Subcellular Localization of Nox-Containing Oxidases Provides Unique Insight Into Their Role in Vascular Oxidant Signaling
Originally, reactive oxygen species (ROS) were thought to be key participants in cellular injury. However, it is rapidly becoming realized that many of the individual species, such as hydrogen peroxide, are important mediators in a diverse array of cellular signaling processes. These processes include physiological regulation associated with the cellular sensing of oxygen tension, forces derived from stretch and shear, the control of cellular growth, and death.1–4 One of the most active areas under investigation in cellular regulation is the subcellular localization and organization of signaling mechanisms. In the article in this issue of Atherosclerosis, Thrombosis, and Vascular Biology,5 Hilenski and colleagues provide the first report on the subcellular localization of the Nox-1 and Nox-4 subunit–containing NAD(P)H oxidases in vascular smooth muscle cells on caveolae and focal adhesions, respectively. These observations have important implications for the organization of oxidant signaling mechanisms thought to be involved in the control of fundamental physiological processes, including the role of Nox-1 expression in promoting cell growth3–5 and roles for Nox oxidase activation by p47phox subunit binding in the sensing of cellular stretch forces.6
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Redox Control by Each Nox Is Likely to Be Localized
Localization of ROS-producing oxidases influences the function of both the signaling mechanisms, which activate the specific oxidases, and the regulatory pathways they control. Because there appear to be specific mechanisms of activation for the individual oxidases that may depend on processes such as the binding of subunits including p47phox and rac,3–5 there need to be ways to control the activation mechanisms for these systems in the subcellular regions where the oxidase is localized. For example, it is not known whether the signaling systems that promote p47phox and rac binding are present at the sites where the Nox-containing oxidases are located or whether these subunits are controlled by a more generalized mechanism of cellular activation. Because caveolae and focal adhesion are thought to be important sites for the localization of components of signaling systems, the organization of proteins present at these sites is likely to be an important factor contributing to cellular function.
Second-messenger–type cellular regulatory systems are controlled by the local activities of enzymes that both generate and metabolize the second messenger and by the proximity of systems regulated by the second messenger. By analogy, the localized activities of oxidases generating ROS, enzymes, and/or antioxidants that metabolize ROS and the proximity of redox sensor sites modified by individual ROS that control signaling should determine responses that are observed. The diversity of redox-linked signaling systems influenced by Nox-derived ROS is likely to be the result of several important factors. The major species generated by the Nox oxidases appears to be superoxide anion, the 1-electron–reduced form of molecular oxygen. These oxidases will be an important source of hydrogen peroxide as a result of the superoxide dismutase (SOD) reaction and/or the enzymes that directly generate peroxide from a 2-electron reduction of oxygen. In addition to generating peroxide, superoxide anion has an extremely efficient rate of reaction with nitric oxide (NO) and with certain metal centers, such as iron-sulfur complexes, which can also influence the localized signaling that occurs. Local concentrations of the cytosolic Cu,Zn-form of SOD (SOD-1) and substances such as NO will determine which species are involved in signaling. The diffusion properties of NO as a dissolved gas allow sites of elevated superoxide production to become an active subcellular site of peroxynitrite (ONOO) formation and a source of activation of signaling processes that originate from the generation of ONOO and its derived species (eg, nitrogen dioxide). Thus, high local levels of SOD should shift signaling to peroxide-linked mechanisms because of prevention of the generation of other superoxide-derived species or the actions of superoxide itself on other signaling systems.
