Ectodomain shedding of E-cadherin and c-Met is induced by Helicobacter pylori infection

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Research Article

Ectodomain shedding of E-cadherin and c-Met is induced by Helicobacter pylori infection Wiebke Schirrmeister a,b,1 , Thorsten Gnad b,1 , Thomas Wex a , Shigeki Higashiyama c , Carmen Wolke b,d , Michael Naumann b , Uwe Lendeckel b,d,⁎ a

Clinic of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke-University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany b Institute of Experimental Internal Medicine, Otto-von-Guericke-University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany c Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan d Institute of Medical Biochemistry and Molecular Biology, Ernst-Moritz-Arndt University, Klinikum Sauerbruchstraβe, D-17487 Greifswald, Germany

AR T IC L E I NF O R M AT I O N

AB S TR AC T

Article Chronology:

Helicobacter pylori, a microaerophilic gram-negative bacterium, colonizes the human stomach.

Received 17 March 2009

About 50% of the world's population is infected, and this infection is considered as the major risk

Revised version received 9 July 2009

factor for the development of gastric adenocarcinomas in 1% of infected subjects. Carcinogenesis is

Accepted 24 July 2009

characterized by the process of epithelial-to-mesenchymal transition (EMT), in the course of

Available online 6 August 2009

which fully differentiated epithelial cells turn into depolarized and migratory cells. Concomitant disruption of adherence junctions (AJ) is facilitated by growth factors like hepatocyte growth

Keywords:

factor 1 (HGF-1), but has been also shown to depend on ectodomain shedding of E-cadherin. The

ADAM

aim of this study was to investigate the impact of infection with H. pylori of NCI-N87 gastric

Shedding

epithelial cells on the shedding of E-cadherin and HGF-receptor c-Met. Our results show that

E-cadherin

infection with H. pylori provokes shedding of the surface proteins c-Met and E-cadherin. Evidence

c-Met

is provided that ADAM10 contributes to the shedding of c-Met and E-cadherin.

Gastric epithelial cell

© 2009 Published by Elsevier Inc.

NCI-N87

Introduction Helicobacter pylori is a gram-negative, spiral shaped bacterium which colonizes the human stomach of about 50% of the world's population. Although all infected subjects develop gastritis, 80% of these individuals remain asymptomatic. Severe H. pylori-mediated diseases are duodenal and gastric ulcer disease, gastric cancer and mucosa-associated lymphoid tissues (MALT) lymphomas affecting about 15%, 1% and 0.1%, respectively [1–2]. Accordingly, H. pylori

⁎ Corresponding author. E-mail address: [email protected] (U. Lendeckel). 1 These authors contributed equally to this work. 0014-4827/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.yexcr.2009.07.029

was classified by the World Health Organization (WHO) as a class I carcinogen in 1994 (NIH Consensus Conference, 1994). 350 strains with remarkable genetic differences have been identified to date. The bacterium's chromosome consists of 1.7 million nucleotides and codes for about 1550 genes [3]. Bacterial factors like flagellin [4], ureases, amidases, and lipases [5] allow H. pylori to colonize the stomach, and are able to evoke inflammation of gastric mucosa [6]. Virulence factors encoded by the cytotoxin associated gene pathogenicity island (cagPAI) include CagA and

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proteins in the type IV secretion system (T4SS) which contribute to the development of gastric cancer. The specialized T4SS translocates the 128 kDa CagA protein into the host cell [7], where it gets rapidly phosphorylated by Srctyrosine kinases, c-Src and Lyn [8]. Thus, type I (CagA-positive) strains bear a higher risk for gastric cancer than type II (CagAnegative) strains do [9,10]. Phosphorylation of CagA contributes to the H. pylori induced epithelial–mesenchymal transition [10–12]. By acquiring a migratory phenotype cells become able to leave the epithelial tissue structure. This cell scattering is stimulated among other factors by HGF, which upon binding to the c-Met receptor [13,14] facilitates disruption of E-cadherin mediated cell-cell adhesion [12,15,16]. In tumor tissue, the E-cadherin mediated cell–cell adhesion is decreased due to the disruption of existing adherence junctions (AJs) and loss of E-cadherin expression [17,18]. H. pylori has been shown to decrease E-cadherin expression via induction of IL-1β production/release and to provoke CagA-independent shedding of E-cadherin [18,19]. βcatenin, released from AJs has been proposed to contribute to the activation of Wnt signalling pathway leading to increased expression of Wnt target genes like Axin2, FGF18, MMP-7, cmyc, and cyclin D1 [20–23]. The membrane-spanning protein c-Met consists of α- and βsubunits linked to each other by disulfide bridges. The highly glycosylated 50 kDa α-subunit forms the extracellular growth factor-binding site. The 140 kDa β-subunit forms the transmembrane and intracellular region. Ligand binding leads to dimerization of c-Met [14,24]. c-Met signalling involves activation of MAPK- and STAT pathways [25,26], that lead to “invasive growth”. The latter is important not only during embryogenesis [27–29], but also in inflammation, wound healing, and neoplasia [30]. c-Met triggers EMT trough the MAPK pathway [31]. The H. pylori protein CagA targets intracellularly c-Met receptor binding region and mimics effects of the adapter protein Gab1, thereby prolonging and amplifying the motogenic effect of the activated receptor [12,32–34]. Ectodomain shedding is mainly attributed to members of the ADAM (a disintegrin and metalloprotease) family of proteases. ADAM10 and ADAM17 are probably the best characterized sheddases and were found to be responsible for shedding of NOTCH, Ecadherin, proHB-EGF, EGFR, erbB4, NGFR, c-Met, and L-selectin [35,36]. ADAMs have been implicated in cell adhesion via integrin binding and shedding of adhesion molecules [37,38]. Specifically ADAM15 has been localized in AJs and its accumulation therein increased with VE-cadherin-dependent formation of AJs [39]. Here, we address the question whether infection of NCI-N87 gastric epithelial cells with H. pylori provokes ectodomain shedding of E-cadherin and c-Met and thereby contributes to infection-dependent cell scattering and EMT. Furthermore, the study aims at the identification of individual ADAM family members involved in H. pylori induced shedding.

