Pflügers Arch - Eur J Physiol (2001) 442:479–486 DOI 10.1007/s004240100586

INVITED REVIEW

Josef Pfeilschifter · Wolfgang Eberhardt Karl-Friedrich Beck

Regulation of gene expression by nitric oxide

Accepted: 29 March 2001 / Published online: 2 June 2001 © Springer-Verlag 2001

Abstract Nitric oxide (NO) modulates transcription factors that bind specific cis-regulatory DNA responsible for coordinating the spatial and temporal patterns of gene expression that are initiated by a changing microenvironment. In this way NO helps to orchestrate gene transcription and forms the basis of functional cell responses to accommodate metabolic requirements and to coordinate endogenous defense mechanisms against a variety of stress and disease conditions. There is marked overlap between the signalling pathways triggered by NO, superoxide, and hypoxia. Understanding the redoxbased regulation of signal transduction and gene expression will provide insights into how cell activities are constantly coordinated and how promising new therapies may be developed. Keywords Gene expression · Hypoxia · Nitric oxide · Redox signalling · Superoxide · Transcription factors

Introduction Nitric oxide (NO) is a free radical gas that has raised extreme interest because of its tremendously broad range of physiological and pathophysiological functions [3, 42]. In mammals, the synthesis of NO is catalyzed by NO synthase (NOS), which is constitutively expressed in endothelial cells (eNOS) and neuronal cells (nNOS). A third isoform is induced (iNOS) when cells are confronted with inflammatory stimuli and is capable of the sustained synthesis of high amounts of NO that typically characterize inflammation. NOS catalyzes the oxidation of the amino Dedicated to Professor Klaus Thurau on occasion of his retirement with great appreciation of his continuous support and encouragement J. Pfeilschifter (✉) · W. Eberhardt · K.-F. Beck pharmazentrum frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany e-mail: [email protected] Tel.: +49-69-63016951, Fax: +49-69-63017942

acid L-arginine to give rise to equimolar amounts of citrulline and NO. At low concentrations, NO stimulates activity of the heme enzyme guanylate cyclase and triggers the formation of cyclic GMP (cGMP), an important messenger molecule that mediates the physiological functions of NO, such as vascular homeostasis and signal transduction in the central nervous system. In this context NO is a key signalling molecule that almost exclusively mediates physiological functions and counteracts pathophysiological processes in blood vessels, brain, kidney, heart, and many other organs. However, NO is a double-edged sword: high concentrations of NO produced mainly by iNOS and eventually also by nNOS, in the setting of appropriate microenvironmental conditions, may turn it from friend to foe and cause tissue damage. iNOS can be induced in almost any cell type in the body by two diverse mechanisms. The first comprises exposing cells to inflammatory cytokines such as interleukin 1, tumor necrosis factor α and interferon γ, or bacterial products such as lipopolysaccharides. Moreover, iNOS can be induced by various pathophysiological conditions, including hypoxia, ischemia-reperfusion injury, reactive oxygen species (ROS) or trauma. iNOS induction requires a delay of 6–8 h before the onset of NO production but, once induced, this enzyme is active for hours to days and produces NO in 1000-fold larger quantities than the constitutive enzymes eNOS and nNOS. The higher concentrations of NO produced by iNOS interact with thiol groups or transition-metal-containing proteins and can alter protein function or initiate gene expression to trigger cell adaptation. There is a continuous shift at even higher concentrations of NO towards cell damage or apoptosis, with other factors in the microenvironment of a cell critically influencing the final outcome [3, 8].

NO and signal transduction Extracellular ligands bind to cell surface receptors and trigger cascades of signalling events that are propagated to the nucleus to alter gene expression. In this way fami-

