IMMUNOLOGY & MEDICAL MICROBIOLOGY

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1 IMMUNOLOGY & MEDICAL MICROBIOLOGY RESEARCH ARTICLE Helicobacter pylori cag pathogenicity island-positive strains induce syndecan-4 expression in gastric epithelial cells Ana Magalhães 1, Nuno T. Marcos 1, Ana S. Carvalho 1, Leonor David 1,2,Céu Figueiredo 1,2, Joana Bastos 2,3, Guido David 4 & Celso A. Reis 1,2 1 Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Porto, Portugal; 2 Medical Faculty, University of Porto, Porto, Portugal; 3 Institute of Public Health, University of Porto, Porto, Portugal; and 4 Department of Human Genetics, Katholieke University of Leuven, Leuven, Belgium Correspondence: Celso A. Reis, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr Roberto Frias, s/n, Porto, Portugal. Tel.: ; fax: ; celsor@ipatimup.pt Received 29 September 2008; revised 9 February 2009; accepted 5 May Final version published online 10 June DOI: /j X x Editor: Leif Percival Andersen Keywords Helicobacter pylori ; cag pathogenicity island; syndecan-4; heparan sulfate; gastric disease. Introduction Abstract Helicobacter pylori is recognized as the main cause of gastritis and is associated with gastric carcinogenesis. Syndecan-4 represents the major source of heparan sulfate (HS) in the gastric cells. HS proteoglycans expressed on the cell surface constitute targets for H. pylori at the early stage of infection. The aim of this study was to determine whether H. pylori induction of syndecan-4 expression is affected by the virulence characteristics of the infecting strain, namely the cytotoxic-associated gene (cag) pathogenicity island (PAI). We observed that individuals infected with highly pathogenic H. pylori strains express syndecan-4 in the foveolar epithelium of the gastric mucosa. The association between the cagpai status of the infecting strain and syndecan-4 expression was further demonstrated by infection of gastric epithelial cell lines with a panel of cagpai 1 and cagpai H. pylori strains, showing that expression of syndecan-4 was significantly increased in response to infection with the highly pathogenic strains. Moreover, infection of gastric cells with caga and cage mutant strains further confirmed that syndecan-4 induction is dependent on an intact cagpai. The present study shows that highly pathogenic H. pylori strains induce syndecan-4 expression, both in human gastric mucosa and in gastric cell lines, in a cagpai-dependent manner. Helicobacter pylori is a Gram-negative spiral-shaped microaerophilic bacterium infecting human gastric mucosa worldwide. Individuals infected by H. pylori develop gastritis and up to 10% of infected individuals develop duodenal ulcer disease. Persistent infection with H. pylori may cause chronic atrophic gastritis with development of intestinal metaplasia (IM), dysplasia and gastric carcinoma (Parsonnet et al., 1991; Correa, 1992; Wang et al., 1998; Suerbaum & Michetti, 2002; Mesquita et al., 2006). The role of H. pylori in this carcinogenesis pathway is reinforced by experiments showing that H. pylori induces gastritis, IM and gastric carcinoma in animal models (Watanabe et al., 1998; Fujioka et al., 2000; Zheng et al., 2004). Helicobacter pylori is classified as a carcinogenic agent class I by the International Agency for Research on Cancer. Although the mechanism of H. pylori pathogenicity is not completely understood, bacterial virulence factors and host susceptibility features have been associated with the development of chronic gastric inflammation and gastric carcinogenesis (Cover & Blaser, 1999; Figueiredo et al., 2002; Peek & Blaser, 2002). Bacterial genetic loci contribute to the pathogenic potential, among them the cytotoxic-associated gene A (caga), which is a marker for the presence of the cag pathogenicity island (PAI) and is found in 50 60% of H. pylori isolates in Western countries. The CagA protein is translocated by a type IV secretion system (encoded by the cagpai) into gastric epithelial cells, where it induces changes in the tyrosine phosphorylation states of distinct cellular proteins, leading to activation of the nuclear factor-kb (NF-kB) signaling pathway and promoting interleukin-8 (IL-8) production (Odenbreit et al., 2000, 2002; Tanaka et al., 2003; Hatakeyama, 2004; Bourzac & Guillemin, 2005; Brandt et al., 2005). Other H. pylori virulence markers include the vaca and the baba2 genes, which encode a vacuolating cytotoxin and an adhesin (Ilver et al., 1998), respectively, both associated with the risk of development of gastric disease (van Doorn et al., 1998; Gerhard et al., 1999).

