Proteomic Analysis of Tumor Necrosis Factor-α-Induced Secretome of

Proteomic Analysis of Tumor Necrosis Factor-r-Induced Secretome of Human Adipose Tissue-Derived Mesenchymal Stem Cells Mi Jeong Lee,† Jaeyoon Kim,‡,| Min Young Kim,† Yoe-Sik Bae,§ Sung Ho Ryu,‡ Taehoon G. Lee,| and Jae Ho Kim*,† Medical Research Center for Ischemic Tissue Regeneration & Medical Research Institute, College of Medicine, Pusan National University, Yangsan 626-770, Republic of Korea, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea, Department of Biological Science, Sungkyunkwan University, Choenchoendong, Jangangu, Suwon, Gyeonggido 440-746, Republic of Korea, and NovaCell Technology, Inc., Pohang, Korea Received October 6, 2009

Human adipose tissue-derived mesenchymal stem cells (hASCs) are useful for regeneration of inflamed or injured tissues. To identify secreted hASC proteins during inflammation, hASCs were exposed to tumor necrosis factor-R (TNF-R) and conditioned media derived from hASCs were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified 187 individual proteins as secreted proteins (secretome) in hASC-conditioned media; 118 proteins were secreted at higher levels upon TNF-R treatment. The TNF-R-induced secretome included a variety of cytokines and chemokines such as interleukin-6 (IL-6), IL-8, chemokine (C-X-C motif) ligand 6, and monocyte chemotactic protein-1 (MCP-1). TNF-R also increased expression of various proteases including cathepsin L, matrix metalloproteases and protease inhibitors, and induced secretion of long pentraxin 3, a key inflammatory mediator implicated in innate immunity. TNF-R-conditioned media stimulated migration of human monocytes, which play a key role in inflammatory responses. This migration was abrogated by pretreatment with neutralizing anti-IL-6, anti-IL-8, and anti-MCP-1 antibodies, suggesting that IL-6, IL8, and MCP-1 are involved in migration of monocytes. Taken together, these results suggest that TNFR-induced secretome may play a pivotal role in inflammatory responses and that shotgun proteomic analysis will be useful for elucidation of the paracrine functions of mesenchymal stem cells. Keywords: mesenchymal stem cells • secretome • TNF-R • monocytes • chemotaxis

Introduction Mesenchymal stem cells (MSCs) possess self-renewal capacity, long-term viability, and differentiation potential toward diverse cell types such as adipogenic, osteogenic, chondrogenic, and myogenic lineages,1-5 implicating these cells as attractive therapeutic tools for regeneration of injured or inflamed tissues. MSCs reside in virtually all postnatal organs and tissues including bone marrow and adipose tissue.6 The multilineage differentiation potential of adipose tissue-derived mesenchymal stem cells (ASCs) resembles that of bone marrow-derived MSCs.7 Because of their stem cell-like properties, ASCs are thought to be an excellent tool for mesenchymal tissue regeneration in many diseases that result from impaired function of mesenchymal cells.8-10 In addition, a growing body of evidence suggests that the tissue regenerative ability of ASCs is largely due to indirect paracrine effects of extracellular factors * Corresponding author: Department of Physiology, School of Medicine, Pusan National University, Yangsan 626-770, Gyeongsangnam-do, Republic of Korea. Tel: 82-51-510-8073. Fax: 82-51-510-8076. E-mail: [email protected]. † Pusan National University. ‡ Pohang University of Science and Technology. | NovaCell Technology, Inc. § Sungkyunkwan University.

1754 Journal of Proteome Research 2010, 9, 1754–1762 Published on Web 02/25/2010

secreted from ASCs, rather than direct transdifferentiationmediated regeneration of endogenous cells.1,11 ASCs produce a variety of angiogenic cytokines and, subsequently, stimulate neovascular formation via autocrine and paracrine actions.12-15 Furthermore, ASCs secrete various inflammatory cytokines, chemokines, and extracellular factors including interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1).16-18 Within injured or inflamed tissue, the secretory properties of ASCs may be mainly affected by the surrounding local microenvironments. In spite of pathophysiological significance of the ASC-derived secreted proteome (secretome), the molecular identities of this secretome are unclear. Tumor necrosis factor-R (TNF-R) is induced in damaged and inflamed tissues, and plays a key role in postinjury organ dysfunction such as myocardial infarction and renal failure by inducing various inflammatory cytokines and chemokines.19 TNF-R increases migration of MSCs toward chemokines20 and stimulates invasive capacity of MSCs by stimulating expression of matrix metalloproteases (MMPs).21 These observations suggest that TNF-R may regulate MSC mobilization and subsequent homing to injured tissues. Therefore, it is likely that released TNF-R can affect the secretion of protein factors from 10.1021/pr900898n