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lies of ligands activate specific signal transduction pathways that exist as conserved linear cassettes, enabling a cell to constantly coordinate its activities with changes in the microenvironment. This notion of a linear sequence of signalling events from the membrane to the nucleus is challenged by the ligand NO, which freely diffuses through cell membranes to reach its targets at different intracellular locations and to alter signalling networks by redox-sensitive modifications [22, 40]. Under physiological conditions cells produce only small amounts of NO by the constitutive NOS isoforms and only trace amounts of ROS are available to scavenge NO, thus indicating that direct NO chemistry will dictate functional cell responses [22]. The physiologically most relevant action of NO is the activation of the soluble guanylate cyclase by binding to the enzyme’s heme moiety. The subsequent increase in cGMP level alters the activity of three main target proteins: (1) cGMP-dependent protein kinases, (2) cGMP-regulated phosphodiesterases, and (3) cGMP-regulated ion channels [59]. In a similar way the formation of nitrosyl complexes affects other metalloproteins such as cytochrome P-450, cytochrome oxidase, catalase and NOS itself. Moreover, NO can scavenge superoxide (O2–) and other free radicals, and also inhibits the O2–-driven Fenton reaction and lipid peroxidation; thus, it may have quite remarkable antioxidant features. By contrast, large amounts of NO, as produced by iNOS in an inflammatory setting, often accompanied by a large production of ROS will shift NO chemistry towards indirect effects such as nitrosation, nitration, and oxidation [22]. The interaction of NO with molecular oxygen (O2) or superoxide (O2–) gives rise to the formation of the potent nitrosating agent N2O3 and peroxynitrite (ONOO–), respectively. In addition, S-nitrosothiol adducts are formed by the interaction between N2O3 and certain protein thiol groups and evoke signalling by altering protein kinases and phosphatases, G-proteins, tyrosine kinases, ion channels, and redox-sensitive transcription factors [3, 35, 40]. Detailed discussions of NOtriggered signalling processes are published elsewhere [50, 51].

Modulation of transcription factors by NO The early and rapid mechanisms of NO signalling depend primarily on post-translational modifications of pre-existing cellular proteins; however, the late phases that are required to accommodate the changing microenvironment are mediated by changes in gene expression. This adaptive long-term regulation occurs primarily at the level of transcription and is controlled by transcription factors. In a coordinated fashion, different types of activators and repressors that selectively bind their cognate sites in the regulatory elements of NO-responsive genes are matched and activate or repress transcription. Increasing evidence is accumulating that NO preferentially alters transcription factors that are sensitive to changes in the cellular redox status [3, 35, 40]. Several

Fig. 1 Hypothetical scheme illustrating nitric oxide (NO) modulation of signal flow, leading to the alteration of transcription factor activity and gene expression. (AP-1 activator protein 1, ERK extracellular signal-regulated kinases, JAK Janus protein kinases, MKP-1 mitogen-activated protein kinase phosphatase-1, NFκB nuclear factor κB, NO nitric oxide, O2– superoxide, ONOO– peroxynitrite, p38 p38 mitogen-activated protein kinases, PTP protein tyrosine phosphatase, Ras small GTP-binding protein, ROS reactive oxygen species, RXR retinoid X receptor, SAPK stress-activated protein kinases)

well-defined transcription factors, including nuclear factor κB (NFκB), activator protein 1 (AP-1), the tumor suppressor protein p53, the zinc-finger transcription factors early growth response 1 (Egr-1), Sp-1, the glucocorticoid receptor, the vitamin D3 receptor and the retinoid X receptor as well as the hypoxia-inducible factor 1α (HIF-1α) have been shown to be regulated by the intracellular redox state [3, 5, 30, 33, 35, 40, 48] (Fig. 1). NO is a molecule with both anti-oxidant and pro-oxidant properties depending on the availability and concentration of potential reaction partners, such as O2–, hydrogen peroxide or other ROS. This may explain the disparate observations that, in some cells, including human blood mononuclear cells, mouse macrophages, rat renal mesangial cells, and human endothelial cells, NO donors cause increased activity of NFκB family members [37, 67, 70, 72], whereas in other cell types NFκB activation is inhibited by NO donors [49]. NO-dependent S-nitrosation of a conserved cysteine residue in the guanine-nucleotide-binding domain of p21ras and subsequent activation of this small G-protein leads to NFκB activation [36]. Moreover, stimulation of IκB kinase (IKK) α activity by NO has been observed in endothelial cells and T lymphocytes [11, 67] and may contribute to the NO-triggered amplification of NFκB-mediated gene transcription. In contrast, endogenous NO may function to scavenge ROS, thereby inhibiting the activation of NFκB, as has been suggested for the expression of genes coding for monocyte chemoattractant protein 1 (MCP-1) and macrophage colony stimulation factor (M-CSF) [3, 49]. Alternative mechanisms for NFκB inhibition by NO include increased expression and nuclear translocation of inhibitor protein IκBα, which may displace NFκB from

