Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration

Abstract

Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.

Helicobacter pylori, progranulin, gastric epithelial cells

Introduction

Helicobacter pylori is a Gram-negative, spiral bacillus found in the human stomach of approximately half of the world's population (Peek & Blaser, 2002). Helicobacter pylori infection is usually established early in life and persists throughout life in the absence of treatment (Wilson & Crabtree, 2007). Epidemiologic studies have shown that H. pylori infection is a risk factor for gastritis, ulcerations, and gastric adenocarcinoma (Uemura et al., 2001; Huang et al., 2003) and it has been classified by the World Health Organization as a group 1 carcinogen (Houghton & Wang, 2005). Recent clinical studies have suggested that H. pylori eradication can reduce the risk of gastric cancer (Wong et al., 2004; Take et al., 2005). Furthermore, several investigators reported that host–pathogen interactions play an important role in the final disease outcome, including peptic ulcer and cancer (Israel & Peek, 2006). The responses of epithelial cells to H. pylori infection possibly influencing subsequent progression to cancer are still largely unknown.

Progranulin (PGRN), also called granulin-epithelin precursor, PC cell-derived growth factor, proepithelin, or acrogranin, belongs to a novel class of growth factors that plays a critical role in development, cell cycle progression, cell motility, and tumorigenesis, and which could promote anchorage-independent growth (He et al., 2003; Jones et al., 2003). PGRN is expressed at high levels in rapidly proliferating cells such as skin cells, deep crypts of the gastrointestinal tract, and immune cells (Daniel et al., 2000). Recent studies have shown that PGRN levels are elevated in tumors, and the extent of the increase is associated with an increased potential for the spread of the malignancy (Zhang & Serrero, 1998; He et al., 2003; Jones et al., 2003). Studies have shown PGRN to be involved in multiple steps of the tumor progression cascade, including cellular proliferation, anchorage independence, invasiveness, resistance to anoikis, and promotion of resistance to certain cytotoxic drugs (Bagnato et al., 1997; Zanocco-Marani et al., 1999; He et al., 2003; Fukazawa et al., 2004). Thus, PGRN is well positioned to regulate epithelial responses that may predispose to malignancies. However, the association of PGRN and H. pylori-infection-induced cellular responses with carcinogenic potential remains unclear.

The ability of cancer cells to proliferate and migrate is a critical step in tumor metastasis (Guo & Giancotti, 2004). Helicobacter pylori infection can augment the proliferation rate of gastric epithelial cells (Moese et al., 2002; Al-Ghoul et al., 2004) and attenuate apoptosis in humans and in rodent models of infection (Ding et al., 2008; Yokota et al., 2010). Moreover, H. pylori infection can increase gastric epithelial cell migration. The mechanisms required for these responses are not clearly defined (Peek et al., 2000; Maeda et al., 2002). Of note, PGRN is necessary for tumor growth and actively confers malignancy (Zhang & Serrero, 1998; Lu & Serrero, 2000; Cheung et al., 2004; Chen et al., 2008).

Previous reports have shown that PGRN levels are increased in gastric cancer (Linē et al., 2002). In the present study, we analyzed whether PGRN is upregulated in patients with gastritis and gastric cancer, how H. pylori elicits the upregulation of PGRN in gastric epithelial cells, and the tumor-promoting potential of PGRN in gastric epithelial cells toward this pathogen. We aimed to ascertain the pathogenetic factors of H. pylori indispensable for PGRN induction and the signal pathways stimulated by H. pylori that are relevant for the overexpression of PGRN. This is the first study to examine the changes of PGRN expression in gastric cancer cells before and after H. pylori infection.

Materials and methods

Cell culture and reagents

SGC7901 gastric cancer and immortalized GES-1 cells were maintained in our laboratory. Both cells were cultured in RPMI 1640 (Gibco Life Technologies, Grand Island, NY) with 10% newborn bovine serum (Gibco Life Technologies) in 5% CO₂-air at 37 °C.

