Nicotinamide adenine dinucleotide (NADPH) oxidases are a family of enzymes responsible for generating superoxide anions (O2•-) and other reactive oxygen species such as hydrogen peroxide (H2O2). They are found in various cell types [1]. The family of NADPH oxidases consists of 7 members, which can be grouped by their mode of activation (Figure 1).

The prototype NADPH oxidase in the first group is Nox2 [2]. Nox1, Nox2, and Nox3 assemble at the plasma membrane with cytosolic subunits. The multistep-activation process secures a controlled formation of O2•- and can be initiated by various stimuli, including growth factors, cytokines, and pathogenic molecules. It often involves signaling pathways mediated by protein kinases, such as protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs).

Nox5, Duox1, and Duox2 are Ca2+-activated NADPH oxidases. They produce either superoxide anions or hydrogen peroxide [3]. Nox5 acts as a homodimer, while Duox1 and 2 require maturation, the factors DuoxA1 and 2. Nox5 is absent in mice and other rodents, but present in humans with six gene splice variants, which differ in the sequence of their N-terminal cytoplasmic domains, comprising the four EF-hand motifs [4]. Ca2+-dependent association and dissociation between the N- and C-terminal cytoplasmic domains of the Nox5β variants depend on Ile-113 and Leu-115 between the third and fourth EF-hand motifs. Substitution of those amino acids by alanine impairs the cell surface localization of Nox5β, similar to what is seen with the Nox5ε/S variant, which lacks all EF-hand motifs and does not localize to the plasma membrane [5]. Recently, Nox5 and the Duoxes have been discovered to convert NAADPH into NAADP in T-cells [6]. It remains to be discovered if other NADPH oxidases can use NAADPH as a substrate as well.

The single member of the third group is Nox4. Nox4 is expressed in almost every differentiated cell and itself contributes to cellular differentiation. Recently, a study was published showing Nox4 promotes endothelial differentiation of induced pluripotent stem cells (iPSC) by an epigenetic mechanism involving JmjD6 and H3K27me3. Once differentiated, Nox4-deficient iPSC-derived endothelial cells are less stable undergo apoptosis and get easily dedifferentiated [7]. Such pro-differential and homeostasis stabilizing function is possible because Nox4 constitutively forms hydrogen peroxide at low concentrations. Nevertheless, Nox4 activity can be reduced by phosphorylation [8] and by modulating its association with p22phox. Binding of sirtuin 1 to p22phox prevents its association with Nox4 and promotes degradation of Nox4 [9], while hypoxia-mediated increase in Nox4 expression is a consequence of reduced lysosomal degradation of Nox4 [10]. Nox4 is localized intracellularly and may reside in the endoplasmic reticulum, in mitochondria, in the perinuclear membranes, or even in the nucleus [11].

Reactive oxygen species (ROS), including superoxide anions and hydrogen peroxide, serve as second messengers in cellular signaling. They can modulate the activity of protein kinases, transcription factors, and phosphatases, by oxidizing specific amino acid residues, including cysteine and methionine [12]. The oxidation of kinases can modulate their ability to interact with phosphatases. For example, Nox4-mediated oxidation of Akt secures its association with the protein phosphatase 2A (PP2A) and subsequent dephosphorylation [13]. Furthermore, NADPH oxidases can differentially alter the activity of transcription factors such as nuclear factor-kappa B (NFκB) and activator protein-1 (AP-1) [14]. For example, Nox2 is a potent inducer of NFκB, whereas Nox4 inhibits NFκB activity and expression [15]. Through this mechanism, NADPH oxidases are implicated, if not causal, in macrophage polarization and the subsequent feed-forward of inflammation through the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) [16].

The site of action of NADPH oxidases is a critical determinant of specific redox signaling [17]. Different isoforms of NADPH oxidases are localized to specific subcellular compartments, including the plasma membrane, endosomes, and the endoplasmic reticulum (ER).

