Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells

• Published in: American Journal of Physiology: Cell Physiology 2005-02, Vol.288 (2), p.C450-C457
• Main Authors: Kawahara, Tsukasa, Kohjima, Motoyuki, Kuwano, Yuki, Mino, Hisano, Teshima-Kondo, Shigetada, Takeya, Ryu, Tsunawaki, Shohko, Wada, Akihiro, Sumimoto, Hideki, Rokutan, Kazuhito
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Vesicular Transport - biosynthesis; Adaptor Proteins, Vesicular Transport - drug effects; Animals; Blotting, Northern; Cells, Cultured; Enzyme Activation - drug effects; Enzyme Activation - physiology; Gastric Mucosa - drug effects; Gastric Mucosa - metabolism; Guinea Pigs; Helicobacter pylori; Intracellular Signaling Peptides and Proteins - pharmacology; Lipopolysaccharides - pharmacology; Male; NADH, NADPH Oxidoreductases - biosynthesis; NADH, NADPH Oxidoreductases - drug effects; NADPH Oxidase 1; rac1 GTP-Binding Protein - drug effects; rac1 GTP-Binding Protein - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - analysis; Superoxides - metabolism; Transcription, Genetic
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00319.2004

Departments of 1 Nutritional Physiology and 3 Stress Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima; 2 Medical Institute of Bioregulation, Kyushu University, Fukuoka; 4 Department of Infectious Diseases, National Research Institute for Child Health and Development, Tokyo; and 5 Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan Submitted 6 July 2004 ; accepted in final form 30 September 2004 Primary cultures of guinea pig gastric mucosal cells express NADPH oxidase 1 (Nox1), a homolog of gp91 phox , and produce superoxide anion (O 2 – ) at a rate of 100 nmol·mg protein –1 ·h –1 in response to Helicobacter pylori ( H. pylori ) lipopolysaccharide (LPS) from virulent type I strains. The upregulated O 2 – production also enhances H. pylori LPS-stimulated tumor necrosis factor- or cyclooxygenase-2 mRNA expression, which suggests a potential role for Nox1 in the pathogenesis of H. pylori -associated diseases. The H. pylori LPS-stimulated O 2 – production in cultured gastric mucosal cells was inhibited by actinomycin D as well as cycloheximide, suggesting that the induction is regulated at the transcriptional level. The LPS treatment not only increased the Nox1 mRNA to a greater extent but also induced expression of the message-encoding, Nox-organizing protein 1 (NOXO1), a novel p47 phox homolog required for Nox1 activity. In addition, H. pylori LPS activated Rac1; i.e., it converted Rac1 to the GTP-bound state. A phosphoinositide 3-kinase inhibitor, LY-294002, blocked H. pylori LPS-induced Rac1 activation and O 2 – generation without interfering with the expression of Nox1 and NOXO1 mRNA. O 2 – production inhibited by LY-294002 was completely restored by transfection of an adenoviral vector encoding a constitutively active Rac1 but not an inactive Rac1 or a constitutively active Cdc42. These findings indicate that Rac1 plays a crucial role in Nox1 activation. Thus the H. pylori LPS-stimulated O 2 – production in gastric mucosal cells appears to require two distinct events: 1 ) transcriptional upregulation of Nox1 and NOXO1 and 2 ) activation of Rac1. superoxide anion; phosphoinositide 3-kinase; Toll-like receptor 4; inflammation Address for reprint requests and other correspondence: K. Rokutan, Dept. of Stress Science, Institute of Health Biosciences, Univ. of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan (E-mail: rokutan{at}basic.med.tokushima-u.ac.jp )