The specific inhibitors — MEK1/2 inhibitor PD98059, p38 inhibitor SB203580, and nuclear factor-kappa B (NF-κB) inhibitor BAY11-7082 — were from Invitrogen. Stock solutions were prepared in dimethyl sulfoxide (DMSO) solution. Cells were treated with the above inhibitors after H. pylori stimulation. Controls without inhibitors were treated with media alone and an equal concentration of DMSO.

Immunohistochemical staining for PGRN

Immunohistochemical studies involved archival, formalin-fixed, paraffin-embedded gastric biopsy samples from healthy people with normal pathology and from patients with gastritis and gastric cancer obtained from the Department of Pathology, Shandong University Medical School, China. Sections were stained with PGRN polyclonal antibody according to the manufacturer's instructions. In brief, 5-µm sections were deparaffinized, subjected to antigen retrieval by boiling in 10 mM sodium citrate buffer, pH 6.0, for 15 min, and blocked in 3% H₂O₂ for 10 min. The slides were washed twice in water and twice in phosphate-buffered saline (PBS). Sections were then incubated with goat polyclonal anti-PGRN antibody (1 : 100) (Santa Cruz, CA, USA) at 4 °C overnight. After slides were washed twice in PBS, Polymer Helper (ZSGB-BIO, Beijing, China) was added to the slides, which were then incubated for 1 h at 37 °C and washed twice in PBS. Polyclonal horseradish peroxidase-conjugated anti-goat immunoglobulin G (IgG) substrate (ZSGB-BIO) was added for 1 h at 37 °C, and detection was by the ChemMate EnVision HRP/DAB system (ZSGB-BIO). Slides were counterstained for 3 s in hematoxylin (ZSGB-BIO) and then dehydrated and mounted using Permount (Fisher Scientific). The results are expressed as percentage of positive cells in 200 cells.

Helicobacter pylori culture

The H. pylori strains used in this study were standard strain NCTC26695, standard strain NCTC11637, and Sydney strain 1 (SS1), and were kindly provided by Dr Zhang Jianzhong from the Chinese Disease Control and Prevention Center. Isogenic cagA⁻ mutant cagA⁻ 26695 was constructed within strain 26695 by insertional mutagenesis using piLL570 vector and were selected with kanamycin. All strains were grown in Brucella broth with 5% fetal bovine serum, with shaking at 120 r.p.m. under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂) at 37 °C, harvested by centrifugation, and added to gastric cells at multiplicity of infection of bacteria to cell of 100 : 1. Helicobacter pylori was heat killed by boiling it for 10 min, pelleted by centrifugation, and resuspended in fresh medium before being added to cells.

Plasmid vectors and transfection

pcDNA 3.1(+) Empty was a gift from Andrew Bateman (McGill University Health Center, Canada). Wild-type (WT) cagA/pcDNA3.1(+) plasmid (WT-cagA) was a gift from Yong-Liang Zhu (Zhejiang University, China). To silence endogenous PGRN, an RNA interference oligonucleotide (5′-ACCUCCUCACUAAGCUGCC-3′) was cloned into the pSuper vector to generate pSuper-PGRN-siRNA. FuGENE® HD Transfection Reagent (Roche Applied Science) was used to transfect vector pcDNA 3.1 Empty, pcDNA3.1WT-cagA, pSuper, and pSuper-PGRN-siRNA. All transfection procedures were performed according to the manufacturer's instructions, and all experiments were repeated three times.

RNA extraction and quantitative real-time PCR

Total cellular RNA was extracted with Trizol (Invitrogen) according to the protocol provided by the manufacturer. First-strand cDNA was synthesized from 1 µg total cellular RNA using the RevertAid TM First Strand cDNA Synthesis Kit (Fermentas) with random primers. Thereafter, cDNA was amplified for PGRN and β-actin. The specific primers used were as follows: for PGRN, forward primer 5′-GGACAGTACTGAAGACTCTG-3′ and reverse primer 5′-GGATGGCAGCTTGTAATGTG-3′; for β-actin, forward primer 5′-AGTTGCGTTACACCCTTTCTTG-3′ and reverse primer 5′-CACCTTCACCGTTCCAGTTTT-3′. The real-time PCR reactions involved the ABI7000 Fast Real-Time PCR System with SYBR Premix Ex TaqTM™ according to the specification procedures.

