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Originally published In Press as doi:10.1074/jbc.M604574200 on September 11, 2006

J. Biol. Chem., Vol. 281, Issue 45, 34651-34662, November 10, 2006
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Cag Pathogenicity Island-independent Up-regulation of Matrix Metalloproteinases-9 and -2 Secretion and Expression in Mice by Helicobacter pylori Infection*

Parag Kundu{ddagger}1, Asish K. Mukhopadhyay§, Rajashree Patra§, Aditi Banerjee{ddagger}, Douglas E. Berg, and Snehasikta Swarnakar{ddagger}2

From the {ddagger}Indian Institute of Chemical Biology, Kolkata 700032, India, the §National Institute of Cholera and Enteric Diseases, Kolkata 700010, India, and the Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri 63110

Received for publication, May 12, 2006 , and in revised form, July 19, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Helicobacter pylori cag pathogenicity island (PAI) is a major determinant of gastric injury via induction of several matrix metalloproteinases (MMPs). In the present study, we examined the influence of the cag PAI on gastric infection and MMP-9 production in mice and in cultured cells. A new mouse colonizing Indian H. pylori strain (AM1) that lacks the cag PAI was used to study the cag PAI importance in inflammation. Groups of C57BL/6 mice were inoculated separately with H. pylori strains AM1 and SS1 (cag+), gastric tissues were histologically examined, and bacterial colonization was scored by quantitative culture. Mice infected with either cag+ or cag- H. pylori strains showed gastric inflammation and elevated MMP-3 production. Significant up-regulation of pro-MMP-9 secretion and gene expression in H. pylori infected gastric tissues indicate dispensability of cag PAI for increased pro-MMP-9 secretion and synthesis in mice. In agreement, cell culture studies revealed that both AM1 and SS1 were equipotent in pro-MMP-9 induction in human gastric epithelial cells. Both strains showed moderate increase in MMP-2 activity in vivo and in vitro. In addition, increased secretion of tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1beta, and IL-6 induced pro-MMP-9 secretion and synthesis in AM1 or SS1 strain-infected mice suggesting elicitation of pro-inflammatory cytokines by both cag- and cag+ genotype. Moreover, tissue inhibitors of metalloproteinase-1 expression were decreased with increase in pro-MMP-9 induction. These data show that H. pylori may act through different pathways other than cag PAI-mediated for gastric inflammation and contribute to up-regulation of MMP-9 via pro-inflammatory cytokines.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Helicobacter pylori is a microaerophilic bacterium with an extraordinary ability to chronically infect human stomachs for years together despite gastric mucosal turnover and other host defenses (1). It colonizes more than half of all people worldwide and is still unknown why most remain asymptomatic (2). Persistent H. pylori infection is associated with chronic gastritis and gastric cancer, one of the most lethal of malignancies worldwide (2-4). It may also cause childhood malnutrition and increase the risk or severity of infection by other gastrointestinal pathogens such as Vibrio cholerae, especially in developing countries (5, 6). H. pylori appears to be one of the most genetically diverse of bacterial species and shows significant geographic differences among strains (7-11). Studies of strains from Europe and North America indicated that H. pylori genotypes can be important in colonization and disease outcome (12). Recent studies revealed that Indian H. pylori strains are genetically distinct from European and East Asian strains (9, 10). Variation in the clinical outcome of H. pylori infection seems to be multifaceted and involves a complex interplay between virulence factors, host immune responses, and other features of the H. pylori gastric mucosal niche. Prominent among the H. pylori virulence-associated determinants is the cag pathogenicity island (PAI)3 (13, 14), a 40-kb genome segment that encodes ~30 genes, most important among them is cytotoxin-associated gene-encoded immunodominant cag A protein (15). Mouse model for H. pylori have been used to understand the role of cag PAI for colonization, persistence of infection, and clinical manifestations (16-21).

MMPs are a family of zinc-dependent endopeptidases that selectively degrade or remodel most of the extracellular matrix (ECM) components of gastric mucosa including collagen, and other structural molecules (22, 23). MMPs, specifically gelatinase A (MMP-2, 72 kDa) and gelatinase B (MMP-9, 92 kDa) are responsible for degradation of type IV and V collagen, elastin, and fibronectin (23, 24). MMP-2 is constitutively expressed and may participate in the remodeling of the ECM (23), while MMP-9 is important in degradation of ECM and basement membrane barriers during gastric ulcer and gastric cancer (25-27). In several inflammatory diseases such as rheumatoid arthritis (28) and inflammatory bowel disease (29), MMPs substantially contribute to remodeling of connective tissues and stromal tissue destruction. Increasing evidence in in vitro systems suggest that H. pylori infection stimulates gastric epithelial cells to produce MMPs either directly or indirectly via cytokine synthesis (30-33). However, few studies have demonstrated that H. pylori induced inflammation and gastric damage in vivo are associated with induction of MMPs (33-36). H. pylori infection increased both basal secretion and activation of MMP-3 in human gastric cells (32, 37).

It is believed that H. pylori induced inflammation in the gastric tissues is associated with the production of pro-inflammatory cytokines and appears to be triggered partly by genes located in the cag PAI, which have been demonstrated in different animal models (14, 38, 39). The possibility that cag PAI directly regulate gastric damage has not been rigorously examined. cag+ H. pylori are presumed to cause gastric mucosal damage by translocation of Cag A protein (40), which in turn induces MMPs in response to pro-inflammatory cytokine signaling. Conversely, long term infection by cag PAI-deficient H. pylori cause gastric damage in mice (20, 41, 42). Although there are reports describing the association between the presence of cag PAI and progression of gastric disease (13, 14, 43), the pathogenic role of cag PAI is not yet completely understood.

