A role for NF-kappa B binding motifs in the differential induction of chemokine gene expression in human corneal epithelial cells.
OBJECTIVE
The interleukin (IL)-8 promoter possesses a NF-kappa B-binding site with affinity to p50p65 and p65p65 complexes while the monocyte chemoattractant protein (MCP)-1 promoter's NF-kappa B-binding site has exclusive affinity to p50p65 heterodimers. The purpose of this study was to determine whether the two NF-kappa B sites play a role in the capacity of tumor necrosis factor (TNF)-alpha-stimulated human corneal epithelial cells (HCECs) to produce nanogram amounts of IL-8 in the absence of MCP-1 synthesis.


METHODS
IL-8 and MCP-1 promoters were cloned into luciferase reporter vectors. Site-directed mutagenesis of wild-type promoters was used to mutate the NF-kappa B-binding motif in the wild-type IL-8 reporter plasmid into a motif with exclusive affinity to p50p65 and to mutate the NF-kappa B binding motif in the wild-type MCP-1 reporter plasmid into a motif with affinity to p65p65. Luciferase activity was determined after transfection of reporter vector constructs into TNF-alpha-stimulated HCECs. The chromatin immunoprecipitation assay was used to confirm binding of NF-kappa B subunits to IL-8 and MCP-1 promoters in vivo.


RESULTS
Promoters with affinity to p65p65 homodimers were active in driving the expression of the reporter gene, whereas promoters with affinity to p50p65 heterodimers did not induce significant reporter gene expression. Incorporation of a CCAAT enhancer-binding protein (C/EBP)-binding site immediately upstream of p65p65-binding sites significantly enhanced promoter activity.


CONCLUSIONS
The results suggest that the interaction of p65p65 homodimers and C/EBP transcriptional factors with IL-8 promoters and not MCP-1 promoters account for the capacity of HCECs to produce IL-8 selectively, in the absence of MCP-1 production.
Ritchie
Mary H
MH
Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile, 36688, USA.
Fillmore
Rebecca A
RA
Lausch
Robert N
RN
Oakes
John E
JE
D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D022762:CCAAT-Enhancer-Binding Proteins; D018932:Chemokine CCL2; C497249:DDIT3 protein, human; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051742:Transcription Factor CHOP