 2010 American Chemical Society

Proteomic Analysis of TNF-r-Induced Secretome of hASCs endogenously recruited or exogenously implanted MSCs for tissue regeneration. However, it is unclear whether TNF-R regulates secretion of secretome from MSCs. To determine the effect of TNF-R on the paracrine properties of human ASCs (hASCs), hASCs were presently treated with TNF-R and the secretome profile of the conditioned media derived from hASCs was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

Materials and Methods Materials. Phosphate-buffered saline, R-minimum essential medium, trypsin, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). Anticathepsin L antibody was purchased from EMD Biosciences (San Diego, CA, http://www.emdbiosciences.com). Antipentraxin 3 (PTX3) antibody, human chemokine (C-X-C motif) ligand 6 (CXCL6)/granulocyte chemotactic protein-2 (GCP-2) quantikine ELISA kit, and carrier-free recombinant human TNF-R were purchased from R&D Systems (Minneapolis, MN, http://www.rndsystems.com). Enzyme-linked immunosorbent assay (ELISA) kits and neutralizing antibodies for human IL-6, IL-8, and MCP-1 were purchased from BD Biosciences (Bedford, MA, http://www.bdbiosciences.com). Peroxidase-labeled secondary antibodies and enhanced chemiluminescence Western blotting system were purchased from Amersham Biosciences (Buckinghamshire, U.K., http://www4.gelifesciences. com). Cell Culture. Subcutaneous adipose tissue was obtained from elective surgeries with the patient’s consent as approved by the Institution Review Board of Pusan National University Hospital. hASCs were isolated from subcutaneous adipose tissue as previously described22 and they were positive for CD29, CD44, CD90, and CD105. In brief, adipose tissues were washed at least three times with sterile phosphate-buffered saline and treated with an equal volume of collagenase type I suspension (1 g/L of Hank’s balanced salt solution with 1% bovine serum albumin) for 60 min at 37 °C with intermittent shaking. The floating adipocytes were separated from the stromal-vascular fraction by centrifugation at 300g for 5 min. The cell pellet was resuspended in R-minimum essential medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, and cells were plated in tissue culture dishes at a density of 3500 cells/cm2. The primary hASCs were cultured for 4-5 days until they reached confluence (defined as passage 0). hASCs used in these experiments were passaged 2-5 times. Sample Preparation and Tryptic Digestion. hASCs were cultured in 150-mm diameter culture dishes until they reached subconfluence and were washed twice with Hank’s balanced salt solution to remove the serum component. The cells were incubated in 20 mL of nonsupplemented (no serum, phenol red, or antibiotics) R-minimum essential medium in the absence or presence of carrier-free recombinant human TNF-R (10 ng/mL) for 48 h. Conditioned media were collected and centrifuged at 3000 rpm for 10 min using a MF 300 centrifuge (Hanil Science Industrial, Inchon, Korea) to remove cell debris, filtered through a 0.2 µm filter, and desalted using a hydrophilic-lipophilic balance (HLB) extraction column (particle size 30 µm; Waters, Milford, MA) pre-equilibrated with 5% acetonitrile/0.1% (v/v) trifluoroacetic acid (ACN/TFA). Cartridges were washed with 5% ACN/0.1% TCA and eluted with 70% ACN/0.1% TCA. Eluents were lyophilized and dissolved in 0.1% TCA in ACN, and relyophilized and dissolved in 50 mM

research articles ammonium bicarbonate. For tryptic digestion, each sample was heated at 90 °C for 15 min and 5 µL of 10 mM dithiothreitol was added and incubated in 56 °C for 20 min. Five microliters of 100 mM iodoacetamide was added and incubated at room temperature in the dark for 15 min. To consume any unreacted iodoacetamide, an additional 5 µL of 100 mM dithiothreitol was supplemented. Reduced and alkylated proteins were digested with 500 ng of trypsin (Promega, Madison, WI) for 12 h at 37 °C. LC-MS/MS. All mass analysis were performed by nano-LC MS system consisting of an Agilent 1100 high-pressure liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA) and a QSTAR quadrupole-time-of-flight mass spectrometer (MDS SCIEX, Concord, Ontario, Canada) equipped with a nanoelectrospray ionization source. To achieve highresolution separation, a nanoscale reversed phase chromatography analytical column (ZORBAX C18, 0.1 mm, 0.075 mm i.d.; Agilent Technologies) was used. Mobile phase A consisted of HPLC-grade water containing 0.1% formic acid and mobile phase B consisted of 84% HPLC grade ACN containing 0.1% formic acid. Separation was performed at a flow rate of 250 nL/min and the applied gradient was 0-40% phase B over 60 min. For MS/MS analysis, each scan cycle consisted of one full scan mass spectrum (m/z 400-1500) followed by three MS/ MS events. Dynamic exclusion was activated during 1 min with a repeat count of two. Digested samples were run in duplicate and representative LC-MS/MS data from three independent experiments are shown (Supplementary Table 2). Database Searching. LC-MS/MS data were searched against the UniProt v14.5 using in-house MASCOT software (ver 2.2.04). The following parameters were used: two missed cuts; carbamidomethylation (C) as fixed modification; N-acetyl (Protein), oxidation (M), pyroglutamylation (N-term EQ) as variable modification; and charge states +2, +3, and +4. Windows of mass accuracy of 100 ppm and 0.25 Da were used for precursor ions and MS/MS data, respectively. Peptide identification and protein assembly were performed in multiple stages. Initial peptide filtering was used to determine estimated 1% false discovery rate, which were calculated using the target-decoy method.23 All protein identifications required detection of unique peptides and proteins with more than two spectral counts were selected for further analysis. Proteins identified with a higher MASCOT score in the bovine database than in the human database were considered as serum contamination and removed. Quantitative Analysis of MS Results. To estimate foldchanges of identified proteins between experimental groups, we used a label-free quantitative method based on ion intensity measurement with some modifications.24,25 Briefly, we used the total ion intensity (TII), which is the sum of all fragment ion intensities in a MS/MS spectrum. The values of TII of a protein in each group (TIICON, TIITNF) could be calculated by summation of TIIs of all spectra identified from each condition. Log2 ratio of TIITNF and TIICON was calculated and used for quantitative index. To avoid taking logarithm on zero’s, we set the TIICON or TIITNF as 0.074 which is the half of the smallest value of TII if no peptide was identified in an experimental group. Bioinformatic Analysis. Identified proteins were assessed to define ‘putative secretory proteins’. Classical secretory proteins with signal peptide were predicted by SignalP 3.0 neural network (NN) scoring. Nonclassical secretory proteins without signal peptide were predicted by secretomeP 2.0 mammalian Journal of Proteome Research • Vol. 9, No. 4, 2010 1755

research articles neural network scoring. DAVID 2008 was used for the information mining and functional annotation analysis (http://david. abcc.ncifcrf.gov/). Western Blotting. Confluent, serum-starved hASCs were treated with the appropriate conditions, washed with ice-cold phosphate-buffered saline, and then lysed in lysis buffer (20 mΜ Tris-HCl, 1 mΜ EGTA, 1 mΜ EDTA, 10 mΜ NaCl, 0.1 mΜ phenylmethylsulfonyl fluoride, 1 mΜ Na3VO4, 30 mΜ sodium pyrophosphate, 25 mΜ β-glycerophosphate, 1% Triton X-100, pH 7.4). Lysates were resolved by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and stained with 0.1% Ponceau S solution (Sigma-Aldrich). After blocking with 5% nonfat milk, the membrane was immunoblotted with the antibody of interest. Bound antibody was isualized with the relevant horseradish peroxidase-conjugated secondary antibody using an enhanced chemiluminescence Western blotting system (Amersham Biosciences, Piscataway, NJ). Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The cDNA in 2 µL of the reaction mixture was amplified with 0.5 U of GoTaq DNA polymerase (Promega) and 10 pmol of each sense and antisense primers. The thermal cycle profile was as follows: denaturation for 30 s at 95 °C, annealing for 45 s at 47-55 °C depending on the primers used, and extension for 45 s at 72 °C. For semiquantitative assessment of expression levels, each PCR reaction was carried out for 30 cycles. PCR products were size fractionated on 1.2% ethidium bromide/ agarose gel and quantified under ultraviolet transillumination. The sequences of primers and the expected size of PCR products were listed in Supplementary Table 1. Enzyme-Linked Immunosorbent Assay (ELISA). Cells were seeded in wells of a 24-well culture plate at a density of 1 × 104 cells/well. Confluent, serum-starved hASCs were treated with the appropriate conditions, and incubated for 48 h. Conditioned medium was collected and centrifuged at 15 000g for 30 min to remove particulates. ELISA was carried out according to the manufacturer’s description (Bedford, MA). The absorbance (450 nm) for each sample was analyzed by an ELISA reader (Biotek Instruments, Inc., Winooski, VT) and was interpolated with a standard curve. Isolation of Monocytes from Human Peripheral Blood. Peripheral blood was collected from healthy adult donors using citrate as an anticoagulant. Peripheral blood mononuclear cells were separated on a Histopaque-1077 gradient.26 After two washes with Ca2+- and Mg2+-free Hank’s buffered saline solution, peripheral blood mononuclear cells were suspended in RPMI 1640 medium containing 10% fetal bovine serum and incubated for 60 min at 37 °C to allow monocytes to attach to the culture dish. The adherent cells were carefully washed twice with prewarmed RPMI 1640 medium to remove nonadherent cells, and the attached monocytes were collected. The purity of the prepared monocytes was >95%, as confirmed by fluorescence-activated cell sorting analysis with anti-CD14 antibodyconjugated phycoerythrin. Isolated cells were used immediately. Chemotactic Migration Assay. Chemotactic migration of monocytes was determined using a ChemoTx 96-well disposable chamber (Neuroprobe, Gaithersburg, MD). Briefly, prepared human monocytes were suspended in RPMI 1640 medium (Carlsbad, CA) at a concentration of 1 × 106 cells/ mL, and aliquots (25 µL) of the cell suspension were placed onto the upper well of a chamber contained a 96 well framed filter with 5 µm polyhydrocarbon pores. The lower chamber contained 30 µL/well of assay medium with or without con1756

