Strain dependent rat iNOS promoter activity--correlation to identified WT1 transcription factor binding site

The free radical nitric oxide (NO) has been implicated in cytokine mediated destruction of rat beta-cells in islets of Langerhans. Cytokine mediated NO production is associated with increased expression of the inducible nitric oxide synthase (iNOS). We have previously shown a strain dependent difference between Wistar Kyoto (WKY) and Brown Norway (BN) rats of IL-1beta mediated destruction of islets of Langerhans to be related to expression levels of iNOS and NO production. The aim of the present study was to clone and screen the iNOS gene promoter region from WKY and BN rats for polymorphisms and to functionally test such nucleotide differences. Within the total 2077 bp sequenced from both rat strains we identified three polymorphisms in two separate areas: (i) a GT-repeat polymorphism linked to (ii) a C/T polymorphisms, leading to a WT1 binding site approximately 1650bp upstream the BN iNOS promoter and (iii) a G/A SNP in exon 1. Apart from these polymorphisms the homology between all published rat iNOS sequences including the presently described are about 96%. Promoter activity was detected for both genes in a luciferase assay followed cloning of 2012 bp fragments and transient transfection into RIN cells. For both strains IL-1beta induced dose-dependent activity and strain dependent iNOS promoter activity was demonstrated when WT1 was co-expressed. To our knowledge, this is the first demonstration of functional WT1/iNOS promoter interaction. We conclude that the iNOS promoter is strain-dependently regulated which may relate to quantitatively as well as qualitatively strain dependent differences in transcription factor expression, in this study exemplified by WT1.