Altering gene expression is the most fundamental and effective way for a cell to respond to extracellular signals and/or changes in its environment, in both the short term and the long term [1]. In the short term, transcription factors are involved in mediating responses to growth factors and a variety of other extracellular signals [2]. In contrast, the long-term control of gene expression induced by growth factors and the changes in gene expression, which occur during development, is generally (with few exceptions) irreversible.

During development, the expression of specific sets of genes is regulated spatially (by position/morphogenetic gradients) and temporally. Regulation of the signaling responses is governed at the genetic level by transcription factors that bind to control regions of target genes and alter their expression [1,2]. Transcription factors are endogenous substances, usually proteins, that are effective in the initiation, stimulation or termination of the genetic transcription process. While in the cytoplasm, the transcription factor is incapable of promoting transcription. A signaling event occurs, such as a change of the state of phosphorylation, which results in protein subunit translocation into the nucleus [3,4]. Transcription is a process in which one DNA strand is used as a template to synthesize a complementary RNA. Signal transduction therefore involves complex interactions of multiple cellular pathways [1,2].

In particular, reduction–oxidation/oxygen (redox)-sensitive transcription factors have gained an overwhelming backlog of interest momentum over the years, ever since the onset of the burgeoning field of free radical research and oxidative stress. The reason for this is that redox-sensitive transcription factors are often associated with the development and progression of many human disease states. Their ultimate regulation therefore bears potential therapeutic intervention for possible clinical applications [1,2,3,4].

In the present review, I will focus on elaborating a comprehensive overview of the current understanding of redox/oxidative mechanisms mediating the regulation of transcription factors. These transcription factors regulate a plethora of cellular functions that span the range from anoxia and hypoxia to oxidative stress within the context of oxidant-mediated lung injury.

A crucial transcription factor that is a master regulatory element in sensing hypoxic conditions and in integrating an adapted response via gene expression of oxygen-sensitive and redox-sensitive enzymes and cofactors is hypoxia-inducible factor-1 (HIF-1) (Fig. 1) [43,44,45]. The signal transduction components that link the availability of oxygen to the activation of these transcription factors are poorly defined, but are broadly believed to hinge on the free abundance of oxidants.

HIF-1 consists of two subunits: HIF-1α, which is unique to the oxygen response; and HIF-1β (aryl hydrocarbon receptor nuclear translocator). The stability and activity of HIF-1α, first identified as a DNA-binding activity expressed under hypoxic conditions, increase exponentially when pO₂ is lowered. Whereas HIF-1β is constitutively expressed under normoxic conditions, HIF-1α is rapidly degraded by the ubiquitin–proteasome system. Under hypoxic conditions, however, HIF-1α protein stabilizes and accumulates, thus allowing the het-erodimer to translocate to the nucleus and to bind specific promoter moieties of selective genes encoding erythropoietin (EPO), vascular endothelial growth factor (VEGF), glycolytic enzymes and glucose transporters, as well as cytokines and other inflammatory mediators (Fig. 1) [44,45,46]. It is expected that any reduction of tissue oxygenation in vivo and in vitro would therefore provide a mechanistic stimulus for a graded and adaptive response mediated by hypoxia-inducible factor (Fig. 2).

The role of HIF-1α in oxidant-induced lung injury is less clear, or less prominent, than that of NF-κB. Indirect, but unprecedented and unequivocal, evidence was independently provided by Hellwig-Bürgel and colleagues [47,48,49] and by Haddad and Land [50,51], however, to indicate HIF-1 as a possible regulator of the evolution and propagation of the inflammatory process. The rate of transcription of several genes encoding proteins involved in oxygen and energy homeostasis is controlled by HIF-1. Since EPO gene expression is inhibited by the proinflammatory cytokines, such as IL-1β and TNF-α, while no such effect has been reported with respect to the VEGF gene, Hellwig-Bürgel et al. investigated the effects of these cytokines on the activation of the HIF-1 DNA-binding complex and the amount of HIF-1α protein in human hepatoma cells in culture [47]. Under normoxic conditions, both cytokines caused a moderate activation of HIF-1 DNA binding. In hypoxia, cytokines strongly increased HIF-1 activity compared with the effect of hypoxia alone. Only IL-1β increased HIF-1α protein levels. In transient transfection experiments, HIF-1-driven reporter gene expression was augmented by cytokines only under hypoxic conditions. In contrast to their effect on EPO synthesis, neither IL-1β or TNF-α decreased VEGF production. The mRNA levels of HIF-1α and VEGF were unaffected. Cytokine-induced inhibition of EPO production may thus not be mediated by impairment of HIF-1 function [47].

Hellwig-Bürgel and colleagues subsequently proposed that HIF-1 might be involved in modulating gene expression during inflammation. Furthermore, since VEGF promotes angiogenesis and inflammatory reactions, in a parallel study VEGF mRNA was found detectable in the proximal tubules of inflamed kidneys but not in normal kidneys [48]. In other organs, VEGF gene expression is induced by hypoxia and by cytokines. To identify the cellular mechanisms in control of tubular VEGF production, the effects of hypoxia and IL-1β on VEGF mRNA levels, on VEGF secretion and on activity of HIF-1 in human proximal tubular epithelial cells were assessed. The human proximal tubular epithelial cells were grown in monolayers from human kidneys, and hypoxia was induced by incubation at 3% O₂. Significant amounts of VEGF mRNA and VEGF protein were measured in human proximal tubular epithelial cell extracts and culture media, respectively. Moreover, stimulation of VEGF synthesis at low pO₂ tension and following IL-1β treatment was detectable at the protein level only. Nuclear HIF-1α protein levels and HIF-1 binding to DNA were also increased under these conditions [48].

VEGF induction appears to increase DNA binding of HIF-1 to hypoxia-responsive elements in the VEGF gene promoter. In inflammatory diseases of the kidney, tubular cell-derived VEGF may therefore contribute to microvascular leakage and to monocyte extravasation. Regarding the mechanisms reported, LY-294002 (an inhibitor of phosphatidylinositol 3-kinase) suppressed HIF-1 activation in a dose-dependent manner irrespective of the stimulus. With respect to target proteins controlled by HIF-1, the production of EPO was fully blocked and that of VEGF reduced following inhibition of the phosphatidylinositol 3-kinase pathway [49]. The role of mitogen-activated protein kinase kinases in this process remained ambiguous, because PD-98059 and U-0126 inhibitors did not significantly reduce HIF-1α levels at non-toxic doses [49]. It was proposed that phosphatidylinositol 3-kinase signaling is not only important in the hypoxic induction of HIF-1, but that it is also crucially involved in the response to insulin and IL-1.

Furthermore, evidence that reactive oxygen species (ROS) signaling mediates cytokine-dependent regulation of HIF-1α has been postulated by Haddad and Land [50,51]. In the airway epithelium, recombinant human IL-1β and recombinant murine TNF-α induced, in a time-dependent manner, the nuclear translocation of HIF-1α. This translocation is an effect associated with upregulating the activity of this transcription factor under normoxic conditions. In addition, analysis of the mode of action of IL-1β and TNF-α revealed a novel induction of intra-cellular ROS, including hydrogen peroxide, the superoxide anion (O₂^-•) and the •OH radical [50,51]. The antioxidants dimethyl sulfoxide and 1,3-dimethyl-2-thiourea, purported to be prototypical scavengers of hydrogen peroxideand •OH, attenuated cytokine-induced HIF-1α nuclear translocation and activation in a dose-dependent manner. The NADPH-oxidase inhibitor 4'-hydroxy-3'-methoxy-acetophenone, which may affect mitochondrial ROS production, attenuated cytokine-mediated nuclear translocation and activation of HIF-1α. Furthermore, inhibition of the mitochondrion complex I nicotinamide ADP-dependent oxidase by diphenylene iodonium, which blocks the conversion of ubiquinone to ubiquinol, abrogated IL-1β-dependent and TNF-α-dependent nuclear translocation and activation of HIF-1α. Similarly, interrupting the respiratory chain with potassium cyanide reversed the excitatory effect of cytokines on HIF-1α nuclear translocation and activation [50,51]. These results indicate that a nonhypoxic pathway mediates cytokine-dependent regulation of HIF-1α translocation and activation in a ROS-sensitive mechanism.

