Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells.
Primary cultures of guinea pig gastric mucosal cells express NADPH oxidase 1 (Nox1), a homolog of gp91(phox), and produce superoxide anion (O2-) at a rate of approximately 100 nmol.mg protein(-1).h(-1) in response to Helicobacter pylori (H. pylori) lipopolysaccharide (LPS) from virulent type I strains. The upregulated O2- production also enhances H. pylori LPS-stimulated tumor necrosis factor-alpha 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 O2- 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 p47phox 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 O2- generation without interfering with the expression of Nox1 and NOXO1 mRNA. O2- 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 O2- production in gastric mucosal cells appears to require two distinct events: 1) transcriptional upregulation of Nox1 and NOXO1 and 2) activation of Rac1.
Department of Nutritional Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
Tsukasa
T
Kawahara
Motoyuki
M
Kohjima
Yuki
Y
Kuwano
Hisano
H
Mino
Shigetada
S
Teshima-Kondo
Ryu
R
Takeya
Shohko
S
Tsunawaki
Akihiro
A
Wada
Hideki
H
Sumimoto
Kazuhito
K
Rokutan
D033942:Adaptor Proteins, Vesicular Transport / Q000096:biosynthesis* / Q000187:drug effects; D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005753:Gastric Mucosa / Q000187:drug effects / Q000378:metabolism*; D006168:Guinea Pigs; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000187:drug effects; D000074624:NADPH Oxidase 1; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013481:Superoxides / Q000378:metabolism; D014158:Transcription, Genetic; D020830:rac1 GTP-Binding Protein / Q000187:drug effects* / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; C442865:lipopolysaccharide, Helicobacter pylori; C015482:protein kinase modulator; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D020830:rac1 GTP-Binding Protein