Transcriptional regulation of IL-8 by Staphylococcus aureus in human conjunctival cells involves activation of AP-1.
OBJECTIVE
To identify signal transduction pathways involved in interleukin (IL)-8 expression by human conjunctival cells challenged with Staphylococcus aureus.


METHODS
Conjunctival cells were cultured in the presence of live or heat-killed S. aureus. IL-8 protein and mRNA were determined by ELISA and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs) and NF-kappaB was analyzed by Western blot analysis with phosphospecific antibodies. Conjunctival cells were transfected with wild-type (wt) or mutated IL-8 promoters (IL-8-97, lacking the AP-1 site; IL-8-97 mutant C/EBP; IL-8-97 mutant NF-kappaB; IL-8/AP-1 double mutant for C/EBP and NF-kappaB) or c-Jun-NH(2)-terminal kinase (JNK)-responsive GAL-c-Jun. In further experiments, cells were cotransfected with wt IL-8 promoter and expression plasmids for p38MAPK-responsive C/EBP homologous protein (CHOP) or wt or dominant negative transactivation domain mutant (TAM-67) c-Jun. A protein-DNA binding study was performed by electrophoretic mobility shift assay (EMSA), to identify the transcription factors bound to the IL-8 promoter.


RESULTS
S. aureus induced significant IL-8 expression and synthesis in human conjunctival epithelial cells by activating c-Jun phosphorylation and transactivation potential via JNK. The IL-8 promoter activation was NF-kappaB- and p38MAPK-independent. Transfection and EMSA experiments suggested that only AP-1 transcription factors were necessary for optimal IL-8 expression.


CONCLUSIONS
Human conjunctival epithelial cells possess the ability to respond to Gram-positive S. aureus and to activate the innate immune response by the IL-8 gene expression. These results are the first to delineate the transcription factors involved in S. aureus-induced IL-8 release by conjunctival epithelium.
Venza
Isabella
I
Departments of Surgical Specialties, The University of Messina, Messina, Italy.
Cucinotta
Maria
M
Caristi
Silvana
S
Mancuso
Giuseppe
G
Teti
Diana
D
D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology*; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013211:Staphylococcus aureus / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases