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Significance: G protein-coupled receptors (GPCR) are the largest group of cell surface receptors, which link cells to their environment. Reactive oxygen species (ROS) can act as important cellular signaling molecules. The family of NADPH oxidases generates ROS in response to activated cell surface receptors. Recent Advances: Various signaling pathways linking GPCRs and activation of NADPH oxidases have been characterized., Critical Issues: Still, a more detailed analysis of G proteins involved in the GPCR-mediated activation of NADPH oxidases is needed. In addition, a more precise discrimination of NADPH oxidase activation due to either upregulation of subunit expression or post-translational subunit modifications is needed. Also, the role of noncanonical modulators of NADPH oxidase activation in the response to GPCRs awaits further analyses., Future Directions: As GPCRs are one of the most popular classes of investigational drug targets, further detailing of G protein-coupled mechanisms in the activation mechanism of NADPH oxidases as well as better understanding of the link between newly identified NADPH oxidase interaction partners and GPCR signaling will provide new opportunities for improved efficiency and decreased off target effects of therapies targeting GPCRs.

Background: Cardiovascular diseases have been associated with stress in the endoplasmic reticulum (ER) and accumulation of unfolded proteins leading to the unfolded protein response (UPR). Reactive oxygen species (ROS) such as superoxide and H 2 O 2 derived from NADPH oxidases have been implicated in the pathogenesis of cardiovascular diseases. ROS have also been associated with ER stress. The role NADPH oxidases in the UPR is, however, not completely resolved yet., Aim: In this study, we investigated the role of p22phox, an essential component of most NADPH oxidases, in the UPR of endothelial cells., Results: Induction of ER stress increased p22phox expression at the transcriptional level. p22phox was identified as novel target of the UPR transcription factor ATF4 (activator of transcription factor 4) under ER stress conditions by promoter analyses and ChIP. Depletion of ATF4 and p22phox diminished the levels of superoxide and H 2 O 2 under ER stress conditions. On the contrary, p22phox was instrumental in increasing eIF2α phosphorylation and subsequent ATF4 expression on induction of ER stress by chemicals, oxysterols, or severe hypoxia in vitro and in vivo, leading to increased expression of CHOP and activation of effector caspases., Innovation: p22phox is a novel target of ATF4 in response to ER stress, which can promote the PERK-ATF4 branch of the UPR in vitro and in vivo., Conclusion: p22phox-dependent NADPH oxidases are important mediators of ER stress driving the UPR.

Aims: Hypoxia and reactive oxygen species (ROS) have been shown to play a role in the pathogenesis of pulmonary hypertension (PH), a potentially fatal disorder characterized by pulmonary vascular remodeling, elevated pulmonary arterial pressure, and right ventricular hypertrophy. However, how they are linked in the context of PH is not completely understood. We, therefore, investigated the role of the NADPH oxidase subunit p22phox in the response to hypoxia both in vitro and in vivo., Results: We found that hypoxia decreased ubiquitinylation and proteasomal degradation of p22phox dependent on prolyl hydroxylases (PHDs) and the E3 ubiquitin ligase protein von Hippel Lindau (pVHL), which resulted in p22phox stabilization and accumulation. p22phox promoted vascular proliferation, migration, and angiogenesis under normoxia and hypoxia. Increased levels of p22phox were also detected in lungs and hearts from mice with hypoxia-induced PH. Mice harboring a point mutation (Y121H) in the p22phox gene, which resulted in decreased p22phox stability and subsequent loss of this protein, were protected against hypoxia-induced PH. Mechanistically, p22phox contributed to ROS generation under normoxia, hypoxia, and hypoxia/reoxygenation. p22phox increased the levels and activity of HIF1α, the major cellular regulator of hypoxia adaptation, under normoxia and hypoxia, possibly by decreasing the levels of the PHD cofactors ascorbate and iron(II), and it contributed to the downregulation of the tumor suppressor miR-140 by hypoxia., Innovation: These data identify p22phox as an important regulator of the hypoxia response both in vitro and in vivo., Conclusion: p22phox-dependent NADPH oxidases contribute to the pathophysiology of PH induced by hypoxia.

