Calcium is an important second messenger involved in intra- and extracellular signaling cascades and plays an essential role in cell life and death decisions. The Ca(2+) signaling network works in many different ways to regulate cellular processes that function over a wide dynamic range due to the action of buffers, pumps and exchangers on the plasma membrane as well as in internal stores. Calcium signaling pathways interact with other cellular signaling systems such as reactive oxygen species (ROS). Although initially considered to be potentially detrimental byproducts of aerobic metabolism, it is now clear that ROS generated in sub-toxic levels by different intracellular systems act as signaling molecules involved in various cellular processes including growth and cell death. Increasing evidence suggests a mutual interplay between calcium and ROS signaling systems which seems to have important implications for fine tuning cellular signaling networks. However, dysfunction in either of the systems might affect the other system thus potentiating harmful effects which might contribute to the pathogenesis of various disorders.

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

NADPH oxidases are important sources of reactive oxygen species (ROS), possibly contributing to various disorders associated with enhanced proliferation. NOX4 appears to be involved in vascular signaling and may contribute to the response to hypoxia. However, the exact mechanisms controlling NOX4 levels under hypoxia are not resolved. We found that hypoxia rapidly enhanced NOX4 mRNA and protein levels in pulmonary artery smooth-muscle cells (PASMCs) as well as in pulmonary vessels from mice exposed to hypoxia. This response was dependent on the hypoxia-inducible transcription factor HIF-1α because overexpression of HIF-1α increased NOX4 expression, whereas HIF-1α depletion prevented this response. Mutation of a putative hypoxia-responsive element in the NOX4 promoter abolished hypoxic and HIF-1α–induced activation of the NOX4 promoter. Chromatin immunoprecipitation confirmed HIF-1α binding to the NOX4 gene. Induction of NOX4 by HIF-1α contributed to maintain ROS levels after hypoxia and hypoxia-induced proliferation of PASMCs. These findings show that NOX4 is a new target gene of HIF-1α involved in the response to hypoxia. Together with our previous findings that NOX4 mediates HIF-1α induction under normoxia, these data suggest an important role of the signaling axis between NOX4 and HIF-1α in various cardiovascular disorders under hypoxic and also nonhypoxic conditions.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

HIF-1 (hypoxia-inducible factor-1) has been shown to essentially control the cellular response to hypoxia. Hypoxia stabilizes the inducible α-subunit, preventing post-translational hydroxylation and subsequent degradation via the proteasome. In recent years, clear evidence has emerged that HIF-1α is also responsive to many stimuli under normoxic conditions, including thrombin, growth factors, vasoactive peptides, insulin, lipopolysaccharide and cytokines such as TNF-α (tumour necrosis factor-α), and in many cases reactive oxygen species are involved. One important mechanism underlying these responses is the transcriptional regulation of HIF-1α by the redox-sensitive transcription factor NF-κB (nuclear factor κB), which binds at a distinct element in the proximal promoter of the HIF-1α gene. More recently, NF-κB binding to this site in the HIF-1α 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-κB family at the previously identified consensus site in the HIF-1α promoter and shown that TNF-α can also transcriptionally induce HIF-1α by this previously described pathway. The identification of HIF-1α as a target gene of NF-κB will have important implications for a variety of disorders related to hypoxia–ischaemia and/or inflammation and oxidative stress.