RESEARCH ARTICLE

The effect of serum withdrawal on the protein

profile of quiescent human dermal fibroblasts

in primary cell culture

Federica Boraldi, Giulia Annovi, Chiara Paolinelli-Devincenzi, Roberta Tiozzoand Daniela Quaglino

Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

The effect of serum deprivation on proliferating cells is well known, in contrast its role on pri-mary cell cultures, at confluence, has not been deeply investigated. Therefore, in order to explorethe response of quiescent cells to serum deprivation, ubiquitous mesenchymal cells, as normalhuman dermal fibroblasts, were grown, for 48 h after confluence, in the presence or absence of10% FBS. Fibroblast behaviour (i.e. cell morphology, cell viability, ROS production and elastinsynthesis) was evaluated morphologically and biochemically. Moreover, the protein profile wasinvestigated by 2-DE and differentially expressed proteins were identified by MS. Serum with-drawal caused cell shrinkage but did not significantly modify the total cell number. ROS pro-duction, as evaluated by the dihydroethidium (DH2) probe, was increased after serum depriva-tion, whereas elastin synthesis, measured by a colorimetric method, was markedly reduced in theabsence of serum. By proteome analysis, 41 proteins appeared to significantly change theirexpression, the great majority of protein changes were related to the cytoskeleton, the stress re-sponse and the glycolytic pathway. Data indicate that human dermal fibroblasts in primary cellculture can adapt themselves to environmental changes, without significantly altering cell via-bility, at least after a few days of treatment, even though serum withdrawal represents a stresscondition capable to increase ROS production, to influence cell metabolism and to interfere withcell behaviour, favouring the expression of several age-related features.

Received: September 2, 2007Revised: September 21, 2007

Accepted: September 24, 2007

Keywords:

Dermal fibroblasts / Primary cell culture / ROS production / Serum withdrawal

66 Proteomics 2008, 8, 66–82

1 Introduction

The common genomic response to serum includes induc-tion of genes related to cell cycle, cell motility, extracellularmatrix remodelling, cell–cell signalling and acquisition of amyofibroblast phenotype [1], indicating that serum factorsinterfere with a broad range of cell processes. Serum, in fact,represents a fundamental source of nutrients, cytokines and

adhesive molecules necessary for in vitro cell growth andmaintenance. Serum deprivation is a widespread methodused to synchronize proliferating cell lines in the G0 phaseof the cell cycle, since the absence of mitotic signals, asthrough serum deprivation in G1 phase, leads to a rapid exitfrom the cell cycle into a nondividing state (G0) [2, 3]. Incontrast, in quiescent cultures, serum starvation has beendescribed to cause cell death [4, 5] and to favour the occur-rence of a fibrotic response [6]. There are several reports inthe literature concerning the effects of serum withdrawal onthe expression of single proteins in different cell types [7–9],but a comprehensive analysis of changes occurring in theprotein profile upon serum starvation has been neverapproached.

Correspondence: Professor Daniela Quaglino, Department ofBiomedical Sciences, Via Campi 287, 41100 Modena, ItalyE-mail: quaglino.daniela@unimore.itFax: 139-0592055426

Abbreviation: TRITC, tetramethyl rhodamine isothiocyanate

DOI 10.1002/pmic.200700833

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Proteomics 2008, 8, 66–82 Cell Biology 67

Fibroblasts are ubiquitous mesenchymal cells that areroutinely used as an in vitro experimental model to mirror invivo physiological and pathological conditions [10], moreover,it has to be underlined that there are conditions such as age-ing or fibrosis where fibroblasts may also experience restric-ted supply of nutrients [5], suggesting that growth factorsabundance influences matrix composition [5] and turnover[11], as well as cell metabolism and behaviour [2]. Further-more, it has been demonstrated that, during senescence,human diploid fibroblasts showed a marked decrease inserum response factor binding activity [12], allowing to hy-pothesize that reduced and/or altered response to serumfactors is associated with the ageing cellular phenotype.

Aim of the present study was to investigate the influenceof serum depletion on quiescent human dermal fibroblastsin primary cell culture, by evaluating cell viability, productionof ROS and changes in the protein profile.

2 Materials and methods

2.1 Cells and treatments

Human dermal fibroblasts were taken during surgery afterinformed consent from six clinically healthy females(44 6 7 years), which did not exhibit any sign of genetic,metabolic or connective tissue disorders. The adopted pro-cedure was in accordance with the guidelines of the ethicalcommittee of the Modena University Faculty of Medicine.Biopsies were taken from the upper thigh. Fibroblasts wereobtained from small skin fragments placed in DMEM plus50% FBS until a consistent number of cells appeared to growaround tissue explants. Trypsinized cells were subculturedone or two times, stored in liquid nitrogen and used in theseexperiments between fourth and sixth passages. Unlessotherwise specified, cells were routinely cultured in 75 cm2

flasks (Nunc, Roskilde, Denmark) with 18 mL of DMEMsupplemented with 10% FBS, 2 mM glutamine, 100 IU/mLpenicillin and 100 mg/mL streptomycin. Cells from each in-dividual were kept separate during all experimental proce-dures.

2.2 Cell morphology and cell viability

Dermal fibroblasts were plated into 25 cm2 flasks (Nunc) at adensity of 2.56105 cells and cultured in 5 mL DMEM untilconfluence, thereafter cells were grown for 48 h in the pres-ence or absence of 10% FBS. In order to evaluate their mor-phology, cells were observed at the inverted microscope(Leica DM-IL). Digital images were taken on cells at con-fluence and after 3, 6, 24 and 48 h of treatment. For cellcounting, cells were trypsinized, washed with PBS andstained with the trypan blue dye (Sigma). The number ofdye-excluding cells was counted with a Neubauer chamber.After 48 h of serum depletion, fibroblasts were trypsinized,centrifuged and resuspended in PBS with propidium iodide

at the final concentration of 20 mg/mL. After staining withpropidium iodide for 5 min, cells were analysed on an EPICSXL flow cytometer (Coulter, USA). For all conditions and celllines, viability assays were performed in duplicate.

2.3 Proteome analysis

2.3.1 Sample preparation

Human dermal fibroblasts at confluence were grown for48 h in DMEM 6 10% FBS. Afterwards, cells were detachedfrom flasks by incubation in 0.25% Trypsin in PBS for10 min at 377C. After washes in DMEM plus FBS and pro-teinase inhibitors (1 mM EDTA, 10 mM e-aminocaproic acid,50 mM benzamidine) cells were centrifuged at 10006g for10 min. After supernatant removal, cell pellets were resus-pended in PBS plus proteinase inhibitors, centrifuged at10006g for 10 min, and immediately resuspended in lysisbuffer (8 M urea, 2% CHAPS, 65 mM dithioerythritol, 2%Pharmalyte pH 3–10 and trace amount of bromophenolblue). Protein concentration was determined according toBradford [13].

2.3.2 2-DE

2-DE was performed, essentially as described by Bjellqvist etal. [14], in two independent assays where the six different celllines with and without serum were run in triplicate. Samplescontaining 60 mg (analytical gels) or 1 mg (preparative gels)of protein underwent 2-DE using the immobiline/polyacryl-amide system [14]. IEF was performed on IPGphor system(GE Healthcare, Uppsala, Sweden) at 167C using two differ-ent protocols. For analytical gels, passive rehydratation for16 h, 500 V for 1 h, 500–2000 V for 1 h, 3500 V for 3 h,5000 V for 30 min and 8000 V for 12 h. For preparative gels,a preliminary step at 200 V constant for 12 h was added.Thereafter, IPG strips were reduced (2% dithioerythritol) andalkylated (2.5% iodoacetamide) in equilibration buffer (6 Murea, 50 mM Tris-HCl, pH 6.8, 30% glycerol, 2% SDS).When the equilibration phase was finished, strips wereloaded onto 12% acrylamide vertical gels using an EttanDALTsix electrophoresis unit (GE Healthcare). Analyticalgels were stained with ammoniacal silver nitrate [15],whereas preparative gels for mass spectrometric analysiswere silver-stained as described by Shevchenko et al. [16].

2.3.3 Data acquisition and analysis

To detect significant differences in protein abundance be-tween the two experimental conditions, all silver-stained gelimages were digitalized at 400 dpi resolution using Im-ageScanner (GE Healthcare) and analysed using Melanie 3.0software (GE Healthcare). After background subtraction,protein spots were automatically defined and quantified withthe feature detection algorithm [15]. Spot intensities wereexpressed as percentages (% vol) of relative volumes by inte-

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68 F. Boraldi et al. Proteomics 2008, 8, 66–82

grating the OD of each pixel in the spot area (vol) and divid-ing with the sum of volumes of all spots detected in the gel.Only those spots that, within the same experimental condi-tion, exhibited the same trend of expression in all gelsunderwent further quantitative analysis. Quantitative datawere exported as a text file to be elaborated using MicrosoftExcel program. Mean values, SDs and coefficients of varia-tion were calculated using the Excel-provided formulas. Sta-tistical data were obtained using GraphPad software (SanDiego, CA, USA) and compared by the unpaired t-test. Dif-ferences between treatments were considered significant atp,0.05. Only those spots whose expression appeared sig-nificantly changed upon serum withdrawal were selected forMS analysis.

2.3.4 In-gel destaining and digestion of protein

samples

Spots of interest were manually excised from preparative sil-ver-stained 2-DE gels. Silver-stained gel pieces weredestained as described by Gharahdaghi et al. [17]. Briefly, gelspots were incubated in 100 mM sodium thiosulphate and30 mM potassium ferricyanide, rinsed twice in 25 mMammonium bicarbonate (AmBic) and, once in water, shrunkwith 100% ACN for 15 min, and dried in a Savant SpeedVacfor 20–30 min. All excised spots were incubated with12.5 ng/ mL sequencing grade trypsin (Roche Molecular Bio-chemicals, Basel, CH) in 25 mM AmBic overnight at 377C.Peptide extraction was carried out twice using 50% ACN, 1%TFA and then 100% ACN. All extracts were pooled, and thevolume was reduced by SpeedVac.

2.4 MS

2.4.1 MALDI-TOF MS

The tryptic peptide extracts were redissolved in 12 mL 0.1%TFA. The matrix (CHCA) was purchased from Laser BioLabs(Sophia-Antipolis, France). A saturated solution of CHCA(1 mL) at 2 mg/200 mL in CH3CN/H2O (50:50 v/v) containing0.1% TFA was mixed with 1 mL of peptide solution on theMALDI target and left to dry. MALDI-TOF mass spectra wererecorded on a Voyager DE-PRO (Applied Biosystems, Cour-taboeuf, France) mass spectrometer, in the 700–5000 Damass range using a minimum of 200 shots of laser per spec-trum. Delayed extraction source and reflector equipmentallowed sufficient resolution to consider MH1 of mono-isotopic peptide masses. Internal calibration was done usingtrypsin autolysis fragments at m/z 842.5100, 1045.5642 and2211.1046 Da. PMF was compared to the theoretical massesfrom the Swiss-Prot 49.1 or the NCBI databases using MS-Fit3.1.1 from ProteinProspector 3.2.1 (http://www.expasy.org/tools/). Typical search parameters were: 630 ppm of masstolerance; carbamidomethylation of cysteine residues; onemissed enzymatic cleavage for trypsin; a minimum of fourpeptide mass hits was required for a match; methionine

residues could be considered in oxidized form; no restrictionwas placed on the pI and molecular weight of the protein.The minimum S/N was generally between 5/1 and 10/1,depending on the spectrum quality. Finally, triptic digeststhat did not produce unambiguous protein identificationwere successively subjected to HPLC/MS.

2.4.2 HPLC/MS

Peptides were resuspended in aqueous 5% formic acid andsubsequently eluted onto a 150 mm675 mm Atlantis C18column analytical (Waters, Milford, MA, USA) and separatedwith an increasing ACN gradient from 10 to 85% in 30 minusing a Waters CapLC system. The analytical column (esti-mated flow approx. 200nL/min) was directly coupled,through a nano-ES ion source, to a Q-TOF Ultima Globalmass spectrometer (Waters). Multicharged ions (chargestates 2, 3 and 4) were selected for fragmentation and theacquired MS/MS spectra were searched against the Swiss-Prot/TrEMBL nonredundant protein and NCBI databaseusing the MASCOT (www.matrixscience.com) MS/MSsearch engine. Initial search parameters were the following:enzyme, trypsin; maximum number of missed cleavages, 1;fixed modification, carbamidomethylation of cysteines; vari-able modification parameters, oxidation Met; peptide toler-ance, 0.5 Da; MS/MS tolerance, 0.3 Da; charge state, 2, 3 or 4.We basically selected the candidate peptides with probability-based MOWSE scores that exceeded its threshold, indicatinga significant (or extensive) homology (p,0.05), and referredto them as ‘hits’. The criteria were based on the manu-facturer’s definitions (Matrix Science, Boston, MA, USA)[18]. Proteins that were identified with at least two peptides,both showing a score higher than 40, were validated withoutany manual processing. Those with at least two peptideswhose score was lower than 40 and higher than 20 were sys-tematically checked and/or interpreted manually to confirmor cancel MASCOT suggestions.

2.5 1-D Western blot

Protein extracts were processed, electrophoresed (30 mg pro-teins/lane) on 10-lane 1-D 10% polyacrylamide gel under re-ducing conditions and transferred to NC. Membranes wereblocked in TBS 1 0.1% Tween 20 (TBST) 1 5% nonfat drymilk for 1 h at room temperature. Primary antibodies werediluted in TBST 1 2.5% nonfat dry milk as follows: (i) b-actin 1:5000 (mouse monoclonal AC-15, Sigma, St. Louis,MO); (ii) calreticulin 1:2000 (rabbit polyclonal, Sigma);(iii) enolase 1:30 000 (rabbit polyclonal, ab49343, Abcam,Cambridge, UK). Membranes were incubated with primaryantibodies at room temperature for 60 min. The followingsecondary antibodies were used after washing membranesthree times in TBST: HRP-conjugated sheep anti-mouse Igantibody 1:5000 (GE Healthcare) or donkey anti-rabbit IgG1:20 000 (ab6802, Abcam). Membranes were washed threetimes in TBST and Western blots were visualized using the

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Proteomics 2008, 8, 66–82 Cell Biology 69

ECL plus detection system (GE Healthcare) according tomanufacturer’s protocols. Densitometry analysis of the pro-tein bands was performed using the ImageQuant TL v2005software (GE Healthcare).

2.6 Flow cytometry (FACS)

Cells grown in 25 cm2 flask until confluence were culturedfor further 48 h in DMEM 6 10% FBS.

2.6.1 F-actin staining

Trypsinized cells were centrifuged for 10 min at 10006g,washed in PBS, suspended in 1 mL of 3% paraformaldehydein PBS for 10 min at 47C and centrifuged again for 5 min.Samples were permeabilized by addition of Triton X-100 for10 min at 47C. After a rapid centrifugation, cells were incu-bated for 30 min at 47C with FITC-labelled phalloidin. Afterwashing with PBS, pellets were resuspended in 500 mL ofPBS.

2.6.2 Annexin A2 immunostaining

Cells were trypsinized, centrifuged, fixed in 4% form-aldehyde in PBS for 10 min at room temperature and per-meabilized in 0.05% Tween 20 in PBS for 15 min at roomtemperature. Cells were washed with PBS and incubatedwith primary rabbit anti-annexin A2 antibodies (1:40, Gen-Way Biotech, San Diego, CA) for 1 h at room temperature.After washing with PBS, secondary tetramethyl rhodamineiso-thiocyanate (TRITC)-conjugate goat anti-rabbit IgG anti-bodies (1:200, Sigma) were added for 1 h at room tempera-ture. Cells were carefully washed in PBS and resuspended in500 mL of PBS. Negative controls were established by usingonly secondary antibodies.

2.6.3 Tubulin immunostaining

Cells were trypsinized, centrifuged and incubated in per-meabilization solution (0.5% Tween 20 in PBS) for 10 min atroom temperature. Cells were washed with PBS, exposed for30 min to a blocking solution (1% BSA in PBS) and thenincubated for 1 h at room temperature with primary rabbitanti-tubulin antibodies (1:80, Sigma) in PBS plus 1% BSA.After washing with PBS, secondary TRITC conjugate goatanti-rabbit IgG antibodies (1:200, Sigma) were added for 1 hat room temperature. Cells were carefully washed in PBS andresuspended in 500 mL of PBS. Negative controls wereestablished by using only secondary antibodies.

2.6.4 ROS production

Intracellular levels of the ROS were estimated by flow cyto-metry using the dihydroethidium (DH2) probe (MolecularProbes, Eugene, OR) [12]. Fibroblasts were treated with 1 mM

DH2 for 60 min at 377C, trypsinized and collected in 500 mLof PBS.

For each measurement (F-actin, annexin A2, tubulinand ROS), fibroblasts, once resuspended in PBS, weretransferred to polystyrene tubes and analysed on anEPICS XL flow cytometer (Coulter). Debris and dead cellswere excluded by forward and side scatter gating. Tenthousand events were collected and evaluated for eachcell type using WINMDI 2.8 program. Experiments wereperformed in duplicate and repeated on different cellstrains.

2.7 Fastin assay for elastin

Cells were plated in 35 mm diameter dishes at a density of1.56105 and cultured in DMEM containing 10% of FBSup to confluence. Thereafter, fibroblasts were grown forfurther 48 h in DMEM 6 10% FBS. Cells were trypsinizedwith trypsin-EDTA and centrifuged at 1500 rpm for 5 min.Pellets were resuspended in 0.25 M oxalic acid for 1 h at1007C and then centrifuged at 3000 rpm for 10 min.Supernatants were collected and treated with the FastinElastin Assay kit (Biocolor, Newtownabbey, UK), as recom-mended by the manufacturer. Recovered dye-bound elastinfrom each sample/standard was read in a 96-well plate at513 nm. All measurements were performed in quad-ruplicate.

2.8 L-lactate production

Cells were harvested, counted and centrifuged at 15006g for10 min. Cell pellets were washed in PBS, resuspended inPBS, sonicated and deproteinized by adding an equal volumeof ice-cold 1 M perchloric acid with mixing. Samples werecentrifuged at 15006g for 10 min. Supernatants, after neu-tralization with 1 M KOH, were assayed using a kit fromMegazyme (Wicklow, Ireland). The amount of L-lactate pro-duced by cells was expressed as microgram of lactate/106

cells.

3 Results

3.1 Cell viability and cell morphology

Serum withdrawal did not significantly modify the numberof dermal fibroblasts in culture (data not shown). At themorphological level, after 48 h of treatment, serum-starvedfibroblasts (Fig. 1B), compared to control cells (Fig. 1A),appeared moderately more elongated. No significant evi-dence of cell necrosis or cell apoptosis was observed at theinverted light microscope. Moreover, at the end of treatment,fibroblasts were stained with propidium iodide and the per-centage of dead cells was similar in control (8%) and inserum starved (10%) fibroblasts.

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70 F. Boraldi et al. Proteomics 2008, 8, 66–82

Figure 1. Light microscopy of human dermal fibroblasts culturedfor 48 h after confluence (A) in the presence and (B) in theabsence of 10% FBS. Serum starved cells appear more elongatedcompared to controls. Original magnification: 406.

3.2 ROS production

The intracellular content of ROS, evaluated by the binding ofthe DH2 probe to the anion superoxide, was significantlyincreased by serum withdrawal already after 3 h of serumdeprivation. Values remained similarly increased also at 6, 24and 48 h (Fig. 2).

Figure 2. ROS accumulation in human dermal fibroblasts wasevaluated by flow cytometry using the DH2 probe. Serum depri-vation significantly increases the amount of ROS. A representa-tive experiment, based on the analysis of 10 000 cells, is reportedin (A) Histograms represent the mean values of three experi-ments done in duplicate using all cell lines (B) Data are expressedas fluorescence percentage variation 6 SD in fibroblastsdeprived of serum for 48 h after confluence, compared to cellsgrown in the presence of serum (*p,0.05 –FBS vs. 1FBS).

3.3 Analysis of the protein profile

The total protein content of the cellular monolayers did notreveal any significant difference between the two experi-mental conditions, as evaluated by the Bradford assay. Simi-larly, the number of proteins that were separated by 2-DE wasapproximately of 1800 from each primary cell culture, inde-pendently from the experimental conditions.

In order to exclude the influence of possible high intra-sample variability, the CV (SD normalized spot volumedivided by mean) was evaluated for each cell line from tripli-cate parallel preparations.

As reported in the literature [19], we considered for fur-ther analysis spots with CV values lower than 30%. Con-sistently with the observation that the great majority of pro-teins gave reproducible results in terms of sample prepara-tion, extraction procedures and 2-DE, CV values higher than30% were only obtained for very faint spots and for spotslocated close to the gel edges.

Serum withdrawal determined significant changes in theexpression of 41 spots, as indicated by labels on the repre-sentative gels obtained from confluent fibroblasts grown inthe presence (A) or absence (B) of serum (Fig. 3).

In particular, 11 proteins appeared significantly moreexpressed upon serum deprivation, whereas 30 proteinswere significantly decreased.

Out of the 41 differentially expressed proteins in thewhole cell lysate, we have identified 31 proteins by MS (Table1), the remaining proteins were either in insufficientamounts to be analysed by MS or MS/MS or the MS-com-patible staining procedure failed to reveal them (see alsoTable S1 of Supporting Information).

Only in one case, MS revealed the presence of two dif-ferent proteins in the same spot (#30, Table 1): namelyphosphoglycerate mutase 1 (PGAM1) and peptidyl-prolylcis–trans isomerase B.

It has to be mentioned that, for some proteins, we iden-tified spots that did not match with the predicted Mr or pI,indicating once more the presence of different isoformsand/or fragments as already shown by other Authors [20].

Due to the very low molecular weight and the position ofidentified peptides within the whole protein length (see alsoFigure S1 of Supporting Information), we may suggest that,among those spots that were reproducibly and significantlydifferentially expressed, the following can be protein frag-ments: heat shock cognate 71 kDa protein (#11, Table 1),mannose 6 phosphate receptor binding protein 1 (#16, Table1), glyceraldehyde 3 phosphate dehydrogenase (G3P) (#18,#23, Table 1), T-complex protein 1, subunit eta (#25, Table 1),fructose-bisphosphate aldolase A (#27, Table 1), elongationfactor 1-a1 (#31, Table 1).

In the case of two differentially expressed actin isoformsthat we have identified, the recognized sequences that werespread through the whole molecule and the experimentalmolecular weight, lower than expected, could indicate thepresence of two short forms of the protein [21].

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Proteomics 2008, 8, 66–82 Cell Biology 71

Figure 3. Representative silver-stained 2-D electropherograms of the cell layer of human dermal fibroblasts cultured (A) in the presence of10% FBS or (B) in the absence of serum for 48 h. Identified differentially expressed proteins are indicated by the symbol name. Unidentifiedproteins are marked with open circles or with arrows when upregulated or downregulated after serum withdrawal, respectively.

Protein distribution into functional categories indicatesthat the majority of differentially expressed proteins thatwere identified by MS belong to cell metabolism, cellularcytoskeleton and ER stress response.

In particular, we have demonstrated that serum depriva-tion caused a significant reduction of several proteinsinvolved in the glycolytic pathway (Fig. 4A). Among all dif-ferent glycolytic enzymes we have selected enolase 1 to bevalidated by Western blot (Fig. 4B). Moreover, since Westernblot does not provide information on the real enzymatic ac-tivity, we performed a functional validation, by measuringthe in vitro formation of L-lactate (Fig. 4C), the ultimateproduct of the glycolytic pathway. All data are consistent withthe downregulation of glycolysis upon serum starvation.

Furthermore, as far as the cytoskeletal proteins, by pro-teome analysis we observed a significant different expressionof two isoforms of b-actin, one being upregulated (#12, Table1) and the other downregulated (#8, Table 1). Validation ofthese changes by Western blot, using anti-b-actin antibodies,revealed that serum starvation induced a global significantupregulation of b-actin (Fig. 5A). Moreover, staining offibroblasts with FITC labelled phalloidin demonstrated thatserum withdrawal was also associated to a significant upreg-ulation of F-actin, i.e. the actin cytoskeletal component orga-nized into filamentous structures (Fig. 6A). Similarly toactin, also tubulin was upregulated by serum deprivation indermal fibroblasts, as demonstrated by 2-DE (Table 1) and byFACS analysis, using a polyclonal antibody recognizing all

tubulin isoforms (Fig. 6B). Changes in the expression andorganization of cytoskeletal protein were associated to dif-ferences in the expression of cytoskeletal related proteins ascofilin and transgelin (Table 1).

Moreover, present data indicate that serum withdrawal,as a stress condition, may upregulate markers of ER-stressresponse, as indicated by the increased expression of calreti-culin revealed by 2-DE (Table 1) and by Western blot (Fig. 5B).Interestingly, there was also a dramatic downregulation ofannexin A2 (Table 1) that was also validated by cytofluori-metric analyses (Fig. 6C).

Due to the difficulties in evaluating matrix proteins by2-DE, the deposition of elastin was assessed in vitro by a col-orimetric assay. Data demonstrated that, upon serum with-drawal, there was a significant downregulation of elastinaccumulation in the cellular monolayer (Fig. 7).

4 Discussion

The importance of serum factors for cell growth and cellmaintenance of in vitro proliferating cells is well known [22],as well as the influence of serum withdrawal on the cell cycle[2, 3]. In contrast, few data are available on the effect ofserum deprivation on quiescent cells in primary cell culture.Since, cells in vitro may undergo serum withdrawal due tothe possible interference of serum proteins with technicalprocedures, and mesenchymal cells, in vivo, may experience

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72 F. Boraldi et al. Proteomics 2008, 8, 66–82

Table 1. Identified differentially expressed proteins in quiescent human dermal fibroblasts upon serum withdrawal

Spotno.

Protein nameaccession no.

Symbol MW/pIa) Average fold-change 6 SEb)

Identification methodc)

1 Far upstream element-binding protein1

Q96AE4

FUBP1 67.4/7.1 24 6 1.1 MS/MS – 8%K.IQIAPDSGGLPER.S (Ions score 38) (21)R.IGGNEGIDVPIPR.F (Ions score 24) (21)R.FAVGIVIGR.N (Ions score 24) (21)R.IAQITGPPDR.C (Ions score 25) (21)R.GTPQQIDYAR.Q (Ions score 24) (21)

2 CalreticulinP27797

CALR 48.1/4.2 14.2 6 1.1 MS/MS – 19%R.FYALSASFEPFSNK.G (Ions score 63) (21)K.HEQNIDCGGGYVK.L (Ions score 66) (21)K.IDNSQVESGSLEDDWDFLPPKK.I

(Ions score 36) (31)K.IKDPDASKPEDWDER.A (Ions score 34) (31)R.QIDNPDYK.G (Ions score 23) (21)

3 a-EnolaseP06733

ENOA 47.0/6.9 22.2 6 0.5 MS/MS – 56%R.GNPTVEVDLFTSK.G (Ions score 27) (21)R.AAVPSGASTGIYEALELR.D (Ions score 43) (21)K.LNVTEQEKIDK.L (Ions score 33) (21)K.DATNVGDEGGFAPNILENK.E (Ions score 33) (21)K.AGYTDKVVIGMDVAASEFFR.S

(Ions score 20) (31)K.YDLDFKSPDDPSR.Y (Ions score 20) (31)R.YISPDQLADLYK.S (Ions score 36) (21)K.SFIKDYPVVSIEDPFDQDDWGAWQK.F

(Ions score 26) (31)K.FTASAGIQVVGDDLTVTNPKR.I

(Ions score 41) (31)K.VNQIGSVTESLQAC#K.L (Ions score 28) (21)K.LAQANGWGVMVSHR.S (Ions score 55) (31)K.IGAEVYHNLK.N (Ions score 35) (21)R.LMIEMDGTENK.S (Ions score 28) (21)R.SGETEDTFIADLVVGLCTGQIK.L(Ions score 32) (21)

4 Phosphoglyceratekinase 1

P00558

PGK1 44.4/8.3 22.3 6 0.6 MS/MS – 7%K.ACANPAAGSVILLENLR.F (Ions score 20) (21)K.LGDVYVNDAFGTAHR.A (Ions score 37) (21)

5 Fructose-bisphosphatealdolase A

P04075

ALDOA 39.2/8.3 24.4 6 1.2 MS/MS – 9%K.ELSDIAHR.I (Ions score 40) (21)R.LQSIGTENTEENRR.F (Ions score 26) (31)R.ALANSLACQGK.Y (Ions score 30) (21)

6 Tubulin a-1C chain/Tubulin a-1A chainQ9BQE3/Q71U36

TBA1C/TBA1A

49.8/4.9 14.1 6 0.6 MS/MS – 19%R.AVFVDLEPTVIDEVR.T (Ions score 84) (21)R.GHYTIGKEIIDLVLDR.I (Ions score 38) (31)K.EIIDLVLDR.I (Ions score 48) (21)R.LISQIVSSITASLR.F (Ions score 67) (21)R.FDGALNVDLTEFQTNLVPYPR.I

(Ions score 24) (21)R.IHFPLATYAPVISAEK.A (Ions score 45) (31)

7 Annexin A2P07355

ANXA2 38.4/7.5 211.2 6 2.3 MS/MS – 13%K.GVDEVTIVNILTNR.S (Ions score 29) (21)K.TPAQYDASELK.A (Ions score 31) (21)R.TNQELQEINR.V (Ions score 32) (21)R.DLYDAGVKR.K (Ions score 21) (21)

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Proteomics 2008, 8, 66–82 Cell Biology 73

Table 1. Continued

Spotno.

