Redox biological reactions are now accepted to bear the Janus faceted feature of promoting both physiological signaling responses and pathophysiological cues. Endogenous antioxidant molecules participate in both scenarios. This review focuses on the role of crucial cellular nucleophiles, such as glutathione, and their capacity to interact with oxidants and to establish networks with other critical enzymes such as peroxiredoxins. We discuss the importance of the Nrf2-Keap1 pathway as an example of a transcriptional antioxidant response and we summarize transcriptional routes related to redox activation. As examples of pathophysiological cellular and tissular settings where antioxidant responses are major players we highlight endoplasmic reticulum stress and ischemia reperfusion. Topologically confined redox-mediated post-translational modifications of thiols are considered important molecular mechanisms mediating many antioxidant responses, whereas redox-sensitive microRNAs have emerged as key players in the posttranscriptional regulation of redox-mediated gene expression. Understanding such mechanisms may provide the basis for antioxidant-based therapeutic interventions in redox-related diseases.

Objective-Reactive oxygen species have been implicated as signaling molecules modulating the activity of redoxsensitive transcription factors such as nuclear factor kappa B (NF-B). Recently, the transcription factor hypoxiainducible factor-1 (HIF-1), known to mediate gene expression by hypoxia, has been found to be also activated by nonhypoxic factors in a redox-sensitive manner. We therefore aimed to elucidate the link between these 2 important redox-sensitive transcription factors. Methods and Results-In pulmonary artery smooth muscle cells, reactive oxygen species generated either by exogenous H 2 O 2 or by a NOX4-containing NADPH oxidase stimulated by thrombin activated or induced NF-B and HIF-1␣. The reactive oxygen species-mediated HIF-1␣ induction occurred on the transcriptional level and was dependent on NF-B.Transfection experiments with wild-type or mutant HIF-1␣ promoter constructs revealed the presence of a yet unidentified NF-B binding element. Gel shift analyses and chromatin immunoprecipitation verified binding of NF-B to this site. Furthermore, reactive oxygen species enhanced expression of plasminogen activator inhibitor-1, which was prevented by dominant-negative IB or mutation of the HIF-1 binding site within the plasminogen activator inhibitor-1 promoter. Conclusion-These findings show for the first time to our knowledge that reactive oxygen species directly link HIF-1␣and NF-B, implicating an important pathophysiological role of this novel pathway in disorders associated with elevated levels of reactive oxygen species. Key Words: hypoxia-inducible factor Ⅲ NADPH oxidase Ⅲ nuclear factor kappa B Ⅲ reactive oxygen species Ⅲ thrombin O xidative stress has been implicated to play an important role in the pathophysiology of many cardiovascular diseases including systemic and pulmonary hypertension and atherosclerosis as well as in tumor progression and vascularization. [1][2][3] Moderate levels of reactive oxygen species (ROS), especially superoxide anions and hydrogen peroxide, have been shown to activate signaling cascades mediating the responses to vasoactive peptides, growth factors, cytokines, hormones, and coagulation factors, as well as to physical and chemical stress. ROS participate in the regulation of vascular proliferation, migration, apoptosis, modification of the extracellular matrix, and procoagulant activity. 4 -9 Moreover, ROS can activate angiogenesis, 10 a process known to be primarily mediated by vascular endothelial growth factor under hypoxia. Under hypoxia, vascular endothelial growth factor expression is induced by the transcription factor hypoxia-inducible factor-1 (HIF-1). 11 Aside from vascular endothelial growth factor, HIF-1 regulates Ͼ100 genes encoding for metabolic enzymes, growth factors, and factors contributing to modulation of extracellular matrix and thrombosis such as plasminogen activator inhibitor-1 (PAI-1). [12][13][14][15] HIF-1 is composed of an inducible ␣-subunit (HIF-1␣) and a constitutive ␤-subunit (also termed ARNT). 12 HIF-1␣ contains an oxygen-de...

The endoplasmic reticulum (ER) plays a major role in regulating synthesis, folding, and orderly transport of proteins. It is also essentially involved in various cellular signaling processes, primarily by its function as a dynamic Ca(2+) store. Compared to the cytosol, oxidizing conditions are found in the ER that allow oxidation of cysteine residues in nascent polypeptide chains to form intramolecular disulfide bonds. However, compounds and enzymes such as PDI that catalyze disulfide bonds become reduced and have to be reoxidized for further catalytic cycles. A number of enzymes, among them products of the ERO1 gene, appear to provide oxidizing equivalents, and oxygen appears to be the final oxidant in aerobic living organisms. Thus, protein oxidation in the ER is connected with generation of reactive oxygen species (ROS). Changes in the redox state and the presence of ROS also affect the Ca(2+) homeostasis by modulating the functionality of ER-based channels and buffering chaperones. In addition, a close relationship exists between oxidative stress and ER stress, which both may activate signaling events leading to a rebalance of folding capacity and folding demand or to cell death. Thus, redox homeostasis appears to be a prerequisite for proper functioning of the ER.

