The family of NADPH oxidases (NOXes) has been considered unique in that their sole function is to generate superoxide or hydrogen peroxide, respectively, and that they are responsive to receptor stimulation
[115,9]. Up to date, this family comprises 7 members, which differ in their catalytic subunits as well as in the requirement of regulatory proteins. The initially identified NADPH oxidase contains the NOX2 core unit, which builds together with the p22phox subunit the cytochrome b558. It is also known as the “respiratory burst” enzyme of neutrophils and is a part of the innate immune response. Upon binding of particles, bacteria, fungi or soluble inflammatory mediators to specific receptors on the neutrophil cell surface, NOX2 is activated and mediates release of large amounts of ROS
[108]. This activation is regulated by cytosolic subunits p47phox, p67phox, p40phox and the Rac GTPase, which need to be phosphorylated by calcium activated protein kinase C (PKC) in order to translocate to the plasma membrane and join the NOX2/p22phox complex
[30].
The majority of neutrophil-activating receptors induce extracellular calcium entry as an early signaling response to activate effector functions, including phagocytosis, degranulation, and chemotaxis
[108]. These membrane receptors induce generation of inositol 1,4,5-trisphosphate (IP3) which activates IP3Rs and Ca2+ release from the intracellular stores which is important for phagocytosis
[137]. Depleted stores are reloaded by the sarco/endoplasmic reticulum Ca2+ ATPase SERCA, whereby calcium influx into the cell is enhanced through store-operated calcium channels
[23]. This Ca2+ influx is also required for neutrophil ROS generation by stimulating Ca2+-dependent recruitment of S100A8/A9 proteins which act as Ca2+ sensors and can interact with flavocytochrome b558 and p67phox to promote ROS generation
[25]. Moreover, Hv1 voltage-gated proton channels have been shown to extrude the protons and compensate the charge generated by NADPH oxidases, thereby enhancing the driving force for extracellular Ca2+ entry and sustaining NADPH oxidase activity
[46].
Similar to the NOX2 containing enzyme in neutrophils, NOX1 activity in keratinocytes has been described to be dependent on calcium in response to UVA light
[153]. NOX1 activity requires the recruitment of cytosolic activators similar to NOX2, suggesting that calcium might also act in resembling way. Moreover, it has been recently shown that NOX1 can directly be phosphorylated by the calcium activated PKCβ1 suggesting that calcium may via this way enhance NOX1 activity
[138].
Apart from these more indirect ways of calcium-dependent NOX activation, the NOX5 as well as the DUOX1 and DUOX2 containing enzymes have been shown to be calcium-binding proteins, which require calcium for ROS generation. NOX5 contains an N-terminal regulatory domain (called NOX5-EF) with four EF-hands. When Ca2+ binds to this domain, hydrophobic residues can interact with the C-terminal catalytic domain and activate the enzyme
[7]. Besides of EF-hands, NOX5 can bind calcium-activated calmodulin to the C-terminal domain, leading to a conformational change and increased N-terminal enzymatic activity. Furthermore calcium-activated calcium/calmodulin-dependent kinase II (CAMKII) can positively regulate NOX5 activity
via the phosphorylation of Ser475
[111]. Calcium-dependent NOX5 activity has been found to contribute to vascular proliferation and vessel formation
[10], to proliferation in different cancer cell lines
[3] and also might play a role in kidney disease
[76] and in coronary artery disease
[61].
Two other family members, dual oxidase 1 (DUOX1) and 2 (DUOX2) have been originally identified in the mammalian thyroid gland. DUOX1 is also highly expressed in airway epithelial cells and DUOX2 in the salivary glands and gastrointestinal tract. Dual oxidases contain an EF-hand calcium-binding cytosolic region similar to that in NOX5 and an N-terminal, extracellular domain with considerable sequence identity with mammalian peroxidases. DUOX enzymes are activated by calcium and release hydrogen peroxide rather than superoxide. In the thyroid, hydrogen peroxide produced by DUOX2 is utilized by thyroperoxidase as an electron acceptor to generate protein-bound iodothyronines (T3 and T4)
[109,27,88]. Recently, it was shown that epidermal wounding induces a calcium flash which activates hydrogen peroxide production via DUOX1 and subsequently the recruitment of immune cells to migrate to the wound
[122]. Similarly, calcium flashes have been shown to trigger DUOX-dependent hydrogen peroxide in zebrafish after mechanical injury, resulting in leukocyte recruitment
[107]. Genetic studies in Drosophila have demonstrated that DUOX can generate microbicidal ROS in the gut epithelia
[91].
Recent studies suggested a cross-talk between NADPH oxidases and mitochondrial ROS generation. For example, NOX2 was shown to stimulate mitochondrial ROS production by activating reverse electron transfer in angiotensin-II induced hypertension, while mitochondrial superoxide induced by activation of mitochondrial ATP-sensitive K+ channels has been demonstrated to stimulate NOX2, contributing to the development of endothelial oxidative stress and hypertension
[106,43]. Although the exact mechanisms of this cross-talk are not clear yet, these findings might explain some discrepancies found in the literature regarding the sources of ROS. Since both ROS generating systems are sensitive to calcium, they show the importance of the calcium-ROS cross-talk under (patho)physiological conditions.
