Essential role of MD-2 in TLR4-dependent signaling during Helicobacter pylori-associated gastritis.
TLR4, a member of pattern recognition receptors, is the main receptor of LPS. MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. Helicobacter pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated in this study the role of MD-2 in TLR4-dependent signaling in H. pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H. pylori infection and four gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-kappaB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H. pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-kappaB and IL-8 promoter upon stimulation with H. pylori LPS. Live H. pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H. pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H. pylori LPS in the stomach.
Ishihara
Shunji
S
Department of Gastroenterology and Hepatology, Shimane University, School of Medicine, Izumo, Japan. si360405@shimane-med.ac.jp
Rumi
Mohammad Azharul Karim
MA
Kadowaki
Yasunori
Y
Ortega-Cava
Cesar Francisco
CF
Yuki
Takafumi
T
Yoshino
Nagisa
N
Miyaoka
Youichi
Y
Kazumori
Hideaki
H
Ishimura
Norihisa
N
Amano
Yuji
Y
Kinoshita
Yoshikazu
Y
D000328:Adult; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005756:Gastritis / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D017930:Genes, Reporter; D016481:Helicobacter Infections / Q000276:immunology* / Q000378:metabolism; D016480:Helicobacter pylori / Q000276:immunology / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D000954:Antigens, Surface; D016209:Interleukin-8; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors