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Molecular Characterization of Helicobacter pylori VacA Induction of IL-8 in U937 Cells Reveals a Prominent Role for p38MAPK in Activating Transcription Factor-2, cAMP Response Element Binding Protein, and NF-B Activation¹

Junzo Hisatsune^*, Masaaki Nakayama^*, Hajime Isomoto, Hisao Kurazono, Naofumi Mukaida, Asish K. Mukhopadhyay^2,*, Takeshi Azuma^¶, Yoshio Yamaoka^||, Jan Sap^#, Eiki Yamasaki^**, Kinnosuke Yahiro^**, Joel Moss^** and Toshiya Hirayama^3,*

^* Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan; Department of Endoscopy, Nagasaki University School of Medicine, Nagasaki, Japan; Department of Applied Veterinary Medicine and Public Health, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan; Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; ^¶ Department of Gastroenterology, Kobe University School of Medicine, Kobe, Japan; ^|| Department of Medicine-Gastroenterology, Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Houston, TX 77030; ^# Copenhagen Biocenter-Biotechnology and Innovation Centre, University of Copenhagen, Copenhagen, Denmark; and ^** Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined the mechanisms by which VacA enhanced IL-8 production by promonocytic U937 cells, which demonstrated the greatest VacA-induced IL-8 release of the cells tested. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), IB ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile), Ca²⁺ entry (SKF96365), and intracellular Ca²⁺ channels (dantrolene) blocked VacA-induced IL-8 production. Furthermore, an intracellular Ca²⁺ chelator (BAPTA-AM), which inhibited VacA-activated p38 MAPK, caused a dose-dependent reduction in VacA-induced IL-8 secretion by U937 cells, implying a role for intracellular Ca²⁺ in mediating activation of MAPK and the canonical NF-B pathway. VacA stimulated translocation of NF-Bp65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-B pathway. In addition, small interfering RNA of activating transcription factor (ATF)-2 or CREB, which is a p38MAPK substrate and binds to the AP-1 site of the IL-8 promoter, inhibited VacA-induced IL-8 production. VacA activated an IL-8 promoter containing an NF-IL-6 site, but not a mutated AP-1 or NF-B site, suggesting direct involvement of the ATF-2/CREB binding region or NF-B-binding regions in VacA-induced IL-8 promoter activation. Thus, in U937 cells, VacA directly increases IL-8 production by activation of the p38 MAPK via intracellular Ca²⁺ release, leading to activation of the transcription factors, ATF-2, CREB, and NF-B.

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.

¹ This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, Ministry of Education, Culture, Sports, Science and Technology Japan. J.M. was supported by the Intramural Research Program, National Institutes of Health/National Heart, Lung, and Blood Institute.

² Current address: National Institute of Cholera and Enteric Diseases, Kolkata 700010, India.

³ Address correspondence and reprint requests to Dr. Toshiya Hirayama, Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523, Japan. E-mail addresss: hirayama{at}net.nagasaki-u.ac.jp

⁴ Abbreviations used in this paper: PAI, pathogenicity island; 2-AG, 2-arachidonoyl glycerol; ATF, activating transcription factor; BAY11-7082, (E)-3-(4-methylphenylsulfonyl)-2-propenenitrile; BHA, butylated hydroxyanisole; DAPI, 4',6'-diamidino-2-phenylindole; iVacA, heat-inactivated VacA; NC-siRNA, negative control small interfering RNA; ROS, reactive oxygen species; siRNA, small interfering RNA.

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