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Lee et al. ditioned medium from hASCs. After incubation for 2 h at 37 °C, nonmigrated cells on the upper side of the filter were removed by scraping. Cells that had migrated across the filter were dehydrated, fixed, and stained with hematoxylin. The stained cells in five randomly chosen high power fields (HPF; ×400) in that well were counted. Statistical Analysis. The results of multiple observations are presented as mean (SD. For multivariate data analysis, group differences were assessed with one-way or two-way ANOVA, followed by post hoc comparisons tested with Scheffe’s method. A value of p < 0.05 was considered significant.

Results Identification of TNF-r-Induced Secretome in hASCs. To identify proteins secreted from hASCs upon TNF-R treatment, serum-starved hASCs were incubated with serum-free medium in the absence or presence of 10 ng/mL TNF-R for 2 days, and the conditioned medium was subjected to LC-MS/MS analysis for protein identification. Relative abundance was based on the number of peptide matches in untreated and TNF-R-treated cells. After single peptide matched proteins were excluded, 187 proteins were identified from comparative proteomic analysis of hASC conditioned medium (Supplementary Table 2). The identified proteins were analyzed for the possibility of secretion using SignalP and SecretomeP. In total, 142 (75.9%) proteins were considered as ‘putative secretory proteins’; 102 proteins were considered to be secreted through a classical pathway (endoplasmic reticulum/Golgi apparatus-dependent pathway), as a signal peptide was predicted by SignalP, while 40 proteins were through nonclassical pathway, predicted by SecretomeP. For the comparative analysis of secretomes in response to TNF-R treatment, label-free quantitative approach was adopted and the relative abundance was based on the sum of total ion intensity of peptides matched in the untreated cells and the TNF-R-treated cells. The identities and relative abundance of the secretome are summarized in Supplementary Table 2 and the subcellular localization and molecular function of the whole secretome are summarized in the Supplementary Table 3. We classified 118 features as TNF-R-induced proteins, of which Log2 ratio values are higher than 2. An abbreviated list of the TNF-R-induced proteins is presented in Table 1. These proteins included numerous inflammatory cytokines and chemokines such as IL-6, IL-8/CXCL8, MIP-2R/CXCL2, CXCL5, CXCL6/GCP2, CXCL10, and MCP-1/CCL2. Representative mass spectra of IL-6 and CXCL2 are shown in Supplementary Figure 1. In addition, various proteases and protease inhibitors, extracellular matrix (ECM) proteins, and proteins involved in immune regulation and cell signaling were identified as TNF-R-induced secretome. The functional annotation analysis of TNF-Rinduced secretome based on gene ontology terms was performed using DAVID 2008 and the complete list of gene ontology annotation was shown in Supplementary Table 4. The data indicate that inflammation related terms such as ‘response to wounding’, ‘response to stress’ and ‘defense response’ were significantly enriched. Characterization of TNF-r-Induced Secretome in hASCs. To validate the secretomic analysis data, the expression levels of the identified proteins in response to TNF-R treatment were determined. Because it was impractical to validate all the proteins in the list, a subset of proteins was chosen for the analysis according to their potential involvement as paracrine factors. The mRNA levels of genes encoding cytokines and chemokines, immune modulators, and protease inhibitors were

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Proteomic Analysis of TNF-r-Induced Secretome of hASCs Table 1. An Abbreviated List of Secreted Proteins That Are Up-Regulated upon TNF-R Treatment Uniprot ID

protein

no. pepa

Log2 ratiob

secretoryc

Q53FK3_HUMAN IL8_HUMAN IL6_HUMAN GDN_HUMAN CXL10_HUMAN C1S_HUMAN CXCL2_HUMAN PAI1_HUMAN A5PLM9_HUMAN CFAD_HUMAN PAI2_HUMAN CCL2_HUMAN CXCL6_HUMAN CFAB_HUMAN CXCL5_HUMAN C1R_HUMAN PTX3_HUMAN

Matrix metalloproteinase 1 (MMP-1) Interleukin-8 (IL-8) Interleukin-6 (IL-6) Glia-derived nexin C-X-C motif chemokine 10 (CXCL10) Complement C1s subcomponent C-X-C motif chemokine 2 (CXCL2) Plasminogen activator inhibitor 1 (PAI-1) Cathepsin L1 Complement factor D Plasminogen activator inhibitor 2 (PAI-2) C-C motif chemokine 2 (CCL2) C-X-C motif chemokine 6 (CXCL6) Complement factor B C-X-C motif chemokine 5 (CXCL5) Complement C1r subcomponent Pentraxin-related protein 3 (PTX3)

28 6 14 17 4 16 6 22 4 3 5 3 3 3 2 12 24

9.748 9.148 9.148 8.80404 8.822 8.631 7.982 7.253 6.591 6.294 6.172 5.845 5.673 4.748 4.746 3.603 3.494

SP+ SP+ SP+ SP+ SP+ SP+ SP+ SP+ SP+ SP+ SPSP+ SP+ SP+ SP+ SP+ SP+

a Number of peptides identified. protein; NP, nonputative.

b

Log2(TIITNF/TIINT).

c

Prediction results of secretion: SP+, classical secretory protein; SP-, nonclassical secretory

Figure 1. Effects of TNF-R on mRNA levels of the hASC secretome. hASCs were treated with serum-free media in the absence (w/o) or presence (TNF-R) of 10 ng/mL TNF-R for 6 h prior to RTPCR determination of mRNA expression levels. Three subsets (cytokines/chemokinse, immune functions, and protease inhibitors) of the hASC secretome were chosen according to their potential involvement as paracrine factors. Representative data from three independent experiments are shown.

examined by RT-PCR. As shown in Figure 1, the expression levels of IL-6, IL-8, LIF, MCP-1, CXCL5, CXCL6, and CXCL10 were increased in TNF-R-treated hASCs. Furthermore, the expression levels of complement B, C1s, C1γ, and D, which are involved in the immune response,27 were elevated in TNFR-treated cells. Consistent with the LC-MS/MS data, expression levels of several protease inhibitors including PAI-1, PAI-2, and glia-derived nexin were up-regulated in response to TNF-R treatment. TNF-r Stimulates Secretion of Cathepsin L from hASCs. Cathepsin L was presently identified as a TNF-R-induced secreted protein by LC-MS/MS analysis. Cathepsins, which are cysteine family proteases, play a key role in the regulation of ECM degradation and remodeling, motility, angiogenesis, and cell signaling.28,29 To confirm the proteomic identification of cathepsin L as a component of the hASC secretome, we next determined the expression levels of cathepsin L by Western blotting. As shown in Figure 2A, the protein levels of cathepsin L in cell lysates increased in a time-dependent manner up to 48 h upon TNF-R treatment. Moreover, TNF-R treatment timedependently increased the secreted levels of cathepsin L in

Figure 2. Effects of TNF-R on expression levels of cathepsin isoforms and matrix metalloprotease-1 in hASCs. hASCs were treated with serum-free media in the absence (w/o) or presence (TNF-R) of 10 ng/mL TNF-R for indicated time periods prior to harvest of cell lysates (A) and conditioned media (B). The expression levels of cathepsin L were determined by Western blotting with anti-cathepsin L antibody. To ensure equal loading of protein between samples, the expression levels of GAPDH were determined by Western blotting. (C) hASCs were treated with serum-free media in the absence or presence of 10 ng/mL TNF-R for 6 h. The mRNA levels of cysteine cathpsin isoforms (cathepsin A, B, C, F, K, O, and S) and matrix metalloprotease-1 (MMP-1) were determined by RT-PCR. Representative data from three independent experiments are shown.

hASC conditioned medium (Figure 2B). We next explored whether TNF-R could regulate the expression of other cathepsin isoforms using RT-PCR. As shown in Figure 2C, TNF-R treatment elevated the mRNA levels of other cathepsin isoforms including cathepsin A, B, C, F, K, O, and S. In addition, MMP-1 was identified in the TNF-R-induced secretome by LC-MS/MS analysis. Consistently, the mRNA level of MMP-1 was upregulated by TNF-R treatment (Figure 2C). TNF-r Increases Secretion of Pentraxin 3 from hASCs. Pentraxin 3 (PTX3) is the prototype of the long pentraxin family, and is induced by various inflammatory signals including TNFR, lipopolysaccharide and IL-1.30 We identified PTX3 as a TNFR-induced secretome by LC-MS/MS analysis. To confirm the proteomic identification of PTX3 as a component of the hASC secretome, the expression levels of PTX3 in hASCs in response Journal of Proteome Research • Vol. 9, No. 4, 2010 1757

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Figure 3. Effects of TNF-R on PTX3 expression levels in hASCs. hASCs were treated with serum-free media in the absence (-) or presence (+TNF-R) of 10 ng/mL TNF-R for indicated time periods prior to harvesting of cell lysates (A) and conditioned media (B). The expression levels of PTX3 and GAPDH were determined by Western blotting. Representative data from three independent experiments are shown.

to TNF-R treatment were examined using Western blotting. As shown in Figure 3A, the expression levels of PTX3 timedependently increased upon TNF-R treatment. To assess whether TNF-R can induce secretion of PTX3 from hASCs, PTX3 levels in hASC conditioned medium were determined by Western blotting. A time-dependent increase was evident (Figure 3B). TNF-R-induced secretion of PTX3 was present at 3 h after TNF-R treatment and was sustained until 24 h, while secretion of cathepsin L only occurred 24 h after TNF-R treatment (Figure 2B). TNF-r Stimulates Secretion of Inflammatory Cytokines and Chemokines. To confirm that TNF-R could stimulate expression of inflammatory cytokines and chemokines from hASCs, IL-6, IL-8, and MCP-1 concentrations in conditioned medium were determined by ELISA. TNF-R treatment dosedependently increased the secreted levels of IL-6 (Figure 4A) and IL-8 (Figure 4B). Furthermore, the secreted levels of IL-6 (Figure 4C) and IL-8 (Figure 4D) were time-dependently increased up to 48 h after TNF-R treatment. In addition, TNF-R treatment stimulated secretion of MCP-1 from hASCs (Figure 4E). Using LC-MS/MS analysis, CXCL6/GCP-2, a CXC chemokine with a similarity to other CXC chemokines such as IL-8, was identified as a TNF-R-induced secreted protein. However, whether CXCL6 was induced by TNF-R treatment remained unclear. To confirm the LC-MS/MS data, the effects of TNF-R on the secreted levels of CXCL6 were assessed. As shown in Figure 4F, TNF-R treatment time-dependently increased the secreted levels of CXCL6 in hASCs. These results support the proteomic identification of inflammatory cytokines/chemokines as TNF-R-induced secretome. Migration of Human Monocytes by IL-6, IL-8, and MCP-1 Secreted from hASCs in Response to TNF-r Treatment. Monocytes migrate along the gradient of chemokines and accumulate within vascular and necrotic hypoxic areas of diseased tissues such as at the sites of ischemia, chronic inflammation, healing wounds, and bacterial infection.31 To explore whether hASC TNF-R-conditioned medium (TNF-R CM) could regulate chemotactic activity of monocytes, isolated human monocytes were allowed to migrate toward different concentrations of hASC TNF-R CM in a multiple chamber chemotaxis assay system. TNF-R CM dose-dependently increased the migration of hu1758

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Figure 4. Effects of TNF-R on hASC secretion of IL-6, IL-8, MCP1, and CXCL6. Serum-starved hASCs were treated with the indicated concentrations of TNF-R for 48 h and conditioned media were subjected to ELISA for determination of the secretion levels of IL-6 (A), IL-8 (B), and MCP-1 (E). Serum-starved hASCs were treated with serum-free media in the absence (w/o) or presence (TNF-R) of 10 ng/mL TNF-R for the indicated time periods and conditioned media were examined for the secretion levels of IL-6 (C), IL-8 (D), and CXCL6 (F) using ELISA. Data represent mean (SD (n ) 4). Asterisk (*) indicates p < 0.05 vs respective controls by one-way ANOVA and Scheffe’s post hoc test.

man monocytes compared with control CM (Figure 5A). Chemotactic migration of monocytes is up-regulated by various cytokines/chemokines such as IL-6, IL-8, and MCP-1.32,33 To assess whether IL-6, IL-8 and MCP-1 could be involved in the monocytes chemotaxis, the effect of anti-IL-6, anti-IL-8, and anti-MCP-1 neutralizing antibodies on TNF-R CM-induced monocyte migration was ascertained. IL-6-, IL-8- or MCP-1induced migration of monocytes and the cell migration was completely blocked by preincubation with their neutralizing antibodies (Figure 5B). As shown in Figure 5C, TNF-R CMinduced monocyte migration was markedly abrogated by preincubation of the TNF-R CM with anti-IL-6, anti-IL-8, or anti-MCP-1 neutralizing antibodies. To consolidate the involvement of these chemokines in the TNF-R CM-induced cell migration, we next examined the effects of the neutralizing antibodies on the cell migration induced by WKYMVm, which has been reported to stimulate migration of monocytes by the activation of the G protein-coupled receptor FPRL1.34 However, these neutralizing antibodies had no influence on WKYMVminduced cell migration, suggesting specific involvement of IL6, IL-8, and MCP-1 in the TNF-R CM-induced monocyte migration.

Proteomic Analysis of TNF-r-Induced Secretome of hASCs

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Figure 5. IL-6-, IL-8, and MCP-1 are responsible for the TNF-R CM-stimulated migration of human monocytes. (A) hASCs were exposed to serum-free medium in the absence or presence of 10 ng/mL TNF-R for 48 h prior to harvesting conditioned media. Human monocytes were loaded onto the upper chambers of a ChemoTx96-well chamber and the indicated concentrations of TNF-R CM or control CM from hASCs were added into the lower chambers. Cell migration was measured after 2 h incubation. (B) IL-6, IL-8, and MCP-1 (each 10 ng/mL) were preincubated with the anti-IL-6, anti-IL-8, and anti-MCP-1 neutralizing antibodies for 1 h, added into the lower chambers, and then migration of monocytes toward the lower chambers was determined. (C) TNF-R CM (10%), control CM (10%), and WKYMVm (1 µM) were preincubated with anti-IL-6, anti-IL-8, and anti-MCP-1 neutralizing antibodies for 1 h, then migration of monocytes toward the lower chambers was determined. Data represent mean (SD (n ) 4). Asterisk (*) indicates p < 0.05 vs respective controls by twoway ANOVA and Scheffe’s post hoc test.