To better understand mechanisms by which H. pylori induce gastric damage, we compared cag+ and cag- strains for tissue injury and MMP-9 secretion in mice and in cultured cells. Keeping in mind the possibility that Indian H. pylori strains might differ from Western strains most studied to date, we screened 50 strains from endoscopic samples of ulcer patients in Kolkata to find a naturally occurring cag- strain. Here we report that Indian strain AM1 (cag-) and reference strain SS1 (cag+) each damages mouse gastric tissues during infection and augments production of MMP-3. Also, each strain markedly increases secretion and synthesis of pro-MMP-9 and to a lesser extent increases active MMP-2 activity at the level of secretion and synthesis both in mice and in cultured cells. The up-regulation of pro-MMP-9 by H. pylori is directly associated with down-regulation of tissue inhibitors of metalloproteinase (TIMP)-1 expression and increased secretion of pro-inflammatory cytokines like interleukin (IL)-1beta, IL-6, and TNF-{alpha}. We hypothesize that initial stages of infection by both cag- and cag+H. pylori may propagate through a common pathway and that process responses to pro-inflammatory signals for MMP-9 up-regulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
H. pylori Strains and Culture—Two unrelated mouse-adapted H. pylori strains were used: SS1 (19, 44), and AM1 (Indian strain). SS1 is much used as the standard mouse-adapted strain for experimental infection (The Sydney Strain). The strain AM1 was isolated from an endoscopic sample of an ulcer patient in Kolkata, India as mixed infections. Both strains of H. pylori were grown on brain-heart infusion agar (BHI; Difco Laboratories, Detroit, MI) supplemented with 7% sheep blood, 0.4% isovitalex, and the antibiotics amphotericin B (8 µg/ml), trimethoprim (5 µg/ml), and vancomycin (8 µg/ml) (referred to here as BHI agar). Nalidixic acid (10 µg/ml), polymyxin B (10 µg/ml), and bacitracin (200 µg/ml) were added to this medium when culturing H. pylori from mouse stomachs. The plates were incubated at 37 °C under 5% O2, 10% CO2, 85% N2. In all experiments, overnight grown cultures on BHI agar plates were used.

DNA Methods—Chromosomal DNA was extracted from different H. pylori strains to characterize the genotype. Chromosomal DNA was prepared by the CTAB (hexadecyltrimethyl ammonium bromide) extraction method (45), from confluent BHI agar plate cultures. The H. pylori 26695 strain was used as a control in the polymerase chain reaction (PCR) assay. Specific PCR was carried out in 20-µl reaction volumes using 10 pmol of each primer per reaction, 0.25 mM of each dNTP, 1 unit of Taq polymerase (Takara, Shuzo, Japan), and 10 ng of DNA (from both a pool of colonies and single colony isolates) in a standard PCR buffer (Takara). PCR was carried out for 30 cycles of: (i) denaturation at 94 °C for 1 min, (ii) primer template DNA annealing at 55 °C for 1 min, and (iii) DNA synthesis at 72 °C for a time based on expected fragment size (1 min/kb). The presence or absence of the cag PAI was scored by PCR with specific primers (Table 1) using DNA from different cultured strains. After amplification, 3 µl of each reaction mixture was separated by electrophoresis on a 2% agarose gel, and the amplified gene products were visualized under uv light after staining with ethidium bromide. DNA markers were obtained from New England Biolabs.


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  		TABLE 1
Details of PCR primers used for analysis of vacA alleles, cagA, and cag PAI empty site in SS1 and AM1 strains of H. pylori

 
H. pylori Infection in C57BL/6 Mice—C57BL/6 mice bred inhouse with free access to food and water were used in all experiments. Experiments were designed to minimize animal suffering and to use the minimum number associated with valid statistical evaluation. Animals were anesthetized by ketamine (12 mg/kg b.w.) followed by cervical dislocation for killing. Animal experiments were carried out following the guidelines of the animal ethics committee of the institute. Animals of both control and experimental groups were kept separately under standard controlled conditions and were fasted for 6 h with free access to water before each inoculation. Mouse infection was done using a modification of the Lee et al. (19) method. Briefly, overnight grown bacterial cultures were harvested in 10 mM phosphate-buffered saline (PBS). Groups of mice (12 mice per group) were intragastrically inoculated by orogastric gavage twice in a period of 3 days with about 108 CFU/mouse/inoculation. Groups of mice inoculated with SS1, AM1, heat-killed bacteria, or PBS were kept separately with free access to water and food. One day after final inoculation (3 days post-primary inoculation), half of the mice from each group were fasted for 6 h, and then sacrificed. The other half from each group was sacrificed 9 days later (13 days post-primary inoculation).

Histology—The body and the pyloric parts of control and 10-day infected mouse stomach were sectioned for histological studies. The tissue samples were fixed in 10% formalin and embedded in paraffin. The sections (5 µm) were cut using microtome, stained with hematoxylin and eosin (26), and assessed under an Olympus microscope (1 x 70). Images were captured using Camedia software (E-20P 5.0 Megapixel) at original magnification 10 x 10, 20 x 10, and 40 x 10 and processed in Adobe Photoshop version 7.0.

Tissue Extraction—The body and the pyloric parts of stomach were suspended in PBS containing protease inhibitors (Sigma), minced, and incubated for 10 min at 4 °C. The suspension was then centrifuged at 12,000 x g for 15 min, and the supernatant was collected as PBS extracts. The pellet was then extracted in lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, and protease inhibitors) and centrifuged at 12,000 x g for 15 min to obtain Triton X-100 extracts. Both PBS and Triton X-100 extracts were preserved at -70 °C for future studies. A small part of the stomach was minced in PBS followed by low speed centrifugation, and the supernatant was used for bacterial culture. Bacterial colonization was scored by quantitative culture.

Gelatin and Casein Zymography—For assay of MMP-9 and -2 activities, tissue extracts were electrophoresed in a 8% SDS-polyacrylamide gel containing 1 mg/ml gelatin (Sigma), under non-reducing conditions. The gels were washed twice in 2.5% Triton X-100 (Sigma) and then incubated in calcium assay buffer (40 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 10 mM CaCl2) for 18 h at 37 °C. For assay of MMP-3 activity, tissue extracts were electrophoresed in casein gel instead of gelatin. Gels were stained with 0.1% Coomassie Blue followed by destaining (46). The zones of gelatinolytic or caseinolytic activity came as negative staining. Standard MMP-9 and MMP-2 enzymes were purchased from Chemicon, Hampshire, UK. H. pylori-infected human gastric tissue extract was used as MMP-3 standard. Quantification of zymographic bands was done using densitometry linked to proper software (Lab Image).