Materials and methods Bacteria H. pylori wild-type strain P1 and its isogenic strains cagA and virB7 were used. Six days before the experiment bacteria were inoculated on agar plates containing vancomycin or vancomy-

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cin/chloramphenicol, respectively. Bacteria were cultivated for three days under microaerophilic conditions (37 °C) and removed with a swab into PBS (with Ca2+ and Mg2+). Bacteria were then cultivated on fresh agar plates for another three days under the same conditions. For the experiments, bacteria were re-suspended in PBS (with Ca2+ and Mg2+), and concentration (bacteria/ml) was determined by measuring the optical density (λ = 580 nm). For heat killed bacteria suspension, bacteria were incubated at 72 °C for 10 min, followed by a 5 min incubation at 95 °C.

Cell culture NCI-N87 cells were obtained from ATCC, Manassas, USA. Cells were maintained in RPMI 1640 (PAA, Cölbe, Germany) with 10% FCS, Gentamicin (Refobacin® 80 mg, 5 μg/ml) and Levofloxacin (Tavanic®, 1 μg/ml). 48 h prior to the experiments, cells were seeded into 6 well plates at a concentration of 500,000 cells/ml in the same media as mentioned above. 16 h prior to stimulation/infection medium was removed, cells were washed twice with PBS (w/o Ca2+ and Mg2+) and fresh, serum and antibiotic free medium was added. This procedure was repeated 30 min before the experiment. Cells were infected with H. pylori and heat killed H. pylori P1 at a “multiplicity of infection” (MOI) of 100 for 3 h, 6 h and 24 h. Selective ADAM inhibitors KB-R7785 ([4-(N-Hydroxyamino)-2 (R)-isobutyl-3(S)-methylsuccinyl]-L-phenylglycin-N-methylamid) (provided by S. Higashiyama) and TAPI-2 (TNF-α processing inhibitor-2; N-(R)-(2-(Hydroxyaminocarbonyl)methyl)-4methylpentanoyl-L-t-butyl-glycine-Lalanin-2amino-ethyl-amid) (Biomol GmbH, Hamburg, Germany) were preincubated at concentrations of 1 μM and 96 μM, respectively for 30 min.

Apoptosis assay The Anexin-V-FLUOS staining kit (Roche Diagnostics, Penzberg, Germany) apoptosis assay was performed following the manufacturer's instructions. Apoptosis was determined in H. pylori infected cells and non-infected controls after 24 h. The fractions of viable, necrotic and apoptotic cells were detected and quantified using flow cytometry (BD FACSCalibur Flow Cytometer™, Franklin Lakes, USA).

Transfection 600,000 cells/well or 750,000 cells/well were seeded into a 6 well plate 24 h prior transfection. Cells were transfected with HP GenomeWide siRNA (Qiagen, Hilden, Germany) against ADAM10 (Hs_ADAM10_1_HP) or ADAM19 (Hs_ADAM19_1_HP), and with a Control (non-sil.) siRNA (Qiagen) using the transfection reagents ICAFectin 442 (Eurogentec, Cologne, Germany) and siLentFect lipid reagent (BioRad, Hercules, USA), respectively. 30 min prior transfection, medium was removed, cells were washed two times with PBS (w/o Ca2+ and Mg2+) and 2.5 ml (ICAFectin 442) or 900 μl (siLentFect) serum-free medium was added. For ADAM10 transfection, the following mix was prepared: solution (A) siRNA (20 μM, 1.25 μl) + 250 μl DMEM; solution (B) ICAfectin (15 μl) + 250 μl DMEM. For ADAM19 transfection the mix was prepared in the following way: solution (A) siRNA (20 μM, 2.5 μl) + 47.5 μl Optimem; solution (B) silentfect (5 μl)+ 45 μl Optimem. Solutions A and B were mixed together and incubated at room temperature for 15 or 30 min prior to the addition to the cell culture medium. 24 h post

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Table 1 – Sequences of primers used for RT-PCR. Gene PSA ADAM9 ADAM10 ADAM12 ADAM15 ADAM17 ADAM19 ADAM20

Sequence

Product size (bp)

Supplier

5′-AGGTGCGCTATGCTGCTGTA-3′ 5′-AGCAGCAACCTCTAACGCAA-3′ 5′-GCTAGTTGGACTGGAGATTTGG-3′ 5′-TTATTACCACAGAGGGAGCAC-3′ 5′-AATTCTGCTCCTCTCCTGGGC-3′ 5′-TATGTCCAGTGTAAATATGAGAGG-3′ 5′-GCTGATGAAGTTGTCAGTGC-3′ 5′-GAGACTGACTGCTGAATCAG-3′ 5′-CAAATATAGGTGGCACTGAGGAG-3′ 5′-TAGCAGCAGTTCTCCAAAGTGTG-3′ 5′-ATGAGGCAGTCTCTCCTATTCCTGAC-3′ 5′-AAGTGGCTCTATGTTATATTCGGCCC-3′ Hs_ADAM19_1_SG QuantiTect Primer Assay 5′-CTGATAGAGCATTCTACAGTGC-3′ 5′-TGCTGTGATAGCTAATGCTT-3′

