Up-regulated Smad5 Mediates Apoptosis of Gastric Epithelial Cells Induced by Helicobacter pylori Infection

The gastric pathogen Helicobacter pylori activates epithelial cell signaling pathways, and its infection induces changes in the expression of several genes in infected human gastric tissues. Recent studies have indicated that the ability of H. pylori to regulate epithelial cell responses depends on the presence of an intact cag pathogenicity island (cagPAI). We investigated altered mRNA expression of gastric epithelial cells after infection with H. pylori, both cagPAI-positive and cagPAI-negative strains, by cDNA microarray, reverse transcription PCR, and Northern blot analysis. Our results indicated that cagPAI-positive H. pylori strains (ATCC 43504 and clinical isolated strains) significantly activated Smad5 mRNA expression of human gastric epithelial cells (AGS, KATOIII, MKN28, and MKN45). We further examined whether the up-regulated Smad5 was related to apoptosis of gastric epithelial cells induced byH. pylori. Smad5 RNA interference completely inhibitedH. pylori-induced apoptosis. These results suggest that Smad5 is up-regulated in gastric epithelial cells through the presence of cagPAI of H. pylori and that Smad5 mediates apoptosis of gastric epithelial cells induced by H. pyloriinfection. The gastric pathogen Helicobacter pylori activates epithelial cell signaling pathways, and its infection induces changes in the expression of several genes in infected human gastric tissues. Recent studies have indicated that the ability of H. pylori to regulate epithelial cell responses depends on the presence of an intact cag pathogenicity island (cagPAI). We investigated altered mRNA expression of gastric epithelial cells after infection with H. pylori, both cagPAI-positive and cagPAI-negative strains, by cDNA microarray, reverse transcription PCR, and Northern blot analysis. Our results indicated that cagPAI-positive H. pylori strains (ATCC 43504 and clinical isolated strains) significantly activated Smad5 mRNA expression of human gastric epithelial cells (AGS, KATOIII, MKN28, and MKN45). We further examined whether the up-regulated Smad5 was related to apoptosis of gastric epithelial cells induced byH. pylori. Smad5 RNA interference completely inhibitedH. pylori-induced apoptosis. These results suggest that Smad5 is up-regulated in gastric epithelial cells through the presence of cagPAI of H. pylori and that Smad5 mediates apoptosis of gastric epithelial cells induced by H. pyloriinfection. Helicobacter pylori is a human pathogen that infects the gastric mucosa and causes an inflammatory process leading to gastritis, gastric ulceration, duodenal ulceration, mucosa-associated lymphoid tissue lymphoma, and gastric cancer (1Wotherspoon A.C. Ortiz-Hidalgo C. Falzon M.R. Isaacson P.G. Lancet. 1991; 338: 1175-1176Abstract PubMed Scopus (1758) Google Scholar). The pathogenesis of gastroduodenal diseases caused by this bacterium is not well understood. Since the whole genome ofH. pylori was sequenced in 1997, several putative virulence factors, including VacA (2Hennig E.E. Butruk E. Ostrowski J. Biochem. Biophys. Res. Commun. 2001; 289: 103-110Crossref PubMed Scopus (27) Google Scholar), IceA, OipA (3Yamaoka Y. Kikuchi S. el-Zimaity H.M. Gutierrez O. Osato M.S. Graham D.Y. Gastroenterology. 2002; 123: 414-424Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), HrgA (4Ando T. Wassenaar T.M. Peek Jr., R.M. Aras R.A. Tschumi A.I. vanDoorn L.J. Kusugami K. Blaser M.J. Cancer Res. 2002; 62: 2385-2389PubMed Google Scholar), lipopolysaccharide, and the neutrophil-activating protein (5Satin B. Del Giudice G. Della Bianca V. Dusi S. Laudanna C. Tonello F. Kelleher D. Rappuoli R. Montecucco C. Rossi F. J. Exp. Med. 2000; 191: 1467-1476Crossref PubMed Scopus (274) Google Scholar), have been elucidated. The cag pathogenicity island (cagPAI), 1The abbreviations used are: cagPAI, cag pathogenicity island; siRNA, short interference RNA; RT, reverse transcription; TGF, transforming growth factor; BMP, bone morphogenetic protein(s) a complex of genes coding ∼30 proteins, has been reported to be a major virulence factor of H. pylori. The cagPAI is acquired by horizontal transfer and is found in about 50–70% of H. pylori isolates in Western countries and in more than 90% of H. pyloriisolates in Asian countries, including Japan (6Maeda S. Yoshida H. Ikenoue T. Ogura K. Kanai F. Kato N. Shiratori Y. Omata M. Gut. 1999; 44: 336-341Crossref PubMed Scopus (158) Google Scholar, 7Mizushima T. Sugiyama T. Komatsu Y. Ishizuka J. Kato M. Asaka M. J. Clin. Microbiol. 2001; 39: 2463-2465Crossref PubMed Scopus (101) Google Scholar). This lesion codes for the type IV secretion machinery system forming a cylinder-like structure connected to epithelial cells (8Covacci A. Telford J.L. Del Giudice G. Parsonnet J. Rappuoli R. Science. 1999; 284: 1328-1333Crossref PubMed Scopus (961) Google Scholar). Many virulence gene products or other interactive proteins might be transferred into the host cells via this system. Peptic ulceration and gastric cancer occur in some people with H. pylori infection, but the majority remain asymptomatic. Although differences among the degrees of gastric mucosal damage caused by different strains should be an important factor for development of various clinical outcomes, these strain differences do not provide a complete explanation for individual differences in H. pylori infection-induced gastric mucosal injury. Therefore, it is presumed that host responses also play an important role in the outcome of H. pylori infection, interacting with virulence factors and environmental factors. Recent studies have shown that H. pylori induced various cellular responses, proliferation, apoptosis (9Wagner S. Beil W. Westermann J. Logan R.P. Bock C.T. Trautwein C. Bleck J.S. Manns M.P. Gastroenterology. 1997; 113: 1836-1847Abstract Full Text PDF PubMed Scopus (304) Google Scholar), cytoskeletal rearrangement (10Segal E.D. Falkow S. Tompkins L.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1259-1264Crossref PubMed Scopus (248) Google Scholar), modification of intracellular signaling molecules (11Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. Science. 2002; 295: 683-686Crossref PubMed Scopus (866) Google Scholar), vacuolation (12Morbiato L. Tombola F. Campello S. Del Giudice G. Rappuoli R. Zoratti M. Rapini E. FEBS Lett. 2001; 508: 479-483Crossref PubMed Scopus (24) Google Scholar), and cytokine secretion (13Ogura K. Takahashi M. Maeda S. Ikenoue T. Kanai F. Yoshida H. Shiratori Y. Mori K. Mafune K.I. Omata M. Dig. Dis. Sci. 1998; 43: 2738-2743Crossref PubMed Scopus (36) Google Scholar). In this study, we investigated the altered gene expression of host cells infected with cagPAI-positive or cagPAI-negative H. pylori strain and the association between the altered gene expression and the cellular responses. Biopsy specimens were obtained from Japanese patients in Hokkaido University Hospital and were cultured on H. pylori-selective agar plates (Eiken Chemical Co., Ltd., Tokyo, Japan) under microaerophilic conditions (5% O2, 10% CO2, 85% N2, at 37 °C; Aaero Pack Systems, Mitsubishi Gas Chemical, Osaka, Japan) for up to 5 days. Biopsies were obtained with informed consent from all patients under protocols approved by our ethics committee. The organisms were identified as H. pylori by spiral morphology and positive oxidase, urease, and catalase reactions. One colony on the agar was collected and cultured again under the same microaerophilic conditions in brain heart infusion broth (Nissui, Osaka, Japan) containing 5% (v/v) horse serum for up to 3 days. Aliquots were stored at −80 °C in 10% phosphate-buffered saline containing 20% (v/v) glycerol. After thawing of aliquots of the frozen culture, bacterial suspensions were cultured at 37 °C in brain heart infusion broth containing 10% fetal calf serum (Invitrogen) under microaerophilic conditions as described above on a gyratory shaker at 170 rpm for 24–36 h to the plateau phase. The human gastric cell lines AGS, KATOIII, MKN 28, MKN 45 were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and were maintained in a complete medium consisting of RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mml-glutamine, 0.2 mg of ampicillin/ml, and 100 μg of kanamycin/ml. Human gastric epithelial cell lines were cultured in RPMI 1640 containing 10% fetal calf serum without antibiotics and used at a final concentration of 5 × 105/ml. Bacterial suspensions were cultured at 37 °C in brain heart infusion broth containing 10% fetal calf serum under microaerophilic conditions as described above on a gyratory shaker at 170 rpm for 24–36 h to the plateau phase. The bacteria were then suspended in sterile phosphate-buffered saline. After centrifugation, the bacteria were resuspended at a final concentration of 1×107 colony-forming