Western blot analysis

Cells were washed twice with ice-cold PBS and lysed in a buffer (50 mmol L⁻¹ Tris-HCl, pH 7.5, 150 mmol L⁻¹ NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS), containing protease inhibitors (Sigma Inc., St. Louis, MO). Equal proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were probed with antibodies against PGRN, β-actin, C-CagA [1](Santa Cruz), MEK1/2, Phospho-MEK1/1 (Ser217/221), phospho-p38MAPK (Thr180/Tyr182), p38MAPK (Beijing Biosynthesis Biotechnology Co. Ltd, China), then anti-goat or anti-rabbit horseradish peroxidase-conjugated IgG. The Chemilucent ECL Detection System (Millipore) was used to detect the signals. Bio-Rad quantity one 1-d analysis software (Bio-Rad, CA) was used to analyze the Western blot results.

Colony formation assay

Anchorage-independent growth was assessed by colony-formation ability (Zanocco-Marani et al., 1999). The cells were transfected with pSuper or pSuper-PGRN-siRNA for 72 h. Thereafter, cells were infected with or without H. pylori for 3 h and planted into six-well plates (300 per well for each cell line) and incubated for 10 days (GES-1) or 14 days (SGC7901). Plates were stained with Giemsa and the number of colonies with more than 50 cells was counted.

Wound healing migration assay

Confluent GES-1 cell monolayers on six-well tissue culture plastic dishes were transfected with pSuper or pSuper-PGRN-siRNA for 72 h. Five wounds (500 µmol L⁻¹) were generated in each plate using a thin disposable tip. Cultures were rinsed with PBS and replaced with fresh quiescent medium containing 10% fetal bovine serum (Mauro et al., 1999). Helicobacter pylori was then added to the cells, and wound images were taken at 0 and 16 h.

Statistical analysis

All experiments were repeated independently three times, and data were expressed as mean±SD. Student's t-test was used for statistical analysis. P<0.05 was considered statistically significant.

Results

PGRN protein levels are increased in patients with gastritis and gastric cancer tissue

To ascertain the PGRN production in gastric tissue, we performed an immunohistochemical analysis of PGRN in gastric biopsy specimens from patients with different disease conditions (Fig. 1). Normal biopsy specimens showed little immunoreactivity for PGRN (only 14.3% positive cells, Fig. 1a and e); however, biopsy specimens from patients with gastritis showed a marked increase in PGRN protein levels (50.6% positive cells in chronic superficial gastritis, Fig. 1b and e; 79.8% cells positive in chronic atrophic gastritis, Fig. 1c and e). Moreover, gastric cancer tissue showed intense PGRN staining of epithelial cells (88.2% positive cells, Fig. 1d and e).

Figure 1

PGRN expression in gastric specimens. Histochemical staining of formalin-fixed, paraffin-embedded gastric biopsy specimens with PGRN antibody. (a) Normal tissue, (b) chronic superficial gastritis patient (CSG) tissue, (c) chronic atrophic gastritis (CAG) tissue, (d) gastric cancer (GC) tissue. Magnification: × 200. (e) Dot-plot analysis of percentage of cells positive for PGRN calculated from 10 high-powered fields for each tissue. The average of these 10 fields is represented by a single dot in the vertical scatterplot. ^**P<0.01.

Helicobacter pylori infection upregulated PGRN mRNA and protein levels in gastric epithelial cells

To confirm that gastric epithelial cells could be a source of the increased level of PGRN with H. pylori infection, we next investigated whether this bacterium could directly induce PGRN production by SGC7901 and GES-1 cells. Confluent SGC7901 and GES-1 cells were incubated with H. pylori for 0, 3, 6, 12, and 24 h. PGRN mRNA expression was increased 4.17-fold in GES-1 cells and 2.15-fold in SGC7901 cells at 3 h after H. pylori infection as compared with control cells (Fig. 2a). Changes in protein levels were consistent with the mRNA data. Untreated cells exhibited low expression of PGRN, whereas H. pylori infection significantly stimulated the production of PGRN protein in SGC7901 and GES-1 cells in a time-dependent manner (Fig. 2b). Furthermore, to determine whether the promotion of PGRN expression infected by H. pylori is strain specific, we used H. pylori strains 26695, 11637, and SS1 to infect GES-1 and SGC7901 cells for 24 h. PGRN expression level was substantially elevated after infection by all three strains as compared with control cells (Fig. 2c).

