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Zhen Ge, Xian Zhong, Jie-Kai Yu, Shu Zheng, Cancer Institute, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, Zhejiang Province, China Yong-Liang Zhu, Gastroenterological Department, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, Zhejiang Province, China Zhen Ge, James D Watson, Institute of Genome Sciences, College of Life Sciences, Zhejiang University, Hangzhou 310008, Zhejiang Province, China Supported by National High Technology 2006AA02Z341 & NSFC 30430730 Correspondence to: Professor Shu Zheng, 88 Jiefang Road, Cancer Institute of Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, Zhejiang, China. zhengshu@zju.edu.cn Telephone: +86-571-87784501 Fax: +86-571-87214404 Received: August 25, 2007 Revised: October 8, 2007
Abstract AIM: To identify the protein expression differences related to the CagA-induced ERK pathway activation in AGS cells.
METHODS: Human AGS cells transfected with cagA and blank vector were treated with specific mitogen-activated protein kinase kinase (MEK) inhibitor. Total cell proteins were combined by strong anion exchange (SAX2) and weak cation exchange (CM10) ProteinChip arrays and analyzed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) proteomics technology. Protein expression profiles were compared with those of inhibitor-untreated cagA transfectants. SwissProt/TrEMBL database searching for differentially expressed proteins was carried out using the TagIdent tool with the pI and mass information.
RESULTS: When a total of 16 proteins that showed expression differences in inhibitor-untreated cagA transfectants were compared with vector transfectants, three proteins with m/z 4229, 8162 and 9084 were found to have no expression differences after treatment with MEK inhibitor, while the other 13 maintained the same expression differences after inhibitor treatment. Seven pieces of meaningful matching information for the three proteins were obtained from database searching.
CONCLUSION: Biomarkers with m/z 4229, 8162 and 9084 are ERK1/2 phosphorylation dependent, and therefore are the downstream molecules of ERK1/2 in the ERK/MAPK signaling pathway. The three biomarkers may be important cancer-associated proteins according to SwissProt/TrEMBL database information.
© 2008 WJG. All rights reserved.
Key words: CagA; ERK pathway; SELDI-TOF-MS; ProteinChip
Peer reviewer: Frank A Anania, Professor, Emory University School of Medicine, Division of Division Digestive Diseases, 615 Michael Street, Room 255 Whitehead Biomedical Research Building, Atlanta, GA 30322, United States
Ge Z, Zhu YL, Zhong X, Yu JK, Zheng S. Discovering differential protein expression caused by CagA-induced ERK pathway activation in AGS cells using the SELDI-ProteinChip platform. World J Gastroenterol 2008; 14(4): 554-562 Available from: URL: http://www.wjgnet.com/1007-9327/14/554.asp DOI: http://dx.doi.org/10.3748/wjg.14.554
INTRODUCTION H pylori infection, a primary etiological cause leading to gastritis and peptic ulcer[1], is also a high risk factor for gastric cancer, and is classified as a class I carcinogen by the International Research Agency on Cancer (IRAC)[2-6]. Cytotoxin-associated gene A (CagA) protein, which is encoded by cytotoxin-associated gene pathogenicity island, is the major virulence factor of type I H pylori. Epidemiological studies indicate that CagA plays an important role in the development of H pylori-induced disease, especially in gastric cancer. The CagA antibody titer in gastric cancer patients is higher than in healthy individuals. Similarly, CagA antibody titer is elevated in high-risk populations[7]. Compared with cagA-negative H pylori strains, cagA-positive strains dramatically increased the occurrence of severe gastritis and gastric adenocarcinoma[8-12]. Experimental studies reveal that CagA is translocated into host cells through a type Ⅳ secretion system of H pylori[13]. After being tyrosine-phosphorylated at the C-terminal repeat sequence(s) by c-src/Lyn kinase[14,15], CagA strongly stimulates the mitogen-activated protein kinase (MAPK) signaling pathway in host cells, through interaction with SHP-2, a tyrosine phosphatase containing an SH2 homolog, and results in cell proliferation and differentiation[16]. As one of the major three members of the MAPKs, extracellular signal-regulated kinase (ERK) has been reported to be a key molecule in cell carcinogenesis in various cell types[17-19]. Higashi et al[20] have found that ERK is activated by CagA via SHP-2 recruitment and activation in AGS cells (human gastric adenocarcinoma epithelial cell line). Our previous research further proved that transformation of immortalized gastric epithelial cells by CagA takes place through the ERK/MAPK pathway[21]. However, the precise mechanism of the interactions between CagA and host cells in associated pathogenesis has not been fully elucidated. Recently, gene microarrays have been used to establish the global pattern of gene expression in cagA gene-transfected AGS cells[22]. Nevertheless, gene and protein expression levels cannot easily be correlated or equated since proteins can exist in different posttranslational functional states. Therefore, it is important to gain an overall view of host response to CagA interference at the protein level. However, to date, few studies that exploit the global protein-expression pattern that can reflect host-cell response to CagA have been reported. Based on surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology, the ProteinChip platform has recently been shown to be valuable in establishing protein expression profiles and discovering new biomarkers[23-25]. ProteinChips, on which proteins from biological samples are selectively retained according to their biochemical properties, are analyzed directly by MS to form peak patterns as protein-expression profiles. The location and intensity of every peak in the pattern reflect the molecular weight and abundance of the corresponding protein. Therefore, the patterns can be used to analyze the protein expression differences under different conditions. This innovative technique has certain advantages over other traditional methods such as two-dimensional gel electrophoresis in its small sample volume, high throughput capability, subfemtomole range sensitivity, high resolution at low mass range, and easy operation, thus it has been successfully applied in new biomarker discovery for early diagnosis of cancer and basic research into disease mechanisms[26-29]. The proteome of AGS cells transfected with cagA gene was analyzed using a SELDI-ProteinChip platform in our previous study. Sixteen biomarker proteins involved in CagA induced pathogenesis were identified[30]. In this study, we further investigated the relationship of these protein expression differences and activation of the ERK/MAPK signaling pathway by CagA. Three more pivotal disease-related biomarkers among the 16 were further figured out.
MATERIALS AND METHODS Expression vectors Wild-type (WT) cagA/pcDNA3.1(+) plasmid (WT-cagA) and phosphorylation site mutant cagA/pcDNA3.1(+) plasmid (MT-cagA) were constructed as previously described[31,32]. Briefly, WT-cagA was constructed for encoding CagA proteins containing three tandem Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, and one repeated EPIYA motif sequence with Asp-Phe-Asp (DFD) in the D2 region. MT-cagA was generated from WT-cagA by substituting a phenylalanine codon for a tyrosine codon in the repeated EPIYA motif.
Cell culture and transfection Human gastric adenocarcinoma epithelial AGS cells (ATCC, Rockville, MD, USA) were cultured in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (Gibco) in six-well plates. For ERK-associated analysis, cells were transiently transfected with WT-cagA or blank vectors. For each well, 4 mg plasmids were transfected into 5.5 × 105 cells by using 6 mL Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. WT-cagA transfectants, vector transfectants and untransfected cells were divided into three groups that were respectively treated with the specific mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (Sigma, St. Louis, MO, USA) at 10 mmol/L, DMSO vehicle, or were not treated. Cells were collected after 48 h for protein expression analysis. The transfection efficiency was at least 37%, as determined by the cells transfected with EGFP/pcDNA3.1(+). The expression of cagA gene was confirmed by RT-PCR.
Western blot analysis WT-cagA transfectants, vector transfectants and untransfected AGS cells were incubated in 10% FCS for 0, 1 or 3 h following a 24-h serum-starvation period. After washing three times with PBS, cells were harvested and treated with lysis buffer (20 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 10 mmol/L EDTA, 10 mL/L Triton X-100, 40 g/L SDS, and 100 mL/L glycerol) at 100℃ for 5 min and then centrifuged at 15 000 × g for 5 min. Western blot analysis was performed with standard techniques, using anti-total ERK1/2 antibody (1:1000; Cell Signaling, Beverly, MA, USA), anti-phospho-ERK1/2 antibody (1:1000; Cell Signaling), followed by an anti-mouse secondary antibody (Beijing Zhongshan Biotechnological, China). Samples were separated on 10% sulfate-polyacrylamide gel and electrotransferred onto nitrocellulose membranes (Amersham Biosciences, USA). Immunoreactive bands were visualized with the ECL detection system (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using Kodak X-ray film.
