NF-kappaB- and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages.
Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1beta (IL-1beta) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1beta release and dissected the signaling pathways leading to LPS-induced IL-1beta secretion. We demonstrate that both the NF-kappaB and the C/EBPbeta-binding elements of the IL-1beta promoter drive LPS-induced IL-1beta gene expression. NF-kappaB activation requires the classical TLR4-initiated signaling cascade leading to IkappaB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPbeta activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPbeta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPbeta. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1beta. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1beta gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1beta gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.
Basak
Chaitali
C
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
Pathak
Sushil Kumar
SK
Bhattacharyya
Asima
A
Mandal
Debabrata
D
Pathak
Shresh
S
Kundu
Manikuntala
M
D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D020170:Caspase 1 / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D017930:Genes, Reporter; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D016328:NF-kappa B / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D054462:p21-Activated Kinases; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; D020170:Caspase 1; D007375:Interleukin-1; D008070:Lipopolysaccharides; D008156:Luciferases; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012313:RNA; D011994:Recombinant Proteins; D012694:Serine; D054462:p21-Activated Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C494918:AKT1 protein, human; C515903:PAK1 protein, human