A non-nucleotide-bridged DNA decoy inhibits renal epithelial nitric oxide synthase expression.
A non-nucleotide-bridged DNA decoy inhibits renal epithelial nitric oxide synthase expression.


BACKGROUND
The expression of inducible nitric oxide synthase (iNOS) is subject to strict tissue-specific transcriptional control. In mouse renal epithelium, an interferon-gamma (IFN-gamma)-induced signaling protein, IFN-gamma regulatory factor 1 (IRF-1), appears to mediate the induction of iNOS expression by cytokines and bacterial lipopolysaccharide (LPS).


METHODS
We used a novel technique, namely, blockade of cytosolic IRF-1 activity with a triethyleneglycol-bridged decoy DNA oligonucleotide (ODN) containing the IRF-1 consensus binding sequences present in the iNOS promoter to inhibit iNOS gene expression. Cultured mouse renal epithelial cells were treated with a combination of LPS (1 microg/mL) and IFN (100 U/mL) in the absence or presence of IRF-1 decoy ODN followed by determinations of NO production and iNOS protein and mRNA expression.


RESULTS
Treatment with IRF-1 decoy ODN resulted in concentration-dependent inhibition of NO production and a marked reduction in iNOS protein and mRNA levels. A scrambled ODN failed to affect LPS/IFN-stimulated NO production or iNOS protein and mRNA levels. Transcriptional assays showed that the IRF-1 decoy ODN inhibited transcriptional activity of an iNOS promoter-CAT gene construct.


CONCLUSIONS
Decoy ODN-based techniques effectively inhibit iNOS expression in renal epithelium and represent a potentially useful approach for selective blockade of this enzyme in pathologic conditions associated with excessive NO production.
Amoah-Apraku
B
B
Division of Nephrology and Hypertension, Georgetown University MedicalCenter, Washington, D.C 20007, USA.
Fang
M Z
MZ
Guzman
N J
NJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000494:pharmacology*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D050856:Interferon Regulatory Factor-1; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D010750:Phosphoproteins; D012333:RNA, Messenger; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse