (MMP2) • Secreted protein rich in cysteine protein (SPARC) • Transforming growthfactor-beta (TGFâ)

Introduction

The program of muscle cell differentiation has proven to bea regulatory paradigm for understanding principles of cellulardifferentiation. At the molecular level, this process is regulatedby two families of transcriptional regulators: the muscleregulatory factor (MRF) family, comprised of MyoD, Myf5,myogenin, and MRF4,1-6 and the myocyte enhancer factor 2(MEF2A-D) transcriptional regulatory proteins, which functionas obligatory partners of the MRFs in the differentiation ofcultured myogenic cells.7,8 One key aspect of the control ofmyogenic transcription factor activity is an acute responsive-ness to cellular signaling pathways; an example is the controlexerted over these factors by growth-factor-activated signalingpathways.9,10 Deciphering the link between myogenic transcrip-tion factors and signals secreted from cells within their ‘com-munity’ is regarded as an important task in developmentalbiology.

A thorough understanding of the mechanisms involved inextracellular regulation is a prerequisite to understanding thecontrol exerted by intracellular effectors in the differentiationprogram. To date, a number of extracellular growth factors havebeen found to play a functional role as myogenic regulators.11

For example, members of the IGF 1 and transforming growthfactor-beta (TGFâ) families were observed to have potent, butopposing, effects on myogenesis.10,11 In addition, dramatic

effects exerted by the ‘conditioned’ media (CM) on muscle cellswere documented, indicating that myogenic cells modify theirown extracellular milieu by secreting factors that exert auto-crine and paracrine effects on the differentiation program.12,13

Indeed, it has been suggested that therapeutic use of thesesecreted factors may constitute the basis for a cell-basedtreatment for Duchenne Muscular Dystrophy (DMD) by acti-vating skeletal muscle stem cells.14 Unfortunately, this optimismis not justified by the scarcity of knowledge on these secretedfactors.15,16 Characterization of the full spectrum of myogenicsecreted factors and subsequent analysis of their functions area critical first step in better understanding the regulation ofmyogenesis and muscle pathology.

Here, we report the results of a proteomic study to identifysecreted proteins central to skeletal muscle differentiation; thisstudy uses the mouse skeletal muscle cell line C2C12 as a modelsystem and matrix-assisted laser desorption/ ionization (MAL-DI) mass spectrometry (MS) and tandem mass spectrometry(MS/MS) for analysis. The C2C12 muscle cell line, initiallydeveloped by Yaffe et al., has been a cornerstone of workconcerning the differentiation of muscle cells since its intro-duction in the 1960s.17,18 These cells proliferate under highmitogen conditions and differentiate on exposure to lowmitogen containing media. The C212 culture model has led tomany significant advances in our understanding of muscledifferentiation, including characterization of the MRFs andMEF2 transcriptional regulatory proteins in this process.1-8 MS-based protein identification methodologies via tryptic peptidemapping in combination with searches against a proteindatabase are now established practices that have led tomapping of proteomes.19,20 There have been a number of

* To whom correspondence should be addressed. E-mails: (for J.C.McD.)[email protected]; (for K.W.M.S.) [email protected].

† Department of Biology, York University.‡ Centre for Research in Mass Spectrometry, York University.§ Department of Chemistry, York University.

698 Journal of Proteome Research 2007, 6, 698-710 10.1021/pr060448k CCC: $37.00 2007 American Chemical SocietyPublished on Web 01/11/2007

reported studies on the identification of secreted proteins andmapping of secretomes from various cells and cell lines;21-29

however, none has involved skeletal muscle cells. To the bestof our knowledge, this is the first report on secreted proteinsin myogenesis.

Materials and MethodsA general sample workflow is shown in Figure 1.Cell Culture. Mitogenic murine C2C12 myoblasts (American

Type Culture Collection, ATCC) were seeded on 10-cm gelatin-coated (Sigma) culture plates (Fisher Scientific), containing 10

Figure 1. General workflow of preparation of conditioned media and subsequent protein identification by mass spectrometry.

Figure 2. (A) Light microscopic images of C2C12 myoblasts during myogenesis in serum-containing (serum+) versus serum-free (serum-)culture system (left and right panel, respectively). The images were taken at various time points (24, 48, 72, 96, and 120 h) followingswitching to, respectively, 5% horse serum or DMEM/F12 supplemented with 2.5 µg/mL insulin. (B) Parallel set of samples as in panelA, but with myosin heavy-chain staining via immunocytochemistry. Positively stained cells were visualized using a colorimetric substrate(diaminobenzidine) in which brown-staining indicates myosin positive cells.

Secreted Proteins in Skeletal Muscle Development research articles

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mL of Dulbecco’s modified Eagle’s medium (MEM, Invitrogen)supplemented with 10% fetal bovine serum (Hyclone), 2 mML-glutamate (Invitrogen), 50 units/mL penicillin-streptomycin(Invitrogen), and 1 mM sodium pyruvate (Invitrogen). Conflu-ent (90%) myoblasts were committed to differentiating intomultinucleated myotubes by switching them into the dif-ferentiation medium comprising 5 mL of Dulbecco’s MEMsupplemented with 2 mM L-glutamate, 50 units/mL penicillin-streptomycin, 1 mM sodium pyruvate, and 5% horse serum(Atlanta).

For serum-free inoculation, confluent (90%) myoblasts werebriefly washed with versene (Bioshop) and digested with 1 mLof 0.125% trypsin (Gibco) for 1 min. The trypsinization processwas ceased by addition of 5 mL of the serum-free differentiationmedium, comprising 1:1 Dulbecco’s MEM/Ham’s NutrientMixture F-12 (Gibco) supplemented with 2 mM L-gluamine, 50units/mL penicillin-streptomycin, 1 mM sodium pyruvate, and2.5 µg/mL bovine insulin (Sigma). The cells were spun bycentrifugation for 10 min at 153g. The pellet was resuspendedin 5 mL of the serum-free differentiation medium and spunby centrifugation. This washing step was repeated. The result-ing pellet was finally resuspended in 5 mL of the serum-freedifferentiation medium and inoculated directly onto a 10-cmgelatin-coated culture plate. The CM were collected after 24,48, 72, 96, and 120 h of incubation. After the collection of CMat each specific time point, the cells that remained on theculture dish were washed extensively with 10 mL of phosphate-buffered saline (Gibco). This washing step was repeated fourmore times, followed by replenishment of the serum-freedifferentiation medium.

Protein Fractionation by One-Dimensional Sodium Dode-cyl Sulfate Polyacrylamide Gel Electrophoresis (1D-SDS-PAGE). The CM collected were purified by two-step centrifu-gation at 4 °C (98.2g) for 5 min, followed by 12 499g for 30 minto remove cells and debris. The clarified medium was thensyringe-filtered (0.45 µm pore size, Millex-HV) to removeremaining particles. The proteins dissolved in the CM wereprecipitated by addition of acetone (Caledon) at -20 °C to afinal concentration of 80%; precipitation was allowed toproceed overnight. The proteins were then pelleted by cen-trifugation at 4 °C (15 344g for 1 h). After removal of acetone,the proteins were allowed to air-dry. The dried proteins weredissolved in a minimum volume of SDS-gel loading buffer,containing 50 mM Tris-HCl (Bioshop), 2% SDS (Bioshop), 25%glycerol (BDH), and 2.93% â-mercaptoethanol (Bioshop). Theprotein concentration was determined by Bradford Assay (Bio-Rad). Equal amounts of 125-µg protein samples were loadedonto a 12% one-dimensional polyacrylamide gel (20 cm × 20cm, Bio-Rad). The resolved protein bands were visualized bystaining with Coomassie blue (ICN Biomedicals, Inc.).

In-Gel Digests with Trypsin. The SDS-PAGE separationrevealed 52 bands per lane at the time point of 120 h at whichmyotubes were clearly evident (Figures 2 and 3). These bandswere excised, and each was subjected to individual in-gel reduc-tion, alkylation, and trypsin digest. Briefly, proteins in the gelslice were reduced with 30 µL of 50 mM ammonium bicarbon-ate (Sigma)/10 mM dithiothreitol (Sigma) at 56 °C for 15 min.This was followed by alkylation conducted with 30 µL of 100mM iodoacetamide (Sigma)/50 mM ammonium bicarbonatefor 15 min in the dark. Enzymatic digest was then performedovernight with sequencing-grade trypsin (Promega) at 37 °C.

Protein Identification with Mass Spectrometry. Each trypticpeptide mixture thus generated was concentrated, desalted with

ZipTip (Millipore), and subsequently eluted with 1.5 µL of 10mg/mL R-cyano-4-hydroxycinnamic acid (Sigma) in 60% ac-etonitrile (Sigma) and 0.3% trifluoroacetic acid (Sigma) onto aMALDI sample plate. All MS and MS/MS analyses wereperformed on a QSTAR XL hybrid quadrupole/time-of-flight(QqTOF) tandem mass spectrometer (Applied Biosystems/MDSSCIEX) equipped with a nitrogen laser (337 nm) for MALDI.The MS and MS/MS spectra generated were searched againstthe Swiss-Prot and NCBInr databases, respectively, using theMascot software (Matrix Science). The search parameters weretaxonomy, Mus musculus; allowed modifications, carboxy-amidomethylation of cysteine and oxidation of methionine;missed cleavages allowed, one; and peptide and MS/MStolerance, (50 and (100 ppm, respectively, with a peptidecharge of 1+. Secretory proteins identified had to have aminimum of two g10-residue peptides identified with highconfidence and had to have been verified by manual inspectionfor having a consecutive series of matched and abundant y orb ions (g3 residues). The vast majority of proteins wereidentified with three or four peptides.

Nonredundant Data Acquisition. To maximize the numberof secreted proteins identified per MALDI spot, a nonredundantdata acquisition strategy (Figure 4) was implemented in the

Figure 3. 1D-SDS-PAGE separation of conditioned media col-lected at 120 h. Fifty-two bands were revealed after Coomassieblue staining. Excised bands were trypsinized and analyzed usingMALDI MS and MS/MS.

research articles Chan et al.

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selection of precursor ions (protonated tryptic peptides) forMS/MS. All peaks in a given MALDI-TOF mass spectrumexceeding a user-defined threshold (typically S/N g 3) wereemployed in a first-round of identification via tryptic peptidemass fingerprinting. The tryptic peptides of the candidateprotein thus identified were noted and entered into a “candi-date peak list” earmarked for subsequent MS/MS analyses. Theremaining peaks within the range of m/z 900-2000 wereentered into a “remaining peak list”. MS/MS was then per-formed first on the ions in the candidate peak list in adescending order of abundance. Proteins thus confirmed wereclassified as intracellular or secretory. All possible trypticpeptides of a verified intracellular protein were immediatelytransferred from the candidate peak list to the remaining peaklist. By contrast, once a secretory protein was verified, ad-ditional validation was immediately sought via sequencing ofa second and sometimes third tryptic peptide. As before, theremaining tryptic peptides of a secretory protein thus validatedwere also transferred to the remaining peak list. Once all theions in the candidate peak list were analyzed, MS/MS was thenperformed on the ions in the remaining peak list, again in adescending order of abundance. These analyses were per-formed until all peptides within a given sample spot werecompletely consumed. The number of distinct MS/MS analyses(accumulated spectra) per sample spot varied from a low ofseven to a high of 33 (due to different quantities of proteinsavailable in the bands), with an average of 19 analyses per spot.

Myosin Heavy Chain Expression with Immunocytochem-istry. Myosin heavy chain subunits are encoded by distinctmembers of a multigene family expressed at different stagesof muscle development and are the archetypal marker proteinsto indicate cellular differentiation of muscle. To visualizemyosin heavy chain expression in cultured muscle cells, weused the MF20 monoclonal antibody30 which recognizes allsarcomeric myosins. Briefly, after fixation (6 min in 90%methanol), the cells were incubated with the primary antibody(monoclonal supernatant produced in our laboratory) followedby incubation with a horseradish peroxidase-conjugated goatanti-mouse secondary antibody at a dilution of 1:1000 (Bio-Rad). Positively stained cells were visualized using a colori-metric substrate (diaminobenzidine substrate, Sigma) whichresults in a visually brown stain of the positive cells.

ResultsCell Culture and Preparation of Secreted Proteins. A key

determinant of our selection of the mouse C2C12 myoblast asthe skeletal muscle model was due to its discernible phenotypicchanges during myogenesis (Figure 2). The choice of 120 h forCM collection was based on a compromise between prominentmyotube formation (as revealed by prominent fusion of multi-nucleated myotubes) and cell viability. Avoidance of fetalbovine serum (FBS), a common medium in cell culturing,31 wasa key step, as it is a rich source of proteins thus makingdifferentiation between C2C12 proteins and FBS proteins

Figure 4. Nonredundant data acquisition for maximizing the discovery of secretory proteins.



difficult. We found that C2C12 cells grow and differentiate inthe serum-free culture medium DMEM/F12 supplemented with

2.5 µg/mL of bovine insulin (Figure 2), which had been reportedto be a potent mitogen, survival, and differentiation factor for

Table 1. Summary of Secretory Proteins



Table 1 (Continued)



muscle cells.32-35 Comparable morphological changes to theC2C12 myoblasts were apparent during myogenesis in theserum-containing and serum-free media: both cell culturesystems showed the same onset time for differentiation (48 h)and maturation time for terminal differentiation (120 h), asindicated by the partial alignment of myoblasts and fusion ofmyotubes, respectively (Figure 2). Insulin (supplied exog-enously) and insulin-like growth factor (secreted endogenously)act through the IGF-1 receptor in cultured muscle cells. IGF-1is a critical paracrine factor in differentiating muscle cells invitro and in vivo. Thus, although the addition of insulin toserum-free media is required for cell survival and maintenance,

this added factor is something to which the cells are normallyexposed in vivo. In normal culture media (both growth anddifferentiation conditions), insulin and IGF-1 are critical com-ponents of the media, and in vivo, the cells are exposed to highcirculating levels of insulin (especially in the embryonic andfetal phases of development).

As detailed earlier, the CM collected at 120 h were precipi-tated with 80% acetone, followed by dissolution in the SDS-gel loading buffer and 1D-SDS-PAGE separation. The resultsof experiments in which we have analyzed the expression of amuscle marker gene (myosin heavy chain) by immunocy-tochemistry (Figure 2B) demonstrate a developmental progres-

Table 1 (Continued)



Table 2. Summary of Intracellular Proteins



sion in myosin gene expression occurring in both the serum-free (serum-) as well as serum-containing (serum+) media.Initial appearance of myosin positive cells (as indicated by thebrown staining) occurred under both serum-containing andserum-free conditions at 72 h. As the differentiation proceededto 96 and 120 h, the caliber and number of multinucleated,elongated myotubes that were positively stained for myosin inboth media were qualitatively equivalent, verifying the efficacyof the serum-free condition for studying muscle differen-tiation.

Mass Spectrometric Analysis of Secreted Proteins. The SDS-PAGE-separated proteins were visualized as discrete bands byCoomassie blue staining (Figure 3). After in-gel tryptic digest,the peptides were extracted for mass spectrometric analysis.We opted to use MALDI MS and MS/MS for this study becauseof its ruggedness in comparison to online electrospray MS/MS. To maximize the number of secretory proteins identified,we implemented the strategy of nonredundant data acquisitionas detailed earlier. In total, we have identified 80 proteins inthe collected conditioned media; 27 have been classified assecretory (Table 1)36-41 and 53 as intracellular proteins (Table2) based on their currently known functions.42-47 Two proteins,fibronectin and procollagen, appeared in more than one regionof the 1D-SDS gel; this strongly suggested the presence ofdegradation products and/or different forms of the proteins.Of the 27 secretory proteins, the vast majority were identifiedwith three or more peptides, thus making these highly confi-dent identifications.

Discussion

Proteins secreted into the extracellular milieu by skeletalmuscle cells, sometimes referred to collectively as the ‘secre-tome’, are potent mediators of cell survival, proliferation,differentiation, and fusion. To begin unraveling the complexbiology underlying the role played by secreted proteins inmyogenesis, the first necessary step is to characterize theproteins involved. A critical initial requirement prior to thisprotein identification is the development of a method forpreparing secretome samples from cultured muscle cellswithout extensive contamination by intracellular or culturemedia-derived proteins. In this study, we have developed aneffective method for myogenic secretome isolation and char-acterization using mass spectrometry. A number of the secre-tory proteins thus identified have previously been reported asproteins secreted from muscle,36,37 indicating the validity of theapproach taken. More importantly, some identified proteinsappear to function as key secreted regulators of the musclegrowth and differentiation program. It is not possible at thisstage to classify unequivocally all of the remainder as secretedproteins, as the possibility exists that these could arise as aresult of cell stress and were released to the conditioned mediasubsequent to apotosis and/or as a consequence of washing.Validation experiments, including localization with taggedproteins, will need to be performed in the future.

