INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.6.3965–3971.2001

June 2001, p. 3965–3971 Vol. 69, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Helicobacter pylori Activates the Cyclin D1 Gene through Mitogen-Activated Protein Kinase Pathway in Gastric Cancer Cells

YOSHIHIRO HIRATA,* SHIN MAEDA, YUZO MITSUNO, MASAO AKANUMA, YUTAKA YAMAJI,KEIJI OGURA, HARUHIKO YOSHIDA, YASUSHI SHIRATORI, AND MASAO OMATA

Department of Gastroenterology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Received 27 November 2000/Returned for modification 2 February 2001/Accepted 26 March 2001

Helicobacter pylori induces cellular proliferation in host cells, but the mechanism remains unclear. Thus, weexamined the effect of H. pylori on cyclin D1, an important regulator of the cell cycle, especially in relation tointracellular signaling pathways. In a Northern blot analysis, cyclin D1 transcription in gastric cancer (AGS)cells was enhanced by coculture with H. pylori strain TN2 in a time-dependent and multiplicity-of-infection-dependent manner. An isogenic mutant form of vacA also increased cyclin D1 transcription, but mutant formsof cagE or the entire cag pathogenicity island did not enhance cyclin D1 transcription. These effects wereconfirmed with a luciferase assay of the cyclin D1 promoter (pD1luc). Cyclin D1 promoter activation by H.pylori was inhibited by MEK inhibitors (U0126 and PD98059), indicating that the mitogen-activated proteinkinase pathway may be involved in intracellular signal transduction. In contrast, transfection of a reporterplasmid having any point mutations of the NF-kB binding sites in the promoter (pD1-kB1M, pD1-kB2M, orpD1-kB1/2M) or cotransfection of dominant negative IkBa did not affect cyclin D1 activation by H. pylori. Inconclusion, H. pylori activates cyclin D1 through the mitogen-activated protein kinase pathway and not throughNF-kB activation in AGS cells. This activation of cyclin D1 is partly dependent on the cag pathogenicity islandbut not on vacA.

Colonization of the human gastric mucosa by Helicobacterpylori induces various diseases, such as atrophic gastritis, pepticulcer diseases, and gastric adenocarcinoma (12, 21, 23, 28, 30,34). It has been recently demonstrated that H. pylori affectsintracellular signal conduction in host cells, leading to theactivation of transcriptional factors (18, 19, 22, 24, 25, 42) andthe induction of proinflammatory cytokines (8, 29, 32, 41). Thecag pathogenicity island (PAI) genes of H. pylori and theirproducts are responsible for the bacterium-host interactions,including activation of the NF-kB and mitogen-activated pro-tein (MAP) kinase pathways (19, 22, 24, 25, 42). The cag PAIgenes have been suggested as the cause of gastric diseases invivo (3, 6, 21), and we confirmed this in the Mongolian gerbilmodel (33).

H. pylori infection is also associated with enhanced cellularproliferation of host cells (9, 14, 16, 36, 37); however, themechanism of cellular proliferation induced by H. pylori infec-tion remains unclear. In mammalian cells, cellular prolifera-tion is regulated in a cell cycle governed by the sequentialformation and degradation of cyclins and cyclin-dependentkinases. Among various cyclins, cyclin D1 regulates passagethrough the restriction point and entry into the S phase (43).Furthermore, cyclin D1 overexpression shortens the G1 phaseand increases the rate of cellular proliferation (15, 38–40).Various factors, such as the MAP kinase cascade (20), NF-kB(13), and the Wnt signal (44, 47), are known regulators ofcyclin D1 expression. In addition, some of these factors arealready known to be activated by H. pylori infection; however,

little is known about the effect of H. pylori infection on cyclinD1 expression. Thus, in this study, we tried to elucidate themechanism of host cell proliferation caused by H. pylori inrelation to cyclin D1 transcription.