Figure 2

Helicobacter pylori upregulates PGRN mRNA and protein levels in gastric epithelial cells. (a) SGC7901 or GES-1 cells were infected with H. pylori [multiplicity of infection (MOI) 100 : 1] for 3 h before analysis of PGRN mRNA expression. Experiments were performed in triplicate with similar results. (b) Helicobacter pylori infection increases PGRN expression in SGC7901 or GES-1 cells. Confluent SGC7901 or GES-1 cells were incubated with H. pylori at MOI 100 : 1 for the indicated times for Western blot analysis of PGRN protein expression. Experiments were performed in triplicate with similar results. (c) Infection with different strains increases PGRN expression in gastric epithelial cells. SGC7901 and GES-1 cells were incubated with different H. pylori strains at a MOI 100 : 1 for 24 h for Western blot analysis of PGRN protein expression. Blots are representative of three separate experiments with similar results. ^*P<0.05, ^**P<0.01.

PGRN upregulation is independent of CagA

Helicobacter pylori has a number of virulence as well as soluble factors. The cag pathogenicity island has been demonstrated to activate an inflammatory response in gastric epithelial cells and also is thought to be important in tumorigenesis (Censini et al., 1996; Higashi et al., 2002; Ernst et al., 2006). To investigate whether CagA was sufficient to induce the upregulation of PGRN, we overexpressed CagA with the pcDNA3.1WT-cagA plasmid in SGC7901 and GES-1 cells. At 48 h after transfection, overexpression of CagA did not induce an increase in PGRN mRNA levels (Fig. 3a). As well, in cells overexpressing CagA, PGRN protein levels did not increase at 48 h after transfection as compared with cells transfected with the control plasmid (Fig. 3b). Furthermore, SGC7901 and GES-1 cells were incubated with the parent strain 26695 and its isogenic cagA⁻ 26695. PGRN induction was significantly increased in cells incubated with the cagA⁻ compared with cells without treatment, and there was no significant difference in PGRN expression level in cells infected by strain cagA⁻ 26695 compared with the wild-type strain (Fig. 3c). The above results suggest that CagA was not sufficient to induce the upregulation of PGRN.

Figure 3

Pathogenetic factors of Helicobacter pylori indispensable for PGRN induction. (a) Real-time PCR analysis of effect of transfection of plasmid pcDNA3.1WT-cagA or control plasmid into SGC7901 and GES-1 cells on PGRN mRNA levels 48 h after transfection. (b) Western blot analysis of PGRN expression in gastric epithelial cells after transfection with CagA plasmid or control plasmid after 48 h. Blots were stripped and reprobed with β-actin as a loading control. Blots are representative of three separate experiments with similar results. (c) SGC7901 or GES-1 cells were incubated with H. pylori cagA⁺ 26695, cagA⁻ 26695 at a multiplicity of infection 100 : 1 for 24 h for Western blot analysis of PGRN protein expression. Blots are representative of three separate experiments with similar results. (d) PGRN protein upregulation by H. pylori-infected gastric epithelial cells is by direct contact with live H. pylori. SGC7901 and GES-1 cells were infected with live bacteria of H. pylori, live bacteria separated by a 0.2-µm filter, or heat-killed bacteria. PGRN protein levels were determined by Western blot analysis at 24 h. NS, No significant; ^**P<0.01.