Peroxide that is derived from Nox-containing oxidases will be metabolized by enzymes including catalase, glutathione peroxidase, and other heme peroxidases. Localized aspects of the metabolism of peroxide by these enzymes are likely to have a major influence on the signaling systems that are regulated by ROS and redox. One aspect of the influence of peroxide metabolizing systems is that they will control the concentration gradient of peroxide in the subcellular region where it is produced. Another important factor is that many of the signaling mechanisms most sensitive to peroxide are regulated by processes that originate from its metabolism by specific peroxide-consuming enzymes.1,2 For example, the metabolism of peroxide by the heme peroxidase reaction of cyclooxygenase activates the metabolism of arachidonic acid to prostaglandins by this enzyme. The metabolism of peroxide by catalase can activate the production of cGMP by soluble guanylate cyclase. As the rate of metabolism of peroxide by glutathione peroxidases and thioredoxin peroxidases (peroxiredoxins) increases, a localized gradient of oxidized glutathione, thioredoxin, and NADP (by the glutathione and thioredoxin reductase reactions) is created. Changes in the redox status of each of these molecules are likely to have a major influence on the subcellular localization of signaling systems regulated by redox. When Nox-derived superoxide promotes the formation of reactive NO-derived species, these species will potentially have a localized influence on signaling both by promoting redox regulation (eg, glutathione oxidation) and by the direct modification of protein function through oxidation, nitrosation, or nitration reactions. Thus, Nox-derived ROS have many ways of regulating localized signaling systems through alterations in the concentration gradients of the species generated and cellular redox systems such as glutathione and NADPH.
Evidence for Localization of Specific Signaling Roles for Oxidases and ROS
Evidence already exists for the compartmentalization within vascular smooth muscle of signaling through different stimuli of ROS generation. For example, although exogenous peroxide and posthypoxic reoxygenation simultaneously activate both cGMP-associated relaxing and prostaglandin-mediated contracting mechanisms that appear to be endothelium-independent in human placental veins, acute exposure to lactate appears to selectively stimulate only the relaxing mechanism activated through the generation of peroxide.1,2 Stretch activates a Nox oxidase–derived peroxide-mediated enhancement of force generation in bovine coronary arteries through an src-dependent epidermal growth factor receptor activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. However, these arteries express relaxing mechanisms when exposed to posthypoxic reoxygenation, exogenous peroxide, and lactate that are more sensitive to peroxide than the ERK-mediated contractile mechanism.1,2,6 These examples suggest that localized sources of ROS generation within vascular smooth muscle cells appear to be tightly coupled to the selective activation of signaling systems that could be located in the region of the specific oxidases that are involved in controlling the response observed.
Potential Importance of the Subcellular Localization of Nox-1 and Nox-4
Hilenski and colleagues5 discuss some of the potential implications of Nox localization. For example, the presence of Nox-1 in caveolae could be involved in growth-promoting actions by angiotensin type-1 receptors. Activation of angiotensin type-1 receptors is associated with translocation to caveolae sites where Nox-1 activation potentially occurs through signaling linked to protein kinase C, Src-family kinases (src), receptor tyrosine kinases, and G proteins (rac; Figure). The localization of Nox-4 in focal adhesions and in the nucleus could be associated with integrin-linked signaling (eg, stretch) and aspects of gene expression (eg, growth, differentiation, inflammatory reactions, senescence, and apoptosis), respectively. Previous work from this group has provided evidence for an initial activation of Nox oxidases by protein kinase C (which phosphorylates the cytosolic p47phox subunit, promoting binding to Nox) and an amplification mechanism of sustained oxidase activation through a rac pathway that involves activation of src by ROS, a transactivation of the epidermal growth factor receptor, and phosphatidylinositol-3-kinase.7 Almost all aspects of the organization of localized systems controlling the activity of each form of Nox and the signaling processes linked to the ROS produced by these oxidases remain to be elucidated. Signaling linked to low levels of oxidase activation are likely to participate in functional responses linked to the sensing of stretch and O2 tension. More prolonged and/or extreme levels of oxidase activation are likely to control gene expression, growth, senescence, and apoptosis. Thus, the observation that Nox oxidases have specific subcellular localization sites provides a unique new insight into their role in multiple signaling processes.
Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol. 2000; 20: 1430–1442.
Taniyama T, Griendling KK. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension. 2003; 42: 1075–1081.
Lassègue B, Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol. 2003; 285: 277–297.
Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 677–683.
Oeckler RA, Kaminski PM, Wolin MS. Stretch enhances contraction of bovine coronary arteries via an NAD(P)H oxidase–mediated activation of the extracellular signal–regulated kinase mitogen-activated protein kinase cascade. Circ Res. 2003; 92: 23–31.
Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.