MATERIALS AND METHODS

Cell culture. Human gastric adenocarcinoma cell line AGS cells were main-tained in Ham’s F12 medium supplemented with 10% fetal bovine serum (LifeTechnologies, Inc., Grand Island, N.Y.) in an incubator with 5% CO2.

Bacterial strains and growth conditions. H. pylori strain TN2 possessing boththe cag PAI and vacA was generously donated by M. Nakao (Takeda ChemicalIndustries, Ltd., Osaka, Japan). Infection with this strain induced gastric cancerin Mongolian gerbils (49). The isogenic mutants cagE-disrupted TN2-DcagE andvacA-disrupted TN2-DvacA were prepared as described previously (22, 32). TN2-DPAI, a strain in which all of the PAI genes are disrupted, was prepared asfollows. A partial fragment of the cagA gene (700 bp) was amplified by PCR andcloned into the plasmid vector pCRII (Invitrogen, San Diego, Calif.). A 700-bpfragment of the cag5 gene, which is localized in cagII, was also amplified andcloned into pCRII containing a cagA gene fragment at the KpnI site oriented inthe same direction. A kanamycin resistance gene cassette was then inserted intothe vector at the BamHI site between cagA and the cag5 fragment. The resultingconstruct was transferred into parental H. pylori cells (strain TN2) by electropo-ration. After selection by kanamycin resistance and Southern blot hybridizationto confirm the disruption of the genes, a clone was selected for use as TN2-DPAI.

These strains were grown in brucella broth with 5% (vol/vol) fetal bovineserum under microaerobic conditions (Campy-Pak Systems; BBL, Cockeysville,Md.), diluted to the desired multiplicity of infection (MOI), and then used forexperiments. The number of bacterial cells in the suspension was quantified byoptical density measurements.

Heat-killed H. pylori was prepared by boiling the bacteria for 30 min. H. pylorifiltrate was prepared by suspending the bacteria in antibiotic-free medium for 30min, pelleting the bacteria by centrifugation, and then filtering the mediumthrough a 0.22-mm-pore-size filter (Nihon Millipore Ltd., Tokyo, Japan).

Plasmids. Cyclin D1 promoter-containing construct pD1luc and vectors pD1-kB1M, pD1-kB2M, and pD1-kB1/2M, possessing mutations in their NF-kB bind-ing sites, were kindly donated by M. Strauss (Humboldt Universitat, Berlin,Germany). Plasmid pD1luc is consistent with an EcoRI/PvuII fragment (1,226bp) of the cyclin D1 promoter. pD1-kB1M possesses mutations in the NF-kBbinding site between nucleotides 2840 and 2831, pD1-kB2M has mutations in

* Corresponding author. Mailing address: Department of Gastroen-terology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo,Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 3-3815-5411, ext. 33056.Fax: 3-3814-0021. E-mail: HIRATAY-INT@h.u-tokyo.ac.jp.

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the NF-kB binding site between nucleotides 233 and 224, and pD1-kB1/2Mpossesses both of these mutations (13, 27). The dominant-negative IkBa expres-sion vector mutant (SS32/36AA) was generously donated by H. Suzuki (Yaman-ouchi Pharmaceutical Co., Ltd., Ibaraki, Japan) (46).

Northern blot analysis. AGS cells were serum starved for 72 h and subse-quently cultured with H. pylori at an MOI of 10 to 300. Cells were harvested atthe times indicated, and total RNA was isolated by using Isogen (Wako, Osaka,Japan) in accordance with the manufacturer’s instructions. Fifteen microgramsof total RNA was loaded onto a 1% agarose–formaldehyde gel, separated byelectrophoresis, and then transferred onto a Hybond N membrane (AmershamPharmacia Biotech, Buckinghamshire, United Kingdom).

Partial cDNA (200 bp) for cyclin D1 was produced by PCR using primers59-ATGGAACACCAGCTCCTGTG-39 (forward) and 59-ACCTCCAGCATCCAGGTGGC-39 (reverse). The DNA sequence of the product was confirmed byusing a cycle DNA sequencing system (Perkin-Elmer Applied Biosystems, FosterCity, Calif.). This cyclin D1 probe was labeled with an AlkPhos Direct LabellingModule (Amersham Pharmacia Biotech) used in accordance with the manufac-turer’s instructions and then hybridized with the membrane for 12 h. Afterseveral washes of the membrane, cyclin D1 mRNA was detected by CDP-StarDetection Reagent (Amersham Pharmacia Biotech), followed by analysis usingthe LAS1000 imaging system (Fuji Photo Film Co., Ltd., Tokyo, Japan).

After the hybridization, the membrane was reprobed with the human gene forglyceraldehyde 3-phosphate dehydrogenase to control for equal loading of theRNA.

Transfections and luciferase assays. AGS cells (1.5 3 105) were seeded intosix-well plates and transfected 24 h later with 200 ng of pD1luc or the mutantvector and the indicated expression vectors using FuGENE 6 Transfection Re-agent (Roche Diagnostic Corporation, Indianapolis, Ind.). Ten nanograms ofRenilla luciferase vector (Promega, Madison, Wis.) was included in each samplefor standardization of transfection efficiency. The total DNA amount was keptconstant by supplementation with empty vectors. After 24 h, H. pylori (strain TN2or an isogenic mutant) was added at an MOI of 10 to 100 and incubated for theindicated times. 12-O-Tetradecanoylphorbol-13-acetate (TPA) (5 to 50 ng/ml)was used as a positive control. When evaluating the effect of the MAP kinasesignaling pathway on activation of the cyclin D1 promoter, the specific MEKinhibitor PD98059 (Wako) or U0126 (Promega) (10) or vehicle (dimethyl sul-foxide) alone was added 2 h before coculture with H. pylori. The cells wereharvested and washed with phosphate-buffered saline and lysed in a luciferaselysis buffer (Piccagene; Toyo Ink, Tokyo, Japan). The lysates were assayed forluciferase and seapansy luciferase activity with a luminometer. After standard-ization with seapansy luciferase activity, luciferase activity was calculated andrepresented as the fold induction, compared with the control, of more than threeindependent experiments.

Statistical analysis. The significance of differences was assessed by analysis ofvariance and, if the F value was significant, by Dunnett’s post hoc tests.

RESULTS

H. pylori increases transcription of the cyclin D1 gene ingastric cancer cells in a time- and MOI-dependent manner.When serum-starved AGS cells were cocultured with H. pyloristrain TN2 at an MOI of 100, the amount of cyclin D1 mRNAwas markedly increased from 60 to 120 min, as observed byNorthern blot analysis (Fig. 1A). A similar increase in thecyclin D1 gene was observed when AGS cells were incubatedwith H. pylori at different MOIs. H. pylori increased transcrip-

tion of the cyclin D1 gene in an MOI-dependent manner up toan MOI of 100 (Fig. 1B).

In the reporter assay using pD1luc, cyclin D1 promoteractivity was enhanced in cells cocultured with H. pylori (Fig.1C) in a time-dependent manner, in the same way as in cells

FIG. 2. Effects of virulence factors of H. pylori on cyclin D1 tran-scription. (A) AGS cells were infected with several isogenic mutantstrains of H. pylori (TN2, TN2-DcagE, TN2-DPAI, and TN2-DvacA) atan MOI of 100 for 90 min. Cyclin D1 mRNA was analyzed by Northernblot analysis. (B) AGS cells were transiently transfected with 200 ng ofpD1luc and infected with different strains of H. pylori at an MOI of 100for 24 h. Luciferase activity is presented as a relative ratio against thebasal level measured in untreated cells. The values shown are themeans 6 the standard deviations of 10 independent experiments. p,P , 0.05 versus wild-type TN2. GAPDH, glyceraldehyde 3-phosphatedehydrogenase.

FIG. 1. H. pylori induces cyclin D1 mRNA and activates the cyclin D1 promoter in AGS cells. (A) AGS cells were serum starved for 72 h andinfected with H. pylori strain TN2 at an MOI of 100 for the times indicated, and cyclin D1 mRNA was measured by Northern blot analysis. (B)AGS cells were serum starved for 72 h and infected with H. pylori strain TN2 at different MOIs for 90 min, and cyclin D1 mRNA was measuredby Northern blot analysis. (C and D) AGS cells which were transiently transfected with 200 ng of pD1luc were infected with H. pylori strain TN2at an MOI of 100 (C) or treated with TPA at 50 ng/ml (D) for the indicated times. (E and F) AGS cells which were transiently transfected with200 ng of pD1luc were infected with H. pylori strain TN2 at different MOIs (E) or treated with several concentrations of TPA (F) for 24 h. (G)Live H. pylori (MOI of 100), heat-killed H. pylori (MOI of 100), or H. pylori filtrate (extracted from the same number of bacteria) was added toAGS cells which were transiently transfected with pD1luc, and the mixture was incubated for 24 h. Luciferase activity is presented as a relative ratioagainst the basal level measured in untreated cells. The values shown are the means 6 the standard deviations of more than five independentexperiments. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

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treated with TPA at 50 ng/ml (Fig. 1D). Cyclin D1 promoteractivity was increased 1.4-fold by 8 h, 3.0-fold by 16 h, and6.4-fold by 24 h of incubation with H. pylori, compared withthat of an uninfected control. MOI-dependent transactivationwas also observed in cells cocultured with H. pylori for 24 h(Fig. 1E), as was observed in cells treated with TPA (Fig. 1F).This activation was observed only when live bacteria wereadded to the cells. Neither heat-killed bacteria nor bacterialfiltrate activated the cyclin D1 promoter (Fig. 1G). Subsequentreporter assays were performed with cells cocultured with liveH. pylori at an MOI of 100 for 24 h.

Effects of virulence factors on cyclin D1 transcription inAGS cells. To assess the relationship between the virulencegenes of H. pylori and cyclin D1 activation, AGS cells werecultured with isogenic mutant strain TN2-DcagE, TN2-DvacA,or TN2-DPAI at an MOI of 100. Northern blot analysis showedthat the induction of cyclin D1 mRNA by TN2-DcagE andTN2-DPAI was significantly less than that by wild-type TN2;however, disruption of the vacA gene did not affect cyclin D1induction (Fig. 2A).

A luciferase reporter assay showed that TN2-DcagE andTN2-DPAI induced 4.5- and 3.7-fold increases in cyclin D1promoter activity, respectively. The induction by TN2-DPAI

was significantly less than that by wild-type TN2 (P , 0.05). Incontrast, TN2-DvacA induced a 6.7-fold increase in cyclin D1promoter activity, a value similar to that of wild-type TN2(Fig. 2B).

MAP kinase activation is required for induction of cyclin D1by H. pylori. Since cyclin D1 expression is regulated by MAPkinase cascades, we examined whether the MAP kinase cas-cade is involved in H. pylori-induced cyclin D1 transcription byusing the specific MEK inhibitors U0126 and PD98059. U0126(5 to 20 mM) and PD98059 (50 to 200 mM) were added to theculture medium of AGS cells 2 h before coculture with H.pylori. Activation of the cyclin D1 promoter by H. pylori wasmarkedly inhibited by each compound in a dose-dependentmanner (Fig. 3A and B), as was observed in the cells treatedwith TPA (data not shown). U0126, at a dose of 20 mM,reduced cyclin D1 promoter activation to 34% (P , 0.05), andPD98059, at a dose of 200 mM, reduced promoter activation to21% (P , 0.05). With the Northern blot analysis, preincuba-tion of the cells with U0126 or PD98059 reduced cyclin D1mRNA induction by coculture with H. pylori (Fig. 3C).