Preparation of total cell lysates
AGS cells of each group were harvested after PBS washing
and resuspended in lysis buffer (8 mol/L urea, 40 g/L CHAPS, 50 mmol/L
DTT, 40 mmol/L Tris-HCl) containing protease inhibitor cocktail (Sigma).
After 30 min lysis on ice, total cell lysates were obtained by
centrifugation at 20 000
× g for 30 min to remove insoluble debris. Protein concentration
was measured using BioRad DC protein assay kit (BioRad, Hercules, CA,
USA) and then adjusted to
SELDI protein profiling Expression profiles of each group were obtained by strong anion exchange (SAX2) and weak cation exchange (CM10) ProteinChip Arrays (Ciphergen Biosystems, Fremont, CA, USA) using an “in situ” protocol provided by the manual. Before sample loading, each spot of SAX2 and CM10 arrays were equilibrated with 3 mL of binding buffer (50 mmol/L Tris-HCL, pH 9.0 for SAX2 and 50 mmol/L sodium acetate, pH 4.0 for CM10). After 5 min incubation for three consecutive periods, 5 mL total cell lysate (diluted to 1.5 mg/mL in binding buffer) was added for chip binding. All arrays were incubated in a humidity chamber for 1 h and each spot was washed three times with 3 mL binding buffer. After rinsing quickly with HPLC water twice, the arrays were air-dried. One microliter of saturated sinapinic acid (Ciphergen Biosystems) solution in 500 mL/L acetonitrile, in water containing 5 mL/L trifluoroacetic acid, was applied twice, followed by air-drying. Mass accuracy was calibrated using the All-in-1 peptide and All-in-1 protein molecular mass standards (Ciphergen Biosystems). MS analysis of each sample was performed using the ProteinChip Biology System Reader (Model PBS Ⅱ; Ciphergen Biosystems) at a laser intensity of 180 and a detector sensitivity of 7.
Data analysis Data of every spectrum were collected and analyzed by ProteinChip Software version 3.0 (Ciphergen Biosystems) with integrated Biomarker Wizard software (Ciphergen Biosystems). In brief, baselines were subtracted and normalization was performed by total ion current normalization function, following the software instructions. Biomarker Wizard was then used to identify corresponding peaks in the spectra. Significant differences (P < 0.05) in peak intensity of particular proteins between two sample groups were calculated by the software using a Mann-Whitney non-parametrical test.
RESULTS ERK activity in transfected cells Western blot results showed that the phosphorylation level of ERK1/2 in WT-cagA transfectants was significantly higher than that in vector transfectants and untransfected AGS cells, after serum stimulation at all time points (Figure 1), which indicates that phosphorylated CagA can strongly enhance the phosphorylation level of ERK1/2 in host cells.
Profiling of cell lysates
Protein profiles
of cell lysates from each group were obtained from SAX2 (strong anion
exchange) and CM10 (weak cation exchange) ProteinChip Arrays. Each type
of array surface retains different groups of proteins depending on the
surface properties (SAX2 captures proteins with low pIs, while CM10
captures proteins with high pIs), therefore assuring that more host
proteins are analyzed. MS analysis was carried out under a laser
intensity of 180 and a detector sensitivity of 7.0, which brought out
the highest resolution determined by previous experiments. Approximately
200 separate protein peaks with a signal to noise ratio > 5.0 and
intensity > 1.0 were obtained in the mass range of 2000-20 000
m/z
Biomarker proteins dependent on activation of ERK/MAPK signaling pathway detected by SELDI-TOF-MS In our previous study[30], we transfected AGS cells with WT-cagA, phosphorylation site mutant cagA, and blank vector. Protein-expression profiles were analyzed using a SELDI-ProteinChip platform, in order to gain an insight into the effect of CagA and CagA phosphorylation on host protein expression. A total of 16 proteins in AGS cells were found have expression differences under the effect of CagA, and 12 of these were CagA phosphorylation dependent (Figure 2, Table 1). These biomarkers may contribute to the host response to CagA and may be involved in CagA-induced pathogenesis. According to the previous results, most changes in expression after cagA transfection are related to CagA phosphorylation. Phosphorylated CagA can interfere with signal transduction of the ERK/MAPK pathway that plays an important role in CagA-induced cell proliferation and transformation. Various downstream molecules involved in the pathway need to be identified. Thus, our next question is which biomarkers among the 16 are caused by CagA-induced activation of the ERK/MAPK signaling pathway. The results may help us to focus on the most important proteins involved in the host response to CagA. After confirming by Western blotting that WT CagA can significantly increase the activation of ERK1/2, we treated AGS cells with MEK inhibitor simultaneously to transfection to investigate if the changes in expression of the 16 biomarkers