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Departments of 1 Biochemistry and Molecular Biology, 2 Neuroscience, and 4 Pediatrics, University of Florida, Gainesville, Florida 32610; and 3 Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Transcription of the human
inducible nitric oxide synthase (iNOS) gene is regulated by
inflammatory cytokines in a tissue-specific manner. To determine
whether differences in cytokine-induced mRNA levels between
pulmonary epithelial cells (A549) and hepatic biliary epithelial
cells (AKN-1) result from different protein or DNA regulatory
mechanisms, we identified cytokine-induced changes in DNase
I-hypersensitive (HS) sites in 13 kb of the iNOS 5'-flanking region.
Data showed both constitutive and inducible HS sites in an overlapping
yet cell type-specific pattern. Using in vivo footprinting and
ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near 
5 kb containing both
constitutive and cytokine-induced HS sites. In both cell types, three
in vivo footprints were present in both control and cytokine-treated
cells, and each mapped within a constitutive HS site. The remaining
footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, performed on chromatin in situ,
identify a portion of the molecular mechanisms regulating transcription
of the human iNOS gene in both lung- and liver-derived epithelial cells.
nitric oxide synthase; deoxyribonuclease I-hypersensitive sites; inflammatory cytokines; lung epithelial cells; liver epithelial cells
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