Protein nameaccession no.

Symbol MW/pIa) Average fold-change 6 SEb)

Identification methodc)

8 Actin 1P60709

ACTB 41.7/5.2 22.8 6 0.8 MS/MS – 45%K.DSYVGDEAQSK.R (Ions score 41) (31)R.VAPEEHPVLLTEAPLNPK.A

(Ions score 31) (21)R.GYSFTTTAER.E (Ions score 22) (21)K.LC#YVALDFEQEMATAASSSSLEK.S(Ions score 23) (31)K.SYELPDGQVITIGNER.F (Ions score 38) (21)K.DLYANTVLSGGTTMYPGIADR.M

(Ions score 41) (21)K.EITALAPSTMK.I (Ions score 31) (21)K.IKIIAPPER.K (Ions score 22) (21)K.QEYDESGPSIVHR.K (Ions score 35) (31)

9 Elongation factor 1 a 1P68104

EF1A1 50.1/9.1 23.8 6 0.5 MS/MS – 22%K.YYVTIIDAPGHR.D (Ions score 65) (21)R.LPLQDVYK.I (Ions score 29) (21)K.IGGIGTVPVGR.V (Ions score 44) (21)K.SGDAAIVDMVPGKPMCVESFSDYPPLGR.F

(Ions score 28) (21)R.QTVAVGVIK.A (Ions score 26) (21)

10 Glyceraldehyde-3-phosphatedehydrogenase

P04406

G3P 35.9/8.5 24.1 6 1.3 MS/MS – 24%K.LVINGNPITIFQER.D (Ions score 42) (21)K.IISNASCTTNCLAPLAK.V (Ions score 105) (21)K.VIPELNGK.L (Ions score 37) (21)K.LTGMAFRVPTANVSVVDLTCR.L

(Ions score 48) (21)R.VPTANVSVVDLTCR.L (Ions score 52) (21)R.LEKPAKYDDIK.K (Ions score 20) (21)R.VVDLMAHMASKE.- (Ions score 18) (21)

11 Heat shock cognate71 kDa protein

P11142

HSP7C 70.8/5.3 23.5 6 0.4 MS/MS – 25%K.VEIIANDQGNR.T (Ions score 40) (21)R.TTPSYVAFTDTER.L (Ions score 87) (21)K.NQVAMNPTNTVFDAK.R (Ions score 79) (21)R.RFDDAVVQSDMK.H (Ions score 18) (21)K.VQVEYKGETK.S (Ions score 42) (21)K.SFYPEEVSSMVLTK.M (Ions score 65) (21)K.EIAEAYLGK.T (Ions score 50) (21)K.TVTNAVVTVPAYFNDSQR.Q

(Ions score 53) (21)K.DAGTIAGLNVLR.I (Ions score 55) (21)R.IINEPTAAAIAYGLDKK.V (Ions score 57) (21)K.STAGDTHLGGEDFDNR.M

(Ions score 48) (21)R.MVNHFIAEFK.R (Ions score 18) (21)

12 Actin 1P60709

ACTB 41.7/5.2 12.1 6 0.4 MS/MS – 36%R.TTGIVMDSGDGVTHTVPIYEGYALPHAILR.L

(Ions score 36) (21)R.GYSFTTTAER.E (Ions score 34) (21)K.SYELPDGQVITIGNER.F (Ions score 43) (21)K.DLYANTVLSGGTTMYPGIADR.M

(Ions score 46) (21)K.EITALAPSTMK.K (Ions score 33) (21)K.IKIIAPPER.K (Ions score 26) (21)K.QEYDESGPSIVHR.K (Ions score 39) (21)

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74 F. Boraldi et al. Proteomics 2008, 8, 66–82

Table 1. Continued

Spotno.

Protein nameaccession no.

Symbol MW/pIa) Average fold-change 6 SEb)

Identification methodc)

13 Tubulin b chainP07437

TBB5 49.6/4.7 13.3 6 0.7 MS/MS – 26%-MREIVHIQAGQCGNQIGAK.F (Ions score 53) (31)R.EIVHIQAGQCGNQIGAK.F (Ions score 39) (31)R.ISVYYNEATGGK.Y (Ions score 70) (21)R.AILVDLEPGTMDSVR.S (Ions score 74) (21)K.GHYTEGAELVDSVLDVVRK.E (Ions score 79) (21)R.KEAESCDCLQGFQLTHSLGGGTGSGMGTLLISK.I

(Ions score 34) (41)K.IREEYPDR.I (Ions score 31) (21)R.IM*NTFSVVPSPK.V (Ions score 69) (21)

14 Triosephosphateisomerase

P60174

TPIS 26.5/6.5 23.9 6 0.8 MS/MS – 77%R.KQSLGELIGTLNAAK.V (Ions score 35) (31)K.QSLGELIGTLNAAK.V (Ions score 69) (21)K.VPADTEVVCAPPTAYIDFAR.Q (Ions score 23) (21)K.IAVAAQNCYK.V (Ions score 59) (21)K.VTNGAFTGEISPGMIK.D (Ions score 47) (21)R.HVFGESDELIGQK.V (Ions score 37) (21)K.VAHALAEGLGVIACIGEK.L (Ions score 51) (31)K.VVLAYEPVWAIGTGK.T (Ions score 66) (21)K.TATPQQAQEVHEKLR.G (Ions score 33) (31)K.SNVSDAVAQSTR.I (Ions score 74) (21)R.IIYGGSVTGATCK.E (Ions score 86) (21)K.ELASQPDVDGFLVGGASLKPEFVDIINAK.Q

(Ions score 24) (31)

15 TransgelinQ01995

TAGL 22.4/8.8 23.7 6 1 MS/MS – 77%K.YDEELEER.L (Ions score 38) (21)R.LVEWIIVQCGPDVGRPDR.G (Ions score 15) (21)R.LGFQVWLK.N (Ions score 11) (21)K.NGVILSK.L (Ions score 20) (21)K.LVNSLYPDGSKPVK.V (Ions score 39) (21)K.VPENPPSMVFK.Q (Ions score 32) (21)K.QMEQVAQFLK.A (Ions score 34) (21)K.AAEDYGVIK.T (Ions score 26) (21)K.TDMFQTVDLFEGK.D (Ions score 64) (21)R.TLMALGSLAVTK.N (Ions score 67) (21)R.EFTESQLQEGK.H (Ions score 44) (21)K.HVIGLQMGSNR.G (Ions score 28) (21)R.GASQAGM*TGYGRPR.Q (Ions score 21) (31)

16 Mannose 6-phosphatereceptor bindingprotein 1

O60664

M6PBP 47.0/5.3 22.1 6 0.5 MS/MS – 7%K.LHQMWLSWNQK.Q (Ions score 31) (21)K.QLQGPEKEPPKPEQVESR.A (Ions score 37) (31)

17 P4HB protein15680282

P4HB 21.2/4.4 13.7 6 0.3 PMF – 23%(K) NFEDVAFDEK(K)(K) VHSFPTLK(F)(K) FFPASADR(T)(R) TVIDYNGER(T)(R) TLDGFKK(F)

18 Glyceraldehyde-3-phosphatedehydrogenase

P04406

G3P 35.9/8.5 22.3 6 0.4 MS/MS – 29%K.LVINGNPITIFQER.D (Ions score 33) (21)K.IKWGDAGAEYVVESTGVFTTMEK.A (Ions score 39) (21)K.AGAHLQGGAK.R (Ions score 32) (21)R.VIISAPSADAPMFVMGVNHEK.Y (Ions score 37) (31)K.VIHDNFGIVEGLM*TTVHAITATQK.T

(Ions score 26) (31)

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Proteomics 2008, 8, 66–82 Cell Biology 75

Table 1. Continued

Spotno.