350

BioTeZ Berlin-Buch GmbH

485

BioTeZ Berlin-Buch GmbH

298

BioTeZ Berlin-Buch GmbH

282

BioTeZ Berlin-Buch GmbH

285

BioTeZ Berlin-Buch GmbH

465

BioTeZ Berlin-Buch GmbH

110 610

Qiagen, Hilden, Germany BioTeZ Berlin-Buch GmbH

transfection, medium was removed, cells were washed twice with PBS, and fresh serum-free medium was added. Stimulation or infection with H. pylori was carried out 48 h after transfection.

Western blot analysis For protein extraction, cells were washed twice with ice-cold PBS (w/o Ca2+ and Mg2+) and lysed in 600 μl RIPA lysis buffer (50 mM Tris–HCl, pH 7.5; 100 mM NaCl; 1% Triton X-100; 10% glycerol; 5 mM EDTA; 10 mM K2HPO4; 0.5% nonidet P-40; 20 mM NaF; 20 mM β-glycerolphosphat; 10 mM Na-pyrophosphat; 1 mM Na2MoO4; 1 mM Na3VO4; 0.1 mM AEBSF; 1× Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany)). The homogenate was incubated on ice for 10 min, drawn through a syringe (0.45 mm, 40 I.U., Omnifix, Braun, Germany), incubated for further 10 min on ice, and finally cleared by 10 min centrifugation for 10 min (12,000 ×g at 4 °C). The supernatant was harvested, and this total cell lysate was immediately frozen at −80 °C. Protein concentration was determined using the modified BCA method (Bicinchoninic Acid Kit for Protein Determination, Sigma, St. Louis, USA). For SDS-PAGE, 25 μg of total protein was loaded on a 7.5% gel. Proteins were transferred on a nitrocellulose membrane (Whatman, Protran, Roth, Karlsruhe, Germany). Membranes were blocked with 5% milk/PBS. Antibodies against c-Met extracellular region (clone DL-21, Upstate, Lake Placid, USA), cMet C-terminus (clone sc-10, Santa Cruz Biotechnology, Santa Cruz, USA), E-cadherin (clone HECD-1, Abcam, Cambridge, UK), ADAM10 (clone sc-28358, Santa Cruz Biotechnology, Santa Cruz, USA) and ADAM19 (clone sc-25988, Santa Cruz Biotechnology, Santa Cruz, USA) were used at 1:1000 dilution in 3% milk/PBS. Anti-mouse, anti-goat and anti-rabbit HRP-conjugated secondary antibodies (Cell Signalling, Boston, USA) were diluted 1:2000 in 3% milk/PBS or 3% BSA/TBS. Supersignal West DURA (Fisher Scientific, Schwerte, Germany) and AGFA CURIX films (AGFA, Bonn, Germany) were used for detection. Blots were quantitatively evaluated by densitometric analysis using the GeneProfiler 3.5 software (Scanalytics, Rockville, USA).

tions. A 1 μg quantity of total RNA was transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). PCR was performed in an iCycler (BioRad, Munich, Germany). A 25 μl reaction mixture consisted of 1× SensiMix (Quantace, Berlin, Germany), 0.5 μl of SYBR-Green I (Quantace), 1 μl cDNA, and 0.3 μmol/l of the specific primers listed in Table 1. Quantities of PSA (puromycin-sensitive aminopeptidase) mRNA were used to normalise cDNA contents. Initial denaturation at 95 °C for 10 min was followed by 40 cycles with denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 30 s. A melting curve was recorded at the end of the program to verify product identity and purity. For ADAM19, a two-step protocol with omission of the elongation step was used. For primer sequences, see Table 1. The cDNA fragment of each primer set included intron-spanning regions resulting in the generation of a larger PCR product from genomic DNA or its exclusion. Therefore, all identified PCR products could exclusively attributed to the corresponding transcript of the sample.

Statistical analysis The experiments were analysed using ANOVA and unpaired t-test. The values of the experiments are expressed as means ± SEM. All statistical decisions were made with a critical probability of P = 5% without α-adjustment.

Results Expression of ADAMs, c-Met and E-cadherin in NCI-N87 cells First, we determined the basic expression of individual ADAMs in NCI-N87 cells by RT-PCR. As shown in Fig. 1, expression of ADAM9, 10, 15, 17, 19 and 20 could be demonstrated at the mRNA level. Expression of ADAM12 could not be detected in NCI-N87 cells.

Shedding of c-Met and E-cadherin after infection with H. pylori Quantitative real-time RT-PCR Total RNA was prepared by using the innuPrep RNA Mini Kit (Jena Analytics, Jena, Germany) according to manufacturer's instruc-

We investigated the impact of infection with H. pylori on the shedding of c-Met and E-cadherin. H. pylori was applied at a MOI of 100 for 3 h, 6 h and 24 h and thereafter shedding was determined

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Fig. 1 – Expression of members of the ADAM family of proteases in NCI-N87 cells. Expression of ADAM9 (485 bp), ADAM10 (298 bp), ADAM12 (282 bp), ADAM15 (285 bp), ADAM17 (465 bp), ADAM19 (110 bp) and ADAM20 (610 bp) in NCI-N87 cells. As shown, ADAMs 10, 15, 17, and 20 are strongly expressed in NCI-N87 cells, whereas ADAMs 9 and 19 are only weakly and ADAM12 is not expressed in this cell line. The ubiquitously expressed puromycin-sensitive aminopeptidase (PSA, 350 bp) was used as positive control.

by immunoblot analysis of cell culture supernatants. To prove viability of the cells after 24 h H. pylori infection, an Anexin-V apoptosis assay was performed. The control showed 3 ± 0.5% apoptotic and 93 ± 0.1% viable cells, whereas the H. pylori infected samples showed 5 ± 0.2% apoptotic and 91 ± 0.4% viable cells (Supplementary Fig. 1). These results provide evidence that H. pylori had no major effect on the viability of the cells and that apoptosis-induced ADAM activity can be neglected. The basic protein expression of c-Met and E-cadherin in these cells was detected using immunoblot analysis. The antibodies used showed no cross-reaction to the bacterial lysate (Supplementary Fig. 2). Within the H. pylori infection a time-dependent increase of the amounts of 95 kDa c-Met ectodomain and 72 kDa E-cadherin ectodomain shed into the supernatant was observed. Maximum amounts of shed ectodomains were observed after 24 h. Simultaneously, a reduction in full-length c-Met (145 kDa) and E-cadherin (120 kDa) was detected in total lysates. Heat killed H. pylori bacteria, however, did not lead to a stimulation of c-Met and Ecadherin shedding (Figs. 2A and B). Quantification of shedding was performed after 24 h. Shedding of c-Met was significantly increased in H. pylori wt infected cells (8.96 ± 2.9% uninfected cells vs. 100% infected cells, P < 0.01) (Fig. 3A). Similarly, a significant increase in shed E-cadherin was detected (1.8 ± 0.9% uninfected cells vs. 100% infected cells, P < 0.01) (Fig. 3B). Administration of KB-R7785 or TAPI-2 markedly reduced the amounts of shed c-Met (39.2 ± 13.1% vs. 38.4 ± 6%, P < 0.01) or E-cadherin (28.2 ± 12.2% vs. 57 ± 6%, P < 0.01), respectively (Figs. 3A and B). We next analysed whether isogenic strains of H. pylori (cagA or virB7) are equally effective in the induction of shedding. There was no difference between wild-type and isogenic mutant strains in the capability of inducing c-Met and E-cadherin shedding (data not shown).

Fig. 2 – Detection of stimulated ectodomain shedding after H. pylori infection. A — Detection and semiquantitative analysis of the 95 kDa c-Met ectodomain in the cell culture supernatant and 145 kDa full-length c-Met in total lysates, and B — detection and semiquantitative analysis of the 72 kDa E-cadherin ectodomain in the cell culture supernatant and 120 kDa fulllength E-cadherin in total lysates of untreated NCI-N87 cells (0 h, 24 h) and after H. pylori (MOI:100) infection for 3, 6 and 24 h. H. pylori strongly stimulates the c-Met and E-cadherin shedding with time-dependent increase in the amount of released ectodomain. As a control, that stimulation of shedding is mediated by viable bacteria and not by secreted bacterial factors, cells were also treated with heat killed (HK) H. pylori for 24 h. Data represent one representative experiment. For illustration maximal shedding activity (after 24 h) was set as 100%, and all other values were calculated in accordance.

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ments resulted in a reduction of transcript levels by 70% and 50% for ADAM10 and 19, respectively. On the protein level, precursor and mature forms of ADAM10 and ADAM19 were reduced by 60– 80% and 30–50%, respectively (Figs. 4A, B, and C). Subsequent analysis of H. pylori induced shedding of c-Met and E-cadherin in siRNA transfected cells revealed a 70% reduction in cMet shedding in ADAM10 knock down cells (26 ± 12.3% vs. 100%, P < 0.01) (Fig. 5A). Shedding of E-cadherin was reduced to 60% in response to knock down of ADAM10 (38.2 ± 15% vs. 100%, P < 0.05) (Fig. 5B). Knock down of ADAM19 showed no effect on the shedding of c-Met and only weakly reduced that of E-cadherin.

Discussion Shedding of c-Met

Fig. 3 – Quantitative analysis of stimulated ectodomain shedding after H. pylori infection. A — Detection of c-Met ectodomain and B — E-cadherin ectodomain in the cell culture supernatant after 24 h H. pylori infection. Preincubation with KB-R7785 (1 μM) and TAPI-2 (96 μM) for 30 min following H. pylori infection for 24 h results in reduced ectodomain shedding. Displayed immunoblots are representative for all 3 experiments. Columns represent the means ± se of three independent experiments, each normalised to its control (=100%). (n = 3, means ± se, statistical significance for infected cells versus untreated/inhibitor pre-treated cells ⁎P < 0.01).

