Inducible nitric oxide synthase in human diseases

Abstract

Since its discovery as a biologically active molecule in the late 1980s, nitric oxide (NO) has been found to play an important role as signal molecule in many parts of the organism as well as cytotoxic or regulatory effector molecule of the innate immune response. The signal molecule NO is synthesized on demand for short periods of time (seconds to minutes) following enzyme activation of constitutively expressed endothelial NO synthase (eNOS) or neuronal NO synthase (nNOS). In contrast, the inducible NO synthase (iNOS) is expressed after cell activation only and then produces NO for comparatively long periods of time (hours to days). Thus, regulated short pulsative synthesis versus constant NO production differentiates between physiological and pathophysiological actions of NO (for review see [1]). As human monocytes in contrast to rodent ones do not produce large amounts of NO when activated in vitro, iNOS expression in human diseases has long been questionable. However, in the last 3 years data have accumulated on iNOS expression in a variety of human diseases or disorders. We here try to review our current understanding of the role of iNOS in human diseases. The iNOS gene is under the transcriptional control of a variety of inflammatory mediators such as cytokines, lipopolysaccharide (LPS), and others (for review see [2]). iNOS cDNAs have independently been cloned from several tissues with only small differences in the deduced amino acid sequences [3–6]. The overall nucleotide sequence identity between human and murine iNOS cDNA is about 80% [3]. Molecular cloning revealed that the iNOS gene is about 37 kb in length [7] and is located on chromosome 17 at position 17cen-q11.2 [8]. The iNOS open reading frame is encoded by 27 exons, with translation initiation and termination in exons 2 and 27, respectively [9]. All intron/exon boundaries of the human iNOS gene conform strictly to the known GT/AG donor/acceptor rule. The structure of the coding region, especially of the cofactor binding sites, is very similar to those of human nNOS [10] and eNOS [11,12]. Southern blot analysis revealed single bands for nNOS and eNOS but multiple bands for iNOS exons 22–26 in humans and apes [13,14] when using the 3′ end of an iNOS cDNA probe. An unprocessed, highly mutated pseudogene has been localized on the same chromosomal region as the functional iNOS gene by fluorescence in situ hybridization (FISH) analysis [15]. Three minor allelic variants have been described [16–18], one bearing pathophysiological significance in resistance against malaria infection [18]. The high homology of iNOS isoforms among different species and various cell types suggests that they are all products of the same gene. However, human iNOS gene transcription in distinct cells is reported to be regulated differently. Significant differences between the human and the murine iNOS promoter region were found by 3′ analysis. Only 1.5 kb of the proximal 5′ flanking region of the murine promoter are necessary to confer inducibility to LPS and interferon-gamma (IFN-γ), whereas the human iNOS promoter is hyporesponsive to LPS/IFN-γ due to nucleotide exchanges in the LPS/IFN-γ-responsive enhancer region (−1083 to −1229) [19,20]. Additionally, the human transcription factor NF-κB, induced by treatment with IL-1β, tumour necrosis factor-alpha (TNF-α) and IFN-γ, binds to the iNOS promoter more weakly than mouse NF-κB does [21]. However, three regions with cytokine-responsive cis-regulatory elements (lying between −3.8 kb and −16 kb in the promoter region) confer cytokine inducibility [22,23]. Interestingly, the human iNOS gene contains a shear-stress responsive element (GAGACC) which is identical to that in human eNOS, but this element does not exist in the murine iNOS promoter [24]. Induction of NO production by this shear-stress element seems to be a key mediator for protection of cardiovascular diseases via inhibition of leucocyte adhesion, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation. At position -226 to -212 an element containing a sequence homology to the human hypoxia-responsive element (HRE) [25] was found in the murine promoter conferring iNOS induction at decreased oxygen tension in IFN-γ-activated macrophages [26]. Induction of the hypoxia-inducible factor-1 by hypoxia and binding to the iNOS-HRE in cooperation with IFN-γ leads to iNOS induction. The necessity for IFN-γ costimulation may help to limit iNOS expression to inflammatory sites with hypoxic conditions. Although the activity of the human HRE is questionable due to two mismatches in the consensus sequence, culture of human hepatoma cells under hypoxic conditions indeed led to iNOS induction [27]. In conclusion, the human iNOS promoter is one of the largest and most complex promoters known today (Fig. 1a,b), indicative of a tightly controlled iNOS gene expression. Schematic structure of the human inducible nitric oxide synthase (iNOS) 5′ flanking region (a), th