Western Blotting—Tissue extracts (100 µg/lane) were resolved by 10% reducing SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (47). The membranes were blocked for 2 h at room temperature in 3% bovine serum albumin solution in 20 mM Tris-HCl, pH 7.4 containing 150 mM NaCl and 0.02% Tween 20 (TBST) followed by overnight incubation at 4 °C in 1:200 dilution of the respective primary antibodies in TBST containing 0.2% bovine serum albumin. The membranes were washed five times with TBST and then incubated with alkaline phosphatase-conjugated secondary antibody (1:2000) for 1.5 h. The bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution (Sigma). Polyclonal mouse reactive anti-MMP-9, anti-MMP-3, anti-TIMP-1, anti-IL-6, anti-TNF-{alpha}, and monoclonal human reactive anti-MMP-9 antibodies were purchased from Santa Cruz Biotechnology, while monoclonal anti-beta-actin antibody and polyclonal mouse reactive anti-IL-1beta were obtained from Sigma.

Reverse Transcriptase-PCR (RT-PCR)—Total cellular RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol and quantified by measuring the absorbance at 260 nm. Complementary DNA was synthesized using 1 µg of total RNA in a 20-µl reaction buffer using Superscript II Reverse Transcriptase (Invitrogen) with an oligo(dT)15 primer (Invitrogen). The cDNA (1 µl) was then amplified in a 20-µl reaction buffer for 35 cycles of denaturation (94 °C for 30 s), annealing (58 °C for 30 s), and extension (72 °C for 60 s) using the following primers: for MMP-9, sense, 5'-GACCCGAAGCGGACATTGTCAT-3', and antisense, 5'-GGAATGATCTAAGCCCAGTGCAT-3', (expected product 755 bp); for MMP-2, sense, 5'-ATGGCTTCCTCTGGTGCTC-3' and antisense, 5'-TCGTAGTGGTTGTGGTTGC-3' (expected product 385 bp); for MMP-3, sense, 5'-GGATTGTGAATTATACACCGGAT-3' and antisense, 5'-GGATAACCTGCTAGCTCCTCGT-3' (expected product 769 bp); and for GAPDH, sense, 5'-TGGGGTGATGCTGGTGCTGAG-3', and anti-sense, 5'-GGTTTCTCCAGGCGGCATGTC-3', (expected product 497 bp). The PCR products were analyzed by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. PCR product sizes were estimated by 100-bp marker (Invitrogen) in each case. The real time RT-PCR was used for quantitative detection of MMP-9 gene in AM1 and SS1 H. pylori-infected gastric tissues. The reaction was carried out in a 20-µl final volume with the iCycler iQ (Bio-Rad) under the following conditions containing 50 ng of cDNA, 10 pmol of each primer, and SYBR Premix Ex Taq (Takara, Japan). Polymerase activation at 95 °C for 3 min followed by 50 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 60 s. A quantitative measure of MMP-9 was obtained through amplification of GAPDH and MMP-9 cDNA in each sample using the same PCR conditions. The amount of MMP-9 expression relative to the total amount of cDNA was calculated as {Delta}Ct = CtGAPDH - Ct MMP-9. The changes of MMP-9 expression in AM1- or SS1-infected samples compared with that of control were expressed as {Delta}{Delta}Ct = {Delta}Ctcontrol - {Delta}CtAM1 or SS1 where CtMMP-9 and CtGAPDH were the fractional cycle number at which fluorescence generated by reporter dye exceeds the fixed level above baseline for MMP-9 and GAPDH cDNA, respectively. The relative expression of MMP-9 gene in the AM1 or SS1 strain of H. pylori-infected samples was calculated as 2{Delta}{Delta}Ct. Each sample was run three times.

Cell Culture and H. pylori Infection—The human gastric epithelial cells (AGS) were maintained in RPMI 1640 (Invitrogen) medium containing 10% fetal bovine serum (FBS) (Invitrogen) and antibiotics/antimycotics at 37 °C in humidified atmosphere containing 5% CO2 (31). Twenty-four hours before infection, cells were transferred into 6-well polypropylene tissue culture plates (Nunc, Roskilde, Denmark). For coculture experiments, viable H. pylori strains AM1 and SS1 grown separately in BHI agar plates were harvested in PBS, centrifuged, and resuspended in antibiotic-free and FBS-free media to a concentration of 1 x 109 colony-forming units/ml and were immediately incubated with the AGS cell line. For all experiments, H. pylori were infected to cells at 80% confluence at a bacteria/cell concentration of 100:1 (48). Infected and uninfected AGS cells were cultured in antibiotic-free and FBS-free media in 6-well plates for either 4 or 24 h. Serum-free media were then collected, centrifuged at 4000 x g to remove cells and bacteria. The media was concentrated 5x by lyophilization to use for gelatin zymography and Western blot.


Figure 1
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  		FIGURE 1.
Histology of mouse gastric tissues 10 days postinfection by cag- and cag+ strains. Different groups of mice were intragastrically inoculated with SS1 and AM1 strains of H. pylori, sacrificed on day 10 postinfection, and stomachs were sectioned for histological studies. Control mice were fed with PBS and kept separately under the same conditions. Histological appearances of control (A), AM1-infected (B), and SS1-infected (C) gastric tissues stained with hematoxylin and eosin and were observed at 10 x 10 magnification. While (D-F) and (G-I) represent control, AM1- and SS1-infected tissues at 20 x 10, 40 x 10 magnification, respectively.

