Pseudomonas aeruginosa induces interleukin-8 (IL-8) gene expression in human conjunctiva through the recruitment of both RelA and CCAAT/enhancer-binding protein beta to the IL-8 promoter.
The purpose of this study was to identify the Pseudomonas aeruginosa-activated signaling pathway leading to interleukin (IL)-8 gene expression and protein synthesis by human conjunctival epithelium. IL-8 protein and mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription-PCR, respectively. Activation of MAPKs and NF-kappaB was analyzed by Western blotting using phosphospecific antibodies. We used transfection with wild-type or mutated IL-8 promoters and cotransfection with transcription factor overexpressing plasmids or small interfering RNAs. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) were performed for in vitro and in vivo protein-DNA binding studies, respectively. P. aeruginosa increased IL-8 expression at the transcriptional level by phosphorylating CCAAT/enhancer-binding protein beta (C/EBPbeta) via p38MAPK and activating NF-kappaB. The simultaneous involvement of RelA and C/EBPbeta and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. Re-ChIP experiments showed that RelA and C/EBPbeta act together at the IL-8 promoter level upon P. aeruginosa infection. Taken together, our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPbeta and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.
Venza
Isabella
I
Departments of Surgical Specialties and Experimental Pathology and Microbiology, University of Messina, 98125 Messina, Italy.
Cucinotta
Maria
M
Visalli
Maria
M
De Grazia
Giuseppina
G
Oliva
Sabrina
S
Teti
Diana
D
D000328:Adult; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003228:Conjunctiva / Q000378:metabolism* / Q000382:microbiology; D004789:Enzyme Activation; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D011552:Pseudomonas Infections / Q000235:genetics / Q000276:immunology*; D011550:Pseudomonas aeruginosa; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; C508606:CXCL8 protein, human; D016209:Interleukin-8; D016755:Proto-Oncogene Proteins c-jun; C498143:RELA protein, human; D051996:Transcription Factor RelA; D048051:p38 Mitogen-Activated Protein Kinases