B Amoah-Apraku1, M Z Fang, N J Guzman. 1. Division of Nephrology and Hypertension, Georgetown University MedicalCenter, Washington, D.C 20007, USA.
Abstract
UNLABELLED: 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.
UNLABELLED: 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.