ADAM10 affects shedding of c-Met and E-cadherin ADAM10 has been reported to mediate shedding of a multitude of proteins and the expression of it was found to be induced by H. pylori infection [53]. Furthermore, expression of ADAM19 is highly up-regulated in human gastric mucosa in response to infection with H. pylori [54]. We found that ADAM10 and ADAM19 expression levels increased after infection of NCI-N87 cells with H. pylori (data not shown). To test the hypothesis that ADAM10 and 19 contribute to the observed H. pylori-dependent shedding of c-Met and E-cadherin, the expression of both proteases was downregulated by siRNA-mediated knock down. Knock down experi-

Pathological activation of c-Met plays a critical role in tumor development and progression. Various tumors were shown to overexpress c-Met which is subject of shedding. Amounts of shed c-Met ectodomain fragments were significantly correlated with malignant potential [40]. Moreover, ectodomain shedding of c-Met can be induced by EGFR ligands, and HGF in turn is able to transactivate EGFR followed by enhanced activation of ERK or PI3K pathways [30]. Petrelli et al. [41] reported HGFR shedding induced by a monoclonal antibody (mab) against c-Met, leading to a double proteolytic cleavage of the ectodomain and the intracellular domain which apparently is not executed by ADAMs. Noteworthy, the shed ectodomain seems to act as a dominant-negative molecule capable of further enhancing the inhibitory effect of mab. Here we demonstrate (I) the expression of c-Met in NCI-N87 gastric epithelial cells and (II) ectodomain shedding of c-Met induced by H. pylori. The fact that this shedding was also observed in response to biochemical activation of ADAMs by APMA (data not shown) strongly implies a role of these proteases for shedding of cMet. The functional relevance of ADAMs in this process is further demonstrated by the use of two ADAM family-specific inhibitors, TAPI-2 and KB-R7785. Both compounds efficiently blocked the APMA- and H. pylori-dependent shedding of c-Met. Applying siRNA-mediated knock down of two potential sheddase candidates, ADAM10 and 19, we were able to prove that ADAM10 is indeed responsible for the H. pylori-dependent shedding of c-Met, while ADAM19 is not. These findings are in line with previous reports demonstrating stimulated shedding of c-Met in different cell lines in response to activation of metalloproteases [36,42]. Furthermore, our results once more support the outstanding role of ADAM10 in ectodomain shedding in general and in tumorigenesis in particular [43–45].

Shedding of E-cadherin Our data show that infection of NCI-N87 cells with H. pylori leads to a substantial shedding of E-cadherin. A similar observation has been made previously by Weydig et al. after infection of MCF-7 cells [19]. Loss of E-cadherin expression is frequently observed in tumors and, as part of EMT, this loss of E-cadherin and concomitant disruption of adherence junctions is a crucial event in carcinogenesis and metastasis [46]. Amounts of Ecadherin ectodomain shed from the tumor cells were found

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Fig. 4 – Efficiency of siRNA knock down. A — Knock down of ADAM10 and B — ADAM19 expression. Cells were treated with siRNA against ADAM10 (siADAM10) and ADAM19 (siADAM19) and after 48 h amounts of mRNA were assessed by quantitative RT-PCR and western blotting. The knock down efficiencies were ∼60 and ∼40%, respectively. C — ADAM10 and 19 expression was semiquantitatively analysed, and expression in ADAM10/19 siRNA versus control siRNA (siMOCK) treated cells was compared. (ADAM10: precursor 100 kDa, mature 80 kDa, active 60 kDa; ADAM19: precursor 115 kDa, mature 87 kDa).

positively correlated with gastric tumor progression [47]. ADAM10 has been identified as the main E-cadherin-shedding protease of primary mouse fibroblasts and HaCaT cells in response to PMA stimulation [48]. In our study, two experimental approaches (inhibitors and siRNA knock down) proved a contribution of ADAM10 for the H. pylori induced shedding of Ecadherin in NCI-N87 cells. Thus, ADAM10 seems to mediate shedding of c-Met and E-cadherin in NCI-N87 cells in response to H. pylori. In contrast, ADAM19-specific gene knock down did not affect at all shedding of c-Met and only slightly reduced that of E-cadherin. This suggests that ADAM19 not significantly contributes to the shedding of these proteins. siRNA-mediated knock down led in part to a reduction of ADAM19 only. Thus, the lacking impact of ADAM19 on shedding observed in this study should be interpreted with caution. It should be noted that both the inhibitors of ADAMs and the ADAM10-specific siRNA led to a partial inhibition of E-cadherin shedding only, suggesting that other sheddases are involved in this process. These proteases may include bacterial proteases as it has been recently reported that the Bacteroides fragilis toxin (BFT) exhibits gammasecretase-dependent E-cadherin-shedding activity [49,50]. Furthermore, proteases secreted by H. pylori such as the T4SS independent serine-protease HtrA might contribute to ectodomain shedding of transmembrane proteins [51].

The pathophysiological role for shedding of c-Met in response to infection with H. pylori remains speculative. The released cMet ectodomain might protect the cell from further motogenic signals through catching HGF ligands and thereby acting as a dominant-negative molecule. Another possibility is that increased shedding of surface proteins/receptors via activation of ADAMs like ADAM10 represents an early defence mechanisms aimed at limiting bacterial adhesion to epithelial surfaces. This view is supported by the observed H. pylori-dependent shedding of other surface proteins such as E-cadherin ([19] and data of this study, or NCAM-L1, ACE-2 (WS and UL, unpublished observation), or epidermal growth factor receptor (EGFR) ligands [52]. Shedding of the latter appeared to be mediated via increased production and release of interleukin-8, which accelerates processing of EGFR ligands (e.g. heparin-binding EGF-like growth factor or amphiregulin) through ADAM activation. This in turn leads to EGF-receptor activation and increased cell proliferation [52]. It could be speculated that increased activity of ADAMs contributes to the carcinogenic activity of H. pylori in susceptible patients via enhancing cell proliferation, disrupting cell–cell and cell–matrix-adhesion, and stimulating cellular motility. A more general role of shedding processes in context with infection by H. pylori is supported by the finding that shedding of c-

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clinical isolates and various cell lines or primary gastric cells are warranted. Precisely how infection with H. pylori leads to the activation of ADAM10 and, possibly other sheddases, remains to be established and represents a limitation of this study. A number of different and, at least in part, rather unspecific stimuli have been shown to increase recruitment of ADAM10 to the membrane and ADAM10mediated shedding of surface proteins. These stimuli include serum factors, peptide growth factors, changes in intracellular calcium concentration, osmotic and mechanical stress, and PKC activation [55]. It is suggested by Yoshimura et al. [53] and our data that enhanced biosynthesis of ADAM10 as detectable at both mRNA and protein levels contribute to increased activity of ADAM10. Taken together, we show that H. pylori infection of gastric epithelial NCI-N87 cells induces shedding of c-Met and E-cadherin, two molecules known to be involved in epithelial tumorigenesis. In addition, we identified ADAM10 as one critical sheddase involved in this process.

Acknowledgments The authors wish to thank Dr. Roland Hartig (Institute for Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany) for his kind support with FACS analyses. The work was supported by the Deutsche Forschungsgemeinschaft by a grant to M.N. and U.L (GRK 1167/-1). All authors on the paper declare that they have no financial interest related to this work.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2009.07.029.

REFERENCES

Fig. 5 – Detection and quantitative analysis of stimulated ectodomain shedding after H. pylori infection of siRNA treated cells. A — Detection and quantification of the c-Met ectodomain fragments in supernatants of cells treated with H. pylori for 24 h after knock down of ADAM10 and ADAM19 expression. B — Detection and quantification of the E-cadherin shedding of ADAM10 and ADAM19 knock down cells infected with H. pylori for 24 h. As a control in each experiment untransfected cells were also infected with H. pylori. Displayed immunoblots are representative for all 3 experiments. Columns represent the means ± se of three independent experiments, each normalised to its control (=100%). (n = 3, means ± se, statistical significance for control siRNA treated cells versus ADAM10 and ADAM19 knock down cells, ⁎P < 0,01; #P < 0.05).

Met and E-cadherin was neither dependent on CagA nor on the presence of a functional T4SS. Thus, it is conceivable that enhanced shedding is provoked by any H. pylori strain independent of their virulence. To address this question, future studies using multiple

[1] N. Uemura, S. Okamoto, S. Yamamoto, N. Matsumura, S. Yamaguchi, M. Yamakido, K. Taniyama, N. Sasaki, R.J. Schlemper, Helicobacter pylori infection and the development of gastric cancer, N. Engl. J. Med. (2001) 784–789. [2] M.R. Amieva, E.M. El-Omar, Host–bacterial interactions in Helicobacter pylori infection, Gastroenterology 134 (2008) 306–323. [3] J.F. Tomb, O. White, A.R. Kerlavage, R.A. Clayton, G.G. Sutton, R.D. Fleischmann, K.A. Ketchum, H.P. Klenk, S. Gill, B.A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E.F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H.G. Khalak, A. Glodek, K. McKenney, L.M. Fitzegerald, N. Lee, M.D. Adams, E.K. Hickey, D.E. Berg, J.D. Gocayne, T.R. Utterback, J.D. Peterson, J.M. Kelley, M.D. Cotton, J.M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W.S. Hayes, M. Borodovsky, P.D. Karp, H.O. Smith, C.M. Fraser, J.C. Venter, The complete genome sequence of the gastric pathogen Helicobacter pylori, Nature 388 (1997) 539–547. [4] H. Leying, S. Suerbaum, G. Geis, R. Haas, Cloning and genetic characterization of a Helicobacter pylori flagellin gene, Mol. Microbiol. 6 (1992) 2863–2874. [5] H. Radosz-Komoniewska, T. Bek, J. Jóźwiak, G. Martirosian, Pathogenicity of Helicobacter pylori infection, Clin. Microbiol. Infect. 11 (2005) 602–610.