Protein nameaccession no.

Symbol MW/pIa) Average fold-change 6 SEb)

Identification methodc)

19 CofilinP23528

COF1 18.3/8.2 23.5 6 1.2 MS/MS – 53%-.ASGVAVSDGVIK.V (Ions score 68) (21)K.AVLFCLSEDKK.N (Ions score 20) (21)K.NIILEEGK.E (Ions score 36) (21)K.EILVGDVGQTVDDPYATFVK.M (Ions score 31) (21)R.YALYDATYETK.E (Ions score 44) (21)K.LGGSAVISLEGKPL.- (Ions score 57) (21)

20 TUBB protein38511503

TUBB 14.4/4.2 13.3 6 0.2 MS/MS – 56%K.EVDEQMLNVQNK.N (ions score 20) (12)K.NSSYFVEWIPNNVK.T (ions score 31) (12)K.TAVCDIPPR.G (ions score 44) (12)K.MAVTFIGNSTAIQELFKR.I (ions score 58) (13)R.ISEQFTAMFR.R (ions score 57) (12)

21 Nascent polypeptide-associated complexsubunit a

Q13765

NACA 23.3/4.5 14.6 6 0.8 MS/MS – 19%K.SPASDTYIVFGEAK.I (Ions score 37) (21)K.IEDLSQQAQLAAAEK.F (Ions score 39) (21)K.DIELVMSQANVSR.A (Ions score 41) (21)

22 Peptidyl-prolyl-cis–transisomerase A

P62937

PPIA 17.8/7.8 23.5 6 1 MS/MS – 32%-VNPTVFFDIAVDGEPLGR.V (Ions score 45) (21)R.IIPGFMCQGGDFTR.H (Ions score 71) (21)K.TEWLDGKHVVFGK.V (Ions score 21) (31)

23 Glyceraldehyde-3-phosphatedehydrogenase

P04406

G3P 35.9/8.5 22.1 6 0.5 MS/MS – 21%K.LVINGNPITIFQER.D (Ions score 38) (21)K.WGDAGAEYVVESTGVFTTMEK.A (Ions score 30) (21)R.VIISAPSADAPMFVMGVNHEK.Y (Ions score 26) (31)

24 Triosephosphateisomerase

P60174

TPIS 26.5/6.5 23.5 6 0.7 MS/MS – 62%R.KQSLGELIGTLNAAK.V (Ions score 40) (31)K.VPADTEVVCAPPTAYIDFAR.Q (Ions score 42) (31)K.IAVAAQNCYK.V (Ions score 54) (21)K.VTNGAFTGEISPGMIK.D (Ions score 62) (21)K.VAHALAEGLGVIACIGEK.L (Ions score 56) (31)K.TATPQQAQEVHEKLR.G (Ions score 52) (21)K.SNVSDAVAQSTR.I (Ions score 83) (31)R.IIYGGSVTGATCK.E (Ions score 87) (21)K.ELASQPDVDGFLVGGASLKPEFVDIINAKQ.-

(Ions score 22) (31)

25 T-complex protein 1,subunit eta

Q99832

TCPH 59.3/7.5 24.8 6 1.5 MS/MS – 11%R.GMDKLIVDGR.G (Ions score 45) (21)K.ATISNDGATILK.L (Ions score 17) (21)K.LLDVVHPAAK.T (Ions score 59) (21)K.TLVDIAK.S (Ions score 36) (21)K.SQDAEVGDGTTSVTLLAAEFLK.Q (Ions score 30) (21)

26 Phosphatidylethanol-amine-bindingprotein 1

P30086

PEBP1 20.9/7.4 23.3 6 0.6 MS/MS – 60%K.WSGPLSLQEVDEQPQHPLHVTYAGAAVDELGK.V

(Ions score 31) (31)K. VLTPTQVK.N (Ions score 26) (21)K.NRPTSISWDGLDSGK.L (Ions score 46) (21)K.LYTLVLTDPDAPSR.K (Ions score 35) (21)K.GNDISSGTVLSDYVGSGPPK.G (Ions score 54) (21)R.YVWLVYEQDRPLK.C (Ions score 39) (21)

27 Fructose-bisphos-phate aldolase A

P04075

ALDOA 39.2/8.3 23.9 6 0.3 MS/MS – 6%K.GILAADESTGSIAK.R (Ions score 41) (21)R.QLLLTADDR.V (Ions score 31) (21)

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76 F. Boraldi et al. Proteomics 2008, 8, 66–82

Table 1. Continued

Spotno.

Protein nameaccession no.

Symbol MW/pIa) Average fold-change 6 SEb)

Identification methodc)

28 TransgelinQ01995

TAGL 22.5/8.8 24.3 6 1.7 MS/MS – 38%K.TDMFQTVDLFEGK.D (Ions score 22) (21)R.TLM*ALGSLAVTK.N (Ions score 43) (21)R.EFTESQLQEGK.H (Ions score 38) (21)K.HVIGLQMGSNR.G (Ions score 27) (21)R.GASQAGMTGYGRPR.Q (Ions score 31) (31)

29 Peptidyl-prolylcis–transisomerase B

P23284

PPIB 22.7/9.3 23.6 6 0.8 MS/MS – 19%K.DFMIQGGDFTR.G (Ions score 28) (21)K.VLEGMEVVR.K (Ions score 55) (21)R.DKPLKDVIIADCGK.I (Ions score 45) (31)

30 Phosphoglyceratemutase 1

P186691

Peptidyl-prolylcis–transisomerase B

P23284

PGAM1

1

PPIB

28.6/6.7

1

22.7/9.3

22.4 6 0.2 MS/MS – 15%R.FSGWYDADLSPAGHEEAK.R (Ions score 25) (31)R.HGESAWNLENR.F (Ions score 31) (21)R.HYGGLTGLNK.A (Ions score 31) (21)MS/MS – 11%K.DFMIQGGDFTR.G (Ions score 34) (21)K.DTNGSQFFITTVK.T (Ions score 40) (21)

31 Elongationfactor 1 a 1

P68104

EF1A1 50.1/9.1 22.1 6 0.6 MS/MS – 13%K.YYVTIIDAPGHR.D (Ions score 28) (21)K.NMITGTSQADC#AVLIVAAGVGEFEAGISK.N(Ions score 21) (31)

For spot #30, fold change refers only to normalised spot volume changes since it was not possible to determine the fold changes of each ofthe two proteins positively identified.a) Abbreviations: MW, theoretical molecular weight (kDa); pI, theoretical pI.b) ‘1’ indicates a significant (p,0.05) increase, whereas ‘2’ indicates a significant (p,0.05) decrease in protein expression in serum

starved fibroblasts compared to control cells.c) For MS/MS sequencing, % coverage, peptide sequence, ion score and charge state are given; For PMF, % coverage and peptide

sequence matched are given; M* – Methionine oxidation; C# – Cysteine carbamidomethylation

changes in growth factors availability and/or responsiveness,as during ageing or as a result of fibrotic processes, aim ofthe present study was to investigate the response of humandermal fibroblasts to serum withdrawal applied for 48 h aftercell confluence. We have used quiescent fibroblasts, in orderto avoid the effect of serum deprivation on the cell cycle andbecause quiescent cells are in a condition more similar to thein vivo situation. In addition, the time length has beenselected in order to assess the reaction of cells upon mod-ulation of early and late responsive genes [23, 24].