We initially assigned the secretory proteins to several func-tional groups, in order to putatively link proteins together in

Table 2 (Continued)



known pathways and to identify possible roles in muscledifferentiation. A number of the proteins segregate into the‘extracellular matrix’ (ECM) class of proteins, including fi-bronectin, nidogen, biglycan, various isoforms of procollagen,moesin, and heparan sulfate proteoglycan (HSPG). Evidence

indicates that the ECM not only acts as a structural supportfor maintaining the cell architecture, but also plays a pivotalrole in both morphogenesis and tissue regeneration via ECMremodeling. This process, in turn, is regulated by ECM-associated proteases.38,39 In particular, the ECM proteins be-

Figure 5. (Continued)



longing to matrix metalloproteinases have been implicated inmuscle differentiation.40 Several of these proteases, includingmatrix metalloproteinase-2 (MMP2) and procollagen C-pro-teinase (PCEP), were found in this study. MMP2 exerts a directdegradation effect on the ECM by cleaving the major ECMcomponents, such as type IV collagen.41-47 PCEP triggers thedegradation of ECM indirectly by promoting the activity of theECM-degrading enzyme, procollagen C-proteinase.48-51

A number of proteins identified belong to the class ofproteins involved in cell migration. During skeletal-muscledevelopment, myoblasts migrate across the basal lamina; thisprocess stops once the differentiation program commences.52

Several secreted proteins that have been identified in this study,including serine protease inhibitors (serpin),53-56 pigmentepithelium-derived factor (PEDF),57-59 annexin-A1 and -A2,60-62

and galectin-1 and -3,63-70 are speculated to be involved in theregulation of myogenesis inasmuch as their capability inregulating cell migration.

An intriguing link between MMP2, secreted protein rich incysteine (SPARC), and cystatin C proteins was identified bycombining our protein identification data (representative MS/MS data shown in Figure 5) with published literature concern-ing the functional properties of these proteins. This putativenetwork of extracellular proteins is based on the followingobservations: MMP2 has been implicated in myoblast migra-tion,43 myotube formation,41 and regulation of satellite cells;45

and SPARC functions as a potent anti-proliferative protein andis highly expressed during the differentiation of myoblasts.37

Both SPARC71,72 and MMP273-77 are common targets of theTGFâ signaling pathway in muscle cells. Moreover, MMP2directly cleaves and activates the SPARC protein.78 In addition,cystatin C, a cysteine protease inhibitor,79 is TGFâ responsive80-82

and is directly associated with MMP2 regulation.83,84 Thus, wepropose a model for this proteomic network in which reciprocalfeedback loops between the respective proteins will contributeto a functional role in the myogenic differentiation program(Figure 6).

In summary, a strategy has been developed for the isolationand identification of secreted proteins during the differentiationof cultured muscle cells. A number of secreted proteins

Figure 5. MS/MS spectra of tryptic peptides that contribute toward identification of three secretory proteins: SPARC, MMP2, andcystatin C.

Figure 6. Proposed network of secretory proteins associated withTGFâ. Positive interactions are indicated by arrowheads, inhibi-tory interactions are indicated by right-angled lines. Direction ofarrows and angled lines indicates the directionality of signaling.Bidirectional lines indicate a proposed feedback loop. In thishypothesis, TGFâ induces MMP2 by increasing its transcriptionand/or prolonging the stability of MMP2 transcripts. A positivefeedback loop is proposed between SPARC and TGFâ: TGFâelicits SPARC expression at the transcriptional level; SPARCinteracts with TGFâ receptor and thus facilitates TGFâ signaling.A negative feedback loop is proposed between cystatin C andTGFâ: TGFâ upregulates cystatin C; however, cystatin C deac-tivates TGFâ signaling by antagonizing cognate receptors of theTGFâ pathway. MMP2 is proposed to generate a cleaved formof SPARC, the physiological role of which is currently unknown.



belonging to several functional groups were identified that holdconsiderable potential as key regulators of the myogenicdifferentiation program. These studies provide a testableframework for identifying mechanistic links within the networkof secreted proteins responsible for the differentiation ofmyogenic cells. The identification and characterization ofsecretory proteins may facilitate the development of smallmolecule-based therapeutic strategies for in vivo and in vitromanipulation of muscle differentiation and growth in a varietyof congenital and acquired myopathies.

Acknowledgment. The authors acknowledge financialsupport in the form of grants from the Canadian Institutes ofHealth Research and the Heart and Stroke Foundation ofCanada (JCMcD), and the Natural Sciences and EngineeringResearch Council of Canada (KWMS). Hardware support wasprovided by the Ontario Research and Development ChallengeFund and Applied Biosystems/MDS SCIEX.

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PR060448K



identification of secreted proteins during skeletal muscle ... · identification of secreted...

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