The liver has a multitude of functions which are necessary to maintain whole body homeostasis. This requires that various metabolic pathways can run in parallel in the most efficient manner and that futile cycles are kept to a minimum. To a large extent this is achieved due to a functional specialization of the liver parenchyma known as metabolic zonation which is often lost in liver diseases. Although this phenomenon is known for about 40 years, the underlying regulatory pathways are not yet fully elucidated. The physiologically occurring oxygen gradient was considered to be crucial for the appearance of zonation; however, a number of reports during the last decade indicating that β-catenin signaling, and the hedgehog (Hh) pathway contribute to metabolic zonation may have shifted this view. In the current review we connect these new observations with the concept that the oxygen gradient within the liver acinus is a regulator of zonation. This is underlined by a number of facts showing that the β-catenin and the Hh pathway can be modulated by the hypoxia signaling system and the hypoxia-inducible transcription factors (HIFs). Altogether, we provide a view by which the dynamic interplay between all these pathways can drive liver zonation and thus contribute to its physiological function.

The enormous number of different liver functions are carried out by parenchymal and four main types of nonparenchymal cells, either alone or in cooperation. Although the liver tissue is uniform on the level of histology, it is heterogenous on the level of morphometry and histochemistry. This heterogeneity is related to the blood supply; cells located in the upstream or periportal zone differ from those in the downstream or perivenous zone in their equipment with key enzymes, translocators, receptors, and subcellular structures and therefore have different functional capacities. This is the basis of the model of metabolic zonation, according to which glucose release from glycogen and via gluconeogenesis, amino acid utilization and ammonia detoxification, protective metabolism, bile formation, and the synthesis of certain plasma proteins such as albumin and fibrinogen occur mainly in the periportal area, whereas glucose utilization, xenobiotic metabolism, and the formation of other plasma proteins such as alpha 1-antitrypsin or alpha-fetoprotein occur predominantly in the perivenous zone. The mor- phologic and functional heterogeneity is the result of zonal differences in the activation of the cellular genome caused by gradients in oxygen, substrate, hormone, and mediator levels, in innervation, as well as in cell-to-cell and cell-to-biomatrix interactions.

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The oxygen sensitive ␣-subunit of the hypoxia-inducible factor-1 (HIF-1) is a major trigger of the cellular response to hypoxia. Although the posttranslational regulation of HIF-1␣ by hypoxia is well known, its transcriptional regulation by hypoxia is still under debate. We, therefore, investigated the regulation of HIF-1␣ mRNA in response to hypoxia in pulmonary artery smooth muscle cells. Hypoxia rapidly enhanced HIF-1␣ mRNA levels and HIF-1␣ promoter activity. Furthermore, inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT but not extracellular signal-regulated kinase 1/2 pathway blocked the hypoxia-dependent induction of HIF-1␣ mRNA and HIF-1␣ promoter activity, suggesting involvement of a PI3K/AKT-regulated transcription factor. Interestingly, hypoxia also induced nuclear factor-B (NFB) nuclear translocation and activity. In line, expression of the NFB subunits p50 and p65 enhanced HIF-1␣ mRNA levels, whereas blocking of NFB by an inhibitor of nuclear factor-B attenuated HIF-1␣ mRNA induction by hypoxia. Reporter gene assays revealed the presence of an NFB site within the HIF-1␣ promoter, and mutation of this site abolished induction by hypoxia. In line, gel shift analysis and chromatin immunoprecipitation confirmed binding of p50 and p65 NFB subunits to the HIF-1␣ promoter under hypoxia. Together, these findings provide a novel mechanism in which hypoxia induces HIF-1␣ mRNA expression via the PI3K/AKT pathway and activation of NFB.

Abstract-The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22 phox -containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-␤ 1 (TGF-␤ 1 ) upregulated HIF-1␣ protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1␣ as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1␣, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22 phox antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1␣, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22 phox -containing NADPH oxidase is crucially involved in this response. Key Words: oxygen radicals Ⅲ p22 phox Ⅲ platelets Ⅲ vascular endothelial growth factor Ⅲ plasminogen activator inhibitor-1