2 224 A. Magalhães et al. Heparan sulfate (HS) glycosaminoglycans have been shown to be recognized by H. pylori outer-membrane proteins and to be involved in the adhesion of the bacteria in cell-line models (Wadström et al., 1996; Utt & Wadström, 1997; Guzman-Murillo et al., 2001; Ruiz-Bustos et al., 2001). The syndecans are a family of four type I transmembrane heparan sulfate proteoglycans (HSPGs) that represent the major source of HS on the cell surface. Syndecan expression shows temporal and spatial specificity, but most cells express more than one family member. Syndecan-1 is expressed in most epithelial cells, syndecan-2 is present mostly in mesenchymal, neuronal and smooth muscle cells, syndecan-3 is mostly expressed on the nervous system, while syndecan-4 is more ubiquitously expressed (Zimmermann & David, 1999). The syndecans bind to a wide variety of extracellular molecules such as extracellular matrix components, growth factors, cytokines and microbial pathogens (Götte, 2003), and have been shown to play a multiplicity of physiological roles, as well as being involved in the pathogenesis of several infectious diseases (Freissler et al., 2000; Henry-Stanley et al., 2003; Smith et al., 2006; Bishop et al., 2007). Previous studies have shown that syndecan-4 expression can be enhanced in gastric epithelial cells upon infection with H. pylori or by stimulation of epithelial cells with agonists specific for members of the Toll-like receptor family, which are the main pattern recognition receptors that participate in innate immune responses against microbial pathogens (Smith et al., 2006). Recently, our group has demonstrated by microarray analysis, using the GLYCOv2 array from the Consortium for Functional Glycomics, that the highly pathogenic cagpai-positive H. pylori strain 26695, but not the cagpai-negative Tx30a strain, induced a significant increase in the syndecan-4 level of expression in a gastric cell line (Marcos et al., 2008). However, these previous studies did not evaluate the expression of syndecan-4 in human gastric mucosas upon H. pylori infection and the role that these proteoglycans play in the inflammatory response and pathogenesis process induced by H. pylori. In the present study, we evaluated whether H. pyloriinfected individuals express syndecan-4 in their gastric mucosa and whether highly pathogenic H. pylori strains would selectively induce the expression of syndecan-4 contributing to the gastric pathogenesis process. Materials and methods Human gastric mucosa samples and characterization of H. pylori infection Fifty-five gastric biopsy specimens from the antrum (n = 49) and incisura angularis (n = 6) were collected from patients who were part of a cohort of dyspeptic individuals from northern Portugal (Viana do Castelo) working in a shipyard (Nogueira et al., 2001). Patients provided informed consent and underwent standard gastroscopy in Hospital S. João (Porto, Portugal). All biopsies were fixed in 10% formalin and embedded in paraffin. Sections were stained by modified Giemsa. The evaluation of the caga, vaca and icea genotypes of H. pylori was performed as described previously using DNA extracted from frozen biopsies adjacent to the biopsies that were paraffin embedded and used for histopathological analysis (Nogueira et al., 2001). The detailed information on the characteristics of the individuals participating in this study, including the H. pylori virulence factor profile (caga, vaca and icea), is described in the Supporting Information, Table S1. caga genotyping was used as an indicator of the cagpai status of the infecting strain. Immunohistochemistry Immunohistochemical staining of syndecan-4 was performed in accordance with standard procedures on paraffin-embedded sections (Reis et al., 1999). Briefly, after deparaffination, rehydration and blocking with normal rabbit serum diluted 1 : 5 in phosphate-buffered saline (PBS) containing 10% of bovine serum albumin (BSA), samples were incubated with 10 mgml 1 of the monoclonal antibody 8G3 (David et al., 1992) overnight at 4 1C. The slides were then washed in PBS and incubated for 30 min with biotinylated rabbit anti-mouse