First, we have demonstrated that, in our experimentalmodel, serum deprivation does not significantly increasethe number of dead cells. Interestingly, there are data inthe literature indicating that, in different cell lines, evenfew hours of serum withdrawal cause cell death either bynecrosis or apoptosis [6, 8, 25]. In the present study, 48 hof serum deprivation do not seem to affect cell viability,as shown by cell counting, cell morphology, trypan blueand propidium iodide staining. These findings indicatethat dermal fibroblasts, in primary cell culture, can quitewell adapt themselves to changes of the environmentalconditions, at least for the time-length of these experi-ments.

Never the less, we have noticed a remarkable increase ofROS, indicating that human dermal fibroblasts are in fact inan oxidative stress condition. ROS accumulation has beenobserved also during ageing, in fibrotic processes and afterinsufficient availability of growth factors [26]. An importantsource of ROS is the glycolytic pathway [27] that represents afont of endogenous molecular toxicity since most of the gly-colytic intermediates are potentially noxious and capable tomodify (i.e. glycate) proteins and other macromolecules,producing the advanced glycosylation end-products (AGEs)that are implicated in several age-related pathologies [28].Within this context, one of the beneficial effects of dietaryrestriction on the ageing process has been attributed to itsability to reduce ROS production, by actually lowering gly-colysis [29]. Surprisingly, serum starvation is associated toincreased levels of ROS as well as to downregulation of sev-eral glycolytic enzymes, as demonstrated by the reducedproduction of lactate, the ultimate product of the glycolyticpathway. These findings suggest that insufficient availabilityof serum factors causes oxidative stress with ROS productionand/or accumulation [30] and that serum withdrawal maylower glycolysis as a consequence of oxidative stress occur-rence, as observed in other experimental models [31–34].

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Proteomics 2008, 8, 66–82 Cell Biology 77

Figure 4. Proteome analysisrevealed that several proteins,downregulated in serum-deprived cells, are involved inthe glycolytic pathway (A).Changes in protein expressionare visualized by bar chartsclose to the enzyme name: trio-sephosphate isomerase 1(TPIS1), fructose bisphosphatealdolase A (ALDOA), glycer-aldehyde 3 phosphate dehy-drogenase (G3P), phosphogly-cerate kinase 1 (PGK1), phos-phoglycerate mutase 1(PGAM1), a-enolase (ENOA).Data have been validated byWestern blot in the case ofENOA (B) and by measuring theamount of L-lactate produced bycells cultured after confluence inDMEM 6 10% FBS for 48 h (C)(*p,0.05 2FBS vs. 1FBS).

Since, reduced and/or inadequate nutrients supply mayoccur during ageing or in fibrotic processes, insufficientgrowth factors availability contribute to oxidative stress-induced cell alterations [35]. In particular, ROS play animportant role in skin ageing and photoageing, by activating,in resident fibroblasts, cytoplasmic signal transductionpathways related to growth, differentiation, senescence andconnective tissue degradation [36]. Studies, investigating thein vitro effects of ROS on the elastic component, showed thatROS accumulation was associated to a dose-related increaseof elastin at the mRNA level [37], but it has been also sug-gested that ROS, within few minutes, may degrade tropoe-lastin, being able to cleave, at relatively specific sites, elasticprotein precursor, thus favouring elastolytic changes and/orthe deposition of fragmented elastic fibres [38]. In the pres-ent study, we have shown that serum deprivation is asso-ciated to a significant reduction of elastin accumulation onthe cellular monolayer. This downregulation could be due tothe lack of serum factors positively regulating elastindeposition, however, it has to be mentioned that serumdeprivation is the method of choice when elastin synthesishas to be measured in cultured smooth muscle cells [39].Therefore, it could be hypothesized that serum deprivationcauses reduced elastin deposition, possibly through ROSproduction.

Interestingly, ROS represent important signalling mole-cules, altering for instance DNA binding and transactivation

properties of transcription factors [40], as well as actin cyto-skeleton regulation and remodelling. Superoxide, forinstance, could be transported through actin microfilamentsfrom the plasma membrane, or from the mitochondria, to-wards the nucleus [41], specifically influencing the transportinto the nucleus of transcription-related proteins [42]. Itcould be the case of the far upstream element binding pro-tein 1 [43] that was significantly reduced in fibroblasts afterserum withdrawal.

In different mammalian cells, oxidative stress has beenreported to cause cytoskeletal alterations and increasedplasma membrane peroxidation [44, 45]. In the presentstudy, we have shown that increased expression of cytoskele-tal proteins, namely actin and tubulin, and a remarkabledownregulation of annexin A2, a membrane protein thatplays a role in membrane stability, modulating lipid micro-domains and affecting transport of compounds amongmembrane compartments [46].

There are data in the literature suggesting that ageingand serum deprivation induce a decrease in the rate of DNAsynthesis, an extension of the cytoplasmic microtubularcomplex [47, 48] and a loss of F-actin fibres [8]. In the presentstudy, we have demonstrated, by proteome and FACS analy-sis, that serum starved dermal fibroblasts showed a signifi-cant upregulation of tubulin, of b-actin as well as of falloidin-stained actin molecules. These findings, compared withthose obtained by Tamm et al. [8], could be explained by a

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78 F. Boraldi et al. Proteomics 2008, 8, 66–82

Figure 5. To validate results of proteome analysis, immunoblotexperiments were performed on 1-D SDS-PAGE using antibodiesspecific for b-actin (A) and calreticulin (B). By Western blot a sig-nificant increase in the expression of b-actin and of calreticulinwas observed upon serum starvation. Data are reported as gra-phical representations of relative band intensities expressed asmean 6 SD of three experiments, done in duplicate, with all celllines cultured for 48 h after confluence in DMEM 6 10% FBS(*p,0.05 2FBS vs. 1FBS). Representative immunoblots of thetwo experimental conditions are shown in the insets.

different response of human dermal fibroblasts in primarycell culture compared to BALB/c 3T3 cells, as alreadyrevealed in the case of cell death [25]. Despite the globaldowregulation of b-actin, as demonstrated by Western blot,proteome analysis revealed that two actin isoforms were dif-ferently affected by serum deprivation, one being upregu-lated and the other downregulated. It is not yet fully estab-lished if differential expression of actin isoforms is func-tionally relevant or if they play a role in human diseases [49],but these data underline two major findings: (i) isoforms canbe differently regulated, thus having distinct biologicalfunctions and/or properties; (ii) there are conditions, i.e.serum starvation, that may actually interfere with theexpression of ‘housekeeping’ molecules as b-actin [50].

It could be suggested that changes in the expression ofactin isoforms are sustained by reduced expression of cofilinthat is known to modulate cytoskeletal dynamics [51]. Actinmonomers are polymerized into filaments under physiolog-ical conditions, but spontaneous depolymerization is tooslow to maintain the fast actin filament dynamics, therefore,actin-severing/depolymerizing proteins, like cofilin, canenhance disassembly of the actin filaments thus promotingreorganization and turnover of the actin cytoskeleton [52].

Actin assembly and disassembly in cells is a major adenosinetriphosphate (ATP)-using process and cellular actindepolymerizing factor (ADF)/cofilin activity correlates withactin-turnover rates. Cells under stress could spare ATP formaintaining essential ionic homeostasis by decreasing actinturnover. Reduced expression of cofilin, as observed in thepresent study, associated to increased ROS production andreduced glycolysis, may favour the formation and accumula-tion of actin filaments, i.e. F-actin. Furthermore, cofilinmight also be a nuclear chaperone for actin, possibly mod-ulating the actin binding properties to chromatin remodel-ling complexes [53].

Cytoskeletal changes are major modulators of cell shapeand integrin cell adhesion receptors for proteins of the extra-cellular matrix, thus influencing communication with growthfactor pathways [54], also at the membrane level. Interestingly,upon serum starvation, we observed a significant down-regulation of transgelin, an actin binding protein localized tothe cell membrane and the cytoplasm, which has beenrecently found to be a novel regulator of matrix metallo-pro-teinase (MMP)-9 expression [55]. Diminished transgelinexpression has been already observed in cancer [55], in age-related diseases [56–58] and after oxidative stress [56, 59] beingresponsible for elevated MMP-9 expression and increasedextracellular matrix degradation. Data from the present studyfurther support the association between increased ROS pro-duction, changes in membrane stability and cytoskeletalorganization, reduced transgelin expression, increasedmatrix degradation and diminished elastin accumulation.

Furthermore, the present study demonstrated thatserum deprivation caused a dramatic reduction of annexinA2. As other annexins, annexin A2 binds to negativelycharged phospholipids and, when is relocated to the plasmamembrane, contributes to its mechanical stability and reg-ulates actin filaments turnover [60]. Consistently with pre-vious observations that annexin A2 depleted cells becameenriched in stress fibres [60], we have shown and validated,upon serum withdrawal, a decreased expression of annexinA2 and an increase of F-actin. Moreover, reduced expressionof annexin A2, as already observed in fibroblasts isolatedfrom old individuals [61], could be at the basis of altered dis-tribution of lipid microdomains on the plasma membrane,causing a weakness of membrane resistance and/or alteredsecretion of molecules from cells into the extracellular space[62].

It has been already reported that serum deprivationaffects the turnover of nascent or newly synthesized proteins[63]. Present data revealed a significant decrease of elonga-tion factor 1a. Interestingly, loss of expression of EF1a isaccompanied by the onset of senescence in human fibro-blasts [64] confirming, once more, that altered growth factorsavailability contributes to the ageing phenotype. Talapatra etal. [65] have demonstrated that elongation factor 1-a is also aselective regulator of ER stress and of growth factor with-drawal-induced apoptosis [65]. A reduced expression ofelongation factor 1, as well as of other proteins involved in

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Proteomics 2008, 8, 66–82 Cell Biology 79

Figure 6. Flow cytometry was applied to validate changes in the expression of (A) F-actin, (B) tubulin and (C) annexin A2. Cells were incu-bated with phalloidin, anti-tubulin and anti-annexin A2 antibodies, respectively. Data indicate that serum withdrawal is associated to asignificant increase of (A) F-actin and (B) tubulin and (C) a significant downregulation of annexin A2. Representative experiments based onthe analysis of 10 000 cells are reported in upper panels, whereas histograms representing the mean values expressed in % 6 SD of twoexperiments done in duplicate with all cell lines are shown in the lower panels (*p,0.05 2FBS vs. 1FBS).

Figure 7. Synthesis and deposition of elastin on the cell mono-layer measured by a colorimetric test. Serum deprivation for 48 hafter confluence significantly decreased the amount of elastinproduced by dermal fibroblasts (**p,0.01, 2FBS vs. 1FBS).

protein folding, such as peptidyl-prolyl cis–trans isomerase,may increase the frequency of proteins that are no longerable to adopt their native conformation. Accumulation ofunfolded proteins can initiate an ER specific unfolded pro-tein response ultimately leading to apoptosis [66] or, beforereaching an irreversible point of no return, can be respon-sible for changes in proteolytic activities of proteasomes andlysosomes [67].

Interestingly, alterations of intracellular protein recogni-tion, assembly and turnover have been related to the occur-rence of tissue abnormal protein accumulation in severaldiseases, as in diabetes [68]. Studies on dermal fibroblastshave shown increased levels of heat shock proteins (HSPs) aswell as a downregulation of annexin A2 and of mannose-6-phosphate receptor binding protein, indicating a possiblepathogenetic role of these chaperone-like proteins in diabetic

complications [68]. It has to be mentioned that, similarly todata from Tessari et al. [68], in the present study we havenoticed that serum starvation was associated to a down-regulation of annexin A2 and of mannose-6-phosphatereceptor binding protein, a membrane glycoprotein thatplays a major role in the trafficking of lysosomal enzymesfrom the trans-Golgi network to the endosomal–lysosomalsystem [69]. In contrast, we failed to demonstrate changes inthe expression of HSPs. However, it is worth mentioningthat, in the present investigation, we observed a significantupregulation of calreticulin, a molecular chaperone beingpart of the primary quality control of protein folding withinthe ER, helping to regulate protein processing, folding ofmembrane and secretory proteins in the ER, calcium home-ostasis and ER-associated apoptotic pathways [70, 71]. Besideits role as a chaperone, calreticulin can be also considered astress response protein [72]. Present findings indicate thatserum withdrawal represents a stress condition, as shown bythe increased production of ROS, however, classical stress-related proteins (i.e. HSP 27, 70, 90) did not appear to beupregulated in serum-deprived fibroblasts. As calreticulinexpression is induced in response to perturbations of normalER function [73], it could be suggested that serum-starvationrepresents a stress condition that specifically targets the ER,possibly through ROS production and/or accumulation, as itoccurs also in the ageing process [74]. Moreover, it has beenrecently hypothesized that calreticulin can modulate cellapoptosis [75], therefore, its increased expression couldrepresent a first sign of the apoptotic pathway activation thatmay reach completion after longer serum starvation.

In conclusion, we have presented evidence that, upon48 h of serum starvation, confluent human dermal fibro-blasts can adapt themselves to environmental changes,

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80 F. Boraldi et al. Proteomics 2008, 8, 66–82

without altering cell viability, although it can be observed asignificant increase in ROS production, and changes in theprotein profile, being membrane stability, cytoskeleton, gly-colysis and ER stress response the major pathways involved.These changes should be taken into account for a betterevaluation of cell phenotype and behaviour, when serumwithdrawal is applied to cell cultures, even if cells are alreadyat confluence. Moreover, dermal fibroblasts represent a goodexperimental model allowing to investigate the response ofquiescent cells to serum deprivation and to better under-stand the diminished responsiveness to serum factors, that,for instance, occurs during senescence [76]. Finally, datafrom the present study highlight the pathways that may pre-lude to irreversible changes and to cell death after a pro-longed serum withdrawal.

Work supported by grant 2004059221 from MIUR and bygrant Elastage 018960 from EU. The Authors gratefullyacknowledge the invaluable technical expertise of Dr. MichelBecchi and Dr. Isabelle Zanella-Cleon from IBCP (CNRS, Lyon,France); and of Dr. Adriano Benedetti from CIGS (University ofModena and Reggio Emilia, Modena, Italy).

The authors have declared no conflict of interest.

5 References

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[2] Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G. et al., Thetranscriptional program in the response of human fibroblaststo serum. Science 1999, 283, 83–87.

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