The hypoxia-inducible factor-2alpha (HIF-2alpha) contributes to the vascular response to hypoxia. Hypoxia inhibits prolyl hydroxylation of the N-terminal transactivation domain (N-TAD), thus preventing binding of the von Hippel-Lindau protein (pVHL) and proteasomal degradation; additionally, hypoxia inhibits asparagyl hydroxylation of the C-TAD, thus diminishing cofactor recruitment. Reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) have been shown to control vascular functions and to promote vascular remodeling. However, whether HIF-2alpha, ROS, and NOXs are linked under such nonhypoxic conditions is unclear. We found that activation of NOX4 by thrombin or H(2)O(2) increased HIF-2alpha protein because of decreased pVHL binding in pulmonary artery smooth muscle cells (PASMCs). Thrombin, H(2)O(2), and NOX4 overexpression increased HIF-2alpha N-TAD and C-TAD activity, which was prevented by ascorbate treatment or mutation of the hydroxylation sites in the TADs. HIF-2alpha also mediated induction of plasminogen activator inhibitor-1 and the proliferative response to thrombin, H(2)O(2), or NOX4 overexpression. Thus, ROS derived from NOX4 in response to thrombin stabilize HIF-2alpha by preventing hydroxylation of the N- and C-TAD, thus allowing formation of transcriptionally active HIF-2alpha, which promotes PASMC proliferation. Together, these findings present the first evidence that HIF-2alpha is critically involved in the ROS-regulated vascular remodeling processes.

HIF-1 (hypoxia-inducible factor-1) has been shown to essentially control the cellular response to hypoxia. Hypoxia stabilizes the inducible alpha-subunit, preventing post-translational hydroxylation and subsequent degradation via the proteasome. In recent years, clear evidence has emerged that HIF-1alpha is also responsive to many stimuli under normoxic conditions, including thrombin, growth factors, vasoactive peptides, insulin, lipopolysaccharide and cytokines such as TNF-alpha (tumour necrosis factor-alpha), and in many cases reactive oxygen species are involved. One important mechanism underlying these responses is the transcriptional regulation of HIF-1alpha by the redox-sensitive transcription factor NF-kappaB (nuclear factor kappaB), which binds at a distinct element in the proximal promoter of the HIF-1alpha gene. More recently, NF-kappaB binding to this site in the HIF-1alpha promoter has been shown also under hypoxic conditions. Thus these two major pathways regulating the responses to inflammation and oxidative stress on the one hand, and hypoxia on the other hand, appear to be intimately linked. In this issue of the Biochemical Journal, a study by van Uden et al. has supported these findings further, in which they have confirmed the binding of several proteins of the NF-kappaB family at the previously identified consensus site in the HIF-1alpha promoter and shown that TNF-alpha can also transcriptionally induce HIF-1alpha by this previously described pathway. The identification of HIF-1alpha as a target gene of NF-kappaB will have important implications for a variety of disorders related to hypoxia-ischaemia and/or inflammation and oxidative stress.

SummaryVascular remodelling isa complex phenomenon associated with restructuring of the vessel wall as a consequence of disruption of vascular homeostasis. Alterations of the vascular wall have been linked to a variety of cardiovascular disorders including atherosclerosis, vascular injury and pulmonary hypertension. Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin (serine proteinase inhibitor) family and acts as an important inhibitor of fibrinolysis by interfering with the plasminogen system. In addition to its anti-fibrinolytic effects, PAI-1 appears to modulate cellular responses linked to vascular remodelling. Since PAI-1 levels have been shown to be altered in various disorders associated with vascular remodelling of the systemic and pulmonary vascular bed, this serpin may playa pivotal role in the pathogenesis of these diseases.

The oxygen sensitive alpha-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-1alpha by hypoxia is well known, its transcriptional regulation by hypoxia is still under debate. We, therefore, investigated the regulation of HIF-1alpha mRNA in response to hypoxia in pulmonary artery smooth muscle cells. Hypoxia rapidly enhanced HIF-1alpha mRNA levels and HIF-1alpha 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-1alpha mRNA and HIF-1alpha promoter activity, suggesting involvement of a PI3K/AKT-regulated transcription factor. Interestingly, hypoxia also induced nuclear factor-kappaB (NFkappaB) nuclear translocation and activity. In line, expression of the NFkappaB subunits p50 and p65 enhanced HIF-1alpha mRNA levels, whereas blocking of NFkappaB by an inhibitor of nuclear factor-kappaB attenuated HIF-1alpha mRNA induction by hypoxia. Reporter gene assays revealed the presence of an NFkappaB site within the HIF-1alpha promoter, and mutation of this site abolished induction by hypoxia. In line, gel shift analysis and chromatin immunoprecipitation confirmed binding of p50 and p65 NFkappaB subunits to the HIF-1alpha promoter under hypoxia. Together, these findings provide a novel mechanism in which hypoxia induces HIF-1alpha mRNA expression via the PI3K/AKT pathway and activation of NFkappaB.