antibody (E0354-Dako- Cytomation, Glostrup, Denmark) diluted 1 : 200 in PBS containing 5% of BSA. Samples were washed with PBS and incubated for 30 min with avidin biotin complex (Vectastain Elite ABC kit, Burlingame, CA) according to the manufacturer s recommendations. Staining was performed with 3,3 0 -diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) containing 0.02% hydrogen peroxide. Slides were counter-stained with Mayer s hematoxylin. Negative controls were performed by replacing the primary antibody with PBS. The immunostaining of gastric mucosas was scored as positive or negative when 4 5% or 5% of the cells from the foveolar epithelium were positive, respectively. The staining intensity was fairly homogeneous in all the positive cases and therefore was not scored. Helicobacter pylori strains characterization and culture Helicobacter pylori cagpai 1 strains 101uk, 127uk, BO265, 111uk, and CM71 and cagpai strains 094uk, 097uk, 131uk and 123uk were obtained from the Department of Molecular Biology, Max-Planck Institut für Infektionsbiology, Berlin, Germany. The H. pylori insertion mutants with inactivation of the caga (60190DcagA), cage (60190DcagE) genes (Bebb et al., 2003) and their parental wild-type strain 60190

3 cagpai positive strains of H. pylori induce syndecan [American type culture collection (ATCC) 49503, cagpai 1 and vaca s1/m1] were obtained from the Institute of Infection, Immunity and Inflammation, University of Nottingham, UK. Helicobacter pylori strains (ATCC , cagpai 1 and vaca s1/m1) and Tx30a (ATCC 51932, cagpai and vaca s2/m2) were obtained from ATCC (Rockville, MD) (Table 1). The strains were grown in tryptic soy agar supplemented with 5% sheep blood (BioMérieux, Marcy l Étoile, France) at 37 1C for 3 days under microaerobic conditions. The cagpai status of the different strains has been characterized previously (Suerbaum et al., 1998; Falush et al., 2003). The vaca genotyping was performed as described previously (Nogueira et al., 2001). Helicobacter pylori infection of gastric cell lines Two human gastric carcinoma cell lines (MKN45 and AGS) were used. MKN45 (Tamura et al., 1996) were obtained from the Japanese Collection of Research Biosources (Osaka, Japan) and AGS (Barranco & Townsend, 1983) were obtained from ATCC. Cell lines were cultured as described previously (Marcos et al., 2004), and 24 h before the infection experiments, cells were transferred to new flasks and cultured in the absence of antibiotics. A panel of H. pylori strains (Table 1) with different virulence profiles, regarding the cagpai and vaca genotypes, was used for infecting Table 1. Helicobacter pylori strains: characterization of their cagpai and vaca status Strain cagpai vaca 094uk s1/m2 097uk s2/m2 101uk 1 s1/m2 111uk 1 s1/m2 123uk s1/m2 127uk 1 s1/m1 131uk s2/m2 CM71 1 s1/m1 BO265 1 s1/m1 Tx30a [ATCC ] s2/m [ATCC 51932] 1 s1/m [ATCC 49503] 1 s1/m1 Suerbaum et al. (1998) and Falush et al. (2003). gastric cell lines. Helicobacter pylori strains were grown for 3 days as described above, collected and resuspended in Brucella broth (Becton and Dickinson, Cockeysville, MD). Bacteria were added to fresh RPMI 1640 media supplemented with 10% fetal bovine serum to obtain an MOI = 100 and incubated with gastric cells for 8 h. Tumor necrosis factor (TNF)-a treatment of gastric cell lines MKN45 and AGS ( cells) were transferred to a T25 t-flask with regular medium 24 h before TNF-a treatment. Both cell lines were then treated with 40 ng ml 1 of TNF-a (PeproTech Inc., Rocky Hill, NJ) or with the equivalent volume of regular medium as control. AGS and MKN45 cells were maintained in culture for 24, 48 and 72 h, followed by RNA extraction as described below. Quantitative real-time PCR Total RNA was extracted using the RNeasy isolation kit (Qiagen, Hilden, Germany) according to the manufacturer s protocol. RNA yield and quality were determined spectrophotometrically and 5.0 mg of total RNA was reverse transcribed using the Superscript III RNAse H Reverse Transcriptase kit (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer s instructions. Real-time PCR was used for quantitative detection of syndecan-4 and