Mechanism of glutamine-mediated amelioration of lipopolysaccharide-induced IL-8 production in Caco-2 cells.
The mechanism of glutamine (Gln)-mediated down-regulation of inflammation in the intestine is poorly understood. We hypothesize that Gln down-regulates lipopolysaccharide (LPS)-stimulated IL-8 production in intestinal epithelial cells via transcription factors that counteract the effect of LPS-mediated increase in IL-8. Caco-2 cells were incubated with different doses of Gln with or without methionine sulfoximine (MS), an inhibitor of glutamine synthetase for 24 h before stimulation by LPS (100 microg/ml for 24 h). Inhibitors of the mitogen activated protein kinase (MAPK) family were added to cells for 1.5 h following stimulation by LPS. The p38 inhibitor SB 203580 resulted in a significant decrease in IL-8 peptide production (p < 0.01). However, p38 MAPK activity increased with Gln (p < 0.05), suggesting that this was not involved with Gln-mediated down-regulation of IL-8. Screening of 54 transcription factors demonstrated that STAT-4 was the only inflammation-related transcription factor that was up-regulated by Gln depletion and down-regulated with Gln supplementation (2-fold increase), paralleling IL-8 production. EMSA analysis confirmed these findings (3.5-fold increase). These results indicate that Gln deprivation enhances IL-8 production by Caco-2 cells after LPS stimulation and that down-regulation of IL-8 production with Gln is associated with alterations in STAT-4 transcription factor binding.
Liboni
Kellym
K
Department of Pediatrics, College of Medicine, University of Florida, PO Box 100296, Gainesville, FL 32610-0296, USA.
Li
Nan
N
Neu
Josef
J
D018938:Caco-2 Cells; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D016209:Interleukin-8 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D040081:Protein Array Analysis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D050797:STAT4 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004268:DNA-Binding Proteins; D007093:Imidazoles; D016209:Interleukin-8; D008070:Lipopolysaccharides; D011725:Pyridines; D050797:STAT4 Transcription Factor; C494090:STAT4 protein, human; D015534:Trans-Activators; D005973:Glutamine; D011505:Protein-Tyrosine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580