Comparative proteome analysis of Tumor necrosis factor α-stimulated human Vascular Smooth Muscle Cells in response to melittin
- Hyun-Ji Cho†1,
- Jeong-Han Kang†2,
- Kwan-Kyu Park1,
- Jung-Yoon Choe1,
- Yoon-Yub Park1,
- Yong-Suk Moon1,
- Il-Kyung Chung3,
- Hyeun-Wook Chang4,
- Cheorl-Ho Kim5,
- Yung Hyun Choi6,
- Wun-Jae Kim7,
- Sung-Kwon Moon8 and
- Young-Chae Chang1Email author
© Cho et al.; licensee BioMed Central Ltd. 2013
Received: 16 October 2012
Accepted: 1 May 2013
Published: 7 May 2013
Bee venom has been used to relieve pain and to treat inflammatory diseases, including rheumatoid arthritis, in humans. To better understand the mechanisms of the anti-inflammatory and anti-atherosclerosis effect of bee venom, gel electrophoresis and mass spectrometry were used to identify proteins whose expression was altered in human Vascular Smooth Muscle Cells (hVSMCs) stimulated by tumor necrosis factor alpha after 12 h in the presence of melittin.
To obtain valuable insights into the anti-inflammatory and anti-atherosclerosis mechanisms of melittin, two-dimensional (2-D) gel electrophoresis and MALDI-TOF/TOF were used. The proteome study, we showed 33 significant proteins that were differentially expressed in the cells treated with tumor necrosis factor alpha and melittin. Thirteen proteins were significantly increased in the cells treated with tumor necrosis factor alpha, and those proteins were reduced in the cells treated with melittin. Five of the proteins that showed increased expression in the cells treated with tumor necrosis factor alpha are involved in cell migration, including calreticulin, an essential factor of development that plays a role in transcription regulation. The proteins involved in cell migration were reduced in the melittin treated cells. The observed changes in the expression of GRP75, prohibitin, and a select group of other proteins were validated with reverse transcribed-PCR. It was confirmed that the observed change in the protein levels reflected a change in the genes level. In addition, the phosphorylation of EGFR and ERK was validated by analyzing the protein pathway.
Taken together, these data established that the expression of some proteins was significantly changed by melittin treatment in tumor necrosis factor alpha stimulated the cells and provided insights into the mechanism of the melittin function for its potential use as an anti-inflammatory agent.
The migration and proliferation of human Vascular Smooth Muscle Cells (hVSMCs) are the major causes of the development of advanced lesions in atherosclerosis . The migration and proliferation of hVSMCs is caused by pathological phenomena such as the accumulation of inflammatory cells and the release of pro-inflammatory cytokines [1, 2]. Pro-inflammatory cytokines such as the tumor necrosis factor (TNF)-α have various acts that mediate inflammation, and atherogenesis. Especially, TNF-α is a cytokine that is involved in systemic inflammation. Thus, the primary role of TNF-α is the regulation of immune cells. Moreover, TNF-α can induce apoptotic cell death, and inhibit tumorigenesis and viral replication . On the other hand, the dysregulation of TNF-α production has been implicated in several of human diseases, as well as in atherosclerosis and cancer . Therefore, hVSMCs and TNF-α decisively promote atherosclerosis and inflammation.
Bee venom (BV) is known as a very complex mixture of active peptides that include melittin, phospholipase A2, apamin, adolapin, hyaluronidase, dopamine, and the protease-inhibitor. It has been used in many studies on the biological and pharmacological activities that have anti-inflammatory effects on rheumatoid arthritis [4, 5]. In addition, BV affects pain release, and immune modulatory activity . It has also, been reported to have induced apoptosis and suppressed the signaling pathway in leukemic cells and renal cancer [7, 8]. The major compound of BV is melittin, a 26 amino acid peptide, which forms an amphipathic helix with a highly charged carboxyl terminus . It comprises 52% of BV peptides . Melittin reportedly has multiple effects, such as antibacterial, antivirus, and anti-inflammatory effects, in various cell types [11, 13]. In addition, it has been reported to be capable of cell cycle arrest, growth inhibition, and apoptosis in various tumor cells [12, 13]. However, the mechanisms of the anti-atherosclerosis and anti-inflammatory effects of melittin have not yet been fully explained. Thus, the proteomics method was used to understand the mechanisms of melittin in inflammation-induced hVSMCs. Such studies can be facilitated by comparing the obtained gels, with the 2-DE reference gels representing the typical pattern of the cells being studied under normal conditions. The association of 2-D electrophoresis with MALDI-TOF-TOF mass spectrometry and database interrogations enabled the identification of 33 proteins that were differentially expressed in the hVSMCs after melittin treatment. In particular, various proteins were implicated in the inflammation, regulation of the protein folding, oxidation reduction, and signal transduction.