IL-8 genes. The reaction was carried out in an ABI Prism 7000 (Applied Biosystems, Foster City, CA) in a 20-mL final volume containing 4 ml of cdna, 6 pmol of each primer (Table 2) and 10 ml of POWER SYBR Green (Applied Biosystems). Polymerase activation was performed at 95 1C for 15 min, followed by 40 cycles as described in Table 2. A quantitative measure was obtained through amplification of 18S cdna in each sample using the same PCR conditions. Triplicates were performed for each sample. Statistical analysis Table 2. Real-time PCR conditions and primer sequences for analysis of gene expression in MKN45 and AGS cells Comparison of syndecan-4 expression in the gastric mucosa regarding H. pylori infection status was performed using the Gene Primers Amplicon (bp) PCR Real-time PCR IL-8 for 5 0 -gccttcctgatttctgcagc rev 5 0 -cgcagtgtggtccactctca-3 0 Syndecan-4 for 5 0 -ccggagccctaccagacgat times 15 s, 95 1C rev 5 0 -aggcaccaagggatggacaa s, 60 1C 18S for 5 0 -cgccgctagaggtgaaattc rev 5 0 -cattcttggcaaatgctttcg-3 0

4 226 A. Magalhães et al. w 2 test and Fisher s exact test whenever appropriate. For evaluation of the H. pylori infection assays, a variable that represents the average of the assays for each strain in each cell line was created and H. pylori strains were divided into cagpai-positive and cagpai-negative groups. The comparison between syndecan-4 expression and the cagpai status of the infecting strain was performed using the nonparametric Mann Whitney test. The t-test was used to compare the values for syndecan-4 mrna expression in the infection experiment with the mutant strains and the TNF-a treatment assay. The statistical analysis was performed using STATA s, version 9.2. Results Expression pattern of syndecan- 4 in the human gastric mucosa of noninfected and H. pylori-infected individuals Syndecan-4 expression was characterized by immunohistochemistry in the gastric mucosa of noninfected individuals and individuals infected with H. pylori strains of different pathogenicities (Table S1). Syndecan-4 expression was evaluated based on the membrane expression in the foveolar epithelium of the mucosa, and excluded the diffuse cytoplasmic staining observed in the gastric glands of all the cases. Most of the noninfected individuals (75%) were negative for syndecan-4 expression in the foveolar epithelium (Fig. 1a c and Table 3). Regarding the expression of syndecan-4 in the gastric mucosa of infected individuals, it was possible to distinguish between the infected ones with less pathogenic strains, cagpai, and those infected with highly pathogenic, cagpai 1, H. pylori strains. Expression of syndecan-4 was observed in the foveolar epithelium of the gastric mucosa of most individuals infected with cagpai 1 strains (81%) (Fig. 1g i and Table 3). The pattern of immunostaining was characterized by a baso-lateral and apical membrane labeling of the epithelial cells. In contrast, the majority of the individuals (65%) infected with H. pylori cagpai strains did not show expression of this proteoglycan in their gastric mucosa (Fig. 1d f and Table 3). These observations indicate that the expression of syndecan-4 in human gastric mucosa is significantly associated (P-value = 0.003) with the infection by highly pathogenic H. pylori strains that harbor the cagpai. It is also possible to observe a significant association between syndecan-4 expression and the vaca s1b/m1 genotype (P-value = 0.04), which was predictable, considering the strong association between the presence of caga and vaca s1 and m1 genotypes (Figueiredo et al., 2001). No association was observed between the icea genotype and syndecan- 4 expression. Fig. 1. Immunohistochemical detection of syndecan-4 in human gastric mucosas. Gastric mucosa of a noninfected individual (a c). Gastric mucosa of a cagpai Helicobacter pylori-infected individual (d f). Gastric mucosa of a cagpai 1 H. pylori-infected individual (g i). Syndecan-4 expression was detected using the monoclonal antibody 8G3 (a, b, d, e, g and h); H. pylori visualization (white arrowheads) by modified Giemsa (c, f and i). Micrograph amplification 400 (a, d and g) and 1000 (b, c, e, f, h and i).

