Porins from Salmonella enterica serovar Typhimurium activate the transcription factors activating protein 1 and NF-kappaB through the Raf-1-mitogen-activated protein kinase cascade

• Published in: Infection and immunity Vol. 70; no. 2; pp. 558 - 568
• Main Authors: Galdiero, Massimiliano, Vitiello, Mariateresa, Sanzari, Emma, D'Isanto, Marina, Tortora, Annalisa, Longanella, Anna, Galdiero, Stefania
• Format: Journal Article
• Language: English
• Published: United States 01-02-2002
• Subjects: Animals; Cells, Cultured; Enzyme Activation; Female; Humans; JNK Mitogen-Activated Protein Kinases; Macrophages, Peritoneal - cytology; Macrophages, Peritoneal - drug effects; Macrophages, Peritoneal - metabolism; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mice; Mice, Inbred C3H; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases - metabolism; Mitogen-Activated Protein Kinases - metabolism; Mitogens - pharmacology; NF-kappa B - metabolism; p38 Mitogen-Activated Protein Kinases; Porins - pharmacology; Protein-Serine-Threonine Kinases - metabolism; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins c-raf - metabolism; Salmonella typhimurium - metabolism; Transcription Factor AP-1 - metabolism; U937 Cells
• ISSN: 0019-9567

In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-kappaB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2'-amino-3'-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-kappaB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-kappaB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-kappaB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-kappaB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation. These data suggest different molecular mechanisms of activation induced by porins or by LPS.