Proteomic analysis of human macrophages exposed to hypochlorite-oxidized low-density lipoprotein

Biochimica et Biophysica Acta 1794 (2009) 446–458

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a p a p

Proteomic analysis of human macrophages exposed to hypochlorite-oxidized low-density lipoprotein Jeong Han Kang a, Hyun Su Ryu a, Hyun Tae Kim a, Su Jin Lee a, Ung-Kyu Choi a, Yong Bok Park a, Tae-Lin Huh a, Myung-Sook Choi a, Tae-Cheon Kang b, Soo Young Choi c, Oh-Shin Kwon a,⁎ a b c

Department of Life Sciences and Biotechnology, School of Life Sciences, Kyungpook National University, Daegu, 702-701, Republic of Korea Department of Anatomy, College of Medicine, Hallym University, Chunchon, 200-702, Republic of Korea Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon, 200-702, Republic of Korea

a r t i c l e

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Article history: Received 15 July 2008 Received in revised form 14 October 2008 Accepted 14 November 2008 Available online 6 December 2008 Keywords: THP-1 cell Macrophage Proteome Oxidized low-density lipoprotein Two-dimensional gel electrophoresis

a b s t r a c t The invasion of monocytes through the endothelial wall of arteries and their transformation from macrophage into form cells has been implicated as a critical initiating event in atherogenesis. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) treatment, and can be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). To identify proteins potentially involved in atherosclerotic processes, we performed a proteomic analysis of THP-1 macrophages exposed to oxLDL generated by treatment with native LDL with hypochlorous acid/hypochlorite (HOCl/OCl−). We detected more than a thousand proteins, of which 104 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF) and the NCBI database. The largest differences in expression were observed for bifunctional purine biosynthesis protein, vacuolar protein sorting 33A, breast carcinoma amplified sequence, adenine phosphoribosyltransferase, and tropomyosin alpha 3 chain. Interestingly, many apoptotic proteins such as lamin B1, poly (ADP-ribose) polymerase, Bcl-2 related protein A1 and vimentin were identified by MALDI–TOF analysis. Identities were confirmed by matching the sequence of several tryptic peptides using MALDI–TOF/TOF MS, Western blot analyses and immunofluorescent microscopy. The data described here will contribute to establishing a functional profile of the human macrophage proteome. Furthermore, the proteins identified in this study are attractive candidates for further biomarkers involved in the pathogenesis of atherosclerosis. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as important early events in the pathogenesis of atherosclerosis [1,2]. The upregulation of cell-adhesion molecules greatly increases the adherence of blood monocytes to the endothelium. After adhesion, monocytes migrate into the subendothelial space where they differentiate into macrophages. Uptake of oxidized low-density lipoprotein (oxLDL) by the macrophage through scavenger receptors will lead to foam cell formation [3]. The THP-1 human monocytic cell line is a well-characterized model system with which to study the transformation of macrophages to foam cells [4]. THP-1 monocytes can be induced with phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype, and the

Abbreviations: PMA, phorbol 12-myristate 13-acetate; oxLDL, oxidized low-density lipoprotein; DAPI, 4′-6-Diamidino-2-phenylindole; DIC images, Differential Interference Contrast image ⁎ Corresponding author. Tel.: +82 53 950 6356; fax: +82 53 943 2762. E-mail address: [email protected] (O.-S. Kwon). 1570-9639/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.11.015

resulting macrophages can then be converted to foam cells following treatment with oxLDL [5]. The mechanisms for the generation of oxLDL in vivo are not well defined. Experimentally, LDL oxidation in vitro is usually performed by incubating the lipoprotein with high concentrations of free metal ions such as copper and iron. Oxidation of the LDL-lipid moiety leads to the generation of lipid peroxidation products. Although copper has been shown to be present in atherosclerotic lesions [6], the significance of trace metal mediated LDL oxidation in vivo has been called into question [7]. There is little increase in markers of copper- and hydroxyl radical-mediated protein damage in either lesion LDL fatty streaks, suggesting that free metal ions are unlikely to be involved early in atherogenesis. In contrast, the involvement of chlorinated oxidants which produce little modifications in the LDL–lipid moiety and preferentially affect its protein moiety, notably apolipoprotein B, appears more likely to occur in vivo. This pathway involves myeloperoxidase, which catalyses the production of hypochlorous acid/hypochlorite (HOCl/OCl−) from hydrogen peroxide and chloride ions in activated neutrophils and monocytes [8,9]. Hypochlorous acid generated by myeloperoxidase converts tyrosine to 3-chloroutyrosine and cholesterol to chlorinated compounds. An increasing number of

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studies have demonstrated that LDL oxidized by hypochlorite in vitro is able to mimic fundamental atherogenic processes including adhesion to endothelial cells, activation of phagocytes with increased formation of reactive oxygen species, and formation of foam cells [10–12]. The products of myeloperoxidase were detected at elevated levels in a lesion of LDL and all stages of atherosclerosis, suggesting that reactive tyrosyl radicals generated by myeloperoxidase represented one pathway of LDL oxidation in vivo. Despite studies of the critical roles of oxLDL in foam cell formation and atherogenesis, relatively little is known about the mechanisms by which oxLDL activates macrophages. In an attempt to increase our understanding of these mechanisms, we initiated a search for proteins that are specifically up- or downregulated in oxLDL activated human THP-1 macrophages compared with nonstimulated cells. Gene expression studies have identified many genes to be either upregulated or downregulated in human atherosclerosis. However, protein expression patterns do not always reflect differential gene expression patterns. Furthermore, protein functions can also be influenced by post-translational modifications. Proteomics offers a unique means for analyzing the expressed genome, to provide important clues to the mechanisms involved in this complex process [13]. Recently, Conway and Kinter [14] reported a proteomics study to identify proteins associated with the foam cell formation. They used murine macrophages that had been chronically exposed to copper-oxLDL. However, copper-mediated LDL oxidation seems not to be pathologically relevant. Therefore, in the present study, we investigated the effects of oxLDL generated by treatment with HOCl on the transition of macrophages into foam cells at the protein levels. The associations of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) and database interrogations allowed us to identify various proteins differentially expressed in monocyte-derived macrophage THP-1 cells. Thus, we identified various proteins more especially implicated in regulation of apoptosis, growth, cell mobility, and signal transduction. 2. Materials and methods 2.1. Isolation and oxidation of LDL LDL (density = 1.020–1.063 g/ml) were isolated by sequential flotation ultracentrifugation from human plasma, as described previously [15]. Before oxidative modification, the LDL was dialyzed against phosphate-buffered saline (PBS), filtered through a 0.2 μm Millipore membrane (Millipore, Bedford, MA, USA), and stored in PBS containing 1 mM EDTA at 4 °C. Hypochlorite modification of LDL was essentially performed according to Vicca et al. [16]. LDL oxidation was induced for 30 min at 37 °C with 40 mM HOCl (i.e. corresponding to oxidant/protein molar ratios (R) of 2000/1). OxLDL was dialyzed overnight against 10 mM phosphate-buffered saline (PBS), at pH 7.4. 2.2. Cell culture and treatment Human THP-1 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (v/v), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.15% sodium bicarbonate (w/v), 0.45% glucose (w/v), 1 × 10− 5 M β-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 μg/ml), and kept at 37 °C in an atmosphere of 5% CO2. The cell medium was replaced every 2 days for the duration of the culture before the beginning of cell treatment. Cell were seeded at a density of 5 × 105 cells/ml in medium containing 100 nM PMA (Sigma, St. Louis, MO for 48 h. The medium was then replaced by culture medium with 100 μg/ml of vehicle (50 mM Tris, pH 7, 150 mM NaCl, 0.3 mM EDTA), and LDL(50 μg/ml), which was oxidized with HOCl. Macrophages were transformed into foam cells by incubation with the presence or absence of oxLDL in culture medium for 24 h. Cells were washed three times with PBS, fixed for 30 min with 5% formalin solution in PBS,

