Distinct Subcellular Localizations of Nox1 and Nox4 in Vascular Smooth Muscle Cells

Abstract

Objective-Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91 phox , both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. Methods and Results-We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22 phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22phox , a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. , are important determinants of vascular function, acting not only as signaling intermediates to promote growth and differentiation 1 but also as modulators in pathological processes such as hypertension, atherosclerosis, and diabetic microvascular disease. 2 Major sources of ROS in the vascular wall are NAD(P)H oxidases, 3 multi-subunit enzymes that differ structurally and biochemically from the prototypical phagocyte NAD(P)H oxidase active in host defense. One structural difference is that vascular smooth muscle cells (VSMC) express two proteins, Nox1 and Nox4, 4 which are homologues to the gp91 phox catalytic subunit in phagocytes. 5 Biochemical differences include kinetics of activation, output, and regulation of NAD(P)H-dependent ROS production. 3 In contrast to the activated phagocyte oxidase, which produces large quantities of ROS into an extracellular (phagosomal) compartment in inducible bursts, Nox1 and Nox4 generate low-level, predominantly intracellular ROS constitutively and in response to agonists. 2 Conclusions-The See page 625 and coverAlthough Nox1 and Nox4 generate ROS, overexpression of these two homologues in the same fibroblast background suggests that Nox1 has mitogenic activity and is growthpromoting, 6 whereas Nox4 is implicated in cellular senescence. 7 In VSMC, Nox1 is inducible and upregulated by growth factors and hormones, whereas Nox4 is downregulated by these agonists. 8 Furthermore, in an animal model of restenosis in which O 2 ·Ϫ production is increased, Nox1 and Nox4 are induced at different times after carotid injury, 9 suggesting different functions of the two Nox proteins in redox-sensitive arterial remodeling.The differential expression and growth factor-related responses of multiple Nox proteins in VSMC imply distinct mechanisms of Nox1 and Nox4 regulation. This might involve specific signaling pathways such as the biphasic production of angiotensin II-mediate...

Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

“…There is ample evidence that more than one Nox exists in this cell type and that the different homologues are located in different compartments [34] and serve different functions. In rat VSMCs, Nox1 appears to be required for angiotensin II-and PDGF-induced ROS production and growth [22], and Nox4 has been shown to be important for the maintenance of a contractile, non-proliferative phenotype [23].…”

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“…100 Nox2, Nox4 and Nox5 seem to localize primarily in the perinuclear area associated with membranes on the endoplasmic reticulum and nucleus although Nox2 is also found in the plasma membrane within cholesterol-enriched domains, which may serve as signaling platforms for ROS generation in vascular disease. 101 Vascular smooth muscle cells possess Nox2 (in human resistance arteries) and Nox4, which are major sources of ROS. Nox1, present in low concentrations in basal states, is upregulated in disease.…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Additionally, NOX-mediated effects on e.g. cell proliferation or migration also depend on strict spatial localization of NOX activation in association with the cytoskeleton (70,71) or in membrane rafts (43,72,73), and on direct interactions with target proteins (3,43,55). This allows for effective oxidative signaling in the presence of abundant cytosolic antioxidant mechanisms (Cu/Zn SOD, GSH peroxidase), and assures confined oxidant production and target specificity.…”