induced by WT-cagA disappeared when ERK1/2 was deactivated. The expression levels of three biomarkers with m/z 4229, 8162 and 9084 were found up-regulated significantly in WT-cagA transfectants with 2.25-, 1.71- and 1.64-fold changes compared with vector transfectants under inhibitor-free conditions (Figure 3, panels 1-3). The fold changes were very similar to those in our previous experiment (Figure 2, panels 1-3). In DMSO-treated conditions, they displayed the same expression pattern (Figure 3, panels 10-15) as under inhibitor-free conditions. However, when treated with MEK inhibitor during transfection, the peak intensity of these three proteins in WT-cagA transfectants was found not to be significantly different (P > 0.05) from that of vector transfectants (Figure 3, panels 4-9). This indicates that the expression differences of biomarkers with m/z 4229, 8162 and 9084 were caused by activation of the ERK/MAPK signaling pathway. The expression differences for the other 13 proteins existed in both ERK1/2-active and -inactive conditions (Figure 4, Table 2). Thus, they were independent of ERK1/2 activation. The biomarkers with m/z 4229, 8162 and 9084 that were identified as ERK1/2 downstream molecules involved in CagA-induced pathogenesis are worthy of further investigation.
Database searching for the three ERK-related biomarkers A search in the SwissProt/TrEMBL database using the TagIdent tool, along with the pI and mass information was performed to get matching proteins for the three biomarkers with m/z 9084, 4229 and 8162. All together, 22 pieces of matching information were obtained from the database. Seven of these were shown to be proteins related to apoptosis, antimicrobial defense, chemotaxis, proliferation, differentiation and carcinogenesis, which may be closely associated with the ERK/MAPK signaling pathway (Table 3). The existence of these proteins was validated at the protein or transcript level in a series of studies. The other 14 were predicted to be proteins or proteins lacking the references providing insightful functional information. Therefore, the three biomarkers with m/z 9084, 4229 and 8162 have great potential to be identified as cancer-associated proteins in future research.
DISCUSSION Translocation of CagA protein into gastric epithelial cells through the type Ⅳ secretion system is considered to be one of the most important processes in the progression of H pylori diseases. In this host-bacterial interaction period, many host proteins and signaling pathways are implicated[33]. In order to gain an overall view into the host response to CagA, the innovative proteomic technology SELDI-ProteinChip platform was employed to investigate the expression changes in gastric epithelial AGS cells. The strongpoint of this technology is that it can detect proteins in fmol quantities, and it allows fast, high-throughput analysis, especially in the low-molecular-weight range, which is essential for biomedical research, and cannot be easily achieved by conventional methods. Following this method, 16 biomarkers have been identified in our previous study as being involved in CagA-host cell interactions, and 12 of these are dependent on CagA phosphorylation. This result provides a primary view of CagA-induced pathological changes at the protein level. To further investigate the role of CagA-phosphorylation-associated functions in gastric carcinogenesis, we focused our interest on the role of the ERK signaling pathway. It has been verified by clinical pathological tests that the level of phosphorylated ERK in gastric cancer tissue is higher than that in the gastric mucous membrane. The level is also higher in cancer tissue of H pylori-positive gastric cancer patients than that in H pylori-negative patients[34]. Besides, it has been reported that cagA-positive H pylori is more powerful than cagA-negative strains for ERK activation[35]. Thus, ERK/MAPK pathway activation and subsequent cell proliferation, differentiation, as well as morphological changes, are among the most important pathogenic factors in CagA-induced diseases. However, this complicated process includes multi-level regulation that has rarely been understood. In the present study, we tried to investigate if the expression changes of the 16 host proteins induced by CagA have some relationship with the activation of the ERK pathway. The phosphorylation level of ERK1/2 in WT-cagA transfectants was confirmed to be much higher than that in vector transfectants and blank control cells. MEK inhibitor U0126 that specifically inhibits ERK1/2 phosphorylation was employed to eliminate the changes induced by ERK activation during the transfection of WT-cagA. The relationship of the 16 biomarkers and ERK pathway can be determined by comparing their expression levels under ERK-inhibited and ERK-activated conditions. On the other hand, protein profiles indicated that protein expression levels were influenced by DMSO as the solvent of U0126. Therefore, we first compared profiles of WT-cagA transfectants and vector transfectants under U0126-free conditions, U0126 treatment, and DMSO treatment. Only the proteins exhibited similar expression differences between WT-cagA transfectants and vector transfectants under both U0126 free and DMSO treated conditions, but they exhibited no expression differences under the U0126 treated condition, and thus were considered to be biomarkers that related to ERK activation. In this manner, among the 16 biomarkers, three with m/z 4229, 8162 and 9084 were identified as ERK pathway downstream proteins related to CagA pathogenesis. These three biomarkers will be characterized in our future research. Most biomarkers discovered so far are peptides or small proteins with low abundance. They are probably metabolic products, abnormally cleaved proteins, modified proteins, neurokinins or cytokines that exist in disease states. The biomarkers detected in our study are also likely small molecules of this kind, which can hardly be detected by traditional methods, but are essential for associated research. While the SELDI-ProteinChip platform is very rapid for protein profiling, it does not allow direct purification and functional identification. A search in the SwissProt/TrEMBL database using the TagIdent tool with pI and mass information was performed to obtain matched information for each biomarker. The search results suggested that these three biomarkers are probably peptides of the following type: b-defensin homologs, which might be involved in host defense to bacterial infection; BCL2-like protein, which might function as an apoptosis inhibitory protein; chemokines; and tumor-related proteins, which might be responsible for cell proliferation and differentiation induced by ERK/MAPK pathway activation, and contribute to CagA-induced carcinogenesis in the gastric epithelium. The matching information provides helpful references for obtaining corresponding antibody which will be employed in future biomarker identification. The biochemical properties provided by the selective ProteinChip surfaces and SELDI-TOF-MS for each biomarker are also useful for the purification process. Further purification and identification will elucidate the function of related small molecules in CagA pathogenesis, and may therefore discover new therapeutic targets.
COMMENTS Background
H pylori
infection is an important causative factor of gastritis, peptic ulcer
and gastric cancer. CagA, the major virulence factor of typeⅠH
pylori, is injected by the bacterium into
gastric epithelial cells, and has been shown by epidemiological and
experimental studies to be closely associated with the development of
Research frontiers CagA is translocated into host cells through a type Ⅳ secretion system of H pylori. After tyrosine phosphorylation at the C terminus, CagA strongly interferes with signal transduction, which results in cell proliferation and differentiation. Various studies have indicated a relationship between CagA, ERK/MAPK pathway activation and gastric cancer. In this study, a proteomic approach was employed to detect host cell proteins related to CagA-induced ERK pathway activation.
Innovations and breakthroughs A new proteomic approach based on ProteinChip array and SELDI-TOF-MS was employed to investigate the global protein expression changes in gastric epithelial cells induced by CagA. Three biomarkers were further identified as downstream molecules of ERK1/2 in the ERK/MAPK signaling pathway. Due to the high sensitivity and resolution in the low-molecular-weight range of SELDI-ProteinChip technology, biomarkers discovered in this study are mainly low-mass and/or low-abundance disease-related proteins, which are difficult to detect by traditional methods.
Applications These results provide new targets for further understanding the biological function and pathogenesis of CagA, as well as new therapeutic targets.
Terminology CagA: cytotoxin-associated gene A protein encoded by cytotoxin-associated gene pathogenicity island is the major virulence factor of type I H pylori. ERK: extracellular signal-regulated kinase. ERK pathway is one of the major three members of the MAPK signaling pathways. ERK has been reported as a key molecule in carcinogenesis in various cell types. ProteinChip array: the surface of array selectively retains proteins from biological samples according to their biochemical properties, and can be analyzed directly by MS. SELDI-TOF-MS: surface-enhanced laser desorption/ionization time-of-flight MS detects proteins in fmol quantities, and allows fast, high-throughput analysis, especially in the low-molecular-weight range.
Peer review This is a state-of-the-art, well-written paper that has used a proteomic approach to understand CagA protein biology as it relates to gastric cancer.
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S- Editor Liu Y L- Editor Kerr C E- Editor Liu Y
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