5 cagpai positive strains of H. pylori induce syndecan Table 3. Expression of syndecan-4 in superficial foveolar epithelium of the gastric mucosa Syndecan-4 expression (antibody 8G3 reactivity) H. pylori status cagpai status Negative (%) Positive (%) ) H. pylori noninfected (n = 8) 6 (75%) 2 (25%) o H. pylori infected (n = 47) cagpai (n = 26) 17 (65%) 9 (35%) cagpai 1 (n = 21) 4 (19%) 17 (81%) Fig. 2. IL-8 mrna expression in MKN45 and AGS cells infected with different Helicobacter pylori strains. MKN45 cells (a) and AGS cells (b) noninfected (control) or infected with each H. pylori cagpai strain (094uk, 097uk, 123uk, 131uk and Tx30a) or H. pylori cagpai 1 strain (101uk, 111uk, 127uk, CM71, BO265 and 26695). Real-time PCR reactions were performed in triplicate. IL-8 expression levels, were normalized to 18S expression levels and results indicate the average fold difference relative to uninfected cells. Characteristics of the cag PAI and vaca virulence factors of H. pylori strains used in infection assays The characteristics of the H. pylori strains used are described in Table 1 and included seven cagpai 1 and five cagpai strains. Regarding the vaca genotyping status, there were five strains s1/m1, four strains s1/m2 and three strains s2/ m2. Modulation of syndecan-4 expression profiles in gastric cell lines MKN45 and AGS induced by different H. pylori strains To test the hypothesis that syndecan-4 expression in gastric epithelial cells is associated with the cagpai status of H. pylori, MKN45 and AGS cells were infected with a panel of cagpai 1 and cagpai H. pylori strains (Table 1). The IL-8 (a) Fold difference (IL-8/18S) (b) Fold difference (IL-8/18S) gene was used as a control known to be induced by H. pylori (Fig. 2). Quantification of syndecan-4 mrna expression by realtime PCR in both cell lines, MKN45 and AGS, showed that syndecan-4 expression levels were significantly increased upon infection with cagpai 1 strains. In MKN45 cagpai 1 H. pylori strains induced a five- to 23-fold increase of syndecan-4 mrna expression while most of the cagpai induced levels similar to noninfected cells. Helicobacter pylori strain 097uk was an exception among the cagpai strains inducing levels similar to 127uk and CM71 cagpai 1 strains (Fig. 3a). Similarly, cagpai 1 H. pylori strains induced a four- to 21-fold increase in AGS, whereas the majority of cagpai strains induced lower levels of syndecan-4 mrna expression. The exceptions were H. pylori strain 097uk, which induced levels similar to the cagpai 1 strains 111uk, 127uk and 26695, and H. pylori strain 094uk, which induced levels similar to cagpai 1 strain (Fig. 3b). The

6 228 A. Magalhães et al. (a) Fold difference (syndecan-4/18s) (b) Fold difference (syndecan-4/18s) Control 094uk 097uk 123uk 131uk Tx30a 101uk 111uk 127uk CM71 BO Control 094uk 097uk 123uk 131uk Tx30a 101uk 111uk 127uk CM71 BO Mann Whitney test showed that the differences in the levels of syndecan-4 expression observed in both cell lines whenever the cells were infected with either cagpai 1 or cagpai strains were statistically significant (MKN45 P = and AGS P = 0.018). Upon infection of MKN45 and AGS cells with the isogenic mutants for caga and cage of the H. pylori strain 60190, syndecan-4 expression was significantly different in comparison with the parental strain (Fig. 4). Both 60190cagA and 60190cagE strains led to decreased induction of syndecan-4, indicating the relevance of these genes in the modulation of syndecan-4 expression. Compared with the parental strain, the more marked decrease in induction was observed with the 60190cagE strain that had a behavior similar to the cagpai strains studied. TNF-a effect in the modulation of syndecan- 4 expression in gastric epithelial cells Because H. pylori gastritis is associated with increased gastric mucosal production of TNF-a (Crabtree et al., 1991) and previous studies have demonstrated that syndecan-4 expression in endothelial cells was induced by TNF-a (Zhang et al., 1999), we analyzed whether TNF-a treatment of gastric epithelial cell lines would lead to increased syndecan-4 expression. Evaluation by quantitative real-time PCR showed induction of syndecan-4 expression in both MKN45 and AGS cell lines when cultured in the presence of TNF-a. In MKN45 cells, a 6.3-fold increase was observed at 48 h and a 2.5-fold increase at 72 h, when compared with the respective controls (Fig. 5). In AGS cells, a 4.4-fold increase was observed at 24 h, a 2.6-fold increase at 48 h and a 3.8-fold increase at 72 h after treatment when compared with the controls without TNF-a treatment (Fig. 5). Discussion Fig. 3. Syndecan-4 mrna expression in MKN45 and AGS cells infected with different Helicobacter pylori strains. MKN45 cells (a) and AGS cells (b) noninfected (control) or infected with each H. pylori cagpai strain (094uk, 097uk, 123uk, 131uk and Tx30a) or H. pylori cagpai 1 strain (101uk, 111uk, 127uk, CM71, BO265 and 26695). Real-time PCR reactions were performed in triplicate. Syndecan-4 expression levels were normalized to 18S expression levels, and results indicate the average fold difference of independent infection experiments relative to uninfected cells (Mann Whitney test, P = for MKN45 and P = for AGS). The virulence factors of H. pylori and the host inflammatory response have been shown to be relevant in the development of gastric lesions induced by the bacteria. In the present study, we evaluated the expression of syndecan-4 in human gastric mucosa taking into account H. pylori infection, with a particular focus on the cagpai status of the bacterial strain. Strains possessing the cagpai are strongly associated with severe gastric inflammation, ulceration and an increased risk of gastric cancer(odenbreit et al., 2000; Figueiredo et al., 2002). Our results showed that H. pylori-infected individuals express syndecan-4 in the foveolar epithelium of gastric mucosa and this expression is dependent on the cagpai status of the infecting strain. Moreover, we demonstrate, using in vitro infection models, that the pathogenicity characteristics of H. pylori strains are relevant for the induction of syndecan-4 expression in gastric cell lines. As

7 cagpai positive strains of H. pylori induce syndecan Fig. 4. Syndecan-4 mrna expression in MKN45 and AGS cells infected with caga and cage Helicobacter pylori mutant strains. MKN45 cells and AGS cells noninfected (control), infected with the H. pylori strain or the mutant 60190cagA and 60190cagE strains. Real-time PCR reactions were performed in triplicate. Syndecan-4 expression levels were normalized to 18S expression levels, and results indicate the average fold difference relative to uninfected cells. P o 0.01 and P o Fig. 5. TNF-a modulation of syndecan-4 expression in human gastric cell lines. Analysis by real-time PCR of syndecan-4 mrna expression in MKN45 and AGS cells cultured in the absence (control) or presence of 40 ng ml 1 of TNF-a. Real-time PCR reactions were performed in triplicate. Syndecan-4 expression levels were normalized to 18S expression levels, and the results indicate the average fold difference compared with untreated cells. P o 0.05 and P o Fold difference (syndecan-4/18s) Fold difference (syndecan-4/18s) Control cag cag Control shown in Fig. 3, the syndecan-4 expression pattern induced by H. pylori strains was similar in both the gastric cell lines analyzed, with cagpai 1 strains inducing higher expression levels of syndecan-4 when compared with cagpai strains. The cagpai-positive strain induced a higher increase of syndecan-4 in MKN45 when compared with AGS. These results are consistent with a previous study that showed that MKN45 cells were more sensitive to the H. pylori strain than AGS cells in terms of syndecan-4 induction (Smith et al., 2006). In order to confirm the cagpai dependence of syndecan-4 induction, we performed infection assays of the gastric cell lines using the 60190cagA and 60190cagE mutant strains. The comparison of syndecan-4 levels showed that these mutant strains induced a significantly lower syndecan-4 expression as shown in Fig. 4. The cage mutant strain induced levels similar to those induced by the cagpai strains, suggesting that syndecan-4 expression depends on the CagE, a structural component essential for a functional type IV secretion system. Together, these results demonstrate that induction of syndecan-4 is partially mediated through the signaling pathways activated by CagA injection into gastric cells. CagA, once injected into the gastric epithelial cells, induces changes in the phosphorylation of distinct cellular proteins, leading to activation of the NF-kB signaling pathway. This activation leads to expression of proinflammatory cytokines, such as TNF-a, IL-8 and IL-1 (Jung et al., 1997; Maeda et al., 2001; Brandt et al., 2005; Seok-Yong et al., 2006). Previous studies have demonstrated that microbial-induced syndecan-4 expression is mediated through the activation of NF-kB (Smith et al., 2006), and that this activation plays an important role in the TNF-a-dependent induction of syndecan-4 in endothelial cells (Zhang et al., 1999). Considering that TNF-a overexpression is observed cag cag

