The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91 phox /Nox2, a membrane-integrated protein that forms a heterodimer with p22 phox to constitute flavocytochrome b 558 . The cytochrome becomes activated by interacting with the adaptor proteins p47 phox and p67 phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47 phox and p67 phox , designated p41 nox and p51 nox , respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41 nox interacts with p22 phox via the two tandem SH3 domains, as does p47 phox . The protein p51 nox as well as p67 phox can form a complex with p47 phox and with p41 nox via the C-terminal SH3 domain and binds to GTPbound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47 phox and p67 phox activate the phagocyte oxidase via the same interactions. Indeed, p41 nox and p51 nox are capable of replacing the corresponding classical homologue in activation of gp91 phox . Nox1, a homologue of gp91 phox , also can be activated in cells, when it is coexpressed with p41 nox and p51 nox , with p41 nox and p67 phox , or with p47 phox and p51 nox ; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41 nox is normally in an active state. Thus, the novel homologues p41 nox and p51 nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

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The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)Hdependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.