Proteome Changes in Human Monocytes upon Interaction with Calcium Oxalate Monohydrate Crystals Nilubon Singhto,†,‡ Kitisak Sintiprungrat,†,‡ Supachok Sinchaikul,§ Shui-Tein Chen,§,| and Visith Thongboonkerd*,†,⊥ Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Department of Immunology and Immunology Graduate Program, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Institute of Biological Chemistry and Genomic Research Center, Academia Sinica, Taipei, Taiwan, Institute of Biological Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan, and Center for Research in Complex Systems Sciences, Mahidol University, Bangkok, Thailand Received February 26, 2010
Monocytic infiltration in renal interstitium is commonly found surrounding the site of calcium oxalate (CaOx) crystal deposition in the kidney. Monocytes are supposed to eliminate the deposited crystals. However, effects of CaOx crystals on the infiltrating monocytes remain unknown. Therefore, this study investigated the altered cellular proteome of human monocytes in response to interaction with CaOx monohydrate (COM) crystals. After 24-h culture with or without 100 µg/mL COM crystals, U937 cells were harvested and subjected to 2-DE analysis with Deep Purple fluorescence staining (n ) 5 gels/ group; each was derived from independent culture). Spot matching, quantitative intensity analysis, and statistics revealed 22 differentially expressed proteins (9 up-regulated and 13 down-regulated proteins), which were successfully identified by Q-TOF MS and MS/MS analyses, including those involved in cell cycle, cellular structure, carbohydrate metabolism, lipid metabolism, mRNA processing, and protein synthesis, stabilization, and degradation. Randomly selected changes [up-regulated ALG-2 interacting protein 1 (Alix), elongation factor-2 (EF-2), and down-regulated β-actin] were confirmed by Western blot analysis. Our data may help to understand how monocytes interact with COM crystals. These processes are proposed to cause subsequent inflammatory response in kidney stone disease through oxidative stress pathway(s). Keywords: calcium oxalate • CaOx • COM • monocytes • proteome • proteomics
Introduction Circulating monocytes represent approximately 5-10% of leukocytes in human peripheral blood.1 In response to inflammation, monocytes migrate from the blood circulation to inflammatory site(s) by stimulation of chemokines and metabolic and immune stimulants, leading to host defense mechanisms.2 On the other hand, monocytic infiltration can enhance progression of several kidney diseases.3,4 For example, circulating monocytes infiltrate into renal interstitium as a result of tubulointerstitial inflammation and acute kidney injury.5,6 The recruitment of monocytes at the site of renal tubulointerstitial * To whom correspondence should be addressed. Visith Thongboonkerd, MD, FRCPT Head of Medical Proteomics Unit, Office for Research and Development, Siriraj Hospital, Mahidol University, 12th Fl. Adulyadej Vikrom Bldg., 2 Prannok Rd., Bangkoknoi, Bangkok 10700, Thailand. Tel/Fax: +662-4184793. E-mail:
[email protected] or
[email protected]. † Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University. ‡ Department of Immunology and Immunology Graduate Program, Faculty of Medicine Siriraj Hospital, Mahidol University. § Academia Sinica. | National Taiwan University. ⊥ Center for Research in Complex Systems Sciences, Mahidol University.
3980 Journal of Proteome Research 2010, 9, 3980–3988 Published on Web 06/07/2010
inflammation depends on chemotactic factors such as monocyte chemoattractant protein-1 (MCP-1),7,8 γ-interferon inducible proteins-10 (IP-10),9 and regulated upon activation: normal T cell expressed/secreted (RANTES).10 In kidney stone disease, calcium oxalate monohydrate (COM) crystal is the predominant type of crystalline composition of stone matrix. After adhering onto apical surface of renal tubular epithelial cells, COM crystals can be internalized into the cells and then externalized at the basal site to promote interstitial damage and inflammation.11,12 Furthermore, COM crystals can induce renal interstitial cells to produce several chemokines such as MCP-1, RANTES, interleukin-10 (IL-10), and macrophage inflammatory proteins (MIP), which then attract circulating monocytes and recruit them into the inflammatory locales.13 Indeed, association between monocytes and COM crystals has been studied in animal models and cell culture. In mouse models, COM crystals deposited at renal interstitium were surrounded by monocytes and multinucleated giant cells.14 Stimulation of renal tubular epithelial cells by CaOx crystals resulted in production of MCP-1, which is the potent chemoattractant for monocytes.13 A recent study of kidney stone disease in mouse model using microarray technique revealed that 10.1021/pr100174a
2010 American Chemical Society
Proteome Changes in Monocytes by COM Crystals differentially expressed genes upon stone formation and deformation were related to chemotaxis (CCL6), monocyte maturation (CD14), phagocytosis and antigen presentation (genes encoding capthesin S and MHC class II), and anti-inflammation (genes encoding nuclear protein 1 and serine peptidase inhibitor).15 Even with this knowledge, the molecular mechanisms of interaction between human monocytes and COM crystals in renal interstitium, particularly at protein level that directly govern cellular function, remain poorly understood. In the present study, we applied a proteomic approach to define responses of monocytes to COM crystals. Human monocytic cells (U937) were exposed to 100 µg/mL COM crystals for 24 h and changes in cellular proteomes were evaluated by 2-DE followed by Q-TOF MS and MS/MS analyses. The proteomic data were then confirmed by conventional immunological method.
Materials and Methods Preparation of COM Crystals. COM crystals were prepared as described previously.16 Briefly, 10 mM calcium chloride dihydrate (CaCl2 · 2H2O) was mixed with 10 mM sodium oxalate (Na2C2O4) to make their final concentrations to 5 mM and 0.5 mM, respectively, in a buffer containing 90 mM Tris-HCl and 10 mM NaCl (pH 7.4). The solution was then mixed and incubated at room temperature (RT) overnight. COM crystals were harvested by centrifugation at 3000 rpm for 5 min. Supernatant was discarded and the crystals were resuspended in absolute methanol. After another centrifugation at 3000 rpm for 5 min, absolute methanol was discarded and the crystals were dried at 37 °C overnight. Purity of the obtained COM crystals as well as their typical morphology were confirmed by inverted phase-contrast light microscope (Olympus CKX41, Olympus Co. Ltd.; Tokyo Japan). Cell Culture and Intervention with COM Crystals. Human monocytic cells (U937) were cultured with RPMI 1640 medium (Gibco; Grand Island, NY) supplemented with 10% (v/v) heatinactivated fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (SIGMA; St.Louis, MO) in 75 cm2 tissue culture flask (n ) 5 individual culture flasks for each condition). The cultured cells were maintained in humidified incubator with 5% CO2 at 37 °C. The medium was refreshed every three days. For crystal-cell interaction, dried COM crystals were decontaminated by UV light radiation for 30 min. Subsequently, U937 monocytic cells at a density of 1 × 106 cells/mL were maintained in fresh medium with or without 100 µg/mL COM crystals for 24 h. The interaction between cells and crystals was examined under an inverted phase-contrast microscope (Olympus Co. Ltd.). Protein Extraction. After incubation with or without COM crystals, the cells were harvested by centrifugation at 1500 rpm, 4 °C for 5 min. The cell pellets were washed three times with PBS and collected by another centrifugation at 10 000 rpm, 4 °C for 2 min. To dissolve adhered COM crystals, cell pellets were resuspended in 0.5 M EDTA/PBS and incubated at 4 °C for 30 min. The cells were then washed again with PBS three times and cellular proteins were extracted with a lysis buffer containing 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate (CHAPS), 120 mM dithiothreitol (DTT), 2% ampholytes (pH 3-10), and 40 mM Tris-HCl (at 4 °C for 30 min). Unsolubilized nuclei, cell debris, and particulate matters were removed by centrifugation at 10 000 rpm, 4 °C for 5 min. Protein concentrations were
research articles determined by the Bradford method using Bio-Rad protein assay (Bio-Rad Laboratories; Hercules, CA). 2-DE and Staining. Cellular proteins derived from individual samples were resolved by 2-DE (n ) 5 gels derived from individual culture flasks for each condition). Equally loaded proteins (150 µg/sample) were premixed with rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 120 mM DTT, 40 mM Tris-base, 2% ampholytes (pH 3-10), and a trace of bromophenol blue (to make the final volume of 150 µL), and then rehydrated onto Immobiline DryStrip (nonlinear pH gradient of 3-10; GE Healthcare; Uppsala, Sweden) at RT for 16 h. The first dimensional separation or isoelectric focusing (IEF) was performed in Ettan IPGphor III System (GE Healthcare) at 20 °C, using a stepwise mode to reach 9083 Vh with limiting current of 50 mA/strip. After completion of the IEF, the strips were first equilibrated for 15 min in an equilibration buffer containing 6 M urea, 130 mM DTT, 112 mM Tris-base, 4% SDS, 30% glycerol and 0.002% bromophenol blue, and then in another similar buffer that replaced DTT with 135 mM iodoacetamide, for further 15 min. The second dimensional separation was performed in 12% polyacrylamide gel using SE260 mini-Vertical Electrophoresis Unit (GE Healthcare) at 150 V for approximately 2 h. Separated proteins were stained with Deep Purple fluorescence dye (GE Healthcare) at RT for 1 h and then rinsed 3 times (5 min each) with deionized water. The resolved protein spots in individual stained 2-D gels were visualized using Typhoon 9200 laser scanner (GE Healthcare). Spot Matching, Quantitative Intensity Analysis, and Statistics. Image Master 2D Platinum software (GE Healthcare) was used for matching and analysis of protein spots in 2-D gels. Parameters used for spot detection were (i) minimal area ) 10 pixels; (ii) smooth factor ) 2.0; and (iii) saliency ) 2.0. A reference gel was created from an artificial gel combining all of the spots presenting in different gels into one image. The reference gel was then used for determination of existence and difference of protein expression between gels. Background subtraction was performed, and the intensity volume of each spot was normalized with total intensity volume (summation of the intensity volumes obtained from all spots within the same 2-D gel). Intensity volumes of individual spots were then compared between COM-treated samples versus controls using Unpaired Student t-test (SPSS; version 13.0). Significantly differed protein spots were subjected to in-gel tryptic digestion and identification by mass spectrometry. In-Gel Tryptic Digestion. The protein spots whose intensity levels significantly differed between groups were excised from 2-D gels, washed twice with 200 µL of 50% acetonitrile (ACN)/ 25 mM NH4HCO3 buffer (pH 8.0) at room temperature for 15 min, and then washed once with 200 µL of 100% ACN. After washing, the solvent was removed, and the gel pieces were dried by a SpeedVac concentrator (Savant; Holbrook, NY) and rehydrated with 10 µL of 1% (w/v) trypsin (Promega; Madison, WI) in 25 mM NH4HCO3. After rehydration, the gel pieces were crushed and incubated at 37 °C for at least 16 h. Peptides were subsequently extracted twice with 50 µL of 50% ACN/5% trifluoroacetic acid (TFA); the extracted solutions were then combined and dried with the SpeedVac concentrator. The peptide pellets were resuspended with 10 µL of 0.1% TFA and purified using ZipTipC18 (Millipore; Bedford, MA). The peptide solution was drawn up and down in the ZipTipC18 10 times and then washed with 10 µL of 0.1% formic acid by drawing up Journal of Proteome Research • Vol. 9, No. 8, 2010 3981
research articles
Singhto et al.
Figure 1. Morphological changes of U937 monocytic cells in response to COM crystals. After incubation of U937 monocytic cells (A) without or (B) with COM crystals (100 µg/mL) for 24 h, cellular morphology was evaluated under an inverted phase-contrast microscope with original magnification power of 400× for both panels. (Inset, B) Zoomed-in image demonstrating the induction of extruded pseudopodia on the surface of monocytes after interaction with COM crystals.
and expelling the washing solution three times. The peptides were finally eluted with 5 µL of 75% ACN/0.1% formic acid. Protein Identification by Q-TOF MS and/or MS/MS Analyses. The trypsinized samples were premixed 1:1 with the matrix solution containing 5 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN, 0.1% (v/v) TFA and 2% (w/v) ammonium citrate, and deposited onto the 96-well MALDI target plate. The samples were analyzed by Q-TOF Ultima mass spectrometer (Micromass; Manchester, UK), which was fully automated with predefined probe motion pattern and the peak intensity threshold for switching over from MS survey scanning to MS/MS, and from one MS/MS to another. Within each sample well, parent ions that met the predefined criteria (any peak within the m/z 800-3000 range with intensity above 10 count ( include/exclude list) were selected for CID MS/MS using argon as the collision gas and a mass dependent (5 V rolling collision energy until the end of the probe pattern was reached. The MS and MS/MS data were extracted and outputted as the searchable .txt and .pkl files, respectively, for independent searches using the MASCOT search engine (http:// www.matrixscience.com), assuming that peptides were monoisotopic. Fixed modification was carbamidomethylation at cysteine residues, whereas variable modification was oxidation at methionine residues. Only one missed trypsin cleavage was allowed, and peptide mass tolerances of 100 and 50 ppm were allowed for peptide mass fingerprinting and MS/MS ions search, respectively. Western Blot Analysis. To confirm the proteomic data, a total of 30 µg of proteins derived from each sample were resolved with 12% SDS-PAGE at 150 V for approximately 2 h using SE260 mini-Vertical Electrophoresis Unit (GE Healthcare). The separated proteins were then transferred onto nitrocellulose membranes (Whatman; Dassel, Germany) using a semidry transfer apparatus (Bio-Rad; Milano, Italy) at 75 mA/gel for 1 h. Nonspecific bindings were blocked with 5% (w/v) skim milk in PBS for 1 h. Thereafter, the membranes were incubated at 4 °C with mouse monoclonal anti-Alix (1:500), rabbit polyclonal anti-EF-2 (1:1000), mouse monoclonal anti-β-actin (1:1000), or mouse monoclonal anti-GAPDH (1:2000). All these antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were diluted in 1% (w/v) skim milk/PBS. After overnight incubation and subsequent washes, the membranes were incubated with rabbit antimouse IgG or swine antirabbit IgG conjugated with horseradish peroxidase (HRP) (1:2000-4000 in 1% (w/v) skim milk/PBS) (DAKO; Glostrup, Denmark) at RT for 1 h. Immunoreactive protein bands were 3982
Journal of Proteome Research • Vol. 9, No. 8, 2010
then visualized with SuperSignal West Pico chemiluminescence substrate(PierceBiotechnology;Rockford,IL)andautoradiography.
Results U937 monocytic cells (approximately 1 × 106 cells/mL) were cultured with or without 100 µg/mL COM crystals for 24 h to evaluate response of human monocytes to COM crystals as determined by changes in cellular proteome. After 24-h incubation, the culture supernatant was removed and the cells were washed several times with PBS. The cells were then subjected to morphological examination and proteome analysis. For morphological examination, crystal-cell interaction and morphological changes were evaluated using an inverted phasecontrast microscopy. After an exposure with COM crystals, U937 monocytes extended pseudopodia-like structure from the cells, unlike the untreated cells that remained typically round shape. Interestingly, COM crystals clearly interacted on the surface of monocytic cells (Figure 1). We selected the 24-h incubation time-point for subsequent proteome study because we observed some crystal-cell interaction and morphological changes at this time-point, whereas earlier time-points did not show obvious changes. In contrast, later time-points were associated with obvious cytotoxic effects with cell detachment. As the aim of this study was to observe changes in the cellular proteome as cellular response of monocytes to COM crystals, we thus focused our study to the 24-h time-point. For proteome analysis, the cells were lyzed and cellular proteins recovered from controlled and COM-treated cells were resolved by 2-DE (n ) 5 gels derived from individual culture flasks for each condition). Using equal amount of total protein (150 µg/sample/gel), Deep Purple fluorescence dye with a laser scanner detected approximately 800 protein spots in each 2-D gel (Figure 2). Spot matching, quantitative intensity analysis, and statistics revealed 22 differentially expressed protein spots between the two groups. Among these, 9 proteins were increased (1.60-3.09 folds), whereas the other 13 proteins were decreased (0.26-0.75 folds) in COM-treated cells. All of these altered protein spots were identified by Q-TOF MS and/or MS/ MS and their identities, identification scores, quantitative data, and other related information are summarized in Table 1. Some of the differentially expressed proteins including two up-regulated proteins and one down-regulated protein were randomly selected for validation by Western blot analysis. This conventional immunological method widely used for protein analyses nicely confirmed the proteomic data. Using GAPDH
a
21 22
19 20
18
17
15 16
13 14
12
9 10 11
8
5 6 7
4
1 2 3
HSP105 beta ALG-2 interacting protein 1 (Alix) Eukaryotic translation elongation factor 2 Chain A, crystal structure of the moesin ferm domain TAIL domain complex Chain A, pyruvate kinase M2 Lamin A/C isoform 2 Heterogeneous nuclear ribonucleoprotein H1 Esophageal cancer associated protein, isoform CRA_c Autoantigen La Beta-actin Chain A, human plasminogen activator inhibitor-2. Guanine nucleotide-binding regulatory protein alpha-inhibitory subunit Acyl-CoA thioester hydrolase Eukaryotic translation initiation factor 3 HSPC263 Proteasome alpha 3 subunit isoform 1 Nascent polypeptide-associated complex alpha subunit isoform b Leukocyte immunoglobulin-like receptor, subfamily B Prohibitin Proteasome (prosome, macropain) subunit, alpha type 6 Proteasome subunit HSPC Hydroxysteroid (17-beta) dehydrogenase 10 isoform 2
protein name
gi|4092058 gi|83715985
gi|4505773 gi|6755198
b
gi|119592630
gi|5031931
gi|6841176 gi|4506183
gi|1906670 gi|4503513
gi|183182
gi|1083506 gi|28336 gi|189545
gi|193787778
gi|35505 gi|5031875 gi|5031753
gi|8569616
gi|3970829 gi|6424942 gi|181969
NCBI IDa
NCBI ) National Center for Biotechnology Information.