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its cognate DNA-binding sites as demonstrated for vascular cellular adhesion molecule 1 (VCAM-1) gene expression in human endothelial cells [61]. Direct inhibition of the DNA-binding activity of NFκB family proteins through S-nitrosation by NO has been observed in cell-free systems using recombinant p50/p65 proteins [41]. NO reportedly modifies a crucial cysteine residue within the N-terminal region of the transcription factor thought to be responsible for DNA binding, dimerization, and nuclear translocation. From the data published so far, it is clear that NO differently modulates NFκB activity depending on the cell type investigated, the activating stimulus applied (which in turn determines the duration of the NO signal and the amount of NO being generated), and the cellular microenvironment, particularly the formation of ROS. Nevertheless, it is worth noting that in vivo NO has been observed to activate NFκB-mediated gene expression in the initiation phase of the inflammatory response following hemorrhagic shock [23]. Moreover, iNOS knock-out mice have abnormal interleukin-1-induced nuclear translocation of the p65 component of NFκB and abnormal NFκB-DNA binding, which are reversed by treatment with an NO donor [69]. In vitro, NO directly modifies AP-1, a heterodimeric transcription factor belonging to the basic domain leucine zipper family [63, 70]. Single conserved cysteine residues within the DNA-binding domains of c-Fos (Cys 154) and c-Jun (Cys 272), the major components of AP1, seem to be responsible for redox regulation of AP-1 activity [46]. Recently, Klatt et al. [31] reported on the NO-induced S-glutathionylation of c-Jun at Cys-269 in the DNA-binding domain that inhibits DNA binding. In intact cells, NO modulates AP-1-mediated gene transcription also indirectly by stimulating soluble guanylate cyclase, and cGMP in turn may account for the observed increase in junB and c-fos gene expression [53]. Moreover, NO has also been reported to modulate the activity of upstream protein kinase cascades triggering AP-1 activity [52] and subsequent gene expression [70]. Other molecular mechanisms by which NO may directly influence transcription factors involve the destruction of zinc-sulfur clusters and the release of zinc from various proteins including the zinc-storing protein metallothionein and zinc finger transcription factors. NO-mediated S-nitrosation of critical cysteine thiol groups and subsequent oxidation, e.g., disulfide formation, causes the loss of in vitro DNA binding capacity, as demonstrated for the yeast transcription factor LAC9 [34] and various mammalian transcription factors such as Egr-1, Sp-1, the glucocorticoid receptor, the vitamin D3 receptor and the retinoid X receptor [33, 35, 56]. Yet another transcription factor that carries a single candidate cysteine residue in the transactivation domain is HIF-1α, which belongs to the basic helix-loop-helixper-arnt-sim family. Together with HIF-1β it forms a heterodimer and regulates gene expression to accommodate a variety of systemic and cellular responses for homeostatic adaptations to hypoxic conditions [60]. Remark-

ably, there is a certain overlap between NO- and hypoxia-induced gene regulation (see below) and this is, at least partially, due to cross-talk between NO and HIF-1α. Hypoxia stabilizes HIF-1α protein and enhances the transactivation activity of HIF-1α, whereas NO’s mechanism of action under normoxia and hypoxia has not yet been fully elucidated. A prototypic hypoxia-inducible gene is vascular endothelial growth factor (VEGF) and there are reports that exogenous NO either down-regulates or up-regulates the expression of VEGF, depending on the redox status of the cell system investigated. More importantly, endogenous NO enhances VEGF production in vascular smooth muscle cells [13], and in keratinocytes in the context of cutaneous wound healing [18]. Recently, Kimura et al. [30] demonstrated that an HIF-1-binding site and its downstream HIF-1 ancillary sequence within the hypoxia-responsive element are required as cis-elements for the transcriptional activation of VEGF by hypoxia or NO. Other transcription factors identified to undergo increased or decreased DNA binding upon exposure of cells or cell extracts to exogenous or endogenously produced NO include heat shock factor 1 [74], Oct-1 [38] and iron-regulatory proteins (IRPs) (see [5], for review [48]). Taken together, it is obvious that NO signalling pathways target gene transcription through multiple promoter regulatory sites. In addition, a novel pathway for gene silencing based on the activation of DNA methyltransferase by NO and the subsequent modification of CpG island methylation has been reported [24]. The activation of DNA methyltransferase is most probably due to S-nitrosation of critical cysteine residues by NO. These observations further demonstrate that NO can have direct effects on gene expression, although an ultimate “NO-responsive promoter element” has yet to be identified.