Discussion MSCs are considered to be highly useful for tissue regeneration due to their multipotent differentiation potential. However, there is mounting evidence that paracrine factors secreted by MSCs are largely responsible for some of the therapeutic effects of MSCs.35,36 Therefore, identification of protein factors secreted from MSCs is necessary to understand the paracrine effects of MSCs on tissue regeneration. Shotgun proteomic analysis using LC-MS/MS is a powerful strategy for identification of protein factors secreted from cells.37 Using LC-MS/MS technology, we identified 187 proteins from hASC conditioned medium and classified 118 features as TNF-R-induced proteins. A study utilizing two-dimensional gel electrophoresis and tandem mass spectrometry compared protein profiles secreted from hASCs and differentiated adipocytes, and identified 77 features exhibiting a g2-fold induction upon adipocyte differentiation of hASCs.38 The identified proteins, which included cathepsin L, PTX3, and plasminogen activator inhibitor-1, were also presently identified. In another study, conditioned medium of MSCs derived from human embryonic stem cells was analyzed using LC-MS/MS and 132 proteins were identified as secretome of MSCs.39 However, the secretome profiles did not include most key paracrine factors including cytokines, chemokines, and growth factors. By using LC-MS/MS and ELISA analyses, we presently showed that the expression levels of cytokines and chemokines such as IL-6, IL-8 and MCP-1 are quite low in quiescent cells and are markedly induced in

response to TNF-R treatment. Therefore, these results suggest that shotgun proteomic analysis using LC-MS/MS is highly useful for identification of agonist-induced secretome of MSCs. TNF-R is a pleiotropic cytokine that plays a key role in tissue injury by initiating of a highly complex biological cascade involving chemokines and cytokines.19 In the present study, we identified numerous inflammatory cytokines and chemokines as TNF-R-induced secretory proteins of hASCs. These include IL-6, IL-8/CXCL8, MIP-2R/CXCL2, CXCL5, CXCL6/GCP2, CXCL10, and MCP-1. Using RT-PCR and ELISA analyses, we confirmed TNF-R-induced expression of IL-6, IL-8, MCP-1, and CXCL6. TNF-R stimulates expression of IL-6 and IL-8 in bone marrow stromal cells.40 Furthermore, TNF-R increases expression of various chemokines and their receptors in human MSCs.41 These results support the present demonstration that TNF-R induces secretion of inflammatory cytokines and chemokines from hASCs. Monocytes migrate into injured tissues including the sites of ischemia, chronic inflammation, healing wounds, and bacterial infection.31 In the present study, we demonstrated that TNF-R CM elicits chemotactic migration of human monocytes. Neutralizing antibodies against IL-6, IL-8, or MCP-1 markedly attenuated the migration of monocytes induced by TNF-R CM but not the FPRL1 agonist WKYMVm, suggesting specific roles of IL-6, IL-8, and MCP-1 in TNF-R CM-induced monocyte migration. IL-6 has been reported to have pro- as well as antiinflammatory properties in acute-phase and immune responses Journal of Proteome Research • Vol. 9, No. 4, 2010 1759