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stained with Oil Red O for 30 min, and counterstained with DiffQuick staining kit (IMEB, Inc.). Finally, the cells were examined by light microscopy. Human monocyte-derived macrophage (HMDM) were kindly provided from Dr. In-San Kim (School of Medicine, Kyungpook National University, Korea) [17]. 2.3. Extraction of proteins from monocyte-derived macrophages Cells were washed three times with 25 mM Tris, pH 7.4 and scraped in buffer containing 50 mM Tris pH 8.6, 10 mM EDTA, 65 mM dithiothreitol (DTT) and a protein-inhibitor cocktail tablet (complete TM; Roche Diagnostics, Meylan, France). Cells were harvested at 1400 ×g for 10 min. Harvested cells were mixed with lysis buffer containing 9.5 M urea, 2% CHAPS, 0.8% carrier ampholytes (Pharmalyte; Amersham Pharmacia Biotech, Piscataway, NJ), and 1% DTT and a protein-inhibitor cocktail tablet. After incubation at room temperature for 1 h and sonication for 3 min, the samples were centrifuged for 15 min at 10,000 ×g, and the supernatants were directly applied to a 180 mm immobilized pH gradient (IPG) strip for isoelectric focusing (IEF). Protein concentrations were determined with commercial Bradford reagent (Bio-Rad, Hercules, CA), and the samples were stored at −70 °C until use. 2.4. Two-dimensional gel electrophoresis (2-DE) 2-DE was performed in an Ettan IPGphor IEF System (Amersham Pharmacia Biotech) using 180 mm pH 3–10 and pH 4–7 Immobiline DryStrips (180 mm × 3 mm × 0.5 mm) for the first dimension, and 12% SDS–polyacrylamide gels for the second dimension. SDS–PAGE was performed in an Ettan DALTsix Larger Vertical System (Amersham Pharmacia Biotech). IPG–IEF can be simplified by the use of the integrated IPGphor system, in which rehydration with sample solution and IEF are performed in a one-step procedure. Initial rehydration was for 12 h at 20 °C. IEF was then carried out using the following voltage program: 500 V (gradient over 1 h), 1000 V (gradient over 1 h), 8000 V (fixed for 4 h) at 50 μA/strip. The IPG strips were equilibrated for 30 min in 125 mM Tris (pH 6.8) containing 6 M urea, 30% glycerol (v/v), 2% SDS (w/v), and 65 mM DTT and then for a further 30 min in the same buffer, except that DTT was replaced with 260 mM iodoacetamide. The IPG strips were then sealed with 0.5% agarose in SDS running buffer at the top of slab gels (260 mm × 200 mm × 1.5 mm). The second-dimension electrophoresis was performed in the Ettan DALTsix Larger Vertical System at 180 V/gel and room temperature for 8 h. The gels were then fixed with 50% ethanol containing 3% phosphoric acid for 30 min and stained with 0.02% Coomassie Brilliant Blue (CBB)–G250, 3% phosphoric acid, 17% ammonium sulfate, 34% ethanol. Relative molecular weights were determined by simultaneously running standard protein markers (MBI Fermentas, Hanover, MA) in the range 10–170 kDa. The pI values used were those given by the supplier of the immobilized pH gradient strips. Excess dye was washed from the gels with distilled water and the gels were scanned with a UMAX PowerLook 1120 scanner (UMAX Technologies, Dallas, TX). Protein spots were outlined (first automatically and then manually) and quantified using PDQUEST software (Bio-Rad). Three batches of cell proteins extracted from untreated cells and oxLDLtreated cells were subjected to 2-DE. Data of 2-DE experiments are shown as means ± standard deviation (SD). For the differential analysis, statistical significance was estimated with Student's t-test. Values of p b 0.05 were considered significant. 2.5. MALDI–TOF MS and MALDI–TOF/TOF MS To identify the protein spots on gel pieces, they were excised from preparative 2-DE gels, and cut into 1–2 mm pieces. These were added to 100 μl of 25 mM NH4HCO3/50% acetonitrile, incubated for