Gene expression by NO A delicate balance between intracellular oxidants and antioxidants is important for physiological and pathophysiological conditions in the body. Excessive and unbalanced production of NO as well as ROS, resulting from exposure to environmental oxidants, ischemia or inflammation, perturbs the cellular redox balance and primes the mechanisms that counteract cellular damage by inducing a set of gene products that prevent or repair this damage. The proper coordination of this phase of gene expression is obviously critical for the resolution of disease and the return to a healthy condition. High output formation of NO and ROS under inflammatory conditions, and also the smaller and chronic production of these radicals are used for intracellular signalling and gene expression. Recently, the regulation of an increasing number of genes has been shown to be controlled by NO, as summarized in Table 1. This list is steadily growing and it is not the intention of this review to provide a comprehen-

482 Table 1 Compilation of NO-regulated gene products Gene products

Up- or down-regulation

cGMPdependent

Regulation by ROS

Hypoxiaresponsive

Reference

Protective mediators Haem oxygenase-1 Cu/Zn superoxide dismutase DNA-PKcs Heatshock protein 70

↑ ↑ ↑ ↑

no no n.d. no

↑ n.d. n.d. ↑

↑ n.d. n.d. n.d.

[1, 39, 40, 58] [20] [75] [1, 74]

Proinflammatory mediators iNOS Cyclooxygenase 2 (early) Cyclooxygenase 2 (later) Secretory phospholipase A2

↑ ↑ ↓ ↑

no yes n.d. no

↑ ↑ n.d. n.d.

↑ n.d. n.d. n.d.

[1, 4, 43, 45, 60] [1, 12, 64] [12] [55]

Chemokines and Cytokines Interleukin 8 MIP-2 MIP-1α MCP-1 M-CSF TNFα

↑ ↑ ↑ ↓ ↓ ↑

n.d. no n.d. n.d. n.d. yes

↑ ↑ ↑ n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d.

[1, 7] [1, 72] [1, 44] [3] [49] [17, 68]

Matrix and matrix-metabolizing enzymes MMP-9 ↓ TIMP-1 ↓ t-PA ↓ PAI-1 ↓ Collagen ↑ Fibronectin ↑ Laminin ↑ SPARC ↓

no no no yes n.d. n.d. n.d. no

↑ ↑ ↑ ↑ ↑ n.d. n.d. n.d.

n.d. n.d. n.d. ↑ n.d. n.d. n.d. n.d.

[15, 16] [15, 16] [14] [14, 60, 67] [1, 9, 66] [66] [66] [71]

Adhesion molecules ICAM-1 VCAM-1 E-selection

↓ ↓ ↓

no no n.d.

↑ ↓ ↓

n.d. n.d. n.d.

[1, 28] [1, 61] [1, 32]

Growth factors, hormones, receptors VEGF ↑ FLT-1 ↓ Erythropoietin ↓ Adrenomedullin ↑ TGFβ ↓ Angiotensin II type 1 receptor ↓

no yes n.d. n.d. no no

↑ n.d. ↑ n.d. ↑ n.d.

↑ ↑ ↑ ↑ ↑ n.d.

[1, 18, 19, 40] [19, 60] [1, 60, 65] [25, 26] [1, 9] [27]

Signalling devices ILK MKP-1

no yes

n.d. ↑

n.d. ↑

[2] [1, 21, 39, 62]

↓ ↑

n.d.: not determined

sive survey of all NO-targeted genes. We rather have primarily, but not exclusively, selected those genes reported to be regulated by NO in the kidney. The changes associated with the exposure of cells to exogenous NO or endogenously produced NO lead to the activation or silencing of genes and subsequently to the up- or downregulation of the respective mRNA species encoding a heterogeneous panel of protective antioxidant defense enzymes, structural proteins, proinflammatory mediators, and signalling devices. One of the first genes identified as a target for transcriptional regulation by NO was iNOS, suggesting that NO modulates its own biosynthetic machinery. Inhibition of NO synthesis clearly reduced IL-1β-stimulated

iNOS expression, suggesting that NO functions in a positive feedback loop that speeds up and strengthens its own biosynthesis [45]. This potent amplification mechanism may form the basis for the excessive formation of NO in acute and chronic inflammatory diseases. Indeed, the in vivo relevance of the NO-induced up-regulation of iNOS has been reported in animal models of endotoxininduced multiple organ dysfunction [54] and ischemic preconditioning in hearts of conscious rabbits [29]. Obviously, the up-regulation of iNOS is a natural response of the heart to a brief ischemic stress and NO itself upregulates myocardial iNOS transcript levels. Moreover, inhalation of NO primes lung macrophages to release ROS and NO in mice [73]. Moreover, other enzymes

