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

Background. The expression of inducible nitric oxide synthase (iNOS) is sub ject to strict tissue-specific transcriptional control. In mouse renal epit helium, an interferon-gamma (IFN-gamma)induced signaling protein, IFN-gamma regulatory factor 1 (IRF-1), appears to mediate the induction of iNOS expr ession by cytokines and bacterial lipopolysaccharide (LPS). Methods. We used a novel technique, namely, blockade of cytosolic IRF-1 act ivity with a triethyleneglycol-bridged decoy DNA oligonucleotide (ODN) cont aining the IRF-1 consensus binding sequences present in the iNOS promoter t o inhibit iNOS gene expression. Cultured mouse renal epithelial cells were treated with a combination of LPS (11 mu g/mL) and IFN (100 U/mL) in the ab sence or presence of IRF-1 decoy ODN followed by determinations of NO produ ction 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 mRN A levels. A scrambled ODN failed to affect LPS/IFN-stimulated NO production or iNOS protein and mRNA levels. Transcriptional assays showed that the IR F-1 decoy ODN inhibited transcriptional activity of an iNOS promoter-CAT ge ne construct. Conclusions. Decoy ODN-based techniques effectively inhibit iNOS expression in renal epithelium and represent a potentially useful approach for select ive blockade of this enzyme in pathologic conditions associated with excess ive NO production.