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10 min to remove Coomassie Brilliant Blue, then rinsed thoroughly. The gel pieces were then dehydrated and dried thoroughly in a vacuum centrifuge for a few minutes. The dried gel pieces were rehydrated with 20 μl of 50 mM NH4HCO3 (pH 8) containing 20 μg/ ml trypsin (Promega, Madison, WI) and the proteins in the gel pieces were digested overnight at 37 °C. After digestion was complete, the supernatant was transferred to another tube, and the gel pieces incubated with 20 μl of 50% acetonitrile/5% formic acid for 10 min at room temperature. The extract was transferred to the primary supernatant, and the extraction procedure repeated once more. The extracted digests were evaporated to dryness in a vacuum centrifuge. The digests were redissolved in 2 μl of 0.1% trifluoroacetic acid (TFA). The solution was agitated on a vortex mixer and centrifuged. The supernatant was desalted with ZipTip C18 (Millipore, Billerica, MA). Supernatant (1 μl) was mixed with 1 μl of 10 mg/ml α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% TFA and the mixed solution was spotted onto a 96-spot MALDI target. MALDI–TOF analyses were performed on an Applied Biosystems 4700 proteomics analyzer (Applied Biosystems, Foster City, CA). The MALDI–TOF mass spectrometer was operated in a positive-ion, delayed-extraction (200 ns delay time) reflector mode using external calibration. The presented spectra generally represented the sum of 200 laser shots under 20 kV voltage, 67% grid. Results were analyzed using 4000 Series Explorer software version 4.0 (Applied Biosystems) to obtain accurate masses (±50 ppm) for all the peptides in the tryptic digest. The resulting peptide mass fingerprints, together with the pI and relative molecular mass values (estimated from 2-DE gels), were used to search the Swiss-Prot (updated 10/10/2007, number of entries in the database: 285,335) and NCBI protein databases (updated 10/10/ 2007, number of entries in the database: 553,940) with a special search tool [MS-FIT from Protein Prospector V 4.27.2 (http:// prospector.ucsf.edu)], which compares the experimentally determined tryptic peptide masses with theoretical peptide masses calculated for proteins contained in the Swiss-Prot or NCBI protein databases. Search parameters were ±50 ppm peptide mass tolerance and one maximum missed cleavage. The search parameters allowed for N-terminal pyroGlu, oxidation of Met, and N-terminal acetylation. Some proteins were subjected to MALDI–TOF/TOF MS (4700 Proteomics Analyzer, Applied Biosystems) with the objective of identifying the respective proteins using a database search. Data were acquired in the MALDI reflector mode with five spots of calibration standard (ABI4700 Calibration Mixture). All mass spectra were searched using the MASCOT (Matrix Science, London, U.K.) V 2.1 engine against the NCBI database. The data base was searched using the following parameters: enzyme, trypsin; missed cleavages, 1; peptide tolerance, 50 ppm; MS/MS tolerance, 0.2 Da; peptide charge, 1+, 2+, and 3+; fixed modification, carbamidomethyl (C); variable modifications, N-acetyl (Protein), oxidation (M), Pyro_glu (N-termQ), Pyro_glu (N-term E). 2.6. Western blot analysis Whole protein extracts (20–30 μg) from treated cells were subjected to 12% SDS–PAGE. Proteins were then transferred to a Protran nitrocellulose transfer membrane (Schleicher & Schuell BioScience, Dassel, Germany). For lamin B1 and annexin 1 immunodetection, membranes were blocked for 1 h at room temperature in PBS containing 0.5% Tween-20 and 5% nonfat dried milk, and then incubated with a goat polyclonal anti-human lamin B1 antibody (1:1000 in blocking buffer, 2 h at 4 °C) and a mouse monoclonal antibovine annexin 1 antibody (1:3000 in blocking buffer, 2 h at 4 °C), respectively. The membrane was incubated with the appropriate secondary antibody (1 h at room temperature) and the proteins were visualized with an enhanced chemiluminescence (ECL) detection system (Amersham Bioscience, Little Chalfont, Buckinghamshire, UK).

The densities of immunoreactive bands were measured by the Quantity One 1D image analysis software program (Bio-Rad). 2.7. Immunofluorescent microscopy Cells were cultured and treated on poly-L-lysine-coated coverslips before being fixed in 4% paraformaldehyde (10 min at room temperature). After a 5 min wash with 2 mg/ml glycine in PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. After two washes with PBS, the cells were blocked with 10% normal goat serum in PBS for 1 h in a humidified chamber. Then, the cells were incubated with vimentin human anti-goat-polyclonal antibody and lamin B1 human anti-rabbit-polyclonal antibody (1:100, diluted in PBS containing 2% normal goat serum) overnight at 4 °C with gentle shaking. The cells were then washed three times before being incubated with Alexa-488 labeled rabbit anti-goat IgG and Alexa-647 labeled goatanti-rabbit IgG secondary antibodies (Molecular Probes, Carlsbad, CA) for 60 min and with DAPI (to stain the nuclei) for 3 min at room temperature. Finally, the cells were washed five times with PBS containing 0.05% Tween 20 and 1% BSA; mounted in anti-fade and coverslips were sealed with nail polish. Slides were examined and scanned on a fluorescence microscope. For selected images, both the fluorescent and the DIC images were collected and merged electronically. 2.8. PubGene analysis To identify potential biological relationships among differential expression genes, we used the recently developed PubGene database (http://www.pubgene.org) [18] to identify citation-based gene-network associations. PubGene extracts gene-to-gene cocitations from the over 10 million Medline records available and presents their relationship as a network. As this tool is only available for human genes, we translated the human protein name to human gene symbols. Also, we used protein expression ratios as input for the PubGene tool. A tab-delimited text file with two columns, one with official gene symbols and one with protein expression ratios, was prepared and submitted for online analysis. To score gene neighborhoods, we used the parameter settings “Score depth” = 2, “Neighborhood size” = 3, and “Upregulation” as score criteria. In this case, “Upregulation” corresponded to high negative logarithms, i.e., low P values. For “Calculation scheme”, we used both “By individual gene” and “By gene associations”. 3. Results 3.1. 2-DE patterns of human monocyte-derived macrophage THP-1 cells The effects of oxLDL exposure on the macrophages were studied. THP-1 cells were induced to differentiate into macrophage-like cells in vitro in the presence of PMA for 48 h, and the cells were then incubated with oxLDL(50 μg/ml) for 24 h. Foam cell formation was confirmed by oil red-O staining as described in Materials and methods (Fig. 1). Compared with macrophages (Fig. 1A), many lipid-filled vacuoles appeared in the cytoplasm of oxLDL-treated THP-1 macrophages (Fig. 1B). The proteomes of the macrophages cultured in the absence and presence of oxidized LDL were compared. We used 2-DE in conjunction with quantitative image analysis and mass spectrometry to investigate changes in the protein expression profiles of macrophages treated with HOCl–oxLDL. Three gels per sample were processed simultaneously and analyzed with PDQUEST 2-D software (Bio-Red). Fig. 2 shows 2-DE images of monocyte-derived macrophage THP-1 cells. More than 2500 spots were detected. Of those proteins with molecular weight ranging from 14 kDa to 97 kDa, 60% had acidic pIs, whereas 40% of polypeptide spots fell within the alkaline region. There were few protein spots over 97 kDa, and no spots were detected with a pI N 9.5.

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HOCl–oxLDL. On the other hand, lamin B1 and Bcl2 related protein A1 decreased threefold and twofold, respectively. Thus, in all cases, the Western blot results confirmed the changes observed with proteomic analysis. 3.3.3. Immunofluorescent microscopy To examine the change in vimentin and lamin B1 protein expression after HOCl–oxLDL treatment, we used immunofluorescent confocal microscopy. The intracellular vimentin and lamin B1 in macrophages were stained with Alexa-488 and Alexa-647-labeled antibodies, respectively. The DNA-specific dye DAPI was included to reveal nuclear morphology. As shown in Fig. 4A, treatment of macrophages with HOCl–oxLDL seemed not to affect the morphology, but vimentin filaments expression was upregulated in a dosedependent manner. The intermediate filament component vimentin, which was not detectable throughout the cytoplasm, had considerably increased after oxLDL treatment. Vimentin was gathered in a dense ring around the nucleus. On the other hand, lamin B1 protein was downregulated. Lamin B1 is highly expressed on nuclear membrane, but HOCl–oxLDL treatment caused considerable decrease. We also investigated the change in vimentin and lamin B1 protein expression in human monocyte-derived macrophage after HOCl–oxLDL treatment (Fig. 4B). Expression of vimentin was upregulated in a dose-dependent manner, whereas lamin was dramatically downregulated. These data are perfectly consistent with the results from PMA-induced THP-1 cells (Fig. 4A) and Western blot analysis (Fig. 3B and C).

Fig. 1. Oil Red O staining of THP-1 macrophages. THP-1 monocytes were induced to differentiate in vitro by PMA treatment for 48 h, and then the macrophages were incubated with oxLDL for 24 h to form foam cells. (A) oxLDL-untreated THP-1 macrophage and (B) HOCl-oxLDL-treated THP-1 macrophages were stained with Oil Red O, in order to access the degree to which lipid accumulation had taken place (magnification ×400).

3.3.4. PubGene analysis We also performed a Literature gene networks with PubGene analysis. The Expression Literature gene networks tool is designed to aid in the task of categorizing and gathering relevant information on genes implicated in an expression analysis study [18]. The literature network obtained from PubGene contained 19 of the submitted genes (17%). As shown in Fig. 5, we found the top-scoring networks from search using the Expression Literature gene networks tool. 4. Discussion 4.1. Classification of the differentially expressed proteins

3.2. Identification of differentially expressed proteins by MS Protein spots from the 2-DE gel were subjected to trypsin digestion and MALDI–TOF analysis. At least 150 spots were differentially expressed more, and proteins were successfully identified for 104 of these. Table 1 lists the most strongly differentially expressed proteins, which were up- or downregulated more than twofold by the oxLDL treatment. More than 95% of spots had a sequence coverage exceeding 10%. Identification was validated by agreement between the apparent Mr and pI determined from 2-DE gels and the theoretical values of the identified proteins (ΔMr b 20% or ΔpI b 0.5). 3.3. Confirmation of differentially expressed proteins 3.3.1. MALDI–TOF/TOF To confirm MALDI–TOF results from identified spots and obtain unambiguous identification for those spots that were not identified by their PMF, MALDI–TOF/TOF analysis was additionally performed on the same spots. Several selected protein spots are identified with high confidence, and the results are summarized in Table 2. 3.3.2. Western blot analysis From the protein list given in Table 1, four proteins were selected for confirmation experiments using Western blot analysis. As shown in Fig. 3, the expressions of annexin I and vimentin were increased by

Human THP-1 monocytic cells can be differentiated into macrophages in the presence of PMA. In this study, the macrophage-like THP-1 was incubated for 24 h and treated with oxLDL generated by treatment with HOCl, and the differentially expressed proteins were identified by 2-DE and MALDI–TOF. The macrophage is known to be one of the most dynamic cells in the body. Therefore, it is not surprising that such a wide rage of proteins involved in various cellular pathways was identified. Of all the differentially expressed spots, 104 showed consistent differences in their expression patterns. The differentially expressed proteins of chlorinated oxLDL-treated macrophages in this study are quite different to those of copper-mediated oxidation [14]. The differences of two studies are not only induction method but also duration of oxLDL exposure. In consequence, only few differentially expressed proteins were overlapped. The differentially expressed proteins were classified in terms of their physiological functions using information from PubMed (www. ncbi.nlm.nih.gov/PubMed) and the Swiss-Prot/TrEMBL protein knowledgebase (http://au.expasy.org/sprot). As shown in Fig. 6A, a large number of the proteins identified were metabolic proteins, and other groups were involved in cell growth or maintenance and the many other proteins. The cellular distribution of the proteins identified is also shown in the Fig. 6B. The most commonly identified location was the cytoplasm (38%), and the next most populated nucleus (14%).

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4.1.1. Metabolic proteins Various proteins involved in the regulation of several metabolic pathways were identified. A large group of the proteins identified were metabolic proteins (22%), such as aldehyde dehydrogenase, and carbonyl reductase. The aldehyde dehydrogenase (ALDH) family is a family of several isoenzymes that are important in cellular defense against exogenous toxic aldehydes and endogenous aldehydes, such as those derived from lipid peroxidation [19]. The latter appear to influence cell growth and differentiation in some tumor cell lines. In this study, ALDH was downregulated by HOCl–oxLDL treatment. There is evidence suggesting that the catalytic properties of this enzyme are changed during ontogenesis, and that aging is accompanied by their reduced activity [20]. It has been proposed that the expression of this enzyme is controlled by aldehydes, as well as by factors inducing the free radical process. Thus, a decrease in the rate of endogenous aldehyde utilization in the macrophage may be one of the key factors responsible for apoptosis-inducing signals.

NADPH-dependent carbonyl reductase is a ubiquitous oxidoreductase that metabolizes prostaglandins, steroids, aromatic and aliphatic aldehydes and ketones, and quinines from polycyclic aromatic hydrocarbons [21]. Several lines of evidence suggest that carbonyl reductase represents a significant pathway for the detoxification of reactive aldehydes derived from lipid peroxidation and that this protein in humans is essential for neuronal cell survival and to confer protection against oxidative stress-induced brain degeneration [22]. Our results show that carbonyl reductase was downregulated twofold in human macrophages treated with HOCl–oxLDL. Under conditions of oxidative stress, accumulation of abnormal protein adducts of reactive aldehydes overwhelms antioxidant defenses, resulting in the cellular growth inhibition and induction of apoptosis. 4.1.2. Proteins for cell growth and maintenance Incubation of cultured cells with toxic concentrations of oxLDL induces progressive changes in cell morphology. The cytoskeletal or related proteins were downregulated in PMA-induced THP-1 cells

Fig. 2. Protein expression maps of HOCl–oxLDL-treated THP-1 macrophages. PMA-induced THP-1 cells were incubated for 24 h with HOCl–oxLDL. (A) Proteins from whole cells were separated on a pH 3–10 IPG strip in the first dimension and on an SDS–polyacrylamide (12%) gel in the second dimension. The middle sections show a representative Coomassiestained gel of proteins derived from control cells. Around the typical control gel, enlarged areas of gels derived from control cells, cells exposed to HOCl–oxLDL are shown. (B) Proteins were separated on a pH 4–7 IPG strip followed by SDS–(12%) PAGE. Protein spots significantly affected by HOCl–oxLDL treatments are marked by arrows. The numbers indicated on the gels correspond to the gel numbers given in Table 1.

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Fig. 2 (continued).

after stimulation with HOCl–oxLDL. Of the total number of proteins identified, 14% are involved in cell growth and maintenance. Although the significance of apoptosis in atherosclerosis remains unclear, it has been proposed that apoptotic cell death contributes to plaque instability, rupture, and thrombus formation. Numerous studies indicate that oxLDL cytotoxicity is partly attributed to induction of apoptosis in many cell types [23–25]. However, some macrophages undergo necrosis when exposed to oxLDL. Both types of cell death have been shown to occur in vivo in human atherosclerotic plaques [26]. In this study, we found that most of the downregulated proteins in HOCl–oxLDL-treated macrophage cells are associated with apoptosis. Stephanie et al. reported that the mechanism of HOCl–oxLDLmediated apoptosis involves the two pathways of apical caspase activation in monocytic U937 cells: death receptor-mediated caspase 8 and mitochondria-mediated caspase [16,27]. This converges in activation of executing caspases, including caspase 3, and apoptosis [16]. In this study, we detected the apoptosis related proteins, lamin B1, poly (ADP-ribose) polymerase (PARP) and Bcl-2 related protein A1. The nuclear lamina is a dense intermediate filament polymer underlying the nuclear envelope of higher eukaryotes [28,29]. It interacts closely with both the inner membrane of the nuclear envelope and the underlying chromatin, and these associations are

thought to be important in maintaining the stability of the interphase nucleus. Interactions between peripheral heterochromatin domains and the nuclear lamina may be important in the control of gene expression and gene silencing [30]. B-type lamin is to a large extent ubiquitously expressed in all animal cells. Here, the expression of lamin B1 was downregulated fourfold by HOCl–oxLDL treatment. Moreover, we confirmed by Western blotting and confocal microscopy, as shown in Fig. 3B and Fig. 4, respectively. The precise roles of lamin in many cellular processes including chromatin organization, transcription regulation, and aging remain to be determined. However, the information will lead to further understanding of the functions of lamins in atherosclerosis. Proteins of the PARP family have wide array of functions, covering virtually every aspect of DNA metabolism and function, most notably with the response to DNA damage and oxidant induced cell death [31]. PARP10 exists in both cytoplasm and nucleus, but only nucleolar PARP10 acquires Cdk dependent phosphorylation through late-G1 to S phase, and from prometaphase to cytokinesis in the nucleolar organizing regions. The PARP activity of PARP10 depends on phosphorylation by Cdk2/cyclinE in vitro. Cdk phosphorylated PARP10 is absent in growth-arrested cells. Therefore, PARP10 functions in cell proliferation may serve as a marker for proliferating cells [32]. Our

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Table 1 Differentially expressed proteins in oxLDL-treated THP-1 macrophages No.

Protein

Localization

MOWSE score

PI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Metabolism Protoporphyrinogen oxidase Glycogenin-1 Geranylgeranyl pyrophosphate synthetase Pyrophosphatase Carbonyl reductase Steroid 21-hydroxylase Glutaryl-CoA dehydrogenase Acetoacetyl-CoA thiolase Pseudouridine synthase I Acyl-coenzyme A dehydrogenase Thymidine kinase 2 Pyridoxal phosphate phosphatase Adenylate kinase isoenzyme 6 Protein phosphatase 2C gamma isoform Apolipoprotein A-IV precursor Spermidine aminopropyltransferase Dimethylargininase 2 Carbonyl reductase Glycogenin-1 Aldehyde dehydrogenase Bifunctional purine biosynthesis protein Aldehyde dehydrogenase 2 Aldolase C Carbonyl reductase

Mitochondrial Cytoplasmic Cytoplasmic Mitochondrial Cytoplasmic Membrane Mitochondrial Mitochondrial Nuclear Mitochondrial Mitochondrial Cytoplasmic Nuclear Cytoplasmic Extracellular Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Mitochondrial Cytoplasmic Cytoplasmic

1110 798 1440 1790 5780 1098 6789 1260 610 7.646e + 04 769 1300 6900 4670 1560 755 5790 4100 1053 1.134e + 04 2300 3200 1560 7980

8.4 5.3 5.8 6.7 5.8 7.3 8.3 9.0 7.6 7.9 8.7 6.0 4.5 4.3 5.3 4.9 5.7 5.8 5.3 6.0 6.3 6.4 6.4 5.8

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Cell growth/maintenance Ras GTPase-activating protein 3 Oviductal glycoprotein Dysbindin Lamin B1 26S proteasome non-ATPase regulatory subunit 12 Nuclear protein HCC-1 Golgi/nuclear envelope protein Tropomyosin 2 Poly (ADP-ribose) polymerase 10 GRP 78 Tropomyosin alpha 3 chain Tropomyosin beta chain Tropomyosin alpha 3 chain EPB4.1 Actin-related protein 3 Arylsulfatase E precursor

Membrane Extracellular Cytoplasmic Membrane Cytoplasmic Nuclear Membrane Cytoskeleton Nuclear Endoplasmic reticulum Cytoskeleton Cytoskeleton Cytoskeleton Nuclear Cytoplasmic Golgi apparatus

1840 1930 555 2045 340 900 1.134e + 04 1760 1450 1690 2100 1.334e + 04 1670 340 650 1300

41 42 43 44 45 46 47 48 49 50

Immunoresponse Interferon regulatory factor 8 Sarcoma antigen NY-SAR-88 Immunoglobulin heavy chain variable region V beta immunoglobulin Tumor necrosis factor, alpha-induced protein 1 MHC class I antigen NY-REN-41 antigen Breast carcinoma amplified sequence NF-kappa-B essential modulator Annexin A1

Nuclear Cytoplasmic

51 52 53 54 55 56 57 58 59

Signal transduction Inositol-trisphosphate 3-kinaseA Ras-related protein Rap-2c Insulin-like growth factor binding protein 1 Frizzled 5 precursor Phosducin-like protein Calsenilin Annexin A3 Guanine nucleotide binding protein Bcl-2-related protein A1

60 61 62 63 64 65 66

Transport protein ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit precursor Extracellular Beta-1,3-galactosyltransferase 3 Vacuolar protein sorting 33A Mitochondrial Carnitine O-palmitoyltransferase II Membrane Cytochrome P450 26B1 Cytoplasmic Alanine-glyoxylate aminotransferase 2 Mitochondrial Tocopherol-associated protein 2 Membrane

MW Accession (kDa) no. P50336 P46976 O95749 BAA84701 O75828 AAB59440 Q92947 P24752 Q9Y606 CAI22389 O00142 Q96GD0 Q9Y3D8 O15355 P06727 P52788 O95865 O75828 P46976 P49189 P31939 AAT41621 P09972 O75828

12 16 12 18 26 15 19 15 11 18 31 14 25 16 16 11 23 17 12 13 29 25 17 15

3 ± 0.3 −2 ± 0.3 −2.5 ± 0.5 −2.5 ± 0.5 −3.5 ± 0.2 −2.2 ± 0.3 3 ± 0.6 2 ± 0.5 −2.7 ± 0.3 2.2 ± 0.2 2.1 ± 0.3 −2 ± 0.5 −2 ± 0.2 3.5 ± 0.4 2.3 ± 0.2 −4.1 ± 0.4 −4 ± 0.2 −2 ± 0.4 −2 ± 0.3 −2 ± 0.3 −45 ± 3 −4 ± 0.7 −2 ± 0.2 −2.1 ± 0.3

6.9 95 9.1 72 4.6 39 5.1 66 7.5 52 6.1 23 5.6 54 4.7 32 4.9 100 5.1 72 4.7 32 4.7 32 4.7 32 5.4 97 5.6 47 6.5 65

Q14644 Q12889 Q96EV8 P20700 O00232 P82979 AAM95335 P07951 Q53GL7 P11021 P06753 P07951 P06753 P11171 P61158 P51690

10 18 19 31 11 15 25 17 11 24 16 14 22 11 22 25

−2.5 ± 0.2 2.5 ± 0.5 −2.5 ± 0.5 −2 ± 0.2 3 ± 0.3 2.9 ± 0.5 −2.4 ± 0.4 −2.1 ± 0.5 −2 ± 0.3 2 ± 0.3 −2.2 ± 0.5 −2 ± 0.4 −12.5 ± 3 −2 ± 0.4 −3.5 ± 0.6 −5.4 ± 0.7

Cytoplasmic Cytoplasmic Cytoplasmic

200 1780 636 1008 1609 545 459 1576 1398 4560

6.4 9.4 7.9 8.3 8.3 4.7 4.8 5.1 5.6 6.6

48 30 13 10 36 15 26 19 48 38

Q02556 AAO65178 CAD60381 CAA41621 Q13829 AAD02036 AAD42874 Q8TDM0 Q9Y6K9 P04083

11 24 56 56 15 26 27 20 13 32

2 ± 0.3 4 ± 0.2 7.5 ± 0.8 5.5 ± 0.6 2.1 ± 0.2 −5.7 ± 0.7 −3.3 ± 0.5 −19 ± 3 −2 ± 0.2 2 ± 0.2

Cytoplasmic Membrane Extracellular Membrane Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic

1933 874 1560 430 1770 555 1580 1880 1980

7.6 4.9 5.6 8.7 4.7 5.2 5.6 6.2 5.3

51 20 13 64 34 29 36 44 20

P23677 Q8BU31 CAA33110 Q13467 Q13371 Q9Y2W7 P12429 P38405 Q16548

13 46 44 19 18 22 25 14 27

−2 ± 0.2 −2.5 ± 0.3 −2.5 ± 0.5 2 ± 0.3 2 ± 0.4 −3.7 ± 0.5 −2.1 ± 0.5 −2.8 ± 0.5 −2 ± 0.2

1700 1560 2469 1789 708 1320 2790

5.2 7.7 6.5 8.4 8.7 8.1 5.8

56 39 67 73 57 57 46

P06576 O75752 Q96AX1 P23786 Q9NR63 Q9BYV1 Q9UDX4

32 14 19 13 16 11 19

2 ± 0.3 −4 ± 0.6 −26 ± 3 4.2 ± 0.5 3.8 ± 0.7 4.6 ± 0.8 −2.5 ± 0.4

Membrane Membrane

50 39 34 31 30 55 48 45 44 50 31 31 20 59 45 41 29 30 39 53 64 56 39 30

Sequence Change coverage (%) fold

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453

Table 1 (continued) No.

Protein

Localization

MOWSE score

PI

67 68 69

Multidrug resistance-associated protein Sodium-calcium exchanger isoform Adenine phosphoribosyltransferase

Mitochondrial Cytoplasmic Membrane

1680 1610 1790

6.5 18 5.0 100 5.8 19

AAB71757 AAD26362 P07741

32 19 52

−2 ± 0.3 −2.1 ± 0.4 −17 ± 4

70 71 72 73 74 75 76 77

Transcription regulation FUSE binding protein 2 TGIF2LX mRNA turnover protein 4 homolog Eukaryotic translation initiation factor 4E Tripartite motif-containing 45 Pre-B-cell leukemia transcription factor-2 HLA-B associated transcript 1 Polyamine-modulated factor 1

Cytoplasmic Golgi apparatus

1100 990 720 2730 1316 1770 600 1200

6.8 9.1 8.3 6.0 8.3 7.2 4.8 5.0

86 26 27 25 64 45 18 19

AAC50892 Q8IUE1 Q9UKD2 AAH12611 Q9H8W5 P40425 CAI18284 Q5TCK0

14 17 33 18 11 18 23 51

2 ± 0.3 2 ± 0.4 2 ± 0.3 −2 ± 0.3 −2.5 ± 0.3 −10 ± 2 −2 ± 0.3 2 ± 0.5

78 79 80 81 82 83 84

Binding protein Unnamed protein product Mesoderm induction early response 1 CD2 tail binding protein Syntaxin-binding protein 3 Vimentin Vimentin Vimentin

Extracellular Cytoplasmic Nuclear Cytoplasmic

1.134e + 03 860 550 1200 2.134e + 04 3744 1678

5.0 4.3 4.5 8.3 5.1 4.8 5.1

83 53 37 67 53 41 53

BAA91700 CAI23428 O95400 O00186 P08670 AAA61281 P08670

13 15 14 10 37 24 28

5.5 ± 0.5 7 ± 0.7 −2 ± 0.4 −2.3 ± 0.7 3.3 ± 0.5 −3.7 ± 0.3 2.5 ± 0.3

85 86 87 88

Protein folding Chaperonin containing TCP1 Protein disulfide-isomerase A3 Protein disulfide-isomerase A2 Atropin-1 interacting protein 2

Cytoskeleton Cytoskeleton Cytoskeleton Cytoplasmic

1090 2.434e + 05 1.134e + 03 889

6.3 6.0 4.8 6.7

17 56 58 98

CAI14169 P30101 Q13087 O00308

19 12 45 18

−2 ± 0.1 −2 ± 0.5 −3.3 ± 0.5 −2 ± 0.3

89 90

Cell adhesion Catenin delta-1 Protocadherin alpha 3 precursor

Cytoplasmic

2.234e + 03 5.9 108 2210 5.0 100

O60716 Q9Y5H8

16 16

−2 ± 0.3 −2 ± 0.2

91 92 93 94 95 96 97 98 99 100 101 102 103 104

Unknown function Semaphorin 3D precursor Fibroleukin precursor Breast carcinoma amplified sequence 1 KIAA1967 protein Unnamed protein Hypothetical protein OTTHUMP00000017295 Tyrosine-protein kinase ZAP-70 Unnamed protein product ADAMTS-like protein 1 precursor OTTHUMP00000059267 Hypothetical protein Ficolin 3 precursor FYVE-finger protein EIP1

Endoplasmic reticulum 980 Cytoplasmic 400 1864 Nuclear 2310 Membrane 4200 2.134e + 03 Extracellular 1.144e + 04 Cytoplasmic 710 Membrane 3210 Cytoplasmic 1980 1.037e + 02 Extracellular 1980 Cytoplasmic 960 Cytoplasmic 650

O95025 Q14314 O75363 AAH18269 BAB14480 CAB94885 CAI19661 P43403 BAC87240 Q8N6G6 CAI18822 CAD97809 O75636 Q96D51

11 17 10 10 14 16 31 10 13 16 22 21 17 14

3 ± 0.1 2 ± 0.4 −2 ± 0.5 −9 ± 0.5 −2 ± 0.6 −5.5 ± 0.4 −10 ± 2 2.7 ± 0.2 2.3 ± 0.3 −2.4 ± 0.4 −2 ± 0.5 −3.8 ± 0.2 −2.6 ± 0.4 −2 ± 0.4

Nuclear Nuclear Nuclear Nuclear Cytoplasmic

Nuclear Nuclear

7.9 7.1 5.0 4.7 7.1 7.2 5.1 7.8 9.0 6.3 5.3 5.5 6.2 5.6

MW Accession (kDa) no.

89 50 61 40 44 30 23 69 76 58 24 20 32 59

Sequence Change coverage (%) fold

Differentially expressed proteins (ratio ≥ 2) that were upregulated or downregulated (−) in response to HOCl–oxLDL treatment for 24 h are listed. The MS spectra of protein digests were compared with the NCBI database using the MS-FIT database-searching program. Protein names and functions have been assigned according to PubMed and Swiss-Prot/ TrEMBL. The fold change columns correspond to the expression of each protein relative to its expression in control cells. Results are means of three independent experiments performed for each condition. The spot numbers are identical to those given in Fig. 2.

results showed that the expression of PARP10 was downregulated twofold in the macrophages. Inhibition of PARP activity by caspasedependent cleavage may be linked to the induction of apoptosis.

The many members of the Bcl-2 gene family are key regulators of cell survival, apoptosis, and necrosis [33–35]. Based on their structural and functional properties they can be divided into three groups:

Table 2 Identification of differentially expressed proteins spots by MS/MS analysis Spot no.

Protein

PI

MW (kDa)

Accession no.

20

Aldehyde dehydrogenase

6.0

53

P49189

Change fold −2 ± 0.3

36 69 82

Tropomyosin beta chain Adenine phosphoribosyltransferase Vimentin

4.7 5.8 5.1

32 19 53

P07951 P07741 P08670

−2 ± 0.4 −27 ± 4 3.3 ± 0.5

87

Protein disulfide-isomerase

4.8

58

Q13087

−3.3 ± 0.5

Sequence TIPIDGDFFSYTR TFVQEDIYDEFVER ELGEYGLQAYTEVK IQLVEEELDR SFPDFPTPGVVFRDISPVLKDPASFR EEAENTLQSFR FADLSEAANR EMEENFAVEAANYQDTIGR DLLIAYYDVDYE K KTFSHE LSDFGLESTAGEIPVVAIR FVMQE EFSR FLQDYFDGN LKR ELSDFISYLQR

454

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Fig. 3. Western blot analysis of selected proteins that are differentially expressed in THP-1 macrophages. Upper panels show the expression levels of annexin 1 (A), lamin B1 (B), vimentin (C), and Bcl-2 related protein A1 (D) in HOCl–oxLDL-treated THP-1 cells for 24 h. The bottom panels show the densities of immunoreactive bands measured by the Quantity One 1-D analysis software program. The values shown are the means ± SD of triplicate independent samples.

antiapoptotic Bcl-2-type proteins, proapoptotic Bax-type proteins, and proapoptotic BH3-domain-only family members [33–36]. The Bcl2-related protein A1 functionally acts as an antiapoptotic regulator. We detected a twofold downregulation of A1 in PMA-induced THP-1 cells treated with HOCl–oxLDL. The decreased expression of this protein was also confirmed by Western blot analysis as shown in Fig. 3D. The downregulated A1 may no longer provide a cytoprotective function, and this could be one of the factors that contribute to oxLDLinduced necrosis and apoptosis. Vimentin, a marker of differentiated cells, was also observed in oxLDL-treated THP-1 cells. It is well documented that cells under

stress produce vimentin, a class III intermediate filament protein involved in cell division and phosphorylated during reorganization of the cytoskeleton, to recover from the shock. This protein is highly abundant in human monocytes, in activated macrophages, and particularly in multinucleated giant cells. It has been reported that vimentin is strongly expressed in foam cells [37] and smooth muscle cells of human atheromatous plaques [38]. Increased expression of vimentin is a late event in the differentiation of human monocytic cells [39,40]. It is generally accepted that oxidized LDL induces macrophage death in vivo and the apoptosis contributes to atherosclerotic lesions. Müller et al. [41] demonstrated that vimentin is

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Fig. 4. Immunofluorescence analyses of vimentin and lamin B1 expression in macrophages. (A) PMA-induced THP-1 cells and (B) human monocyte-derived macrophage (HMDM) were either untreated or treated for 24 h with HOCl–oxLDL (50 or 100 μg/ml). Cells were double stained with anti-vimentin antibody (green) or anti-lamin B1 antibody (red) and DAPI (blue), examined by fluorescent microscopy.

Fig. 5. Literature gene networks by PubGene analyses. The top-scoring networks from search by protein associations were the literature neighborhoods of adenine phosphoribosyltransferase. Proteins showing upregulation in the submitted file are represented by network nodes in shades of red whereas downregulated proteins are shown in shades of green. In other respects the networks shown in the Expression Literature tool are similar to those presented by the Network Browser. The values shown on network edges (connectors between nodes) indicate the number of MEDLINE records that link each of the cocited proteins.