Objective - Reactive oxygen species have been implicated as signaling molecules modulating the activity of redox-sensitive transcription factors such as nuclear factor kappa B (NF-kappa B). Recently, the transcription factor hypoxia-inducible 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 H2O2 or by a NOX4-containing NADPH oxidase stimulated by thrombin activated or induced NF-kappa B and HIF-1 alpha. The reactive oxygen species-mediated HIF-1 alpha induction occurred on the transcriptional level and was dependent on NF-kappa B. Transfection experiments with wild-type or mutant HIF-1 alpha promoter constructs revealed the presence of a yet unidentified NF-kappa B binding element. Gel shift analyses and chromatin immunoprecipitation verified binding of NF-kappa B to this site. Furthermore, reactive oxygen species enhanced expression of plasminogen activator inhibitor-1, which was prevented by dominant-negative I kappa B 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 alpha and NF-kappa B, implicating an important pathophysiological role of this novel pathway in disorders associated with elevated levels of reactive oxygen species.

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 factorB (NF B) nuclear translocation and activity. In line, expression of the NF B subunits p50 and p65 enhanced HIF-1 mRNA levels, whereas blocking of NF B by an inhibitor of nuclear factorB attenuated HIF-1 mRNA induction by hypoxia. Reporter gene assays revealed the presence of an NF B 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 NF B 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 NF B.

The hypoxia inducible factors (HIF1, HIF2 and HIF3) are a family of transcription factors activated by low oxygen tension. They are essential for the normal development of the circulatory system. However, HIF activation in a number of pathological conditions is correlated with worse prognosis. The HIF proteins are heterodimers composed of a tightly regulated alpha subunit and a constituitively expressed beta subunit (also known as Aryl Nuclear Translocator protein, ARNT). HIF1α and HIF2α have been well characterised and shown to be regulated mostly at the protein level via oxygen dependent degradation under normoxia. At low oxygen levels the alpha subunit is stabilised and forms a heterodimer with ARNT. This is followed by DNA binding and formation of the transcription complex to bring about the gene expression. HIF3α is the last member of this family to be discovered and is structurally similar to HIF1α and HIF2α. Coimmunoprecipitation and bimolecular fluorescence assay showed that HIF3α is a novel interaction partner for both ARNT and HIF1α. Luciferase reporter gene assays showed that expression of HIF3α results in downregulation of HIF activity. Furthermore, endothelial cells expressing HIF3α showed decreased proliferation and reduced angiogenic activity under stimulation. HIF3α is therefore an inhibitory component of the HIF pathway. Interestingly, we found that non-hypoxic stimuli such as thrombin are also able to activate HIF1α. Luciferase reporter gene assays using deletion constructs from the HIF1α promoter revealed that an NFκB site was a major determinant for this upregulation. Since NFκB is an inflammatory mediator whose activity is related to oxidative stress, we investigated the link between these two transcription factors. Our studies showed that thrombin is able to increase HIF1α levels through a redox sensitive mechanism in which NFκB is activated and brings about expression of HIF1α by binding to its promoter. Together these studies show novel mechanisms by which the HIF pathway is regulated and may offer new therapeutic strategies to modify the pathological course of a number of diseases where HIF activation is undesirable.

approximately 200-250 words) Pulmonary hypertension is characterised by hypoxia, increased shear stress and thrombin levels which result in uncontrolled growth of endothelial cells, pulmonary artery smooth muscle cells and fibroblasts. Hypoxia Inducible Factor-1 (HIF1) is a transcription factor composed of α- and β- subunits which accumulates under hypoxic conditions and is responsible for many of the effects seen in pulmonary hypertension. Hypoxic regulation occurs mainly via protein stabilisation of the α-subunit (HIF1α). However we observed that non-hypoxic stimuli such as thrombin are also able to induce HIF1α accumulation in PASMC. We therefore decided to investigate the mechanism by which thrombin is able to upregulate HIF1α. Northern blot showed that HIF1α mRNA was increased in response to thrombin which was completely abrogated by pre-treatment with actinomycin-D, indicating that a transcriptional mechanism is involved. Treatment of cells with the antioxidants ascorbic acid or n-acetylcysteine also diminished response to thrombin indicating that upregulation was via a redox mediated pathway. Luciferase reporter gene assays using 1.2 kb sequence 5' to the HIF1a coding sequence (p12-Luc) showed increased activity in response to thrombin. Deletion constructs revealed that an NFkB site was a major determinant for this upregulation. Since NFκB is an inflammatory mediator whose activity is related to oxidative stress we decided to investigate this link further. Immunofluorescence showed that the p50 subunit of NFκB translocates to the nucleus in PASMC in response to thrombin. Its activity was also increased as measured by Luciferase activity. Moreover inhibition of NFκB not only decreased basal activity of the HIF1α promoter but also resulted in complete lack of induction by thrombin, which were also seen at rna and protein level. Mutagenesis of the NFκB binding site on the p12-Luc construct confirmed these results whilst gel shift analysis showed that NFκB is indeed able to bind this consensus sequence. We here show that thrombin is able to increase HIF1α levels through a redox sensitive mechanism in which NFκB is activated and brings about expression of HIF1α by binding to its promoter resulting in crosstalk between two major transcription factor pathways. This is a novel mechanism by which HIF1α is upregulated under non-hypoxic conditions.