spot no.
NA, 38 83, NA
87, 182 NA, 104
71,NA
NA, 43
NA, 111 NA, 66
100, 85 83, 132
NA, 98
NA, 53 NA, 89 95, 145
79, NA
132, 163 69, NA 141, 279
NA, 97
77, 59 NA, 49 114, 47
NA, 5 40, NA
46, 20 NA, 11
64, NA
NA, 6
NA, 10 NA, 5
36, 8 44, 8
NA, 9
NA, 3 NA, 9 33, 11
22, NA
37, 9 19, NA 46, 17
NA, 9
21, 20 NA, 1 28, 5
%cov (MS, MS/MS)b,c
NA, 1 10, NA
8, 4 NA, 2
5, NA
NA, 1
NA, 2 NA, 1
11, 2 10, 2
NA, 2
NA, 1 NA, 2 13, 3
13, NA
17, 4 9, NA 15, 5
NA, 3
14, 2 NA, 1 18, 1
8.60 6.73
5.57 6.34
4.80
4.52
4.90 5.19
0.4314 ( 0.0097 0.1066 ( 0.1066 0.0994 ( 0.0046
0.0243 ( 0.0123 0.5206 ( 0.0479
11.23 0.6872 ( 0.5780 29.84 0.1871 ( 0.0170 27.81 0.0479 ( 0.2020
28.06 0.0681 ( 0.0038 26.17 0.7045 ( 0.4850
0.1040 ( 0.0156 0.0451 ( 0.0115
41.01 0.1481 ( 0.0105 36.88 0.0748 ( 0.0058 6.84 5.17
0.2477 ( 0.0620
0.1103 ( 0.0081
41.01 0.1478 ( 0.0080 5.34
23.37 0.4124 ( 0.0237
0.1793 ( 0.0273 0.0232 ( 0.0146 0.1855 ( 0.0290
46.98 0.1105 ( 0.0110 42.13 0.0796 ( 0.0062 46.94 0.2613 ( 0.0126 6.68 5.22 5.46
0.0965 ( 0.0218 0.0526 ( 0.0062
0.0240 ( 0.0148
6.54 100.12 0.0697 ( 0.0061
31.19 0.0316 ( 0.0062 28.64 0.1808 ( 0.0178
0.0724 ( 0.0165 0.0064 ( 0.0041 0.1365 ( 0.0127
57.09 0.0234 ( 0.0097 65.15 0.0243 ( 0.0046 49.48 0.0789 ( 0.0136
8.00 6.40 5.89
8.92
5.42 6.13 6.41
0.1130 ( 0.0072
COM-treated
34.55 0.0706 ( 0.0093
control
0.0774 ( 0.0030 0.0306 ( 0.0049 0.2320 ( 0.0379
MW (kDa)
92.97 0.0447 ( 0.0133 96.65 0.0149 ( 0.0038 96.25 0.0980 ( 0.0193
pI
intensity (mean ( SEM)
0.36 0.74
0.57 2.08
0.63
0.60
3.05 0.31
0.70 0.60
0.75
1.62 0.29 0.71
0.35
3.09 0.26 1.73
1.60
1.73 2.06 2.37
ratio (COMtreated/ control)
0.010 0.027
0.002 0.037
0.002
0.038
0.021