E XP E RI ME N T AL C E L L R E S EA RC H 31 5 ( 20 0 9) 3 5 0 0– 3 50 8

[6] J.E. Crabtree, Immune and inflammatory responses to Helicobacter pylori infection, Scand. J. Gastroenterol. Suppl. 215 (1996) 3–10. [7] S. Odenbreit, J. Püls, B. Sedlmaier, E. Gerland, W. Fischer, R. Haas, Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion, Science 287 (2000) 1497–1500. [8] M. Stein, F. Bagnoli, R. Halenbeck, R. Rappuoli, W.J. Fantl, A. Covacci, c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs, Mol. Microbiol. 43 (2002) 971–980. [9] J.E. Crabtree, M. Naumann, Epithelial cell signaling in Helicobacter pylori infection, Curr. Signal. Transduc. Ther. 1 (2006) 53–65. [10] A. Covacci, S. Censini, M. Bugnoli, R. Petracca, D. Burroni, G. Macchia, A. Massone, E. Papini, Z. Xiang, N. Figuramand, R. Rappuoli, Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 5791–5795. [11] S. Censini, C. Lange, Z. Xiang, J.E. Crabtree, P. Ghiara, M. Borodovsky, R. Rappuoli, A. Covacci, cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease associated virulence factors, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 14648–14653. [12] Y. Churin, L. Al-Ghoul, O. Kepp, T.F. Meyer, W. Birchmeier, M. Naumann, Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response, J. Cell Biol. 161 (2003) 249–255. [13] M. Stoker, E. Gherardi, M. Perryman, J. Gray, Scatter factor is a fibroblast-derived modulator of epithelial cell mobility, Nature 327 (1987) 239–242. [14] C. Birchmeier, W. Birchmeier, E. Gherardi, G.F. Vande Woude, Met, metastasis, motility and more, Nat. Rev. Mol. Cell Biol. 4 (2003) 915–925. [15] J.P. Thiery, Epithelial–mesenchymal transitions in tumour progression, Nat. Rev. Cancer 2 (2002) 442–454. [16] A.J. Ridley, M.A. Schwartz, K. Burridge, R.A. Firtel, M.H. Ginsberg, G. Borisy, J.T. Parsons, A.R. Horwitz, Cell migration: integrating signals from front to back, Science 302 (2003) 1704–1709. [17] S. Hirohashi, Inactivation of the E-cadherin-mediated cell adhesion system in human cancers, Am. J. Pathol. 153 (1998) 333–339. [18] A.O. Chan, E-cadherin in gastric cancer, World J. Gastroenterol. 12 (2006) 199–203. [19] C. Weydig, A. Starzinski-Powitz, G. Carra, J. Löwer, S. Wessler, CagA-independent disruption of adherence junction complexes involves E-cadherin shedding and implies multiple steps in Helicobacter pylori pathogenicity, Exp. Cell Res. 313 (2007) 3459–3471. [20] E.H. Jho, T. Zhang, C. Domon, C.K. Joo, J.N. Freund, F. Costantini, Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway, Mol. Cell. Biol. 22 (2002) 1172–1183. [21] T. Shimokawa, Y. Furukawa, M. Sakai, M. Li, N. Miwa, Y.M. Lin, Y. Nakamura, Involvement of the FGF18 gene in colorectal carcinogenesis, as a novel downstream target of the beta-catenin/T-cell factor complex, Cancer Res. 63 (2003) 6116–6120. [22] J.R. Miller, The Wnts, Genome Biol. 3 (2002) 3001. [23] A.O. Chan, E-cadherin in gastric cancer, World J. Gastroenterol. 12 (2006) 199–203. [24] E. Gherardi, M.E. Youles, R.N. Miguel, T.L. Blundell, L. Iamele, J. Gough, A. Bandyopadhyay, G. Hartmann, P.J. Butler, Functional map and domain structure of MET, the product of the c-Met protooncogene and receptor for hepatocyte growth factor/scatter factor, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 12039–12044. [25] L. Naldini, E. Vigna, R. Ferracini, P. Longati, L. Gandino, M. Prat, P.M. Comoglio, The tyrosine kinase encoded by the MET

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

[37] [38] [39]

[40]

[41]

[42]

[43]

[44]

3507

proto-oncogene is activated by autophosphorylation, Mol. Cell. Biol. 11 (1991) 1793–1803. P. Longati, A. Bardelli, C. Ponzetto, L. Naldini, P.M. Comoglio, Tyrosines1234–1235 are critical for activation of the tyrosine kinase encoded by the MET proto-oncogene (HGF receptor), Oncogene 9 (1994) 49–57. F. Bladt, D. Riethmacher, S. Isenmann, A. Aguzzi, C. Birchmeier, Essential role for the c-Met receptor in the migration of myogenic precursor cells into the limb bud, Nature 376 (1995) 768–771. C. Schmidt, F. Bladt, S. Goedecke, V. Brinkmann, W. Zschiesche, M. Sharpe, E. Gherardi, C. Birchmeier, Scatter factor/hepatocyte growth factor is essential for liver development, Nature 373 (1995) 699–702. Y. Uehara, O. Minowa, C. Mori, K. Shiota, J. Kuno, T. Noda, N. Kitamura, Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor, Nature 373 (1995) 702–705. K.P. Xu, F.S. Yu, Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing, Invest. Ophthalmol. Vis. Sci. 48 (2007) 2242–2248. A. Gentile, P.M. Comoglio, Invasive growth: a genetic program, Int. J. Dev. Biol. 48 (2004) 451–456. K.M. Weidner, S. Di Cesare, M. Sachs, V. Brinkmann, J. Behrens, W. Birchmeier, Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis, Nature 384 (1996) 173–176. K.A. Furge, Y.W. Zhang, G.F. Vande Woude, Met receptor tyrosine kinase: enhanced signalling through adapter proteins, Oncogene 19 (2000) 5582–5589. C.M. Botham, A.M. Wandler, K. Guillemin, A transgenic Drosophila model demonstrates that the Helicobacter pylori CagA protein functions as a eukaryotic Gab adaptor, PLoS Pathog. 4 (2008) e1000064. D.F. Seals, S.A. Courtneidge, The ADAMs family of metalloproteases: multidomain proteins with multiple functions, Genes Dev. 17 (2003) 7–30. C. Kopitz, M. Gerg, O.R. Bandapalli, D. Ister, C.J. Pennington, S. Hauser, C. Flechsig, H.W. Krell, D. Antolovic, K. Brew, H. Nagase, M. Stangl, C.W. von Weyhern, B.L. Brücher, K. Brand, L.M. Coussens, D.R. Edwards, A. Krüger, Tissue inhibitor of metalloproteinases-1 promotes liver metastasis by induction of hepatocyte growth factor signaling, Cancer Res. 67 (2007) 8615–8623. C.P. Blobel, Metalloprotease-disintegrins: links to cell adhesion and cleavage of TNF alpha and Notch, Cell 90 (1997) 589–592. C.P. Blobel, Remarkable roles of proteolysis on and beyond the cell surface, Curr. Opin. Cell Biol. 12 (2000) 606–612. C. Ham, B. Levkau, E.W. Raines, B. Herren, ADAM15 is an adherens junction molecule whose surface expression can be driven by VE-cadherin, Exp. Cell Res. 279 (2002) 239–247. G. Athauda, A. Giubellino, J.A. Coleman, C. Horak, P.S. Steeg, M.J. Lee, J. Trepel, J. Wimberly, J. Sun, A. Coxon, T.L. Burgess, D.P. Bottaro, c-Met ectodomain shedding rate correlates with malignant potential, Clin. Cancer Res. 12 (2006) 4154–4162. A. Petrelli, P. Circosta, L. Granziero, M. Mazzone, A. Pisacane, S. Fenoglio, P.M. Comoglio, S. Giordano, Ab-induced ectodomain shedding mediates hepatocyte growth factor receptor down-regulation and hampers biological activity, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 5090–5095. D. Nath, N.J. Williamson, R. Jarvis, G. Murphy, Shedding of c-Met is regulated by crosstalk between a G-protein coupled receptor and the EGF receptor and is mediated by a TIMP-3 sensitive metalloproteinase, J. Cell. Sci. 114 (2001) 1213–1220. H. Qi, M.D. Rand, X. Wu, N. Sestan, W. Wang, P. Rakic, T. Xu, S. Artavanis-Tsakonas, Processing of the notch ligand delta by the metalloprotease Kuzbanian, Science 283 (1999) 91. A. Raucci, S. Cugusi, A. Antonelli, S.M. Barabino, L. Monti, A. Bierhaus, K. Reiss, P. Saftig, M.E. Bianchi, A soluble form of the receptor for advanced glycation endproducts (RAGE) is produced