Detection of the differentially expressed proteins in the hVSMCs
To determine the changes in the protein expression after the treatment of the cells with TNF-α or melittin, we used 2-D gel electrophoresis to separate the total cell proteins from the hVSMCs. Each gel was loaded with 400 μg of protein. Approximately 1,000 individual spots were resolved in this manner. It was expected that many individual spots would contain more than one protein and this was borne out by the subsequent analysis using mass spectrometry. It was also found in a number of instances that the same protein was present in multiple spots and was most likely the product of the post-translational modifications or alternative splicing at the mRNA level. Nevertheless, an estimated 900 individual proteins at least were resolved, which provided a representative samples of the cellular proteins and allowed identification of many differentially expressed proteins.
Identification of the differentially expressed proteins with MS
Proteins showing differential expression in hVSMCs in response to melittin
a AC No
MEL + TNF/Con
Serine/threonine-protein phosphatase 2A regulatory subunit
RWD domain-containing protein 3
Oxidative stress-induced growth inhibitor 1
Mitochondrial-processing peptidase subunit beta
Ubiquinone biosynthesis protein COQ9
60 kDa heat shock protein
N-terminal EF-hand calcium-binding protein 1
Calcium ion binding
Protein N-terminal asparagine amidohydrolase
Guanine nucleotide-binding protein G(k) subunit alpha
Signal transducer activity
Polyadenylate-binding protein 3
Poly(A) RNA binding
Uncharacterized protein C1orf55
Calcium-activated potassium channel subunit beta-3
Transcription factor ETV7
LON peptidase N-terminal domain and RING finger protein 1
Eukaryotic translation initiation factor 3 subunit E-interacting protein
Sorting and assembly machinery component 50 homolog
Transmembrane emp24 domain-containing protein 3 precursor
Neutrophil cytosol factor 4
Regulation of actin phosphorylation
cAMP-dependent protein kinase type I-beta regulatory subunit
Neutrophil cytosol factor 4
MAGUK p55 subfamily member 5
Tight junction assembly
Tropomyosin alpha-3 chain
Ankyrin repeat domain-containing protein 45
Copper chaperone for superoxide dismutase
Protein pathway analysis of the differential expression protein in the TNF-α treated cells
Validation of the selected proteins by with RT-PCR
BV has been widely used to relieve pain and treat inflammation in various chronic inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. A major compound of BV, melittin is the principal active component of BV and is a powerful stimulator of phospholipase A2. It also inhibits various protein kinases and is a cell membrane lytic factor. It has also shown potent anti-microbial activity and suppression of infections [14, 15].
In this study, a proteomics analysis of hVSMCs treated with TNF-α or melittin was first performed to identify differentially expressed proteins that are highly affected by melittin in TNF-α stimulated cells. This technique revealed 52 differentially expressed proteins and identified 33 significantly differentially expressed proteins whose expressions had consistently different patterns after TNF-α or melittin treatment. Many of the identified proteins were Stress-70 protein and Annexin A1, which are involved in anti-apoptosis.
Prohibitin 1 (PHB) is multifunctional protein that is localized in cells and mitochondrial membranes as well as in the nucleus [16, 17]. It is implicated in cellular processes such as the mitochondrial function and protein folding , transcription regulation  and proliferation control and suppression of oncogenesis [20, 21]. Expression of PHB decreases during ulcerative colitis and Crohn’s disease, two forms of inflammatory bowel disease [22, 23]. In this study, prohibitin expression was down-regulated in TNF-α treated cells and up- regulated in melittin treated cells.
Annexins have been diversely implicated in cell differentiation and proliferation, extracellular processes such as coagulation, and membrane fusion events such as endocytosis and exocytosis . In addition, annexins are intracellular molecules that are implicated in the down-regulation of inflammation. Annexin-1 was identified recently as a secreted molecule and suggested as a potent inhibitor of inflammation both in vitro, and in vivo. Studies have indicated that annexin-1 is secreted, and subsequent reports have shown that secreted annexin-1 participates in additional anti-inflammatory effects such as detachment of neutrophils from the vascular endothelium [25, 26]. In this study, annexin-1 expression was restored in melittin-treated hVSMCs and melittin was suggested as having anti-inflammatory effects.
Calreticulin binds unfolded glycosylated proteins in the ER and is implicated in many cellular functions such as lectin-like chaperoning, Ca2+ storage and signaling, regulation of gene expression, cell adhesion, wound healing and auto-immunity. Its over-expression on the surface of lung fibroblasts has been reported in response to cytomegalovirus infection [27, 28]. Calreticulin was up-regulated by TNF-α and down-regulated in the melittin-treated hVSMCs.
This is the first report of the proteomic analysis of the effects of melittin treatment on cultured hVSMCs. In this study, the anti-inflammatory mechanism of melittin on the inflammatory process was discovered. And several target molecular of inflammation and proliferation such as prohibitin, annexin-1 and calreticulin were identified. In addition, we found that two major protein pathways using bioinformatics method. The protein pathway analysis showed that NF-κB, and EGFR are the main molecular of inflammation in hVSMCs treated with TNF-α or melittin. It was consistent with other study. Jijon HB et al. reported that TNF-α induced ERK and EGFR activation  and Andrianifahanana M et al. reported that TGF-β induced EGFR activation . According these reports, we suggest that TNF-α induced ERK/NF-kb and EGFR. We believe that this experiment would improve understanding of the anti-inflammatory effects of melittin on inflammatory disease, and atherosclerosis.