Pulmonary vascular remodeling associated with pulmonary hypertension is characterized by media thickening, disordered proliferation, and in situ thrombosis. The p21-activated kinase-1 (PAK-1) can control growth, migration, and prothrombotic activity, and the hypoxia-inducible transcription factor HIF-1alpha was associated with pulmonary vascular remodeling. Here we studied whether PAK-1 and HIF-1alpha are linked in pulmonary vascular remodeling. PAK-1 was expressed in the media of remodeled pulmonary vessels from patients with pulmonary vasculopathy and was upregulated, together with its upstream regulator Rac1 and HIF-1alpha in lung tissue from lambs with pulmonary vascular remodeling. PAK-1 and Rac1 were activated by thrombin involving calcium, thus resulting in enhanced generation of reactive oxygen species (ROS) in human pulmonary artery smooth muscle cells (PASMCs). Activation of PAK-1 stimulated HIF activity and HIF-1alpha expression involving ROS and NF-kappaB, enhanced the expression of the HIF-1 target gene plasminogen activator inhibitor-1, and stimulated PASMC proliferation. Importantly, HIF-1 itself bound to the Rac1 promoter and enhanced Rac1 and PAK-1 transcription. Thus, PAK-1 and its activator Rac1 are novel HIF-1 targets that may constitute a positive-feedback loop for induction of HIF-1alpha by thrombin and ROS, thus explaining elevated levels of PAK-1, Rac1, and HIF-1alpha in remodeled pulmonary vessels.

Reactive oxygen species (ROS) have been implicated in many intra- and intercellular processes. High levels of ROS are generated as part of the innate immunity in the respiratory burst of phagocytic cells. Low levels of ROS, however, are generated in a highly controlled manner by various cell types to act as second messengers in redox-sensitive pathways. A NADPH oxidase has been initially described as the respiratory burst enzyme in neutrophils. Stimulation of this complex enzyme system requires specific signaling cascades linking it to membrane-receptor activation. Subsequently, a family of NADPH oxidases has been identified in various nonphagocytic cells. They mainly differ in containing one out of seven homologous catalytic core proteins termed NOX1 to NOX5 and DUOX1 or 2. NADPH oxidase activity is controlled by regulatory subunits, including the NOX regulators p47phox and p67phox, their homologs NOXO1 and NOXA1, or the DUOX1 or 2 regulators DUOXA1 and 2. In addition, the GTPase Rac modulates activity of several of these enzymes. Recently, additional proteins have been identified that seem to have a regulatory function on NADPH oxidase activity under certain conditions. We will thus summarize molecular pathways linking activation of different membrane-bound receptors with increased ROS production of NADPH oxidases.

Increased levels of reactive oxygen species (ROS) contribute to many cardiovascular diseases. In neutrophils, ROS are generated by a NADPH oxidase containing p22phox and NOX2. NADPH oxidases are also major sources of vascular ROS. Whereas an active NOX2-containing enzyme has been described in endothelial cells, the contribution of recently identified NOX homologues to endothelial ROS production and proliferation has been controversial. The authors, therefore, compared the role of NOX2 with NOX4 and NOX1 in endothelial EaHy926 and human microvascular endothelial cells. NOX2 and NOX4 were abundantly expressed, whereas NOX1 expression was less prominent. NOX2, NOX4, and NOX1 were simultaneously present in a single cell in a perinuclear compartment. NOX2 and NOX4 co-localized with the endoplasmic reticulum (ER) marker calreticulin. Additionally, NOX2 co-localized with F-actin at the plasma membrane. NOX2 and NOX4, which interacted with p22phox, as was shown by bimolecular fluorescent complementation, contributed equally to endothelial ROS production and proliferation, whereas NOX1 depletion did not alter ROS levels under basal conditions. These data show that endothelial cells simultaneously express NOX2, NOX4, and NOX1. NOX2 and NOX4, but not NOX1, equally contributed to ROS generation and proliferation under basal conditions, indicating that a complex relation between NOX homologues controls endothelial function.