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of an organism. Although IL-6 is not a classical chemokine, recent evidence has demonstrated that IL-6 directly stimulates migration of monocytes and T lymphocytes,42,43 supporting the current study that IL-6 is involved in TNF-R CM-induced migration of monocytes. IL-8 is increased during TNF-Rinduced inflammatory responses and recruits monocytes and neutrophils to the inflamed area.44 MCP-1 plays a key role in the recruitment of monocytes from the blood into inflamed sites early in the inflammatory process.45 Intramyocardial injection of MCP-1 into the infarct border zone results in neoangiogenesis and monocyte infiltration in rat heart,46 which leads to the idea that increased expression of MCP-1 in MSCs stimulates angiogenesis within injured tissues. Taken together, the previous and present observations suggest that hASCs play a pivotal role in the recruitment of monocytes through TNFR-dependent secretion of various cytokines and chemokines. The present study demonstrated that TNF-R increases the expression of various proteases and protease inhibitors including cysteine protease cathepsin isoforms, MMPs, and PAIs. Cathepsins have been reported to play a key role in the regulation of ECM degradation and remodeling, motility, angiogenesis, and cell signaling.29 To date, 11 members of the human cathepsin family (cathepsin A, B, C, F, H, K, L, O, S, W and X) have been identified.28,29 We identified cathepsin L as a TNF-R-induced secreted protein by LC-MS/MS analysis and confirmed the expression of cathepsin L by Western blotting of hASC CM. In addition, we observed that TNF-R induced the mRNA levels of cathepsin A, B, C, F, K, O and S isoforms. Cathepsin L is a representative lysosomal cysteine proteinase that is ubiquitously expressed in most cell types.28 However, several reports demonstrated increased secretion of cathepsin L in response to inflammatory cytokines; TNF-R stimulates secretion of cathepsin L in smooth muscle cells, endothelial cells,47 and human fibrobrosarcoma HT 1080 cells.48 These results suggest a possible role of hASC-derived cathepsin L in TNF-R-induced biological responses. The present study identified PTX3 as a TNF-R-induced secretory protein by LC-MS/MS analysis and confirmed the expression levels of PTX3 in TNF-R-treated cell lysate and conditioned medium by Western blotting. Our results show that the expression levels of PTX3 are rapidly increased upon TNF-R treatment in hASCs. PTX3 is a member of the long form of PTX protein and is involved in innate immunity and inflammatory responses by recognizing microbes and activating complement factors, which facilitates pathogen recognition by phagocytes.49 PTX3 levels are normally very low in serum and tissues, but are rapidly increased in response to inflammatory stimulation.50 For example, circulating PTX3 levels are elevated in mice with myocardial ischemia.51 TNF-R induces expression of PTX3 in various cell types including endothelial cells and adipocytes.30,52 Thus, MSCs may play a key role in innate responses in injured tissues through TNF-R-induced secretion of PTX3. We identified complement B, C1s, C1γ, and D as TNF-Rinduced secreted proteins. In support of these results, TNF-R treatment augmented the mRNA levels of the complement factors in hASCs. In contrast to basal mRNA expression of the complement factors in serum-starved hASCs, peptides corresponding to complement B, C1s, and D were not detected in conditioned medium from serum-starved hASCs using LC-MS/ MS analysis, indicating a discrepancy between mRNA levels and secreted protein levels of complement factors. Secreted levels of proteins can be regulated by multiple steps, including transcription, translation, and secretion of expressed proteins, 1760

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Lee et al. and a lack of correlation between the transcriptional profiles and the actual protein levels has been well documented.53 These results support an idea that mRNA levels cannot reflect the actual protein levels of secreted proteins because of posttranscriptional and post-translational effects and proteomic identification of secretome is necessary for elucidation of paracrine function of hASCs. Inflammation is a key response to tissue injury and is critical for tissue regeneration, with many cytokines being associated with this process.54 It has been shown that MSC-derived paracrine factors promote wound healing by recruiting of macrophages and endothelial lineage cells.55 We have presently characterized for the first time the inflammation-associated secretome profiling of hASCs, which include cytokines and chemokines, protease inhibitors, and proteases. Taken together, the present study will provide a clue for understanding of the paracrine effects of hASCs in the regeneration of injured or inflamed tissues. Abbreviations: CXCL6, chemokine (C-X-C motif) ligand 6; ELISA, enzyme-linked immunosorbent assay; hASCs, human adipose tissue-derived mesenchymal stem cells; IL-6, interleukin-6; LC-MS/MS, liquid chromatography-coupled with tandem mass spectrometry; MCP-1, monocyte chemotactic protein-1; MSCs, mesenchymal stem cells; PAI-1, plasminogen activator inhibitor-1; secretome, secreted proteome; PTX3, pentraxin 3; RT-PCR, reverse transcription-polymerase chain reaction; TNFR, tumor necrosis factor-R.

Acknowledgment. This work was supported by the MRC program of MOST/KOSEF (R13-2005-009) and a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (00000033). Supporting Information Available: Sequence information of primers used for RT-PCR; complete list of secreted proteins that are secreted from hASCs in the absence or presence of TNF-R; biological function and localization of the hASC secretome; functional annotation clustering of TNF-Rinduced secretome; representative mass spectra of IL-6 and CXCL2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Phinney, D. G.; Prockop, D. J. Concise review: mesenchymal stem/ multipotent stromal cells: the state of transdifferentiation and modes of tissue repair--current views. Stem Cells 2007, 25, 2896– 2902. (2) Pittenger, M. F.; Mackay, A. M.; Beck, S. C.; Jaiswal, R. K.; Douglas, R.; Mosca, J. D.; Moorman, M. A.; Simonetti, D. W.; Craig, S.; Marshak, D. R. Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284, 143–147. (3) Chamberlain, G.; Fox, J.; Ashton, B.; Middleton, J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 2007, 25, 2739–2749. (4) Caplan, A. I. Mesenchymal stem cells. J. Orthop. Res. 1991, 9, 641– 650. (5) Short, B.; Brouard, N.; Occhiodoro-Scott, T.; Ramakrishnan, A.; Simmons, P. J. Mesenchymal stem cells. Arch. Med. Res. 2003, 34, 565–571. (6) da Silva, M. L.; Chagastelles, P. C.; Nardi, N. B. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J. Cell Sci. 2006, 119, 2204–2213. (7) Zuk, P. A.; Zhu, M.; Ashjian, P.; De Ugarte, D. A.; Huang, J. I.; Mizuno, H.; Alfonso, Z. C.; Fraser, J. K.; Benhaim, P.; Hedrick, M. H. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 2002, 13, 4279–4295.

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