Cell culture and biological reagents
Human Vascular Smooth Muscle Cells (hVSMCs) were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI medium (Invitrogen, Gland island, USA) containing 10% fetal bovine serum. The cells were maintained at 37°C. The hVSMCs were grown on culture plates to 60%-70% confluence in complete medium containing 10% FBS for 12 hours, and then changed to serum-free medium after washing twice with medium. Then, the cells were incubated with TNF-α or melittin at various concentrations.
Protein extraction and Two-dimensional gel electrophoresis
hVSMCs were washed three times with ice-cold PBS. Cells were lysed with a buffer containing 5 mM EDTA, 9.5 M urea, 4% (v/v) CHAPS, 65 mM DTT and protease inhibitors (Complete kit, Roche Diagnostics, Germany) for 1 h at 24°C. Cellular debris was removed by centrifugation for 15 min at 20000 × g at 4°C. Protein samples were stored at −70°C. Protein concentrations were quantified using a commercial Bradford Kit (DC reagent kit, Bio-Rad).
2-D electrophoresis was performed using an established procedure . Whole cell lysate (400 μg) was added to immobilized pH 3–10 linear gradient strips (ReadyStrip IPG strip, Bio-Rad). After finished IEF, the IPG strips were incubated in equilibration buffer containing 37.5 mM Tris–HCl (pH 8.8), 6 M urea, 2% (w/vol) SDS, 30% (v/v) glycerol and 2% (w/vol) DTT or 2.5% (w/vol) iodoacetamide for 30 min. The equilibrated IPG strips were transferred onto 12% Duracryl gels (180 × 160 × 1.5 mm) for SDS–PAGE. We stained gels with sensitive colloidal coomassie G-250 according to Neuhoff et al. . To check the reproducibility of the data, three independent experiments were performed on each cell lysate. For the differential analysis, statistical significance was estimated with Student’s t-test. Values of p < 0.05 were considered significant.
Protein identification was performed as described previously study . In brief, proteins were in-gel digested with trypsin and extracted from coomassie stained 2-D gel pieces in according to standard procedures. After in-gel digested, the peptides were extracted twice with 0.1% TFA in 50% acetonitrile. Extracts were pooled and lyophilized. The resulting lyophilized tryptic peptides were concentrated and desalted by passing them through C18ZipTip (Millopore, Billerica, MA, USA) following standard procedures. MS analysis was conducted with a MALDI-TOF-TOF mass spectrometer 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA). Data were analyzed using GPS Explorer software (Applied Biosystem) and MASCOT software (Matrix Science, London, UK). NCBInr and human were selected as the database and taxonomy, respectively. Identification was assigned to a protein spot feature if the protein score was calculated to be greater than 50, correlating to a confidence interval of 99%.
Protein pathway analysis
After protein identification, the accession numbers and fold changes of the differentially expressed proteins were tabulated in Microsoft Excel and imported into IPA (Ingenuity System, Montain View, CA, USA). IPA is a software application that enables to identify the biological mechanisms, pathways and functions matching a particular dataset of proteins. IPA is based on a database obtained by abstracting and interconnecting a large fraction of the biomedical literature according to a very strict algorithm. This database integrates protein functions, cellular localization, small molecules and disease inter-relationships. The networks are displayed graphically as nodes, representing individual proteins and edges representing the biological relation between nodes. Using IPA, Canonical pathway analysis utilizes well characterized metabolic and cell signaling pathways which are generated prior to data input and on which identified proteins are overlaid.
Western blot analysis
Whole cell and nuclear lysates were prepared as previously described . Cells were lysed with RIPA buffer. (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% [v/v] NP 40 10 mM NaF and proteases inhibitors). The cells were disrupted and proteins were extracted at 4°C for 30 min. The proteins were electro transferred to PVDF membranes (Invitrogen). Detection of specific proteins was carried out with an enhanced chemiluminescence western blotting kit following the manufacturer’s (Pierce) instructions. Antibodies specific for p-ERK, ERK, EGFR, p-EGFR, NF-κB and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
After treatment of cells with TNF-α or melittin, Total RNA was isolated from each preparation using the Trizol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was carried out using a commercial kit (Superscript II RNase H-reverse transcriptase, Invitrogen) and total RNA (1 μg) from hVSMCs, according to the manufacturer's protocol. Gene expression was analyzed using specific primers. Amplified products were resolved by 1.0% (w/v) agarose gel electrophoresis and visualized by staining with ethidium bromide. We quantified the actual mRNA level of each gene by using Eagle Sight densitometry software (Version 3.21; Stratagene, La Jolla, CA).
This work was supported by a grant (PJ00953402) from Biogreen 21 Program, Rural Development Administration, Republic of Korea and also by the National Research Foundation of Korea (NRF, Personalized Tumor Engineering Research Center) grant funded by the Korea government (MEST)(NO. 2012–0000481).
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