 
Statistical Analysis—Densitometry data are fitted using Sigma Plot. Data are presented as the mean ± S.E. Statistical analysis was performed using the Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Histology of Mouse Gastric Tissues 10 Days Postinfection by cag- and cag+ H. pylori Strains—Because H. pylori infection is responsible for inflammation in gastric tissues and degradation of gastric ECM as well, we were prompted to investigate the possible changes in tissue integrity. Different groups of mice were inoculated with PBS, SS1 strain, and AM1 strain of H. pylori separately and were sacrificed at day 10 postinfection. Histological examination of mouse gastric tissues revealed that infection with either SS1 or AM1 strains caused inflammation in gastric pit cells along with disruption in submucosa and muscularis mucosa compared with control (Fig. 1, A-F). Overall damage in epithelial mucosa was more prominent in SS1-than in AM1-infected mice compared with control. Glandular atrophy and infiltration of inflammatory cells, mostly lymphocytes, were also detected in the gastric tissues of SS1 or AM1 strain infected mice (Fig. 1, G-I). These results suggest that infection by the strains SS1 or AM1 has the capacity to cause gastric inflammation although the severity of damage was more pronounced in SS1-than in AM1-infected C57BL/6 mice.

The Genetic Makeup of Mouse Colonizing Indian H. pylori Strain, Named AM1—The presence of vacAs1 versus vacAs2 alleles at the 5'-end of vacA gene was determined based on the sizes of PCR products (259 bp versus 286 bp, respectively) generated with vacAs region-specific primers. Fig. 2A shows that both SS1 and AM1 strains yielded a 286-bp fragment, which indicate the presence of s2 alleles. The alleles of the vacA middle (m) region, which determines the cell type specificity of vacuolating cytotoxin action, were also studied by PCR. The presence of vacAm1 versus vacAm2 alleles was determined based on sizes of PCR products generated with vacA middle region-specific primers. Both strains yielded a 642-bp fragment indicating that they carried vacAm2 alleles (Fig. 2B). A 350-bp product indicative of the cag PAI was obtained with primers specific for the cagA gene from SS1 but not from AM1 (Fig. 2C). Instead, AM1 yielded a 550-bp product expected of a cagA empty site with the primers designed from the flanking region of the cag PAI, which indicate complete absence of the cag PAI (Fig. 2D).

Up-regulation of pro-MMP-9 Activity in Mice Requires H. pylori Colonization—Because MMPs are responsible for degradation of variety of ECM molecules, we examined whether colonization of live SS1 has any effect on pro-MMP-9 activity in C57BL/6 mice. Fig. 3, A and C show that colonization of the live H. pylori SS1 strain in mouse gastric mucosa caused significant up-regulation (~6-fold) in pro-MMP-9 activity on day 1 postinfection at the level of secretion compared with that observed in heat-killed bacteria and was further increased by ~25% on day 10. These results suggest that interaction with the live H. pylori SS1 strain rather than the protein or lipid components of heat-denatured bacteria is essential for higher pro-MMP-9 secretion (Fig. 3A). The changes in the activity at the level of secretion prompted us to determine pro-MMP-9 activity at the level of synthesis during infection by H. pylori. Analysis of Fig. 3, B and D indicate that synthesized pro-MMP-9 activity was increased by ~5.5-fold and ~7.5-fold in live compared with dead bacteria on days 1 and 10 postinfection, respectively. Interestingly, Fig. 3 shows that both secreted and synthesized MMP-2 activities were moderately (~2-fold) increased in gastric tissues infected with live H. pylori SS1 strain compared with that observed in heat-killed bacteria-treated mice and pro-MMP-2 activity remained unchanged. Our data revealed that H. pylori colonization as well as infection up-regulated pro-MMP-9 and MMP-2 activities at the level of secretion and synthesis in mice.


Figure 2
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  		FIGURE 2.
Genetic status (vacA and cagA) of two strains of H. pylori colonizing in C57BL/6 mice. Marker represents the 100-bp marker in every case. Fully sequenced H. pylori 26695 and Escherichia coli DH5{alpha} strains, used as positive and negative control, respectively. Test for the presence of: A, vacAs1 versus vacAs2 alleles using primers VA1-F and VA1-R; B, vacAm1 and vacAm2 alleles using primers VAG-F and VAG-R; C, cagA gene using primers cag5c-F and cag5c-R; and D, cag PAI empty site using primers Luni1 and R5280.

 


Figure 3
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  		FIGURE 3.
Up-regulation of MMP-9 and -2 activities in mouse gastric tissues infected with live H. pylori strain SS1. Different sets of C57BL/6 mice were intragastrically inoculated with heat-killed and live SS1 strains separately and were sacrificed on day 1 and day 10 postinfection. A, gelatin zymography was performed to detect the activities of secreted MMP-9 and -2 in the gastric tissues exposed to dead and live bacteria. Equal amounts (70 µg of protein) of PBS extracts of different tissues were electrophoresed in 8% non-reducing SDS-polyacrylamide gel containing 1 mg/ml gelatin. MMP activity was monitored by gelatinolytic activity as described under "Experimental Procedures." Marker represents purified MMP-9 and MMP-2 enzymes. B, gelatin zymography as described in A was performed using Triton X-100 extracts (70µg of protein) of control and infected gastric tissues. Histographic representation of secreted (C) and synthesized (D) MMP-9 and -2 activities plotted from the above zymogram and three other representative zymograms from independent experiments in each case are shown. Activities were measured by using Lab Image densitometry program. Error bars, ± S.E. *, p < 0.001; Figure 3, p < 0.01 versus the dead bacteria (C and D).

