FUNCTIONAL REQUIREMENT OF THE HYPOXIA-RESPONSIVE ELEMENT IN THE ACTIVATION OF THE INDUCIBLE NITRIC-OXIDE SYNTHASE PROMOTER BY THE IRON CHELATOR DESFERRIOXAMINE

We have previously reported that a 19-base pair element of the 5'-flan king region of the inducible nitric oxide synthase (iNOS) gene contain ing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE) me diates picolinic acid (PA)- or hypoxia-induced activation of the iNOS promoter in interferon-gamma (IFN-gamma)-treated murine macrophages. T he iron chelator desferrioxamine (DFX) induces the activity of the hum an erythropoietin enhancer in Hep3B cells. We have investigated the in fluence of DFX on the activation of the iNOS promoter and iNOS gene ex pression in ANA-1 macrophages. We have found that DFX induced DNA-bind ing activity to the hypoxia-inducible factor 1 (HIF-1) consensus seque nce of the iNOS promoter and activated the iNOS-HRE in murine macropha ges. These activities of DFX were associated with a synergistic induct ion of iNOS mRNA expression and iNOS transcription in IFN-gamma-treate d ANA-1 macrophages. Functional analysis of the 5'-flanking region of the iNOS gene demonstrated that IFN-gamma plus DFX activated the full- length iNOS promoter and that the iNOS-HRE was required for DFX-induce d iNOS transcriptional activity. We also investigated the role of iron metabolism in the DFX- or PA dependent induction of HIF-1 activity an d iNOS expression, We demonstrate that addition of iron sulfate comple tely abolished DFX or PA induction of HIF-1 binding and iNOS-HRE activ ation and abrogated IFN-gamma plus either DFX- or PA-induced iNOS expr ession. These data establish that DFX is a co-stimulus for the transcr iptional activation of the iNOS gene in IFN-gamma-treated macrophages, and they provide evidence that the iNOS-HRE is required for the DFX-d ependent activation of the iNOS promoter. Furthermore, our results ind icate that the iNOS-HRE is a regulatory element of the iNOS pro meter responsive to iron chelation.