units/ml in RPMI 1640 supplemented with 10% fetal calf serum and used immediately. Gastric epithelial cells alone or cells with bacteria were cultured in tissue culture dishes (Falcon; Becton Dickinson) at 37 °C in a humidified incubator in an atmosphere of 95% air and 5% CO2. The cells were washed with phosphate-buffered saline three times after 4, 8, 12, and 24 h. Total cellular RNA was extracted from the cells by using Isogen reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions, and the amount was measured by absorbance at 260 nm. Poly(A) RNA was isolated from total cellular RNA (100 μg) using an MagExtractor (Toyobo, Tsuruga, Japan) according to the manufacturer's instructions. Total cellular RNA was incubated with oligo(dT) magnetic beads (in the kit), and then nonspecific substance was removed by washing. 2 μg of mRNA was reverse transcribed into cDNA by reverse transcriptase, ReverTraAce (Toyobo), in the presence of a cDNA synthesis primer. Biotin-labeled probes were generated by binding of biotin-16-deoxyuridine triphosphate during synthesis of cDNA. The human cDNA expression filters, human cancer filters (Toyobo) were prehybridized at 62 °C for 30 min in 20 ml of PerfectHyb solution (Toyobo). After denaturalization, cDNA probes were hybridized to the filters overnight at 62 °C. The membranes were washed three times with solution 1 (2× SSC and 0.1% SDS) and three times with solution 2 (0.1× SSC and 0.1% SDS) for 5 min at 62 °C. Specific signals on the filters were detected by using a chemiluminescence detection kit, Imaging High (Toyobo), according to the manufacturer's instructions. CDP-Star was used as the chemiluminescent substrate. Images and quantitative data of gene expression levels were obtained using a Fluor-S Multiimager system (Nippon Bio-Rad Laboratories, Tokyo, Japan) and quantified into intensity of signals by using ImaGene (BioDiscovery, Inc., Los Angeles, CA). 20 μg of total RNA was electrophoresed on a 1% agarose gel containing 6.5% formaldehyde and then transferred onto a nylon membrane. A Smad5 probe was made from human Smad5 cDNA that corresponded to its whole coding region. Each probe was labeled with biotin using Biotin-16-dUTP (Roche Diagnostics, Tokyo, Japan). A human β-actin probe labeled with biotin was used as a positive control. The membrane was hybridized with the labeled probe for 20 h at 62 °C in PerfectHyb (Toyobo). After hybridization, it was washed three times with 2× SSC with 0.1% SDS for 10 min and washed three times with 0.1× SSC with 0.1% SDS for 10 min at 62 °C. Positive bands were detected by using chemiluminescence detection kit (Imaging High; Toyobo), and CDP-Star was used as the chemiluminescent substrate according to the manufacturer's instructions. First strand cDNA templates were synthesized from 2 μg of total RNA using ReverTraAce and a random primer (Toyobo) according to the manufacturer's instructions. An aliquot (0.1 μl) of Taq DNA polymerase and deoxynucleoside triphosphates (Takara Shuzou Co., Ltd., Shiga, Japan) was mixed with 0.5 μl of a first strand cDNA sample and each primer. The primers used were Smad5F (5′-CAACACAGCCTTCTGGTTCA-3′) and Smad5R (5′-TTGACAACAAACCCAAGCAG-3′) for Smad5 amplification. PCR was performed using a thermal cycler (Takara Shuzou) under the following conditions: an initial denaturation for 5 min at 94 °C; 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C; and a final extension at 72 °C for 5 min with the number of cycles at which the band intensity increased linearly with the amount of mRNA used. The PCR product was then run on 1.5% agarose gel. We used two methods to detect apoptosis of epithelial cells induced by H. pyloriinfection. After co-culture of AGS cells with H. pylori for 72 h, DNA was extracted from the control and treated cells using an apoptosis ladder detection kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Each DNA (5 μg) was electrophoresed in 2% agarose gels. The gels were photographed under ultraviolet light, and DNA ladder formation was observed. Next, we carried out quantitative analysis of apoptosis. AGS cells were cultured with H. pylori for 72 h in 96-well plates (2×104cells/well). After centrifugation at 1500 rpm for 5 min, the supernatant was removed, and the pellets were frozen at −80 °C for 15 min. Then the terminal deoxynucleotidyl triphosphate-mediated deoxyuridine triphosphate nick end labeling assay was performed using a apoptosis screening kit (Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The degree of apoptosis was evaluated numerically by measuring the absorbance (490 nm). Two 29-mer DNA oligonucleotides (siRNA oligonucleotide templates) with 21 nucleotides encoding the siRNA and 8 nucleotides complementary to the T7 promoter primer were chemically synthesized, desalted, and purified by reverse phase high pressure liquid chromatography. These sequences were subjected to a BLAST search (NCBI data base) to ensure that only one gene was targeted. Two 21-mer oligonucleotides (sense, 5′-AATTACATCCTGCCGGTGATA-3′ and antisense, 5′-AATATCACCGGCAGGATGTAA-3′) encoding Smad5 had no homology to those of Smad1, 2, 3, 4, and 8 in a BLAST search. The two siRNA oligonucleotide templates were hybridized to a T7 promotor primer and were extended by the Klenow DNA polymerase. The sense and antisense siRNA templates were transcribed by T7 RNA polymerase and were hybridized to create double-stranded siRNA using a Silencer siRNA construction kit (Ambion). The control and H. pylori-treated cells were grown in 96-well plates, and cationic lipid-mediated transient transfections were carried out with 50 ng of siRNA/well using GeneSilencer siRNA transfection reagent (Gene Therapy Systems, San Diego, CA). After incubation at 37 °C for 24 h, Northern blot analysis was performed to assess the effectiveness of RNA interference, and quantitative analysis of apoptosis was carried out as described above. The data are presented as the means ± S.D. The differences were examined by analysis of variance, and pvalues were We first examined changes in gastric cellular mRNA expression in to co-culture with H. pylori 43504 at 8 and 24 h by cDNA in AGS genes were used as to the mRNA Although the majority of genes indicated only the expression of Smad5 mRNA increased with the changes in to genes and at 8, and 24 h, The expression of the other 2, 3, 4, and mRNA including were increased after 24 h co-culture and were not Northern blot analysis was carried out to the of Smad5 Total RNA was extracted from AGS cells treated with H. pylori and AGS cells at 4, 8, 12, and 24 h. Northern blot analysis that H. pylori infection up-regulated Smad5 mRNA expression of AGS cells after h of co-culture We examined several other gastric epithelial cell lines MKN28, and to Smad5 mRNA expression after co-culture with H. pylori by Smad5 mRNA was after co-culture with H. pylori in all gastric epithelial cell lines 3 pylori up-regulated Smad5 expression in AGS cells by Northern blot analysis. H. pylori-induced Smad5 mRNA expression of AGS cells was detected by Northern blot analysis. Total RNA was extracted from the cells with H. pylori for the indicated H. pylori infection up-regulated Smad5 mRNA expression after 8 h of The of was The β-actin probe was hybridized as a pylori up-regulated Smad5 expression in other human gastric epithelial cell lines and in gastric total RNA was extracted from human gastric epithelial cell lines and with H. pylori for the indicated and the expression of Smad5 mRNA was by using the Smad5 mRNA were up-regulated in all of the The of was was as a control in total RNA was extracted from the gastric specimens from patients infected with cagPAI-positive strains or the gastric specimens from and the expression of Smad5 mRNA was by using the Smad5 mRNA were in gastric The of is We whether Smad5 mRNA was in the 10 gastric specimens by Smad5 mRNA was in the gastric specimens from patients infected with cagPAI-positive strains, in the gastric specimens 3 assess the role of cagPAI in Smad5 mRNA we cagPAI-positive strains and and cagPAI-negative strains and T. Sugiyama T. T. Komatsu Y. Ishizuka J. Kato M. Asaka M. 2002; PubMed Scopus Google Scholar, T. S. A. Sugiyama T. K. K. 2001; PubMed Scopus Google by Northern blot analysis. and to Smad5 mRNA cagPAI-positive strains its expression DNA was induced in AGS cells 72 h after been with pylori strains and no DNA was with cagPAI-negative strains and 5 analysis of cellular apoptosis that pylori strains and induced significantly levels of apoptosis in AGS cells than cagPAI-negative strains and AGS cells alone 5 The between the results obtained using cagPAI-positive strains and cagPAI-negative strains or AGS cells alone was The up-regulated Smad5 mRNA expression was by RNA interference in all cagPAI-positive AGS cells the of Smad5 mRNA the of apoptosis was completely in the quantitative apoptosis assay The