483

producing proinflammatory mediators such as cyclooxygenase-2 and secretory phospholipase A2 are also transcriptionally affected by NO [12, 55, 64]. Many of the genes targeted by NO share roles in common physiological or pathophysiological processes. For example, chemokines involved in the attraction of neutrophiles, monocytes, and other inflammatory cells seem to be prime targets for regulation by NO. The gene for IL-8 has been revealed to be under transcriptional control by NO. In human endothelial cells, TNFα-stimulated IL-8 production is inhibited by the NOS inhibitor (L)-NGnitroarginine methylester (L-NAME) in a dose-dependent manner. Moreover, exogenously added NO donors induce IL-8 expression in endothelial and mesangial cells [3, 7]. In addition to IL-8, TNFα is probably induced in a paracrine manner by endothelial and smoothmuscle-derived NO in human neutrophils [17, 68]. Furthermore, the expression of macrophage inflammatory protein 1α (MIP-1α) directly depends on lipopolysaccharide-induced NO synthesis in peripheral blood mononuclear cells [44]. A most convincing demonstration, also in mechanistic terms, of NO-regulated chemokine expression was recently reported for the CXC-chemokine MIP-2 [72]. Walpen et al. [72] reported on an NOand IL-1β-dependent increase in MIP-2 mRNA and protein levels in renal mesangial cells. Moreover, inhibition of IL-1β-induced endogenous NO formation markedly attenuated MIP-2 protein expression. Transfection of a 770-bp MIP-2 promoter-luciferase reporter gene construct into mesangial cells resulted in a 3.5-fold increase in luciferase activity in cells treated with an NO donor, suggesting a transcriptional mechanism for NO-induced MIP-2 expression. Deletion and mutational analysis identified critical nuclear factor NFκB and NF-IL-6 binding sites required for the NO-dependent regulation of MIP-2. In vivo, the inhibition of NO synthesis in the Thy-1.1 model of mesangioproliferative glomerulonephritis by a specific iNOS inhibitor resulted in the marked reduction of MIP-2 expression. Strikingly, infiltration of neutrophils into the glomerulus was attenuated by more than 90% in the rats treated with iNOS inhibitor. Down-regulation of MIP-2 may partly explain the beneficial effect of NOS inhibitors observed in early phases of glomerulonephritis. Another group of target genes for NO action are the extracellular matrix proteins and their metabolizing enzymes, the matrix metalloproteinases and plasminogen activators, such as MMP-9 and t-PA [14, 15, 16], and their endogenous inhibitors TIMP-1 and PAI-1, respectively [6, 15]. In the kidney, accumulation of extracellular matrix is often a hallmark of chronic disease, eventually leading to the development of glomerulosclerosis. In this context, the coordinate expression of proteases and their inhibitors by inflammatory cytokines and NO will allow the fine-tuned regulation of tissue proteolysis and protect against overwhelming tissue destruction. NO also modulates the expression of major matrix components such as collagen, fibronectin or laminin [6, 9], which may also be important for tissue remodeling in chronic

inflammatory kidney diseases. Recently, NO was found to inhibit the expression of another matrix protein, SPARC (secreted protein acidic and rich in cysteine, also known as BM-40 or osteonectin) [71]. The highly glycosylated SPARC protein has a variety of biological activities, and its action as a scavenger of platelet-derived growth factor may be relevant in the course of glomerulonephritis. By modulating SPARC expression, NO may subsequently affect mesangial cell proliferation in the course of glomerular inflammation. The regulation of VEGF, a growth factor mediating angiogenesis and vascular permeability, was found to be modulated by NO in mesangial cells [19], keratinocytes [18], and other cell types [3]. Interestingly, NO-donating agents induce VEGF expression in mesangial cells in a cGMP-independent manner, whereas expression of the VEGF receptor FLT-1 (fms-like tyrosine kinase) is simultaneously decreased in a cGMP-dependent fashion in mesangial cells [19]. Obviously the increased production of VEGF in NO-exposed mesangial cells is destined for export and cannot trigger autocrine feedback loops.