8 230 A. Magalhães et al. in H. pylori-infected gastric mucosas (Crabtree et al., 1991; Jung et al., 1997) and recent data showing that TNF-a expression levels in gastric epithelial cells are increased by infection with cagpai H. pylori Tx30a and even more with the cagpai strain (Marcos et al., 2008), we evaluated whether this cytokine could have a relevant function in the induction of syndecan-4 expression. As shown in Fig. 5, we observed that the expression of syndecan-4 mrna was significantly increased in MKN45 and AGS gastric cell lines upon TNF-a treatment. This direct induction by TNF-a shows that other mechanisms are capable of modulating the expression of syndecan-4. The role of syndecan-4 in the epithelial cell response to H. pylori infection is not fully understood. Several pathogens exploit cell surface HSPGs in order to bind to host cells. Previous studies demonstrated that overexpression of both syndecan-1 and syndecan-4 in HeLa cells led to increased Neisseria gonorrhoeae invasion, which was dependent on the presence of both syndecans (Freissler et al., 2000). Furthermore, it was demonstrated that HS, such as those on syndecan-1, played an important role in adherence and internalization of Listeria monocytogenes by cultured enterocytes (Henry-Stanley et al., 2003). Regarding H. pylori, several studies have pointed to a role of HS glycosaminoglycans in the process of bacteria adhesion to epithelial cells (Wadström et al., 1996; Utt & Wadström, 1997; Guzman-Murillo et al., 2001; Ruiz-Bustos et al., 2001). Furthermore, a study has predicted the HS-binding properties of the VacA cytotoxin, suggesting that HS may play a role in mediating the entry of this toxin into cells (Utt et al., 2001). Recently, syndecan-4 has been suggested as essential in the induction of a conformational change necessary for functional activation of b1 integrin (Saito et al., 2007). Integrins are cell adhesion receptors that mediate cell cell, cell extracellular matrix and cell pathogen interactions and have been proposed as molecules exploited by H. pylori for injection of CagA into gastric epithelial cells (Kwok et al., 2007). These observations suggest that syndecan-4 plays an important role in H. pylori adhesion to the gastric epithelial cells and in the process of type IV secretion system activation and CagA injection. In conclusion, we have demonstrated that H. pylori infection induces syndecan-4 expression, both in human gastric mucosa and in gastric epithelial cell lines, and that this is dependent on the cagpai. Induction of syndecan-4 by highly pathogenic H. pylori strains can contribute towards the higher aggressiveness of these strains, supporting the role of this HS-rich proteoglycan in the gastric pathogenesis process. Acknowledgements We thank Nuno Mendes for technical support. We thank M. Achtman and the Department of Molecular Biology, Max- Planck Institut für Infektionsbiology, Berlin, Germany, and Rachel Thomas, Richard Argent and John C. Atherton from the Institute of Infection, Immunity and Inflammation, University of Nottingham, UK, for the H. pylori strains. We thank Maria Oliveira and Emerson Bernardes for helpful discussions. This work was supported by Fundação para a Ciência e a Tecnologia (FCT, POCI/SAU-OBS/56686/2004, POCI/QUI/56393/2004, PTDC/SAU-MII/64153/2006 and PDTC/CTM/65330/2006) financiado no âmbito Programa Operacional Ciência e Inovação 2010 do Quadro Comunitário de Apoio III e comparticipado pelo FEDER; Association for International Cancer Research (AICR Grant ); Fundação Calouste Gulbenkian (project FC ) and FLAD. A.M. (Ref. SFRH/BD/36339/2007) and N.T.M. (Ref. SFRH/BD/11764/2003) acknowledge FCT for financial support. References Barranco SC & Townsend CM Jr (1983) Establishment and characterization of an in vitro model system for human adenocarcinoma of the stomach. Cancer Res 43: Bebb JR, Letley DP, Thomas RJ, Aviles F, Collins HM, Watson SA, Hand NM, Zaitoun A & Atherton JC (2003) Helicobacter pylori upregulates matrilysin (MMP-7) in epithelial cells in vivo and in vitro in a Cag dependent manner. 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