 
Infection by cag- Strain of H. pylori Increases MMP-9 and -2 Secretion and Expression—We next examined whether cag- strain of H. pylori could up-regulate pro-MMP-9 and -2 activities as observed for cag+ SS1 strain, at the level of secretion and synthesis. Because no significant changes were observed in pro-MMP-9 activity between PBS and heat-killed bacteria-treated mice (data not shown), we used PBS-treated mice as control in the following experiments. Different groups of mice were intragastrically inoculated with PBS or strain AM1 and sacrificed at day 1 and day 10 postinfection. Fig. 4A shows that secreted pro-MMP-9 activity was increased significantly at day 1 postinfection, and the activity was further increased at day 10 compared with either day 1 or day 10 control. Mice treated with PBS showed no changes in pro-MMP-9 and MMP-2 activities with increasing day postinoculation (Fig. 4A). The activity of secreted pro-MMP-9 in infected tissues was increased by ~8-fold compared with the control at day 1, and the activity reached ~9.5-fold at day 10 postinfection (Fig. 4B). In addition, the activity of active MMP-2 was elevated by ~2.5-fold on day 1 and further increased to ~3-fold at day 10, whereas no significant changes were observed in pro-MMP-2 activity. Fig. 4C shows that the secreted pro-MMP-9 protein level was increased significantly with time in infected tissues compared with control. The Western blot for beta-actin was done to confirm equal protein loading.


Figure 4
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  		FIGURE 4.
Up-regulation of secreted MMP-9 and -2 activities in mice infected by strain AM1. Different groups of mice were intragastrically inoculated with H. pylori strain AM1 and sacrificed on day 1 and day 10 postinfection. Control group of mice were inoculated with PBS, kept under the same conditions, and sacrificed on the respective days. A, gelatin zymography as described in the legend to Fig. 3A was performed using PBS extracts of control and infected tissues. B, histographic representation of MMP-9 and -2 activities as measured by Lab Image densitometry values from the above zymogram and four other representative zymograms from independent experiments. Error bars, ± S.E. *, p < 0.001 versus control. C, Western blots described under "Experimental Procedures" were performed using equal amounts of PBS (100 µg of protein) extracts from control and AM1-infected tissues at different days and probed with polyclonal anti-MMP-9 antibody and monoclonal anti-beta-actin antibody.

 
At the level of synthesis, pro-MMP-9 activity was increased by ~6-fold and ~7-fold at day 1 and day 10, respectively in infected gastric tissues compared with control (Fig. 5, A and B). Similarly, synthesized MMP-2 activity was increased by ~2-fold on day 1 and that was further increased by ~25% on day 10, whereas no changes at the level of synthesized pro-MMP-2 activity could be detected (Fig. 5, A and B). Overexpression of pro-MMP-9 protein at the level of synthesis was clearly indicated in infected tissues in the blot (Fig. 5C), and blots for beta-actin confirmed equal loading of proteins. After observing these changes at the protein level, we analyzed the mRNA expression for MMP-9 and -2 genes through RT-PCR.


Figure 5
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  		FIGURE 5.
Up-regulation of synthesized MMP-9 and -2 activities and mRNA expression in mice infected by strain AM1. Different groups of mice were orally fed with AM1 strain of H. pylori and sacrificed on day 1 and day 10 postinfection. Control group of mice were inoculated with PBS, kept under same conditions and sacrificed on the respective days. A, gelatin zymography described in the legend to Fig. 3A was performed using Triton X-100 extracts of control and infected tissues. B, histographic representation of MMP-9 and -2 activities as measured by Lab Image densitometry values from the above zymogram and four other representative zymograms from independent experiments. Error bars, ± S.E. *, p < 0.001; Figure 5, p < 0.05 versus control. C, Western blots described under "Experimental Procedures" were performed using 100 µg of Triton X-100 extracts of the above tissue samples and probed with polyclonal anti-MMP-9 antibody and monoclonal anti-beta actin antibody. D, RT-PCR analysis of MMP-9 and MMP-2 mRNA expression in control and infected mice. RNA extraction and RT-PCR analysis were performed as described under "Experimental Procedures." PCR using GAPDH primers was a positive control.

 


Figure 6
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  		FIGURE 6.
Comparison of MMP-9 and -2 activities and mRNA expression in mouse gastric tissues during H. pylori infection by cag- and cag+ strains. Different strains of H. pylori (SS1 and AM1) were orally fed separately to two groups of mice, and they were sacrificed on day 10 postinfection. Control mice were fed with PBS and kept separately under the same conditions. A, gelatin zymography described in the legend to Fig. 3A was performed using PBS and Triton X-100 extracts of control and infected tissues. Histographic representation of secreted (B) and synthesized (C) MMP-9 and -2 activities in control, strain SS1, and strain AM1 infected mice measured by Lab Image densitometry values from the above zymogram and four other representative zymograms from independent experiments are shown. Error bars, ± S.E. *, p < 0.001; Figure 6, p < 0.01 versus the appropriate control. D, RT-PCR analysis of MMP-9 and MMP-2 mRNA expression in control and infected mice. RNA extraction and RT-PCR analysis were performed as described under "Experimental Procedures." PCR using GAPDH primers was done as a positive control. E, histographic representation of relative expression of MMP-9 transcript in AM1- and SS1-infected mouse gastric tissues as measured by real time RT-PCR.

 
RT-PCR analysis (Fig. 5D) revealed ~6-fold and ~9.5-fold increases in MMP-9 mRNA at day 1 and day 10, respectively, in AM1-infected mouse gastric tissues, whereas an increase in MMP-2 mRNA expression was observed by ~1.2-fold on day 1 and ~1.4-fold on day 10 postinfection. The above results clearly suggest that cag- strain of H. pylori infection increased pro-MMP-9 secretion and synthesis at the protein and gene expression levels.

Comparison between cag- and cag+ Strains of H. pylori Infection for MMP-9 and -2 Secretion and Expression in Mouse Gastric Tissues—Because the cag- strain of H. pylori infection up-regulated pro-MMP-9 and MMP-2 activities in mice at the level of both secretion and synthesis, we conducted a comparative study between cag+ and cag- strains for their ability to up-regulate gelatinase activity. Different groups of mice were inoculated with PBS, AM1 strain, and SS1 strain separately and were sacrificed at day-10 post infection. Fig. 6A shows significant up-regulation of secreted and synthesized pro-MMP-9 activity in mouse gastric tissues infected by cag- as well as cag+ strains at day 10 postinfection. Both AM1 and SS1 strains up-regulated significantly the secreted pro-MMP-9 activity compared with control (Fig. 6B), while secreted MMP-2 activity was enhanced moderately. Similarly, compared with control, AM1 and SS1 infected mice showed marked increase in pro-MMP-9 activity and moderate increase in MMP-2 activity at the level of synthesis (Fig. 6C). No significant changes in secreted or synthesized pro-MMP-2 activity were observed in infected mice compared with control. Fig. 6D shows the RT-PCR analysis of MMP-9 and MMP-2 gene expressions in the gastric tissues of control, AM1-, and SS1-infected mice. An increase in MMP-9 transcript in infected mice by ~10-fold and ~10.5-fold for AM1 and SS1, respectively, compared with control was observed. Whereas, ~1.4-fold increase in MMP-2 mRNA expression was observed for both AM1 and SS1 infected mice. The above results suggest that both cag+ and cag- strains were equipotent in up-regulating pro-MMP-9 and MMP-2 secretion, synthesis, and gene expression in vivo. Using real time RT-PCR, a direct quantitative measurement of MMP-9 expression in AM1- and SS1-infected tissues was expressed in Fig. 6E. The relative expression of MMP-9 was 42- and 91-fold for AM1 and SS1 infected tissues, respectively, compared with control. We used the GAPDH gene as an internal control against which MMP-9 signal was normalized (CtMMP-9 - CtGAPDH = {Delta}Ct) (see "Experimental Procedures").

Infection by Both cag+ and cag- Strain of H. pylori Increases MMP-3 Production in Mouse Gastric Tissues—Because MMP-3 production is specific for H. pylori infected gastric tissues, we took advantage of this and performed casein zymography to monitor MMP-3 activity in AM1- and SS1-infected gastric tissues (Fig. 7A). Both strains, AM1 and SS1 up-regulated secreted pro-MMP-3 activity by ~3-fold and ~4-fold, respectively, compared with control on day 10 postinfection, while active MMP-3 activity was enhanced by ~5-fold and ~6.5-fold, respectively. Fig. 7B shows that secreted MMP-3 protein level was increased significantly in both AM1- and SS1-infected tissues compared with control. Western blot for beta-actin confirmed equal protein loading. RT-PCR analysis of MMP-3 gene expression in the gastric tissues of AM1- and SS1-infected mice showed ~3.5-fold and ~5-fold increase in MMP-3 transcript, respectively, compared with control (Fig. 7C). These results confirm that both AM1 and SS1 strains were able to up-regulate MMP-3 secretion and gene expression in vivo.

Involvement of Regulatory Molecules in the Increased pro-MMP-9 Activity and Expression in Mice Infected with H. pylori Strain SS1 (cag+) and AM1 (cag-)—The presence of inflammatory cells in H. pylori-infected gastric tissues suggested the investigation of regulatory molecules, especially pro-inflammatory cytokines involved in pro-MMP-9 up-regulation. Overexpression of pro-MMP-9 occurred in mice even on day-1 and increased further at day 10 postinfection by cag+ as well as cag- strains compared with that observed for day 10 control (Fig. 8, A and B). Because the activities of MMPs are regulated by TIMPs, we then examined the level of TIMP-1 expression. Interestingly, the expression of TIMP-1 followed a distinct inverse ratio with pro-MMP-9 expression. However, the band for TIMP-1 came at ~120 kDa instead of the expected 29 kDa. The reason possibly because of multimerization of TIMP-1 or its binding to MMPs in tissue extracts. The role of cytokines on MMP-9 expression was then tested through Western blot analysis of PBS extracts followed by probing with anti-IL-1beta antibody, anti-IL-6 antibody, and anti-TNF-{alpha} antibody (Fig. 8, A and B). The level of secreted IL-1beta increased parallel with pro-MMP-9 expression in mice infected either with cag- or cag+ strains at day 1 as well as day 10. The level of secreted IL-6 also increased simultaneously with pro-MMP-9 expression in mice infected either with cag- or cag+ strains. TNF-{alpha} secretion increased significantly with increased pro-MMP-9 expression in cag- or cag+ strains infected mice at day 1 and day 10 postinfection. Western blot for beta-actin was conducted to confirm equal protein loading in the above blots. These results indicate that TIMP-1 and pro-inflammatory cytokines play a major role in the increased pro-MMP-9 activity and expression.


Figure 7
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  		FIGURE 7.
Increased MMP-3 activity and expression in mouse gastric tissues during H. pylori infection by cag- and cag+ strains. Different strains of H. pylori (SS1 and AM1) were orally fed separately to two groups of mice and they were sacrificed on day 10 postinfection. Control mice were fed with PBS and kept separately under the same conditions. A, casein zymography described under "Experimental Procedures" was performed using PBS extracts of control and infected tissues. Densitometric analysis of caseinolytic bands is shown below the zymogram. Band intensities in lane 1 were set to 100%. B, Western blots as described under "Experimental Procedures" were performed using equal amounts of PBS (100 µg of protein) extracts from control and infected tissues and probed with polyclonal anti-MMP-3 antibody and monoclonal anti-beta-actin antibody. C, RT-PCR analysis of MMP-3 mRNA expression in control and infected mice. PCR using GAPDH primers was done as a positive control.

 
H. pylori Induced MMP-9 and -2 Secretions in Gastric Epithelial Cells—Because cag+ and cag- strain infection increased pro-MMP-9 and -2 secretions in mice, we asked whether they would induce gelatinases in vitro in the gastric epithelial cell population. Coculture of SS1 or AM1 H. pylori with AGS cells enhanced the secretion of both MMP-9 and -2 in conditioned media collected after 4 and 24 h (Fig. 9A). The activity of pro-MMP-9 in culture media was increased by ~4.3-fold and ~4.5-fold after 4 h of coculture by AM1 and SS1 strains, respectively, compared with that of control and the activity reached ~8.25-fold and ~9-fold after 24 h (Fig. 9B). The activity of secreted MMP-2 was elevated by ~3.5-fold and ~3-fold after 4 h and ~5-fold and ~5.3-fold after 24 h by AM1 and SS1 strain infection, respectively, compared with that of control (Fig. 9B). Western blot (Fig. 9C) shows increased secretion of pro-MMP-9 protein by AGS cells at 4 h and 24 h postcoculture compared with control. Western blot for beta-actin was done to confirm equal protein loading. These results suggest that cag- H. pylori increase pro-MMP-9 and MMP-2 secretions in vitro and determine gastric epithelial cell types for gelatinase secretion during infection.