Cross-communication in redox signalling Whereas most physiological responses triggered by low concentrations of NO are mediated by the activation of soluble guanylate cyclase and the subsequent production of cGMP as the principal signalling messenger, recent studies have provided a wealth of evidence for alternative signalling cascades in which concentrations of NO are moderate to high. These signals operate in part through the redox-sensitive regulation of transcription factors and gene expression and alter, on a more longterm basis, the capacity of a cell to deal with stress conditions. Indeed, the majority of NO-regulated genes listed in Table 1 are modulated independently of cGMP effector mechanisms, although there are a certain number of exceptions [17]. Cross-communication with other pro-oxidant or anti-oxidant mediators will critically influence the fate of a cell under pathological conditions when iNOS is expressed. In addition to NO, superoxide (O2–) is another inflammatory mediator synthesized by different enzymes, most prominently the NADPH oxidases, xanthine oxidase, cyclooxygenases, lipoxygenases, and the cytochrome P-450 oxidases. Once primed and activated by proinflammatory cytokines such as IL-1β and TNFα most cells, including renal mesangial cells, co-produce NO and O2– or more generally speaking ROS. ROS themselves are potent modulators of signal transduction pathways and gene expression (Table 1) [1]. The interaction of NO and ROS is thought to be highly relevant to the regulation of gene expression (Fig. 1). The free radicals NO and O2– react with each other at a rate close to that simply limited by diffusion and outcompete most other targets of the radicals found in a cell. In this context it is important to note that the ratio between NO and O2– formation determines whether cells live or die by apoptosis or necrosis [57]. As shown

484

in Table 1 quite a number of NO-regulated genes are also affected by ROS. Whereas certain genes, such as that for iNOS, are regulated in a coordinated manner by NO and O2– [4, 45], others are affected in a contrasting manner. For example, IL-1β-induced expression of matrix metalloproteinase-9 (MMP-9) is inhibited by NO [15] but amplified by O2– [16]. The simultaneous production of NO and O2– by many cells exposed to an inflammatory environment and the opposite effects of both radicals on MMP-9 expression may provide a switch-like mechanism, with a subtle change in the O2–/NO ratio resulting in quite dramatic changes in enzyme expression. Interestingly, increased DNA binding activities of NFκB and AP-1 were found to constitute the O2–-induced amplification of MMP-9 expression [16]. As for NO, perturbations of the cellular redox balance, especially in the endogenous thioredoxin and glutathione systems, seem to be of central importance in the redirection of gene expression by ROS signalling [10]. It is difficult to reconcile at a first glance the facts that NO and O2– target identical sets of signalling pathways and transcription factors and, nevertheless, can trigger coordinate or opposite effects on gene transcription. Moreover, both radicals communicate with each other and generate peroxynitrite, complicating the situation further; indeed, contrasting data on NO and O2– effects on transcription factor such as NFκB and AP-1 have been reported (see above). It is likely that some of this apparent complexity may reflect timing differences in the generation of both types of radicals in in vitro and in vivo situations. A dynamic temporal organization of the NO and O2– signals and the ability of a cell to spatially resolve both signals in a differentially or coordinated manner may help to resolve these contradictory observations. The microenvironment of a cell (predominantly NO, balanced NO/O2–, or predominantly O2–), the setting of a threshold (glutathione and thioredoxin systems), and the ability of cells to propagate signals (NO or O2–) across different time scales will trigger appropriate gene expression and the subsequent cell responses. Another interesting facet in redox regulation of gene expression is the fact that hypoxia also targets a wide variety of genes that partially overlap with the NO- or ROS-targeted genes (Table 1). In mammals, the transcriptional response to hypoxia is commonly mediated by HIF-1α and approximately 40 hypoxia-regulated genes have been identified in mammals so far [60]. In this context it is quite intriguing that HIF-1α is also a target for NO and, vice versa, the expression of iNOS is modulated by HIF-1α binding to a hypoxia response element in the 5′-flanking sequence of the iNOS gene [30, 60]. Like NO, hypoxia coordinately up-regulates matrix production, but, unlike NO, hypoxia decreases matrix turnover in renal fibroblasts [47], thus providing an additional level of complexity to redox regulation of gene expression. Finally, studying gene transcription is only a first step and other complementary approaches are needed. Proteomics-based analysis of NO-regulated gene expression and mass spectrometry will provide the additional information required to iden-

tify new therapies emerging from this exciting field of investigation. Acknowledgements The authors’ work was supported by the Deutsche Forschungsgemeinschaft (SFB 553) and the Stiftung VERUM für Verhalten und Umwelt.

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