Proteome Analysis of Rat Bone Marrow ... - ACS Publications

Proteome Analysis of Rat Bone Marrow Mesenchymal Stem Cell Subcultures Betu ¨l C ¸ elebi and Y. Murat Elc¸in* Ankara University, Faculty of Science and Biotechnology Institute, AU-TEBNL, Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara, Turkey Received August 2, 2008

Bone marrow mesenchymal stem cells (BM-MSCs) have the capacity for renewal and the potential to differentiate in culture into several cell types including osteoblasts, chondrocytes, adipocytes, astrocytes, myocytes, oligodendrocytes, and neurons. Albeit previous reports demonstrated some of the effects of extensive subculturing on MSCs, the results still remain controversial. The aim of this study was to generate proteome maps of undifferentiated rat BM-MSCs, and identify differentially regulated proteins during serial subcultures within the first 10 passages. Proteins extracted from Wistar rat BM-MSCs were separated by two-dimensional gel electrophoresis and about 1000 protein spots were detected using the Sypro Ruby dye. Among them, 106 selected spots were digested with trypsin for mass spectrometry analysis, and 31 proteins were successfully identified by MALDI-TOF-MS. Here, 18 differentially expressed proteins are reported for the first time; these proteins are classified into 8 functional categories: metabolism, signal transduction, cell adhesion and growth, cytoskeleton, cell cycle, protein degradation, cell-cell interaction, and ion transfer. These proteins are reported to be involved in cell proliferation and differentiation through different signaling pathways. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of proliferation potential under serial subculturing. The molecular mechanisms of rat BM-MSCs are discussed at the proteome level. Keywords: Mesenchymal stem cells • Multipotent stromal cells • Bone marrow • Rat • Proteome analysis • MALDI-TOF-MS • Self-Renewal • Subculture • Serial passaging • Stem cells

Introduction Adult bone marrow (BM) contains at least two types of stem cells: hematopoietic (HSCs) and nonhematopoietic.1-4 The precursors of nonhematopoietic tissues are referred to as plastic-adherent cells or colony-forming-unit fibroblasts, since they adhere to tissue culture plastic and form fibroblast-like colonies.5,6 The nonhematopoietic subset of stem cells are also referred to as mesenchymal progenitor cells because of their capacity to differentiate into a variety of nonhematopoietic tissues.7-9 Apart from the two characteristics, they have been defined as multipotent stromal cells (or mesenchymal stem cells; MSCs), since they appear to arise from supporting structures found in the marrow and can act as substantial layers for the growth of HSCs in culture.10-12 Hence, BM-MSCs have been assayed for their potential use in cell-based regenerative and gene therapies, for a number of human diseases.13-15 The first successful isolation of fibroblast-like colonies of the bone marrow was described by Friedenstein et al.16 Enhanced expansion of both human (h) and rat (r) BM-MSCs by lowdensity plating was shown by Javazon et al.17 Rat MSCs are more sensitive to plating density, expand rapidly after low* Corresponding Author: Prof. Dr. Y. Murat Elc¸in. Mailing address, Ankara University, Faculty of Science, Department of Chemistry, Biochemistry Division, Degol Caddesi, Tandogan, 06100 Ankara, Turkey; phone, +90(312) 212-6720; fax, +90(312) 223-2395; e-mail, [email protected].

2164 Journal of Proteome Research 2009, 8, 2164–2172 Published on Web 03/26/2009

density plating (reach confluence usually within 4 weeks of culture) but require frequent medium changes.17 Besides, hMSC cultures plated at low density discontinue to expand after 2 to 3 weeks, and the type of culture medium used has a drastic effect on cell expansion behavior; mature hMSCs in cultures can generate cells which are able to differentiate and form single-cell-derived colonies.17 While the existence of mesenchymal stem cells is incontrovertible, many questions remain regarding their self-renewal. Paramount to genomic and transcriptomic analyses, proteomic characterization of stem or progenitor cells would be an ideal way to observe alterations in the developmental or metabolic state of cells or tissues of any kind. The most widely used method for characterizing complex protein mixtures, prior to mass spectrometry (MS) analysis, is the two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-DE) followed by the enzymatic digestion of the separated protein spots.18 The first proteomic analysis of MSCs was performed by Prockop’s group.1 With the use of 2-DE, they compared the properties of hBM mature MSCs with that of a MSC subpopulation called the rapidly self-renewing (RS) cells, which demonstrated greater potential of proliferation and differentiation. Over 30 proteins were identified in fractions enriched for RS cells which were absent in fractions enriched for mature MSCs. 10.1021/pr800590g CCC: $40.75

