A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.

Abstract

SummaryPicolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-y-treated murine macrophages . We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation ofiNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN -'Y and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid-but not LPS-induced CAT activity by more than 70% . Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-y or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRB . Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-y activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression . These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

Hypoxia upregulates hypoxia inducible factor (HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

“…By contrast, under hypoxia or in the presence of iron chelators, the degradation of HIF-1α is prevented [7,11,12]. As a result, this stabilization initiates a multi-step pathway of activation of HIF-1α that includes hypoxia-dependent nuclear translocation and dimerization with ARNT to interact with hypoxia responwww.cell-research.com | Cell Research Qi Fang Li et al 549 npg sive element (HRE) of target genes such as erythropoietin (EPO) [13], vascular endothelial growth factor (VEGF) [14], inducible nitric-oxide synthase [15], heme oxygenase 1 [16], and so on. Taken together, it comes as no surprise that the HIF-dependent hypoxic response pathway plays a prominent role in mediating the consequences of many disease states, including cerebral and myocardial ischemia, pulmonary hypertension, and tumorigenesis [17].…”

Activation of the hypoxia‐inducible factor pathway induced by prolyl hydroxylase domain 2 deficiency enhances the effect of running training in mice

“…Additionally, iNOS is a hypoxia‐inducible gene because hypoxia‐responsive element is present in the iNOS promoter (Melillo et al . 1995). Therefore, increased NO production in Phd2 cKO could also be mediated by inflammatory cells expressing iNOS like macrophages, which needs further investigation.…”

Picolinic acid- or desferrioxamine-inducible autocrine activation of macrophages engineered to produce IFNγ: an approach for gene therapy

“…The Mf vector system described in this paper was designed to generate activated macrophages (Mf), because the therapeutic gene (IFNg) is an Mf activator and is expressed in an inducible manner in response to molecules, PA or DFX, which have costimulatory activities on Mf. The inducibility by hypoxia of HREcontaining synthetic promoters was previously characterized in vivo 23,24 in vitro, 9,15,25,11,26 and also in Mf. 12,11 New perspectives in the use of hypoxia-sensitive Mf vectors stem from the observation that stimuli other than hypoxia can activate HRE.…”

“…A common denominator of many pathological lesions is hypoxia, a condition of low oxygen tension that occurs, for example, within the tumor mass or in inflammatory areas. Mf respond to hypoxia 9,10 and can be engineered to express therapeutic genes under these conditions 11,12 using synthetic promoters containing the hypoxia-responsive element (HRE), which is specifically transactivated under hypoxic conditions (reviewed in Lewis et al 10 and Semenza 13 ).…”

“…Therefore, it is important to develop strategies allowing the expression of the hypoxia-driven therapeutic gene under normoxic conditions utilizing stimuli or drugs, other than hypoxia, which can be delivered in a controlled manner. One group of these compounds is represented by metal chelators including desferrioxamine (DFX), 15,16 a drug used to treat iron overload, and picolinic acid (PA), 9 a metabolite of tryptophan. The Mf-activating properties of PA have been studied extensively.…”