All NADPH oxidases localized in the plasma membrane can modulate downstream signaling pathways of membrane bound receptors or the receptors themselves [18]. Superoxide dismutase converts extracellularly produced superoxide to hydrogen peroxide, which can re-enter the same or a neighboring cell and oxidize target proteins at the cell membrane, such as receptor tyrosine kinases [19] or associated components in a receptor complex. For example, ROS activate the alpha subunit of heterotrimeric GTP-binding proteins (Gα(i) and Gα(o)) [20]. At the same time, ROS can transiently inhibit the activity of phosphatases and thereby prolong ligand mediated signaling [21]. Migration can serve as a nice example. When vascular smooth muscle cells are stimulated with platelet derived growth factor (PDGF-BB), Nox1 derived ROS transiently inhibit PP2A, which would otherwise dephosphorylate atypical protein kinase C (aPKC). Through that mechanism, Nox1 forces anterior–posterior axis polarization based on prolonged persistence of Par3/Par6/aPKC complexes to modulate microtubule organizing center positioning microtubule stabilization and ultimately single lamellipodium formation, resulting in directed cell movement [22]. This fits very well to an older publication showing the migration of smooth muscle cells specifically relies on Nox1, while Nox4 has no impact on migration in those cells [23]. Different from receptor mediated ROS formation and phosphatase inhibition, Nox5 modulates cell migration is through its interaction with actin. Pharmacological inhibition of actin polymerization induces a rise in intracellular Ca2+ and Nox5-dependent superoxide production. In tandem, Nox5 oxidizes actin and modifies the ratio of filamentous and monomeric actin. As a consequence, knockdown of Nox5 in the pancreatic cancer cell line PSN-1 impairs cell migration [24].

NADPH oxidases at the plasma membrane can oxidize nearby ion channels, thereby increasing or decreasing their open probability, and the activity of the channel itself can improve the function of the NADPH oxidase [25]. This mode of direct action of ROS on ion channels is also used for intercellular communication. Pató et al. show that ROS is produced by Duox1 in keratinocytes can trans-activate sensory nerve terminals in the skin, thereby alter both the activity of neuronal transient receptor potential ankyrin repeat 1 (TRPA1) nonselective cation channel and of redox-sensitive Kv7.4 M-type potassium channel, expressed in the dorsal root ganglia, the primary sensory neurons. Accordingly, Duox1-deficient animals present with increased sensitivity towards certain noxious stimuli, including mustard oil-induced thermal hyperalgesia, heat injury-induced thermal, mechanical, and plantar incision-induced mechanical hyperalgesia on the paw and formalin-induced nociception [26].

Endosomes are membrane-born vesicles involved in various cellular processes, including internalization and trafficking of membrane-bound receptors and NADPH oxidases. At least for Nox1 and Nox2, endosomal translocation has been suggested. In fact, the originally established concept of Nox2 activation was its role within the neutrophil phagosomes [27]. Nox2-centered NADPH oxidase produces oxidants that support optimal antimicrobial activity by phagocytes [28]. The charge created by electron transfer would terminate NADPH oxidase activity within less than 100 ms and needs to be compensated. In phagocytes voltage-gated proton channel Hv1 compensates electron transfer and corrects charge inequity [29]. Formation of phagosomes represents a concept different from static localization of NADPH oxidases at the plasma membrane. Rather, it suggests a dynamic, site specific signaling mediated by NADPH oxidases. This concept continues in cytokine-induced translocation of NADPH oxidases, e.g. in trafficking of toll like receptor (TLR3, 8, and 9) from the endoplasmic reticulum to endosomes and subsequent signaling in the human monocytic cell line MonoMac1 (MM1). Endosomal Nox2 is needed for the early response of monocytes to stimulation with Poly I:C, R848, and CpG and induction of TNF-α mRNA and TNF-α secretion [30]. Different from monocytes, Nox1-mediated intra-endosomal ROS formation in response to TNFα-treatment of smooth muscle cells appears to be dependent on activity of the chloride-proton antiporter chloride channel-3 (ClC3), and activity of ClC3 is essential for TNFα-induced proliferation of those cells [31]. Accordingly, in those endosomes charge compensation is mediated by Clˉ outflow from Nox1, which contains endosomes.

The classic concept of plasma membrane born endosomes can be extended to endocytosis of extracellular vesicles, or exosomes. Bone marrow-derived macrophages can produce exosomes containing active Nox2 complexes. Those can be incorporated into the endosomes of injured dorsal route ganglions (DRGs) and are required for DRG neurite outgrowth [32]. Once incorporated, the Nox2-containing endosomes contribute to intracellular signaling pathways. The phosphatase and tensin homologue (PTEN) is oxidized and thereby inhibited, which subsequently activates the PI3K–Akt signaling pathway and forces DRG axonal outgrowth. In a similar manner, adipose tissue serves as an endocrine organ. Obese adipocytes can secrete exosomes that contain the constitutively active NADPH oxidase Nox4. Those, when injected into pregnant mice, can be taken up by the placentas of the animals, where they induce oxidative DNA damage with subsequent senescence of placental cells and reduced litter sizes [33]. Although not yet recognized as a general signaling pathway, the formation of NADPH oxidase containing exosomes and their incorporation into endosomes are at least involved in cell–cell communication and may contribute to inter-organ communications, e.g. the brain–heart or brain–gut axis.

The endoplasmic reticulum (ER) is a significant cellular organelle involved in protein synthesis, folding, and calcium storage. The NADPH oxidase Nox4 is localized in the ER [34], and H2O2 generated in the ER can affect several cellular processes related to protein homeostasis and calcium signaling [35]. Redox regulation of sarco- and endoplasmatic reticulum calcium ATPase (SERCA) by ER-localized NADPH oxidases can affect calcium handling in the ER, influencing calcium signaling pathways [36]. Further, hydrogen peroxide can modulate the activity of ER-resident proteins, including protein disulfide isomerases (PDIs) and ER chaperones. This redox regulation is crucial for the proper folding and maturation of proteins in the ER lumen and may regulate the ER stress response. ER bound Nox4 has been described to play a role in autophagy. Autophagy promotes cell survival by providing energetic substrates for the production of ATP and by favoring the turnover of damaged proteins and dysfunctional organelles. Two examples of Nox4 promote autophagy are RANKL-induced autophagy and osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 pathway [37] and autophagy of energy-deprived cardiomyocytes through suppression of prolyl hydroxylase 4 and subsequent activation of the protein kinase RNA-activated-like ER kinase pathway [38].

Besides Nox4, Duox2 is present at the ER, in intracellular structures reminiscent of trafficking vesicles, and occasionally at the plasma membrane, when co-localized with DuoxA2 [39]. The role of Nox5 in the ER remains to be defined. In vitro studies have confirmed that Nox5 promotes proliferation and survival and reduces apoptosis in several cancer cells. It plays a role in the vasculature, neurological diseases, and diabetes [40]. Whether or not all this relates to its potential ER localization is uncertain.

NADPH oxidases can directly and indirectly influence mitochondrial function and ROS production and vice versa. Silencing of Nox2 and subsequent upregulation of Nox4 have been shown to enhance glycolysis and oxidative phosphorylation rates, together with an enhanced production of mitochondrial ROS and a decrease in mitochondrial DNA copy number, indicating mitochondrial dysfunction [41]. By a mechanism called ROS-induced ROS release ROS formation by NADPH oxidases in response to a stimulus can be further increased by mitochondrial ROS formation in a feedforward fashion. ROS generated by NADPH oxidases may trigger the opening of redox-sensitive mitoKATP channels [42]. The increased potassium influx causes matrix alkalinization, swelling, and mild mitochondrial uncoupling and subsequent ROS production, which ultimately impair mitochondrial function [43]. Mitochondria-derived ROS then, can e.g. activate or prolong existing activity of the mitogen-activated protein kinase (MAPK) pathway [44].

An interesting speculation is that activation of NADPH oxidases by mitochondria may be based on mitochondrial chain uncoupling and subsequent increases in intracellular Ca2+ [45]. Ca2+ activates PKC, which phosphorylates cytosolic subunits of Nox1 and 2 and eventually results in increased ROS formation by those NADPH oxidases [46,47].