456

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Fig. 6. Classification of the differentially expressed proteins identified. Pie charts representing the distribution of the 104 identified proteins according to their biological function (A) and cellular localization (B). Assignments were made based on information from the NCBI (www.ncbi.nlm.nih.gov/PubMed) and the Swiss-Prot/TrEMBL protein knowledgebase (http://au.expasy.org/sport) websites.

cleaved into fragments of 48–50, 46, 29 and 26 kDa during apoptosis of human macrophages. In this study, three fragments were detected with different intensities. Caspase cleavage vimentin has been shown to disrupt intermediate filaments and promote apoptosis [42]. The increased expression of this protein was also validated by Western blot analysis and confocal microscopy, as shown in Figs. 3C and 4. The vimentin network is detectable in the form of dense aggregates closely associated with the nucleus. Thus, our results support the hypothesis that HOCl–oxLDL may induce apoptosis. Actin-related proteins are particularly important in this cell type in regulating actin filament length and concentration. We identified various actin-related proteins that regulate actin-driven assembly, such as actin-related protein 3 and tropomyosin 2. Some of these actin-binding proteins are essential for the reorganization of actin filaments as a cellular response to various growth factors, chemoattractants, and tumor target binding, and could play crucial roles in human disorders. The actin-related protein 3 is localized at the periphery of migrating cells, especially at the leading edge [43–45]. Actin-related protein 3 is involved in the nucleation and polymerization of actin filaments in cell motility processes, such as intracellular movement of organelles, amoeboid motility, and spatial control of neutrophilic chemotaxis. In this study, we detected a 3.5-fold downregulation of actin-related protein 3 in the macrophages treated with HOCl–oxLDL. 4.1.3. Signal transduction proteins OxLDL induces the activation of a large array of signaling pathways, some of them potentially cytotoxic, such as ceramide release, caspase

activation, dysregulation of cytosolic calcium homeostasis, and proteasome dysfunction [46]. Of the proteins identified in this study, 9% are involved in cell signal transduction. Most of these proteins were downregulated in PMA-induced THP-1 cells with HOCl–oxLDL treatment. We detected a twofold downregulation of inositol-trisphosphate3-kinase A (ITPKA) in PMA-induced THP-1 cells treated with HOCl– oxLDL. ITPKA regulates the calcium (Ca2+) level within the cell by releasing Ca2+ from intracellular stores, and is responsible for regulating the levels of a large number of inositol polyphosphates that are important in cellular signaling. This has led investigators to speculate the ITPKA may be related to carcinogenesis by the modulation of inositol polyphosphates and Ca2+ homeostasis [47]. Aberrant rises in intracellular calcium can produce seizures and excitotoxic neuronal death [48]. Changes in the expression of ITPKA may be implicated in the control of cellular homeostasis including cell survival. Therefore, ITPKA may be a potential biomarker for a number of differentiating cells such as THP-1 macrophages. Annexin I is involved in exocytotic processes, as well as in the regulation of intracellular vesicular trafficking. Recent works have shown that annexins may play a pivotal role at sites of tissue damage in resolving the inflammatory episode and preventing autoimmune responses [49]. We have previously reported that annexin I is upregulated in monocytic THP-1 cells stimulated with oxLDL [50]. In the present work, annexin I expression was also elevated in the macrophages treated with HOCl–oxLDL. The change of the expression was confirmed by Western blotting (Fig. 3A), which is consistent with the results from 2-DE.

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4.2. PubGene analysis We analyzed connectivity of all differentially expressed genes using the PubGene literature co-citation network. The connectivity map may be used not only to identify key proteins among differentially expressed proteins, but also to interpret the fundamental disease processes. A small number of the differentially expressed proteins were highly connected as shown in Fig. 5, while most proteins had few or no connections. The most highly connected protein was APRT, the key enzyme that converts adenine to adenosine monophosphate in the purine salvage pathway. Cooccurrence reflects biologically meaningful relationships, thus the connected proteins may have more disease relevance than unconnected proteins. 5. Conclusions The molecular mechanisms involved in oxLDL-induced transformation of macrophages to foam cells are unclear. Exposure of macrophage cells to oxidative stress causes dramatic changes in the global gene expression pattern. In the present study, the proteins differentially expressed after HOCl–oxLDL treatment in THP-1 macrophages were analyzed using 2-DE and MALDI–TOF MS. The protein expression patterns of oxLDL-treated macrophages in this study are quite different to those of monocytes that we have previously shown [50]. These proteins are related to cellular metabolism, cytoskeletal function, and cell signaling. Interestingly, most of the downregulated proteins are associated with the apoptosis of macrophages. As far as we are aware, this is the first proteomic analysis of human macrophage treated with HOCl–oxLDL. Many proteins for which the functions are still unknown were induced or repressed by the stimuli. Therefore, the next step should be to clarify the comprehensive view of oxLDL-induced transformation of macrophages to foam cells by analyzing the exact function of the proteins. The data described here are expected to provide valuable information in our understanding of the pathophysiological mechanism of atherosclerosis. Acknowledgments This work was supported by a Korea Research Foundation Grant (KRF-2005-005-J00104) and Kyungpook National University Research Team Fund. References [1] A. Mertens, P. Holvoet, Oxidized LDL and HDL: antagonists in atherothrombosis, FASEB J. 15 (2001) 2073–2084. [2] R. Stocker, J.F. Keaney, Role of oxidative modifications in atherosclerosis, Physiol. Rev. 84 (2004) 1381–1478. [3] J.A. Berliner, J.W. Heinecke, The role of oxidized lipoproteins in atherogenesis, Free Radic. Biol. Med. 20 (1996) 707–727. [4] J. Auwerx, The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte–macrophage differentiation, Experientia 47 (1991) 22–31. [5] W. Jessup, L. Kritharides, Metabolism of oxidized LDL by macrophages, Curr. Opin. Lipidol. 11 (2000) 473–481. [6] C. Smith, M.J. Mitchinson, O.I. Aruoma, B. Halliwell, Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions, Biochem. J. 286 (Pt 3) (1992) 901–905. [7] R. Stocker, Lipoprotein oxidation: mechanistic aspects, methodological approaches and clinical relevance, Curr. Opin. Lipidol. 5 (1994) 422–433. [8] J.W. Heinecke, Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis, Curr. Opin. Lipidol. 8 (1997) 268–274. [9] G. Marsche, J. Arnhold, M.J. Davies, Modification of low-density lipoprotein by myeloperoxidase-derived oxidants and reagent hypochlorous acid, Biochim. Biophys. Acta 1761 (2006) 392–415. [10] L.J. Hazell, R. Stocker, Oxidation of low-density lipoprotein with hypochlorite cause transformation of the lipoprotein into a high-uptake form for macrophages, Biochem. J. 290 (1993) 165–172. [11] S. Kopprasch, W. Leonhardt, J. Pietzsch, H. Kuhne, Hypochlorite-modified lowdensity lipoprotein stimulates human polymorphonuclear leukocytes for enhanced production of reactive oxygen metabolites, enzyme secretion, and adhesion to endothelial cells, Atherosclerosis 136 (1998) 315–324.

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