 2009 American Chemical Society

Proteomics of Rat MSC Subcultures Contrarily, over 10 proteins were identified in fractions enriched for mature MSCs that were not detected in fractions enriched for RS cells.1 Wang et al. investigated the effects of transforming growth factor-β (TGF-β) on the hMSC proteome and identified about 30 altered proteins.19 Ye et al. investigated the effects of 5-azacytidine (5-aza) on rat bone marrow MSCs; they identified 34 proteins with MALDI-TOF-MS analysis and 9 proteins showed distinct regulation in MSCs after 5-aza treatment.20 Sun et al. characterized the proliferation and osteogenic potential of hMSCs during serial subcultures; they identified proteins that were differentially regulated in hMSCs during passages and osteogenic differentiation using proteome analysis.21 Zhang et al. generated proteome maps of undifferentiated and osteogenically induced hMSCs on day 3 and day 7 of culture.22 The osteogenic differentiation of hMSCs has also been studied by Salasznyk et al. through the comparison of the proteomes of undifferentiated MSCs with MSCs differentiated into osteoblasts and with physiologically differentiated human osteocytes (hOSTs). Their study has demonstrated that MSCs which differentiated into osteoblasts shared 64% overlap in spot pattern with that of undifferentiated MSCs and 78% overlap with physiologically differentiated hOSTs, thus, representing a shift to a committed osteogenic phenotype.23 Nengseng et al. identified 37 proteins involved in different cell processes in Chinese miniswine BM-MSCs at passages three to five.24 Some groups have focused on MSCs isolated from umbilical cord blood (UCB) which represents an alternative to BM-MSCs. Feldmann et al. identified 205 proteins expressed by the UCBMSCs.25 It is possible to state that studies on the proteomic characterization of MSCs are still limited; thus, investigations devoted to systemic proteomic analysis are needed to further understanding of stem cell biology. Here, we have produced and compared the 2-D gel proteome maps of rat BM-MSCs sampled from subcultures between passages 0 and 10, identified detectable proteins by using the MALDI-TOF-MS, and characterized differentially expressed ones. Spectrums were analyzed by using the Protein Lynx Global Server (PLGS) system and the proteins were identified using the Swiss-Prot database.

Materials and Methods Rat Mesenchymal Stem Cell Isolation and Culture. Bone marrow MSCs were isolated from 8-10 weeks old male Wistar rats weighing 150-200 g (n ) 12). All protocols involving animals were conducted according to the standards of international regulations. Rats were anesthetized by an injection of avertin. Under anesthesia, BM was aspirated from the tibias and femurs into Hank’s Buffered Salt Solution (HBSS; Biochrom AG, Berlin, Germany) containing 2% heparin (100 U/mL), and the mononuclear cells were obtained using the density gradient solution (Polymorphoprep; Axis-Shield, Norton, MA) by centrifugation. Cells were plated at a density of ∼2000 cells/cm2 in two parallel T-75 cell culture plates (one for proteomic analysis and the other for subculturing), and cultured in Minimum Essential Medium (R-MEM, Gibco, Paisley, U.K.) supplemented with 20% fetal bovine serum (FBS; Hyclone, Thermo, Rockford, IL), 2 mM L-glutamine, 100 U/mL penicilin, and 100 µL/mL streptomycin (all from Sigma, St. Louis, MO) at 37 °C humidified atmosphere with 5% CO2 for 3 days before the first medium change. Medium was then changed twice a week. At ∼80-85% confluence, cells were trypsinized with 0.25% trypsin-EDTA solution (Sigma), and cell viability was checked by trypan blue dye exclusion test and transferred to

research articles fresh flasks up to 10 passages. The phase contrast images of rat MSC morphology were regularly collected using a Nikon TS 100 inverted microscope with 20× objective and a Nikon digital CCD camera. MSC subcultures were collected as cell samples for proteomic analysis. MTT Assay. The mitochondrial dehydrogenase activity of all subcultures was evaluated by an MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide]-based assay (Sigma). Briefly, 30 mL of fresh MTT solution (5 mg/mL) was added to cultures inside 270 mL of R-MEM (without serum), and were incubated at 37 °C and 5% CO2 for 4 h. The MTT taken up by the cells and reduced in the mitochondria to the insoluble dark blue formazan product was quantified by reading the optical density values at 550 nm using a spectrophotometer. Preparation of Protein Samples. For proteomic analysis, passages at confluence were washed three times with ice-cold phosphate-buffered saline (PBS); then, the cells were removed from the cultures using a cell scraper (Orange Scientific, Brainel’Alleud, Belgium) and dissolved in lysis buffer containing 0.5% Triton-X 100 (Sigma), 50 mM Tris-HCl (pH 7.5-8), 10 mM tris(2-carboxyethyl)phosphine (TCEP; Pierce, Thermo), and complete protease inhibitor (Roche, Mannheim, Germany), and finally sonicated for 30 s. The samples were then centrifuged at 18 000g for 10 min to remove any insoluble cell debris. Protein amounts of the cell lysates were determined by the Bradford method (Bio-Rad, Hercules, CA); the remaining protein solutions were stored at -80 °C for further proteomic analysis. Protein Separation and Image Analysis. For trichloroacetic acid (TCA) precipitation, 100 µL of 10% TCA was mixed with 1000 µg of soluble protein, followed by incubation on ice for 15 min, then centrifuged at 18 000g for 10 min, and washed two times with 25% acetone by a dilution of 1:1 (w/w). After centrifugation for 5 min at 18 000g, the pellet was ready for the next step. 2-DE experiments were carried out according to the manufacturer’s instructions (Bio-Rad). For the first dimension, 300 µL of rehydration buffer consisting of 7 M urea (BioRad), 2 M thiourea (Sigma), 1% dithiothreitol (DTT; Fermentas, Glen Burnie, MD), 4% CHAPS (Sigma), and 0.5% (v/v) IPG buffer (Fluka, Buchs, Germany) containing 500 µg of proteins was subjected to isoelectric focusing (IEF) on an 17 cm long IPG strip, pH 3-10 linear gradient (Bio-Rad) at 20 °C.22 IPG strips were actively rehydrated with the sample mixture at 50 V for 15-16 h to enchance protein uptake. With Protean IEF Cell (Bio-Rad), proteins were focused using the following protocol: 250 V for 15 min, then 10 kV for 3 h at rapid ramping mode, and the final phase at 10 kV for about 6 h (until reaching 60 kVh). All IEF steps were carried out at 20 °C. Before carrying out the 2-DE, IPG gel strips were placed in an equilibration buffer consisting of 6 M urea (Bio-Rad), 30% glycerol (Bio-Rad), 2% SDS (Sigma), 50 mM Tris-HCl (pH 8.8), and 2% DTT for 15 min while being shaken. The strips were then transferred to the same equilibration buffer in which DTT was replaced with 2.5% iodoacetamide (Sigma) and shaken for a further 15 min. The strips were then transferred onto the second-dimensional 1.5 mm thick, 4% stacking, and 10% running polyacrylamide gels and sealed in place with 0.5% lowmelting point agarose (Sigma). Separation in the second dimension was carried out using Protean Plus Dodeca Cell system (20 cm × 25 cm, Bio-Rad) at 30 mA/gel until the bromophenol blue dye marker reached the bottom of the gel. The temperature of the electrophoresis system was kept at the range of 11-12 °C. The protein spots of the protein extracts Journal of Proteome Research • Vol. 8, No. 5, 2009 2165

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were visualized by Sypro Ruby dye according to the protocol described by Sigma. Spot detection and matching was automatically performed using the PDQuest 7.2 software (Bio-Rad). Protein spots at passage 0 (which were not observed in any other subculture) were selected by Match analysis; spots at passage 5 were selected according to a 2-fold up-regulation of proteins at passage 4; and spots of passage 5 were selected arbitrarily according to a 2-fold down-regulation of proteins expressed between passages 0 and 4.20,22 In-Gel Digestion of Proteins Separated by 2D-Gel Electrophoresis. Spots of interest were selected from image analysis, then were identified by mass spectrometric analysis. Each gel was cut into small pieces and placed into 96-microplate wells. After cutting the spots from the gels by Bio-Rad’s Spot-Cutter, the gel fragments were washed with double-distilled H2O. For trypsinization, gel fragments were swelled in 10 mM DTT (Fermentas) in 0.1 mM NH4CO3 (Sigma), and incubated for 30 min at 37 °C to reduce protein, and the gels were shrunk using acetonitrile (Sigma). After removing the supernatant, 55 mM iodoacetamide and 0.1 mM NH4CO3 (both from Sigma) were added. Then, the gels were washed with 0.1 mM NH4CO3 for 15 min and dried in acetonitrile. Later, the gels were rehydrated in the digestion buffer (50 mM NH4CO3 containing 12.5 ng/µL trypsin) at 4 °C for 30-45 min, and 15 µL of NH4CO3 was added onto each gel. As the last step, the gels were incubated at 37 °C for 16 h. An aqueous solution containing 50% acetonitrile and 5% formic acid (ACN, Riedel de Hae¨n, Seelze, Germany) was added to recover the peptides from the digestion mixture and sonicated for 10 min; then, the peptides were lyophilized. The digested peptides were analyzed using a matrix-assisted laser desorption/ionization time-of-flight delayed-extraction mass spectrometer (MALDI-TOF-MS) (Waters Corporation, Milford, MA). MALDI-TOF-MS. The dried extracts were reconstituted with the matrix solution composed of 10 mg R-cyano-4-hydroxycinnamic acid (recrystallized CHCA, Sigma) in 1 mL of 49.5% acetonitrile, 49.5% ethanol (Sigma), and 1% of 0.1% TFA (Sigma). Then, 1.5 µL of the dissolved sample was spotted onto a target plate holder. After air-drying, the digested peptides were analyzed using the MALDI-TOF-MS. Mass spectra were obtained over the m/z range of 800-3000 Da. The spectrometer was run with the following settings: 15 kV source voltage; 3 kV pulse voltage; 1850 V MCP detector voltage; 500 V reflectron voltage; and TLF delay of 500 ns. Spectra were internally calibrated using trypsinized alcohol dehydrogenase (147 kDa; Sigma). Glu-Fibrinopeptide B (Glu-Fib; 1570 Da; Sigma) was used as the external standard. Proteins were identified by their peptide mass fingerprint (PMF) with Protein Lynx Global Server and Swiss-Prot (http://www.expasy.ch). The search parameters were allowed for fixed modification, carbamidomethylation of cysteine, variable modifications, and oxidation of methionine. Statistics. The data were expressed as the mean ( standard deviation (SD) for three gel sets in proteome analysis, and for six independent experiments in proliferation rate studies. Unpaired Student’s t-test was employed to establish the difference between groups. A value of P < 0.05 was considered to be statistically significant.

large polygonal cells in primary culture (P0 cells). During initial growth, they formed colonies, that is, the colony-forming-unit fibroblasts. However, some phenotypic differences were observed between primary and subcultured cells; the majority of the cells demonstrated spindle morphology in ongoing subcultures. Trypan blue dye exclusion test confirmed >97% cell viability at P0-P1, and no significant change was observed in subsequent cultures. Additionally, MTT assay demonstrated that the cells retained their mitochondrial dehydrogenase activity for the duration of the experiments, with slight decrease after passage 6 onward (data not given). Representative phase contrast images of rat BM-MSCs at different stages of culture are presented in Figure 1. The cells were plated at same density (∼2000 cells/cm2) and subcultured at similar confluence levels. Following initial adherence, the cells started to proliferate on the plastic substrate (Figure 1A); later spreaded and demonstrated typical elongated fibroblastic phenotype (Figure 1B). Figure 1C shows a representative confluent culture ready for subculturing, and Figure 1D, accumulation of the extracellular matrix in dense cultures which raised complications during trypsinization. Results showed differential proliferation rate of MSCs during the 10-passage-period, under standard culture conditions. These findings are presented in Figure 2. Time to reach confluence took about 13 days in P0, which eventually dropped to 6-8 days between P1 and P4. Then, it increased to 11 days in P5, and stayed at 12-14 days during subsequent subcultures. Protein Expression in Different Passages. IPG strips of pH 3-10 range were used in first dimension, and three analytical gels were performed for each group (Figure 3). We evaluated the proteins of samples retrieved from different passages. As the result, we were able to find a number of distinct proteins of rat MSCs: passage 0 proteins which were differentially expressed compared to that of all other passages; passage 5 proteins which decreased (at least 2-fold) and finally discontinued its expression; and up-regulated proteins (at least 2-fold) at passage 5 compared to that of passage 4 (Figure 4). A total of 18 protein spots were chosen for protein identification and expression of spot intensity appeared at different levels. For passage 0, 37 proteins were marked, with Match method analysis set parameters; only 5 were identified. Between passage 4 and 5, three out of seven proteins with 2-fold expression changes were identified. At passage 5, 62 proteins were down-regulated, or not expressed in subsequent subcultures; among these, 10 were identified (Figure 5). Functional Categories of Identified Proteins. Identified proteins were classified into eight functional categories: metabolism, signal transduction, cell adhesion and cell growth, cell cytoskeleton, cell-cell interaction, cell cycle, protein degradation, and ion transfer. These proteins have been reported to be involved in cell proliferation and differentiation through different signaling pathways. The largest protein group was the ion transport proteins; the second largest consisted of two different classes, namely, the metabolic and signal pathway proteins; the third group included cell-cell interaction, protein degradation, cell adhesion, and growth proteins. The expression of cytoskeletal and cell cycle proteins was not significantly altered during passages (Table 1 and Figure 6).

Results

Discussion

Morphology, Viability, and Proliferation Rate of Rat BM-MSCs. The shape of the rat bone marrow adherent cells was heterogeneous, consisting of both fibroblastoid cells and

While the existence of mesenchymal stem cells is undisputed, many questions remain unanswered regarding the molecular mechanisms of self-renewal and differentiation. It has been

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Proteomics of Rat MSC Subcultures

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Figure 1. Representative phase contrast microscopy images of rat BM-MSCs at different stages of culture (Hoffman modulation). Initial adherence and expansion of the cells on plastic substrate (A). Spreading of the typical elongated fibroblastic phenotype (B). Confluent culture ready for subculturing (C). Accumulation of the extracellular matrix observed in dense cultures which are not subcultured (D). Scale bar: 100 µm.

Figure 2. Proliferation rate of rat BM-MSCs under standard culture conditions, in terms of reaching confluence level during subcultures. Plating density: ∼2000 cells/cm2. Data are presented as mean ( SD; n ) 6; P < 0.05 statistical significance for θ, θ and Ψ, Ψ.

reported by several groups that, after a certain number of population doublings and passages, MSCs undergo cellular senescence with decreasing proliferation and show changes in cell morphology.26,27 We have found that beginning from passage 5-6, the expansion tendency of rat MSC cultures decreased but the morphological characteristics remained unchanged up to the 10th passage. A proteomic characterization of stem or progenitor cells would be an ideal way to show changes in the developmental or metabolic state of cells or tissues of any species. The most widely used method applied to complex protein mixtures prior to mass spectrometry analysis is the 2-DE. The aim of the study was to evaluate the proliferation and self-renewal of rat BM-MSC subcultures under standard culture conditions; thus, a proteomic approach based

on 2-DE and MALDI-TOF-MS was performed which resulted in the identification of 18 differentially expressed proteins given below: Metabolic Pathway Proteins. Metabolic reactions and related enzymes are vital for the viability, growth, and function of all cell types including MSCs. In our study, phosphatidylcholine-sterol acyltransferase (Lecithin cholesterol acyltransferase; LCAT) precursor, squalene monooxygenase required for lipid biosynthesis, as well as sorbitol dehydrogenase required for the glucose pathways were differentially expressed in passages P0 and P5. LCAT, a central enzyme in the extracellular metabolism of the plasma lipoproteins, is a glycoprotein enzyme responsible for the formation of cholesteryl ester in plasma via transfer of the sn-2 fatty acid from phosphatidylcholine to the 3-hydroxy group of cholesterol in plasma,28 and sorbitol dehydrogenase (SDH), a member of the medium-chain dehydrogenase/reductase protein family and the second enzyme of the polyol pathway of glucose metabolism, converts sorbitol to fructose strictly using NAD+ as coenzyme29 and was only expressed at passage 0 in our study. Huck et al. have evaluated the formation and degradation of pentitols in human fibroblasts and erythrocytes.30 Pentitol dehydrogenase, catalyzing the conversion of xylitol and sorbitol to D-xylulose and D-fructose, respectively, was found in lysed erythrocytes, in agreement with described activities of NAD+-linked xylitol dehydrogenase and sorbitol dehydrogenase in human erythrocytes; however, no metabolism of sorbitol and xylitol was found in the lysed fibroblasts.30 In our study, the expression of squalene monooxygenase decreased 2-fold at passage 5. This is an enzyme found in the endoplasmic reticulum membrane which converts squalene into squalene 2, 3-epoxide playing a significant role during cholesterol synthesis.31,32 Journal of Proteome Research • Vol. 8, No. 5, 2009 2167

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Figure 3. Typical 2-DE profiling of rat BM-MSCs located in different passages (P0-P10). The protein samples were prepared and separated as described in Materials and Methods, and the gels were subjected to Sypro Ruby staining and image analysis.