gastric pathogen H. pylori activates epithelial cell signaling after the signaling are The host pylori infection might be of with to the various clinical of infection by this We that H. pylori the Smad5 expression of gastric epithelial cells and that the Smad5 is in H. pylori-induced apoptosis of gastric epithelial In it was found that the presence of intact cagPAI is for apoptosis of epithelial We that a or system of and bone morphogenetic proteins from infected H. pylori or AGS cells are in the of Smad5 In human gastric epithelial AGS had and and of Smad5 mRNA expression was after of and by Northern blot analysis not Although and in from H. and AGS cells were measured by differences were not found not mRNA and mRNA were not up-regulated after co-culture with H. pylori in cDNA H. pylori not or which was examined by BLAST search (NCBI data it is that a or of or from AGS infected with H. pylori or of or from H. pylori is in of Smad5 The cagPAI a H. type IV machinery S. C. M. Rappuoli R. A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar), and this is for the of via an process in human gastric cells Blaser M.J. J. 1998; Google Scholar, A. C. E. A. A. S. M. J. 1997; Google Scholar). has been shown that is from the H. pylori into host cells via the type IV machinery and that it a complex with the homology 2 in a and the (11Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. Science. 2002; 295: 683-686Crossref PubMed Scopus (866) Google Scholar, E.D. J. J. Falkow S. Tompkins L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). These suggest that protein or gene through the type IV machinery is a for induced by H. pylori it is not that the of gastric epithelial cells is on the presence of We examined the Smad5 expression of AGS cells using cagPAI-positive and cagPAI-negative strains and that of the gastric mucosa infected with H. pylori. Our results indicated that cagPAI-positive H. pylori strains were to Smad5 mRNA expression and to apoptosis of the infected epithelial cells but that cagPAI-negative strains were not to Smad5 mRNA expression or apoptosis. Although is the only H. pylori protein to from the bacterium into the cell via the type IV secretion it be that transfer of genes or gene products through the type IV machinery might be for of the Smad5 gene in host has been reported that in a role in the of K. 2000; Google and that a of with had in the gene and were at increased of J. B. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). has been no on Smad5 expression in the role of Smad5 in or is not proteins have of about different have been identified in and be into three and K. 1997; PubMed Scopus Google Scholar). Each of the a different role in signaling be further into two those after by and Smad5 to the K. 2000; PubMed Google Scholar). Smad5 was isolated as and was in signaling and C. M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). A. C. A. 1997; PubMed Scopus Google that Smad5 the formation of the and in In an antisense oligonucleotide study, Smad5 was shown to the growth in cells E. D. E. C. R. 1998; PubMed Google Scholar), and N. S. T. M. T. T. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google that Smad5 apoptosis in the H. L. Science. 1996; PubMed Scopus Google Scholar), and also induced apoptosis in human cell via of and C. M. K. H. Y. Y. T. T. Y. 2000; PubMed Google Scholar). Many studies have that H. pylori induced apoptosis of gastric epithelial cells S. Yoshida H. Y. Y. Ogura K. Shiratori Y. Omata M. Gut. 2002; PubMed Scopus Google Scholar), that the up-regulated Smad5 mRNA expression might be in the apoptosis of gastric epithelial cells induced by H. pyloriinfection. We also that only cagPAI-positive H. were of of Smad5 mRNA as well as in human gastric Although virulence factors, and have been investigated as factors D. B. C. W. J. 2001; PubMed Scopus Google Scholar), the intracellular signaling of apoptosis induced by H. pylori is Our results indicated that Smad5 might be related to the induced by cagPAI-positive H. as one of the intracellular signaling We the levels of H. pylori-induced apoptosis and after of Smad5 mRNA expression by RNA interference, and it was found that the of apoptosis was to the after the These suggest that Smad5 is a factor for H. pylori-induced apoptosis. In H. Smad5 expression through the presence of cagPAI encoding type IV secretion and up-regulated Smad5 induces responses in infected gastric epithelial