Effect of NF-kB activation on H. pylori-induced cyclin D1promoter transactivation. We examined the role of NF-kBactivation on H. pylori-induced cyclin D1 transcription since

FIG. 3. Role of MAP kinase activation in the induction of cyclin D1 promoter by H. pylori. AGS cells transfected with pD1luc were infectedwith H. pylori at an MOI of 100 or left uninfected in the presence of the MEK inhibitor U0126 (A) or PD98059 (B) or dimethyl sulfoxide (DMSO)(control). Luciferase activity is presented as a relative ratio against the basal level measured in untreated control cells. The values shown are themeans 6 the standard deviations of more than three independent experiments. p, P , 0.05 versus H. pylori with DMSO preincubation. (C)Serum-starved AGS cells were infected with H. pylori at an MOI of 100 for 90 min in the presence of 20 mM U0126, 200 mM PD98059, or DMSOalone. Fifteen micrograms of total RNA was tested for cyclin D1 mRNA by Northern blot analysis. GAPDH: glyceraldehyde 3-phosphatedehydrogenase.

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the type 1 strain of H. pylori can activate NF-kB and cyclin D1transcription is also reportedly regulated by NF-kB. We exam-ined the effect of H. pylori on the promoter constructs contain-ing point mutations in the kB sites (pD1-kB1M, pD1-kB2M,and pD1-kB1/2M). These mutant reporters were transfectedinto AGS cells instead of pD1luc, and luciferase expressioninduced by H. pylori was measured. Coculture with H. pyloricaused about a sixfold increase in the activation of luciferaseexpression on each promoter construct (Fig. 4A).

In addition, we cotransfected the dominant negative form ofIkBa (SS32/36AA) with pD1luc to inhibit NF-kB activation byH. pylori. Although activation of NF-kB was completely inhib-ited by this dominant-negative form of IkBa (22), induction ofthe cyclin D1 promoter was not reduced by the mutant of IkBa

(Fig. 4B). These data indicate that H. pylori transactivates thecyclin D1 promoter independently of NF-kB activation in AGScells.

DISCUSSION

In this study, we demonstrated that live H. pylori transacti-vates cyclin D1, one of the critical regulators of the cell cycle,in AGS cells in a time- and dose-dependent manner. Althoughmany studies have revealed that H. pylori induces cellular pro-liferation (9, 14, 16, 36, 37), the underlying mechanism has notyet been clarified. Our result indicating that H. pylori directlyaffects the cell cycle regulator molecule represents one possiblereason for cellular proliferation. Furthermore, overexpressionof cyclin D1 has been demonstrated to contribute to the on-cogenic transformation of cells in vitro and in vivo (1, 4, 11, 26,48, 50). Although little is known about the relationship be-tween cyclin D1 and gastric cancer, transactivation of cyclin D1and an accelerated cell cycle caused by H. pylori infection canbe among the factors contributing to malignant transforma-tion.

We also demonstrated the effects of virulence genes of H.pylori on cyclin D1 activation. In a Northern blot analysis,TN2-DcagE and TN2-DPAI exerted weaker effects on H. pylori-induced cyclin D1 activation compared with wild-type TN2,while TN2-DvacA induced cyclin D1 transcription at a levelsimilar to that of the wild type. In the reporter assay, strainscontaining all of the cag PAI genes (TN2 and TN2-DvacA)activated the promoter more strongly than did strains partiallyor completely lacking cag PAI genes (TN2-DcagE and TN2-DPAI). Although we did not evaluate the cell cycle directly,these data may be compatible with a previous report by Peek etal. which demonstrated that H. pylori possessing cag PAI ac-celerated the progression of the cell cycle from G1 into G2-Min AGS cells at 6 h (37). We have recently demonstrated in theMongolian gerbil model that TN2-DcagE did not induce anysevere gastric diseases, in contrast to wild-type TN2 and TN2-DvacA, in spite of the similar colonization abilities (33). To-gether with other host responses such as transcriptional factoractivation (18, 19, 22, 24, 25, 42) and apoptosis (17, 37), whichcan be enhanced by cag PAI-positive H. pylori, accelerated cellproliferation may provide one of the reasons for the highprevalence of gastric cancer in patients suffering from cagPAI-positive H. pylori (3, 21, 35).