Direct evidence implicating HIF-1 in lung injury emerged with VEGF, which has been recognized as a potent mediator of endothelial barrier dysfunction and is upregulated during ischemia in many organs [43,44,45,46]. Because ventilated pulmonary ischemia causes a marked increase in pulmonary vascular permeability, it was hypothesized that VEGF would increase during ischemic lung injury.

To test this hypothesis, VEGF expression was measured by northern and western blot analysis in isolated ferret lungs after 45 or 180 min of ventilated (95% or 0% O₂) ischemia [52]. Pulmonary vascular permeability, assessed by measurement of the osmotic reflection coefficient for albumin, was evaluated in the same lungs, as was expression of HIF-1α. The distribution of VEGF as a function of ischemic time and oxygen tension was also evaluated by immunohistochemical staining in separate groups of lungs. VEGF mRNA increased threefold by 180 min of ventilated ischemia, independent of oxygen tension. VEGF protein increased in parallel to VEGF mRNA. Immunohistochemical staining demonstrated the appearance of VEGF protein along alveolar septae after 180 min of hyperoxic ischemia and after 45 or 180 min of hypoxic ischemia. In addition, albumin was not altered by 45 min of hyperoxic ischemia (0.69 ± 0.09 versus 0.50 ± 0.12, respectively), but decreased significantly after 180 min of hyperoxic ischemia and after 45 and 180 min of hypoxic ischemia (0.20 ± 0.03, 0.26 ± 0.08 and 0.23 ± 0.03, respectively) [52]. HIF-1α mRNA increased during both hyperoxic and hypoxic ischemia, but HIF-1α protein increased only during hypoxic ischemia. This implicates VEGF as a potential mediator of increased pulmonary vascular permeability in this model of acute lung injury.

Further elaborating on the mechanisms involving HIF-1 in regulating the inflammatory response, Hierholzer et al. reported that hemorrhagic shock (HS) initiates an inflammatory response that includes increased expression of inducible nitric oxide synthase and production of prostaglandins [53]. Induction of inducible nitric oxide synthase during the ischemic phase of HS may involve the activation of HIF-1. Increased expression of cyclooxygenase-2 during HS contributes to prostaglandin production. The lungs of rats subjected to HS demonstrated a twofold increase in HIF-1 activation and a 7.4-fold increase in expression of cyclo-oxygenase-2 mRNA, as compared with sham controls [53]. It was concluded that the upregulation of inducible nitric oxide synthase and cyclooxygenase-2 during ischemia are two important early response genes that promote the inflammatory response and may contribute to organ damage through the rapid and exaggerated production of nitric oxide and prostaglandins.

Furthermore, in a novel study by Shoshani and colleagues, the identification and cloning of a HIF-1-responsive gene, designated RTP801, was recently reported. Strong upregula-tion of RTP801 by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke [54]. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from hydrogen peroxide-triggered apoptosis via a dramatic reduction in the generation of ROS. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Furthermore, liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death [54]. The biological effect of RTP801 overexpression thus depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.

A hypothetical schematic depicting the role of HIF-1 in lung injury is displayed in Fig. 3.

The molecular response to oxidative stress is regulated by redox-sensitive transcription factors [55,56,57,58,59,60]. The study of gene expression and regulation is critical in the development of novel gene therapies [61,62,63,64,65,66,67,68,69,70]. Recognition of reactive species and redox-mediated protein modifications as potential signals may open up a new field of cell regulation via specific and targeted genetic control of transcription factors, and can thus provide us with a novel way of controlling disease processes [71,72,73,74,75]. Dynamic variation in pO₂ and redox equilibrium thus regulate gene expression, apoptosis signaling and the inflammatory process, thereby bearing potential consequences for screening emerging targets for therapeutic intervention.

None declared.

AM = alveolar macrophage; CF = cystic fibrosis; EPO = erythropoietin; HIF-1 = hypoxia-inducible factor-1; HS = hemorrhagic shock; IL = interleukin; NF-κB = nuclear factor-κB; pO₂ = partial pressure of oxygen; redox = reduction–oxidation; ROS = reactive oxygen species; SLPI = secretory leukocyte protease inhibitor; TNF = tumor necrosis factor; VEGF = vascular endothelial growth factor.