3508

[45]

[46]

[47]

[48]

[49]

[50]

E XP E RI ME N T AL C E L L R E SE A RC H 31 5 ( 20 0 9) 3 5 00 – 3 50 8

by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10), FASEB J. 22 (2008) 3716–3727. N. Gavert, M. Sheffer, S. Raveh, S. Spaderna, M. Shtutman, T. Brabletz, F. Barany, P. Paty, D. Notterman, E. Domany, A. Ben-Ze'ev, Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis, Cancer Res. 67 (2007) 7703–7712. M.A. Huber, N. Kraut, H. Beug, Molecular requirements for epithelial–mesenchymal transition during tumor progression, Curr. Opin. Cell Biol. 17 (2005) 548–558. A.O. Chan, S.K. Lam, K.M. Chu, C.M. Lam, E. Kwok, S.Y. Leung, S.T. Yuen, S.Y. Law, W.M. Hui, K.C. Lai, C.Y. Wong, H.C. Hu, C.L. Lai, J. Wong, Soluble E-cadherin is a valid prognostic marker in gastric carcinoma, Gut 48 (2001) 808–811. T. Maretzky, K. Reiss, A. Ludwig, J. Buchholz, F. Scholz, E. Proksch, B. de Strooper, D. Hartmann, P. Saftig, ADAM10 mediates E-cadherin shedding and regulates epithelial cell–cell adhesion, migration, and beta-catenin translocation, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9182–9187. S. Wu, K.C. Lim, J. Huang, R.F. Saidi, C.L. Sears, Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 14979–14984. S. Wu, K.J. Rhee, M. Zhang, A. Franco, C.L. Sears, Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and gamma-secretase-dependent E-cadherin cleavage, J. Cell. Sci. 120 (2007) 1944–1952.

[51] M. Löwer, C. Weydig, D. Metzler, A. Reuter, A. Starzinski-Powitz, S. Wessler, G. Schneider, Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA, PLoS ONE 3 (2008) e3510. [52] T. Joh, H. Kataoka, S. Tanida, K. Watanabe, T. Ohshima, M. Sasaki, H. Nakao, H. Ohhara, S. Higashiyama, M. Itoh, Helicobacter pylori-stimulated interleukin-8 (IL-8) promotes cell proliferation through transactivation of epidermal growth factor receptor (EGFR) by disintegrin and metalloproteinase (ADAM) activation, Dig. Dis. Sci. 50 (2005) 2081–2089. [53] T. Yoshimura, T. Tomita, M.F. Dixon, A.T. Axon, P.A. Robinson, J.E. Crabtree, ADAMs (a disintegrin and metalloproteinase) messenger RNA expression in Helicobacter pylori-infected, normal, and neoplastic gastric mucosa, J. Infect. Dis. 185 (2002) 332–340. [54] T. Wex, A. Sokic-Milutinovic, T. Kalinski, A. Roessner, P. Malfertheiner, M. Naumann, U. Lendeckel, Expression of ADAM19 in Helicobacter Pylori-mediated diseases and gastric cancer, in: Marilyn B. Tompkins (Ed.), Gastric Cancer Research Trends, NOVA Publishers, 2007, pp. 171–188. [55] P. Saftig, D. Hartmann, ADAM10. A major membrane protein ectodomain sheddase involved in regulated intramembrane proteolysis, in: NM Hooper, U Lendeckel (Eds.), The ADAM Family of Proteases. Proteases in Biology and Disease, Volume 4, Springer, 2005, pp. 85–121.