It is not known, however, how cag PAI genes contribute tothe differences in host response. Possibly only cag PAI-positivestrains transport certain molecules of H. pylori, which directlyactivate intracellular signal transduction. Some of the cag PAIgenes and their products have been thought to form a type IVsecretion system (5, 7). Very recently, CagA protein was re-ported to be transported into AGS cells by this secretion sys-tem and the transportation was blocked by cagE knockout (2,31, 45). However, since the mutant strains used in this studyalso weakly induced cyclin D1 transcription, the proteins trans-ported by this secretion system cannot be the sole moleculesresponsible for cyclin D1 activation.

In the Northern blot analysis, an increased level of cyclin D1mRNA was observed 60 to 120 min after coculture with H.pylori. The time-dependent increase in cyclin D1 mRNA levelswas also observed when MKN-28 cells were cocultured with H.

FIG. 4. Role of NF-kB activation on H. pylori-induced cyclin D1promoter transactivation. (A) AGS cells transiently transfected with200 ng of pD1luc or a mutant reporter (pD1-kB1M, pD1-kB2M, orpD1-kB1/2M) were infected with H. pylori at an MOI of 100 for 24 hor left uninfected. The luciferase activity of each reporter is presentedas a relative ratio against the basal level measured in uninfected cells.The values shown are the means 6 the standard deviations of fiveindependent experiments. (B) AGS cells were transfected with variousamounts of the dominant-negative IkBa expression vector togetherwith pD1luc for 24 h. The total DNA amount was kept constant bysupplementation with an empty vector (pcDNA3). The cells wereinfected with H. pylori at an MOI of 100 for 24 h or left uninfected.Luciferase activity is presented as a relative ratio against the basal levelmeasured in uninfected control cells. The values shown are the themeans 6 the standard deviations of more than five independent ex-periments.

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pylori (data not shown). This time lag suggests an involvementof intracellular signal transduction. Cyclin D1 expression isregulated by many signaling cascades, such as mitogenic stimuliand the MAP kinase cascade, inflammation and NF-kB acti-vation, and Wnt signaling (13, 20, 44, 47). Some of thesesignaling cascades are involved in H. pylori-mediated host re-sponses. Thus, we determined the intracellular signaling path-way for H. pylori-induced cyclin D1 activation.

The present results showing that MEK inhibitors reduced H.pylori-induced cyclin D1 activation suggest the involvement ofMAP kinase cascades. Since the cag PAI mutant strains areknown to be less potent in activating the MAP kinase cascades(19, 24, 25), less activation of cyclin D1, as shown by themutant strains used in this study, is reasonable. We also per-formed in vitro kinase assays to measure MAP kinase activity,and we confirmed that H. pylori had the ability to activate MAPkinase, and this ability was correlated with the status of cagPAI (data not shown). On the other hand, the use of a mutantreporter plasmid of the NF-kB binding site or dominant-neg-ative IkBa expression vector in the present study showed thatNF-kB activation is not required for cyclin D1 transactivationin AGS cells.

In conclusion, we have demonstrated that H. pylori activatescyclin D1 expression and that this activation is partly depen-dent on the cag PAI genes. Furthermore, cyclin D1 expressionwas activated through the MAP kinase signaling pathway butnot by activation of NF-kB. It is possible that other signalingpathways which can be induced by H. pylori exist, as well asanother mechanism for cyclin D1 transactivation. These pos-sibilities should be investigated in future studies.

ACKNOWLEDGMENTS

This study was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science, Sports, and Culture(11557040).

We appreciate the generous donation of plasmid constructs by M.Strauss and H. Suzuki, and we thank Mitsuko Tsubouchi for excellenttechnical assistance.

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