Contact with live H. pylori is required to induce PGRN production by gastric epithelial cells

We next investigated the role played by bacterial factors in PGRN production. SGC7901 and GES-1 cells were directly infected with live H. pylori or co-cultured with H. pylori using 0.2-µm filters to separate the bacteria from the cells. Heat-killed bacteria were also added to the cells. Neither heat-killed bacteria nor soluble factors contained in H. pylori filtrates such as vacuolating toxin stimulated PGRN upregulation, which indicates that viable H. pylori is required for PGRN upregulation. A direct exposure of cells to live H. pylori caused an increase in PGRN production by SGC7901and GES-1 cells (Fig. 3d).

Possible signaling pathways of PGRN induction by H. pylori infection

Signal transduction pathways activated in response to bacterial contact play an important role in the pathogenesis of H. pylori infection. A group of important signaling molecules were found activated by H. pylori, including NF-κB and mitogen-activated protein kinases (MAPKs) (Ding et al., 2008; Saha et al., 2008). To define the molecular pathways mediating H. pylori-induced PGRN upregulation, we next applied the inhibitors to these three factors to SGC7901 cells 2 h before H. pylori infection. Only the p38 inhibitor (SB203580, 10 µM) and MEK1/2 inhibitor (PD98059, 50 µM) were able to significantly inhibit PGRN expression activated by H. pylori and reduce H. pylori-induced PGRN levels to baseline (Fig. 4a). To investigate the participation of the p38 and MEK1/2 pathway in the signal transduction process, phosphorylation of p38, MEK1/2 in response to H. pylori infection was assessed in Western blot studies. Following treatment of SGC7901 cells with H. pylori, we observed a dramatic, time-dependent activation of the p38 and MEK1/2 pathway. The level of phosphorylation of p38 and MEK1/2 increased at 30 min and remained elevated for 2 h after H. pylori infection (Fig. 4b). Thus, the p38MAPK and MEK1/2 signaling pathway may be primarily responsible for the increase of PGRN expression induced by H. pylori in SGC7901 cells.

Figure 4

Possible signaling pathways of PGRN induction by Helicobacter pylori infection. (a) Representative Western blot showing effects of inhibitors on PGRN expression in SGC7901 cells with H. pylori infection. Confluent SGC7901 cells pretreated with BAY11-7082 (5 µM), SB203580 (10 µM) and PD98059 (50 µM) for 2 h before incubation with or without H. pylori at a multiplicity of infection (MOI) 100 : 1 for 6 h. Blots were stripped and reprobed with β-actin as a loading control. Experiments were performed in triplicate with similar results. (b) Activation of the p38 and MEK1/2 pathway by H. pylori. Confluent SGC7901 cells were treated with H. pylori at an MOI 100 : 1 for the indicated period of time (15–120 min). Samples were collected and resolved by Western blot with rabbit anti-phospho-p38MAPK, rabbit anti-Phospho-MEK1/2. The membrane was subsequently stripped and reprobed with rabbit anti-p38MAPK, rabbit anti-MEK1/2 as protein loading controls. Blots are representative of three separate experiments with similar results. Blots are representative of three separate experiments with similar results. NS, Not significant; ^*P<0.05.

Role of PGRN in the proliferation of gastric cancer cells

PGRN can promote anchorage-independent growth (He et al., 2003; Jones et al., 2003) and proliferation of uterine smooth cells and mouse blastocysts (Qin et al., 2005; Matsumura et al., 2006). We examined whether this is also the case in gastric epithelial cells and the role of PGRN in the proliferation gastric cancer cells. For this purpose, we used PGRN-specific siRNA to knockdown PGRN expression in SGC7901 and GES-1 cells. The cells were first transfected with pSuper and pSuper-PGRN-siRNA. Efficient silence of PGRN expression in cells was verified by Western blot analysis (Fig. 5a). The repression of PGRN in SGC7901 cells and GES-1 cells led to a significant reduction in foci number as well as size (Fig. 5b and c). Regardless of whether they were infected with H. pylori, SGC7901 and GES-1 cells all showed good proliferation after transfection with control plasmid in cologenic analysis, but knockdown by PGRN-specific siRNA almost attenuated the proliferative activities induced by H. pylori infection.

Figure 5

siRNA analysis of promotion of cell growth due to production of PGRN by Helicobacter pylori infection. (a) Western blot analysis of PGRN expression in gastric epithelial cells transfected with pSuper and pSuper-PGRN-siRNA plasmid. Blots are representative of three separate experiments with similar results. (b) GES-1 and SGC7901 cells were transiently transfected with pSuper or pSuper-PGRN-siRNA plasmid for 72 h and co-cultured with H. pylori at an multiplicity of infection 100 : 1 for 3 h, and the clonogenic potentials of the these cells were then assessed. Experiments were performed in triplicate with similar results. (c) Mean foci numbers from three experiments. HP, Helicobacter pylori. ^*P<0.05.

Requirement of PGRN for cell migration in response to H. pylori infection

The growth factor PGRN has recently been shown to promote wound healing migration of breast cancer cells (He et al., 2003; Tangkeangsirisin & Serrero, 2004; Monami et al., 2006). To determine whether H. pylori infection promotes cell migration in a PGRN-dependent manner, GES-1 cells were treated with PGRN-specific siRNA to knockdown PGRN expression and were infected with H. pylori. Wounds were then induced in cell monolayers and measured over 16 h. Knockdown of PGRN significantly altered cell motility in uninfected cells (Fig. 6a and b). Although H. pylori infection increased wound healing compared with that in uninfected cells, the healing was still decreased by knockdown of PGRN (Fig. 6a and b). These data suggest that upregulation of PGRN by H. pylori infection promotes gastric cell migration.

Figure 6

Helicobacter pylori-infection-induced cell migration mediated by upregulation of PGRN. (a) GES-1 cells were transfected with pSuper or pSuper-PGRN-siRNA plasmid for 72 h. Wounds were then introduced into the cell monolayer, and medium or H. pylori (HP) was added. Wound areas were measured at 0 and 16 h after infection. Experiments were performed in triplicate with similar results. (b) Quantification of wound closure for each treatment group in experiments repeated at least five times. ^*P<0.05.

Discussion

PGRN belongs to a novel class of growth factors that play a critical role in development, cell cycle progression, cell motility, and tumorigenesis, and could promote anchorage-independent growth (He et al., 2003; Jones et al., 2003). In addition to its normal regulatory role in wound healing and cellular mitosis, PGRN can function as an autocrine regulator of tumorigenesis, it has an important influence on tumor growth, and it actively confers malignancy (Bagnato et al., 1997; Zanocco-Marani et al., 1999; He et al., 2003). Although PGRN is highly expressed in gastric carcinoma cells, the relation between PGRN and H. pylori-infection-induced cellular responses with carcinogenic potential has remained unclear. In this report, we have shown that infection of gastric epithelial cells by the gastric pathogen H. pylori upregulates PGRN, and the induction of PGRN by H. pylori infection can stimulate gastric epithelial cell proliferation and migration, which may contribute to the process of oncogenic transformation.

Previous reports have shown that PGRN levels are increased in gastric cancer (Linē et al., 2002). Gastritis and gastric cancer tissue showed elevated PGRN levels compared with healthy tissue. As well, gastric epithelial cells infected with H. pylori responded by upregulating PGRN mRNA and protein production. To our knowledge, this is the first report of PGRN upregulation by H. pylori-infected gastric epithelial cells.

The best characterized H. pylori virulence determinant is the cag pathogenicity island. CagA translocates into host cells through a type IV bacterial secretion system and activates various signal transduction pathways, which results in pathological cellular responses such as morphological changes, increased cell proliferation, motility, and apoptosis (Handa et al., 2007). Accordingly, we wondered whether CagA plays a critical role in PGRN upregulation in H. pylori-infected gastric epithelial cells. However, we noted that the transfection of CagA into gastric epithelial cells did not have significant effect on the induction of PGRN. Furthermore, PGRN induction was significantly increased in cells incubated with the cagA⁻ vs. the wild-type strain. CagA was not sufficient to induce the upregulation of PGRN. Other well-recognized virulence factors which are more commonly present in disease-associated H. pylori strains are vacuolating cytotoxin (VacA) and lipopolysaccharide. VacA has multiple effects on epithelial cells, i.e. induction of apoptosis, inhibition of the activation and proliferation of T lymphocytes, and modulation of the T-cell cytokine response. These effects trigger cellular damage and immunosuppressive activities by which H. pylori predisposes to gastric carcinogenesis (Boncristiano et al., 2003; Cover et al., 2003). Similarly, H. pylori lipopolysaccharide can enhance inflammatory reactions and augments cell proliferation (Chochi et al., 2008). As well, PGRN upregulation was not induced by soluble secreted factors, which may include vacuolating toxin and lipopolysaccharide. Only contact with live H. pylori was able to cause increased PGRN production in gastric epithelial cells, as the induction of MMP-7 by H. pylori required adhesion of bacteria to epithelial cells (Wroblewski et al., 2003). Therefore, it is increasingly appreciated that H. pylori–host interactions contribute to the final disease outcome.

A group of important signaling molecules activated by H. pylori is the MAPKs. Helicobacter pylori-induced cathepsin X upregulation depends on the activation of ERK1/2 and JNK/p38 in monocytes and gastric epithelial cells, respectively (Krueger et al., 2009) and ERK1/2 activation is required for H. pylori-mediated PAI-1 upregulation (Keates et al., 2008). The MAPK pathway plays a crucial role in mediating a number of events in host cell physiology, including movement, adhesion, metabolism, and apoptosis (Chang & Karin, 2001; Johnson & Lapadat, 2002). However, which pathway plays a key role in H. pylori-upregulated PGRN is not clear. In the present study, we found that p38 and MEK1/2 activation is required for H. pylori-mediated PGRN upregulation in gastric epithelial cells. However, further experiments are required to determine the precise mechanism by which p38 and MEK1/2 are activated in H. pylori-infected gastric epithelial cells.

Many interventional studies have established a relation between H. pylori infection and gastric cancer. New cancer lesions were detected later in patients cured by eradication therapy than in patients with persistent H. pylori infection (Daniel et al., 2000). Thus, H. pylori eradication might delay the development of gastric cancers. Helicobacter pylori was found to augment the growth of gastric cancers via the lipopolysaccharide-TLR4 pathway, promoting proliferation and progression of gastric cancers (Chochi et al., 2008). In line with this, H. pylori promotes cellular migration and decreases apoptosis by activation of phosphatidylinositol 3-kinase signaling (Nagy et al., 2009). We attempted to evaluate the oncogenic effect of PGRN on gastric carcinoma cells after H. pylori infection. Knockdown of PGRN in gastric epithelial cell line could inhibit proliferation. Hyperproliferation has been reproducibly demonstrated in H. pylori-infected tissue (Fraser et al., 1994); however, regardless of infection with H. pylori, gastric epithelial cells all had good proliferation after transfection with control plasmid in cologenic analysis, but knockdown of PGRN by PGRN-specific siRNA almost attenuated the proliferative activities induced by H. pylori infection.

Cellular migration plays an important role in the invasive potential and metastatic growth of cancers. PGRN could evoke a substantial migration of gastric epithelial cells into the denuded area. Furthermore, H. pylori infection significantly increased wound healing compared with that in uninfected cells, although PGRN interference still reduced the healing. Thus, the overexpression of PGRN by H. pylori infection is a strong tumorigenic factor for gastric cancer.

In summary, our data provide a novel insight into H. pylori-induced host–pathogen interactions related to MAPK signaling. Helicobacter pylori infection activated the p38 and MEK1/2 signal pathway, which resulted in the elevated expression of the growth factor PGRN in gastric epithelial cells. Increased expression of PGRN with H. pylori infection could promote cell proliferation, stimulate cell migration, and increase the malignancy of gastric epithelial cells. Our findings suggest that increased PGRN production with H. pylori infection may contribute to H. pylori-associated tumorigenesis.

Authors' contribution

H.W. and Y.S. contributed equally to this work.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 30972775, 30770118, 30800406, 30800037, 81071313, 81001098, 81000868, and 30971151) and the Science Foundation of Shandong Province, China (no. ZR2009CZ001 and ZR2009CM002).

Author notes

Editor: Nicholas Carbonetti