Figure 4. Differentially expressed proteins between P0-P10 by the rat BM-MSCs. (A) Expression level of proteins that diminish at passage P5. (B) Expression level of proteins at P0. (C) Expression level of proteins that change between passages P4 and P5. Each protein spot is presented by an SSP number.

Ion Transport Proteins. ATP synthase delta chain mitochondrial precursor was only expressed in passage P0 which provided MSCs’ energy need. Inward rectifier potassium channel 13 (Kir7.1) which was down-regulated at passage 5 is 2168

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characterized by a greater tendency to allow potassium to flow into the cell rather than out of it.33 Insulin, a hormone known to increase the message levels of the TSH receptor and thyroglobulin in the thyroid follicle, also increases the levels

Proteomics of Rat MSC Subcultures

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Figure 5. 2-DE gel analysis of proteins extracted from rat BM-MSCs. The first dimension was performed using 500 µg of total soluble proteins on linear gradient IPG strips, pH 3-10. In the second dimension, 10% SDS-PAGE gels were used, and the proteins were visualized using Sypro Ruby. (A) Passage 0, (B) passage 4-5, (C) passage 5. The arrows represent proteins differentially up-regulated (g2-fold) between passages 4 and 5, and also determined by the Arbitrary method for passage 5, and by Match analysis for passage 0.

of Kir7.1 in the FRTL-5 cells. Sequence analysis of the promoter regions of rat Kir7.1 gene for cis-acting elements revealed the presence of CRE and AP-1 sites, suggesting the regulatory effect of cAMP on the expression of the Kir7.1 gene.34 Cytochrome P450 7b1 fragment, which plays a role in the lipid metabolism, was down-regulated 2-fold at passage 5. It is known that cytochrome P450 7b1 (oxysterol 7 L-hydroxylase) and cytochrome P450 8B1 (sterol 12 L-hydroxylase) genes may represent adaptive responses to minimize cytotoxic bile salt levels of the liver.35 Mineralocorticoid receptor (MR), which was upregulated at passage 5, is a receptor for both mineralocorticoids (MCs) such as aldosterone and glucocorticoids (GC), for example, corticosterone or cortisol. Pascual-Le Tallec et al. have reported that MR takes part not only in the regulation of sodium and water homeostasis, but also in cardiovascular function, neuronal fate, and adipocyte differentiation.36 The inhibitory role of MCs on the formation of bone marrowderived progenitor cells is known, at least in part, by attenuating VEGFR-2 expression and the subsequent Akt signaling.37 Reduction of MC levels, blockade of MR, and/or co-treatment with antioxidants may, therefore, enhance vascular regenera-

tion.37 Hence, MSCs may well be bound to eliminate or decrease the expression of MR. Signal Pathway Proteins. Somatostatin receptor type 5 (SST5), the receptor for somatostatin-28, which inhibits adenylyl cyclase, is mediated by the G proteins. It is clear that SST5 modulates SST 2 regulation of the ACTH secretion, and predominantly suppresses MAPK pathway activation.38,39 In our study, SST5 was up-regulated at passage 5, compared to passage 4. Chemokine binding protein 2 was down-regulated at passage 5, its function being a receptor for C-C type chemokines including SCYA2 (MCP-1; monocyte chemoattractant protein-1), SCY3/MIP-1-alpha, SCYA4/MIP-1-beta, and SCYA5/RANTES. Welle et al. have reported that, like chemokine binding protein-2, glutathione reductase, heat shock transcription factor 2, and several stress response genes were differentially expressed in the young and old muscle.40 A receptor for somatostatins-14 and 28, somatostatin receptor type 3, was down-regulated at passage 5. This receptor is coupled via pertussis toxin sensitive G proteins for the inhibition of adenylyl cyclase. Journal of Proteome Research • Vol. 8, No. 5, 2009 2169

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Table 1. Identities of Differentially Expressed Proteins in Rat Bone Marrow MSCs during Subcultures passage

P0

P4-P5

P5

spot no.

protein identity

accession no.

theoretical Mr/pI

observed Mr/pI

sequence coverage

matched peptides

PLGS score

0002 1303 9101 2205 5710 8401 3604 4204 2602 5304 5405 8507 6608 8301 8404 7105 0608 6706