Figure 8
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  		FIGURE 8.
Involvement of regulatory molecules in the up-regulation of pro-MMP-9 activity and expression in H. pylori-infected C57BL/6 mice. H. pylori strains SS1 and AM1 were orally fed to separate groups of mice and they were sacrificed on day 1 and 10 postinfection. Control mice were fed with PBS, kept under the same conditions, and sacrificed on day 10. Triton X-100 extracts of different tissues were subjected to Western blot described under "Experimental Procedures" and probed with polyclonal anti-MMP-9 antibody and polyclonal anti-TIMP-1 antibody. PBS extracts from tissues described above were subjected to Western blot and probed with monoclonal anti-IL-1beta antibody, polyclonal anti-IL-6 antibody, polyclonal anti-TNF-{alpha} antibody, and monoclonal anti-beta actin antibody. A, representative Western blots are shown in all cases. B, histographic representation of fold changes at protein level as measured by Lab Image densitometry values from the above blots and two other representative blots from independent experiments in each case. Error bars, ± S.E. *, p < 0.001; Figure 8, p < 0.01 versus control.

 


Figure 9
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  		FIGURE 9.
Induction of secreted MMP-9 and -2 activity by H. pylori in AGS cells. AGS cells were cocultured with viable H. pylori strains AM1 and SS1 and incubated for 4 and 24 h, respectively. AGS cells alone were considered as control. A, conditioned media from coculture and control wells were concentrated and analyzed by gelatin zymography as described under "Experimental Procedures" for MMP-9 and -2 activities. B, histographic representation of MMP-9 and -2 activities as measured by Lab Image densitometry values from the above zymogram and four other representative zymograms from independent experiments. Error bars, ± S.E. *, p < 0.001 versus AGS cells alone. C, Western blots described under "Experimental Procedures" were performed using equal volumes of concentrated conditioned media from coculture and probed with monoclonal anti-MMP-9 antibody and monoclonal anti-beta-actin antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The clinical outcome of H. pylori-induced pathology is likely to be determined by a combination of factors including the pathogen virulence factors, host immune responses, and capacity for colonizing new host niches (13, 14, 49). Many putative virulence factors have been identified in H. pylori, and most important among them is the cagA gene (13, 14), a part of cag PAI. There are reports indicating the association between cag PAI and increased severity of disease (14, 38, 50); however, many studies have shown no correlation between the two (51-53). To ascertain the potency of cag- H. pylori in colonization and inflammation in mice, we used naturally occurring cag- strain (AM1) as a representative strain. We found that the cag- Indian strain AM1 colonizes in mice, suggesting cag PAI of H. pylori is not indispensable for colonization. However, the colonization of AM1 strain is ~50-fold less compared with the SS1 strain that harbor the cag PAI (data not shown). We have now demonstrated the increase in MMP-9 and -2 secretion and synthesis in gastric tissues in vivo and in vitro by cag- AM1 strain infection, leading to gastric inflammation.

The pathogenesis of H. pylori-induced gastric disease is not well understood. We asked whether MMP secretion from host cells might contribute to gastric inflammation because MMPs have analogous roles in other inflammatory diseases such as arthritis (28). There is increasing evidence that H. pylori infection can influence the level of MMP-1, -2, -3, -7, and -9, but the mechanisms involved are largely unresolved (30-37, 54, 55). MMP-9 and -2 have the substrate specificity for basement membrane, which is typically composed of type IV collagen (23). We selected MMP-9 because prior observations in human and other cell types have indicated the direct association of MMP-9 in early phases of gastric ulcer and gastric cancer (33, 36). In the present study, we have tested the hypothesis that cag- H. pylori infection causes MMP-9 up-regulation in mouse gastric tissues. We observed, that MMP-3 is markedly increased in gastric tissues infected either with cag+ or cag- H. pylori in mice and is responsible for pathogenesis. Strain AM1 infects gastric tissues and in turn induces pro-MMP-9 and active MMP-2 secretion and synthesis almost as effectively as that of the cag+ strain SS1. The up-regulation of pro-MMP-9 activity occurs on day 1 postinfection, which further increases by ~25% on day 10, suggesting that the initial contact between the bacteria and mouse gastric tissues triggers MMP-9 up-regulation. Furthermore, SS1 strain compared with AM1 strain-infected tissue possesses slightly higher pro-MMP-9 activity, indicating the minor role of other bacterial mechanisms including type IV secretion system of cag PAI (40). Our data indicate that the increase in the level of MMP-9 and -2 mRNA is parallel to that of their secretion in mice infected with either cag+ or cag- H. pylori strains suggesting supplementation of enhanced secretion of gelatinases by their enhanced synthesis. When real time RT-PCR data from three experiments were analyzed, the mean expression of MMP-9 transcript was ~2.5-times higher for SS1-than AM1-infected gastric tissues. We have been unable to detect any up-regulation of pro-MMP-9 or MMP-2 activities in mice inoculated with heat-killed bacteria, indicating that the components of dead bacteria have no effect on MMP activity. Heat-killed bacteria are used instead of just PBS because even though they will not persist, lipopolysaccharide or small protein fragments might a priori have been responsible for the cellular response (56). It is worth mentioning that 5-10% of mice inoculated either with the SS1 or AM1 strains show very little induction in MMP-9 and MMP-2 activities, although carrying high loads of bacteria, and this could be caused by normal physiologic diversity, not the heterogeneity in the mouse colony (57). To our knowledge, ours is the first demonstration that irrespective of the cag PAI presence, H. pylori infection increases the secretion and synthesis of pro-MMP-9 and MMP-2 in mouse gastric tissues.

