The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Abstract

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.

Fig. 1

The vascular NOX isoform-based NADPH oxidase complexes. Cell or subcellular compartment membranes are shown in gray, core proteins in yellow, activator binding proteins in green and organizer binding proteins in blue. All the NOX isoforms shown are membrane proteins and are localized in the plasma membrane (PM). Additionally, NOX1 was found at the plasma membrane in caveolae [147], NOX2 in membranes of phagosomes, and NOX4 in mitochondrial [182] and ER-membranes [191], as well as in the nucleus [97]. Little is known about subcellular localization of NOX5 other than the plasma membrane, but a localization at the ER membrane has been reported [29, 192]. NOX1, NOX2, and NOX4 are associated with p22^phox, but only NOX1 and NOX2 are regulated by the small GTPase Rac. For its activation, the NOX1 enzyme complex requires the assembly of NOX organiser 1 (NOXO1) and NOX activator 1 (NOXA1), but also forms complexes with p47^phox and p67^phox (not shown). The NOX2 enzyme complex requires binding of p47^phox, p67^phox, and optionally p40^phox that can further support the activity. In contrast to NOX1 and NOX2, NOX4 and NOX5 do not depend on any of the ‘classical’ cytosolic NADPH oxidase subunits. Recently, the protein polymerase (DNA-directed) delta-interacting protein 2 (Poldip2) was identified to bind and to increase the activity of NOX4. Further, protein disulfide isomerase (PDI) [23] and a p47^phox analogue tyrosine kinase substrate with 4/5 SH3 domains (Tks4/5) have been reported to bind and activate NOX1 and NOX4 [21, 22]. NOX4 is the only isoform that produces hydrogen peroxide instead of superoxide [17]. The NOX5 protein contains four N-terminal calcium-binding sites that regulate activation of the enzyme. Activity of NOX5 can be further supported by the binding of Hsp90 or Calmodulin to the C-terminus of the protein [24]

Fig. 2

Published NOX4 knock-out (KO) mouse models. a Wild-type NOX4 has six transmembrane helices and cytosolic binding domains for FAD and NADPH at the C-terminus. b Deletion of exons 1 and 2 should delete the complete NOX4 protein [32]. c Deletion of exon 4 only leaves the first transmembrane domain of NOX4. However, hypothetically, this may also result in the formation of a splice variant that contains both FAD and NADPH binding domains and thus has remaining ROS-forming activity [43]. d Another knock-out was generated by conditionally deleting exon 9 of NOX4 in cardiomyocytes, thereby deleting the FAD binding domain, likely leaving a non-functional enzyme [34]. e The fourth published NOX4 KO mouse was generated by deleting exons 14 and 15 that refer to the NADPH binding domain. This likely results in the expression of a non-functional enzyme [33]

Fig. 3

VAS2870 inhibits assembly of NADPH oxidases. NOX1 whole cell homogenates of CaCo-2 cells (native) were prepared and ROS measured as described in presence or absence of VAS3947 (30 μM) [59]. Columns represent means ± SEM of n = 3 experiments normalized to untreated controls. NOX2 membranes of human neutrophils, Rac-2-enriched cytosol fraction as well as recombinant p47^phox and p67^phox were treated with SDS to induce assembly of these subunits as described [71]. VAS2870 (55 μM) or a solvent control were added before (pre-) or after (post-) assembly of NOX2 with its subunits, and NADPH oxidase activity was measured using the cytochome c reduction assay as described [71]. Columns represent means ± SEM of n = 3 experiments normalized to untreated controls. NOX4 whole-cell homogenates of A7r5 cells (native), mainly expressing NOX4 compared to other NOX isoforms, were prepared and ROS measured as described in presence or absence of VAS3947 (30 μM) [59]. Columns represent means ± SEM of n ≥ 5 experiments normalized to solvent treated controls. Untransfected HEK293 cells (not shown) or HEK293 cells stably transfected with human NOX4 (overexpr.) were treated with VAS2870 or solvent control, and H₂O₂ release was measured using Amplex Red. Briefly, Amplex Red (20 μM) and horseradish peroxidase (100 mU/ml) in a phosphate buffer containing VAS2870 (10 μM) or equal volumes of solvent as control were incubated for 10 min at 37 °C in the dark in a 96-well plate. Then, 10⁵ native or human NOX4 overexpressing HEK293 cells were added to the wells, and fluorescence was recorded for 60 min in a Wallac Victor V (Perkin Elmer Life Sciences, Waltham, MA, USA) or Spectramax M2 (Molecular Devices, Sunnyvale, CA, USA) plate reader using 540/590 nm excitation/emission wavelength filters. Columns represent means ± SEM of the AUC of time-dependent fluorescence curves in quadruplicates of n = 4 experiments normalized to non-VAS treated HEK293-NOX4 cells. NOX5 L012 (100 μM) was used to measure NOX5 activity in HEK293 cells stably transfected with human NOX5 beta. VAS2870 (10 μM) or a solvent control was added to the cells in a 96-well plate, and basal chemiluminescence was recorded in a Victor V plate reader with 10 readings per well. Then, NOX5 was stimulated with phorbol myristate acetate (PMA, 1 μM) and the calcium ionophore ionomycin (1 μM), or HBSS as control was added (not shown), and chemiluminescence was measured for 20 readings per well for 60 min. Columns represent means ± SEM of AUC of time-dependent chemiluminescence in quadruplicates normalized to VAS2870 solvent control. (***p < 0.001, **p < 0.01 are significantly different from control values; n.s. p > 0.05 not significantly different from control values; 1-way ANOVA calculated with GraphPad Prism5 for each individual experiment)

Fig. 4

The role of NOX1, NOX2, and NOX4 in disease models. NO, generated by NO-synthases (NOS), activates soluble guanylate cyclase (sGC) by binding to its reduced (Fe²⁺) heme moiety leading to the formation of cGMP from GTP. cGMP mediates protective effects, e.g. vasodilation and anti-inflammation. This signaling pathway is most likely disturbed by NOX1-derived superoxide (O₂ ⁻) as shown in Angiotensin II-induced hypertension and spontaneous hypertensive rats (SHR). Superoxide can either directly interact with NO to form peroxynitrite or oxidize the essential NOS cofactor tetrahydrobioapterin (BH₄) and thus uncouple NOS. Uncoupled NOS forms superoxide itself (not shown). Further, superoxide can oxidize the Fe²⁺ heme of sGC. Thereby, sGC becomes insensitive to NO. These mechanisms most likely account, at least in part, for the acute effects of increased NOX1 activity mediating endothelial dysfunction and the chronic effects that are discussed to cause hypertension. NOX2-derived superoxide is a major signaling molecule in innate immunity mediating host defense. NOX4 is unlikely to directly interfere with the NO/cGMP-signaling pathway as it releases hydrogen peroxide (H₂O₂) and not superoxide. However, in high concentrations, H₂O₂ causes acute cytotoxicity. This mechanism is suggested to be involved in NOX4-mediated effects after acute ischemic stroke, acute effects of pressure overload in heart, and bleomycin-induced cytotoxicity. The lower chronic activity of NOX4 seems to be involved in angiogenesis and wound healing, and thus rather protective