ATP synthase delta chain mitochondrial precursor Phosphatidylcholine sterol acyltransferase precursor Gap junction beta 5 protein connexin 31 Adenylate kinase isoenzyme 1 Sorbitol dehydrogenase Somatostatin receptor type 5 NDRG1 protein N myc downstream regulated gene 1 Junctional adhesion molecule 1 precursor Di N-acetylchitobiase precursor Cathepsin D precursor Inward rectifier potassium channel 13 Squalene monooxygenase Mineralocorticoid receptor Cytochrome P450 7B1 oxysterol 7 hyroxylase Somatostatin receptor type 3 Chemokine binding protein 2 Tubulin beta chain T beta 15 Cell cycle autoantigen SG2NA

P35434 P18424 P28232 P39069 P27867 P30938 Q6JE36 Q9JHY1 Q01460 P24268 O70617 P52020 P22199 Q63688 P30936 O09027 P04691 P58405

17595/4.51 49727/6.5 31047/9.13 21584/7.66 38235/7.27 39971/9.56 42955/5.77 32370/5.81 41531/5.54 44681/5.69 40637/6.10 64024/8.72 106737/7.1 48227/8.3 47151/9.03 43293/8.64 49953/4.78 87111/5.14

17601/5.0 49527/5.5 31026/9.0 21526/5.5 42807/6.9 39944/9.6 42980/5.6 32348/5.9 41504/5.2 44651/6.7 40503/5.8 63983/8.5 106999/7.1 48196/8.3 45817/8.6 43227/7.8 49930/4.6 50410/4.9

4.8 2.3 3.7 4.6 3.0 2.5 2.0 3.0 4.9 2.0 2.5 1.4 1.0 4.1 3.9 2.1 20.0 1.7

1 1 1 1 1 1 1 1 2 2 1 1 1 2 1 1 12 1

4.9 4.9 5.5 3.8 5.1 5.4 5.3 4.4 4.6 4.0 6.5 4.2 8.5 3.7 6.5 5.3 5.0 4.1

a Observed Mr/pI values are based on the 2-DE migration, and the theoretical values were calculated from amino acid sequences. The protein samples were subjected to 2-DE, followed by Sypro Ruby staining and image analysis. Results were quantified from three sets of gels. The spots of interest were excised from the gels and digested with trypsin. The resulting peptides were used in MALDI TOF-MS analysis, and the proteins were identified by searching the Swiss-Prot database using peptide sequences.

Figure 6. Functional categories of identified proteins. The percent of proteins included in each category is indicated; the majority belongs to ion transport, metabolism, and signal pathways’ categories.

Cytoskeletal Proteins. A major constituent of microtubules, the tubulin beta-2B chain, was down-regulated at passage 5. This protein binds 2 mol of GTP, one at an exchangeable site on the beta chain and one at a nonexchangeable site on the alpha-chain. Rautajoki et al. have previously reported that tubulin beta chain protein was down-regulated in IL-4 treated T cells.41 Additionally, Sun et al. have highlighted that tubulin expression decreased with increasing cell passages.21 Cell Adhesion, Growth Proteins. Adenylate kinase isoenzyme 1 is located in the cytoplasm, responsible for the phosphorylation of AMP in erythrocytes and production of ADP, and is involved in the catabolic pathway of adenine nucleotide and homeostasis of guanine nucleotide pools. This small ubiquitous enzyme known to be essential for cell maintenance and growth was expressed only in passage 0. The expression of N-myc downstream-regulated gene 1 (NDRG1) protein was up-regulated at passage 5 compared to passage 4; this protein may have a growth inhibitory role. Ellen et al. have 2170

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previously reported that NDRG1 is up-regulated by cell differentiation signals in various cancer cell lines and suppresses tumor metastasis. Despite its specific role in the Charcot-MarieTooth type 4D disease, this gene attracts interest as a marker of tumor progression and enhancer of cellular differentiation.42 It is likely that a tumor suppressor function of NDRG1 is only the consequence of its up-regulation in differentiating cells and down-regulation under conditions of cell growth. Cell-Cell Interaction Proteins. Gap junction beta-5 protein is a connexin (Cx) of 266 amino acid residues sharing ∼70% amino acid identity both with Cx31 and Cx31.1. These molecules together share a rather restricted expression pattern, being preferentially expressed in the skin, and undetectable in most other tissues. In our study, the gap junction beta-5 protein was only expressed in passage 0. Lack of regular cell-cell interaction is one major cause of neoplastic growth and metastasis. A 10-fold down-regulation of Cx31.1, as well as mutations in the TGF-beta-receptor-II, has previously been reported in head and neck squamos cell carcinoma.43 Junctional adhesion molecule A (JAM-A) precursor was up-regulated at passage 5 compared to passage 4. This protein is known to play a role in epithelial tight junction formation and cell migration,44 and identified as the platelet antigen for the stimulatory monoclonal antibody F11.45 JAM-A mRNA is significantly increased in inflammed tissues, supporting its connection with the immune response. Blocking of JAM-A is known to inhibit basic fibroblast growth factor (bFGF)-induced angiogenesis.46 Protein Degradation Proteins. Cathepsin D (CTSD) precursor, acid protease active in intracellular protein breakdown, has specificity similar to but narrower than that of pepsin A. Unlike Sun et al.47 who did not observe any down-regulation of CTSD mRNA in human MSC subcultures, we determined a decrease in Cathepsin D expression in rat MSCs after passage 5. We also observed the down-regulation of Di-N-acetylchitobiase precursor at passage 5. This protein is involved in the degradation of asparagine-linked glycoproteins. The reducing end of the N-acetyl-beta-D-glucosamine (1-4)N-acetylglucosamine chitobiose hydrolyzate core requires prior cleavage by glycosy-

Proteomics of Rat MSC Subcultures 48

lasparaginase. In agreement with the very low levels of chitobiase enzyme, a similarly low level of chitobiase gene expression in bovine indicates that chitobiase in this species has a minor role in hydrolyzing the reducing end GlcNAc of asparagine-linked glycoproteins within the lysosomes. This is in contrast with some other species, such as humans, that express substantial quantities of this glycosidase. Thus, the extreme range of chitobiase gene expression among species explains why either one or two GlcNAc residues remain intact at the reducing end of stored oligosaccharides when either chitobiase-expressing or chitobiase-deficient species, respectively, suffers from a lysosomal storage disease.49 Cell Cycle Proteins. Down-regulation of the cell cycle and proliferation-associated proteins, such as the T-complex protein 1 alpha subunit (TCP-1alpha), has previously been reported for the late subcultures of human MSCs.47 Our results indicated that Striatin-3 (cell-cycle autoantigen SG2NA), which has a role in the cell cycle, was down-regulated at passage 5. This protein binds calmodulin in a calcium-dependent manner, and may function as scaffolding or signaling protein. Besides, Tan et al. have demonstrated that Striatin-3 gamma inhibits estrogen receptor activity by recruiting a protein phosphatase.50

Conclusion In this study, we have generated proteome maps from the continuous subcultures of rat bone marrow mesenchymal stem cells up to 10 passages. With 2-DE and MALDI-TOF, we have identified differentially regulated proteins potentially involved directly or indirectly in cellular processes. We also point out that some proteins observed in our experiments are associated with the differentiation capacity of MSCs. It seems likely that rat BM-MSCs under unstimulated culture conditions can differentiate into other cell types in a slow and progressive way; however, further studies will be needed to confirm this argument. The acquired protein list of rat BM-MSCs in this study can be integrated into inventory, which may facilitate the identification of the normal proteomic pattern as well as changes in activated or suppressed pathways occurring during proliferation and other experimental conditions.

Acknowledgment. The partial support of the Turkish ¨ BA (to Y.M.E.), TU ¨ BITAK-BAYG (to Academy of Sciences, TU B.C ¸ .), and Ankara University Biotechnology Institute (Ankara, Turkey) is acknowledged. The authors acknowledge Dr. A. E. Elc¸in on cell isolation, and Dr. M.S. Halloran for helpful discussions. We also thank Drs. D.O. Demiralp, and I. Bosg¸elmez for technical support. Supporting Information Available: Table of modification, miscleavage, m/z, peak m/z, peptide m/z, and delta values of differentially expressed proteins in rat bone marrow MSCs during subculture. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Colter, C. D.; Sekiya, I.; Prockop, D. J. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Cell Biol. 2001, 98 (14), 7841–7845. (2) Kassem, M.; Abdallah, B. M. Human bone-marrow-derived mesenchymal stem cells: biological characteristics and potential role in therapy of degenerative diseases. Cell Tissue Res. 2008, 331, 1157–163. (3) Eslaminejad, M. B.; Nikmahzar, A.; Taghiyar, L.; Nadri, S.; Massumi, M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev. Growth Differ. 2006, 48, 361–370.

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