Similar to others (58, 59), we also support the notion that the inflammatory response of the gastric mucosa by H. pylori infection is the key to understanding the biochemical mechanisms for variability in clinical symptoms. Histological examination of mouse gastric tissues revealed that inflammations along with disruption in gastric mucosal cells and reduction in glandular region are more prominent in SS1-than in AM1-infected mice compared with control. Low grade inflammation particularly in the early stages of infection in mice by cag- AM1 may be caused by the absence of cag PAI in AM1. It has been described (20, 60) that cag-positive strains compared with cag-negative strains of H. pylori infection are associated with more severe gastritis in human and in other animal models. Furthermore, it is known that SSI H. pylori having PAI aggravates inflammation in murine host compared with other animal models. Gastric inflammation is a hallmark of H. pylori infection where epithelial cells in gastric mucosa play a major role in colonization. Causal relevance of MMP-9 because of gastric infection was demonstrated by examining MMP-3 production in H. pylori-infected gastric tissues of mouse. Our data indicate that mice infected either with SS1 or AM1 strains show elevated levels of MMP-3 secretion and gene expression in gastric tissues. To determine whether H. pylori infection could induce MMP-9 in gastric epithelial cells in culture, we have done coculture experiments using AGS cells and H. pylori. The present study shows that induction of pro-MMP-9 in AGS cells does not require cag PAI of H. pylori. Our data support the finding that enteric species of the Helicobacter genus lacking cag PAI are capable of inducing MMP-9 in hepatocytes, colon, and bile duct epithelial cells in culture (54). In contrast, Mori et al. (33) reported that H. pylori cag PAI is required for induction of MMP-9 in MKN 45 human gastric epithelial cells. The plausible reason for these differences may be the use of different cell lines, which differ markedly in many respects including surface markers, receptors, cell polarity, and synthetic capability. Thus, the actual effect of cag PAI in infection and inflammation may vary with context, and the notion that only cag+ strains are destructive, may be an oversimplification. Altogether, these results provide further insights into the epidemiologic relevance of the cag genes.

MMP activity is regulated by their endogenous inhibitors TIMPs, which are synthesized by the same cell types that produce MMPs (23, 61). Apart from its function as a MMP inhibitor, TIMP-1 promotes growth of a variety of cells, prevents apoptosis in B cells and induces cell differentiation (62-64). Because complex formation of MMP-9 with TIMP-1 influences MMP-9 activation and in turn affects its enzymatic activity (23), we examined the level of TIMP-1 during infection. Our data show that the increase in the pro-MMP-9 expression is accompanied by a significant decrease in TIMP-1 level suggesting that imbalance in MMP-TIMP level during H. pylori infection may have a role in ECM degradation and subsequently gastric inflammation. We hypothesize that this depletion of TIMP-1 not only facilitates MMP-9 activity but also affects gastric cell regeneration thus causing a dual effect on gastric ECM remodeling. In addition, our data show that the decrease in TIMP-1 protein level is also independent of H. pylori cag PAI.

Role of growth factors, hormones, and cytokines in up-regulation of MMP expression have been reported earlier under pathological conditions (23, 65, 66). The cytokines induced by H. pylori infection include TNF-{alpha}, interferon {gamma}, IL-1beta, IL-6, and IL-8 (32, 67, 68). It addition, earlier studies have shown that H. pylori strains that induce IL-8 secretion is because of the presence of cag PAI especially the cagA gene (38). This has been contested recently by the report of Audibert et al. (69) showing the induction of IL-8 secretion by a strain negative for cag PAI and no secretion of IL-8 for cag+ strain. Because H. pylori induce MMP-9 expression in response to release of pro-inflammatory cytokines in gastric epithelial cells in vitro (32), we tested whether the up-regulation of MMP-9 in H. pylori-infected mice is mediated by IL-1beta, TNF-{alpha}, and IL-6. Our results show that the level of secreted IL-1beta, TNF-{alpha}, and IL-6 are significantly increased in the mouse gastric tissues infected either with cag+ or cag- strains of H. pylori, suggesting that cag PAI is not the sole factor for the induction of pro-inflammatory cytokines, and also indicates that the cells producing MMPs have responded to the increased IL-1beta, TNF-{alpha}, and IL-6 secretions.

In summary, we suggest that cag PAI of H. pylori may not always be essential for colonization and infection as well. Up-regulation of pro-MMP-9 and MMP-2 secretion and synthesis and induction of pro-inflammatory cytokines are independent of cag PAI. Altogether, these data provide an insight into the pathogenicity of both cag- and cag+ H. pylori that may participate in gastric inflammation via MMP-9 induction and MMP-3 production. Obviously, MMP-9 up-regulation by cag+ H. pylori in gastric tissues follows an additional pathway. The idea behind a common pathway for cag+ and cag- strain infection and MMP-9 up-regulation does not necessarily rule out the importance of the cag+ genotype in further disease progression. This work opens a new avenue for an alternate pathogenic mechanism for inflammation underlying cag- H. pylori.


   FOOTNOTES
 
* This work was supported in part by Grants GAP209 of Indian Council of Medical Research, New Delhi and JICA/NICED 054-1061-E-O of Japan International Cooperation Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a senior research fellowship, University Grants Commission, India. Back

2 To whom correspondence should be addressed: Dept. of Physiology, Indian Institute of Chemical Biology, Jadavpur, Kolkata-700032, India. Tel.: 91-33-2473-3491 (ext. 159); Fax: 91-33-1473-5197; E-mail: snehasiktas{at}hotmail.com.

3 The abbreviations used are: PAI, pathogenicity island; BHI, brain heart infusion agar; ECM, extracellular matrix; FBS, fetal bovine serum; IL, interleukin; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase polymerase chain reaction; TIMP, tissue inhibitors of metalloproteinase; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF, tumor necrosis factor. Back



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