Tetsuro Ago

NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart

NAD(P)H oxidases (Noxs) produce O2− and play an important role in cardiovascular pathophysiology. The Nox4 isoform is expressed primarily in the mitochondria in cardiac myocytes. To elucidate the function of endogenous Nox4 in the heart, we generated cardiac-specific Nox4−/− (c-Nox4−/−) mice. Nox4 expression was inhibited in c-Nox4−/− mice in a heart-specific manner, and there was no compensatory up-regulation in other Nox enzymes. These mice exhibited reduced levels of O2− in the heart, indicating that Nox4 is a significant source of O2− in cardiac myocytes. The baseline cardiac phenotype was normal in young c-Nox4−/− mice. In response to pressure overload (PO), however, increases in Nox4 expression and O2− production in mitochondria were abolished in c-Nox4−/− mice, and c-Nox4−/− mice exhibited significantly attenuated cardiac hypertrophy, interstitial fibrosis and apoptosis, and better cardiac function compared with WT mice. Mitochondrial swelling, cytochrome c release, and decreases in both mitochondrial DNA and aconitase activity in response to PO were attenuated in c-Nox4−/− mice. On the other hand, overexpression of Nox4 in mouse hearts exacerbated cardiac dysfunction, fibrosis, and apoptosis in response to PO. These results suggest that Nox4 in cardiac myocytes is a major source of mitochondrial oxidative stress, thereby mediating mitochondrial and cardiac dysfunction during PO.

Nox4 as the Major Catalytic Component of an Endothelial NAD(P)H Oxidase

Background—Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood. Methods and Results—Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro. Conclusions—These findings suggest that Nox4 may function as the major catalytic component of an endothelial NAD(P)H oxidase.

Upregulation of Nox4 by Hypertrophic Stimuli Promotes Apoptosis and Mitochondrial Dysfunction in Cardiac Myocytes

Rationale: NADPH oxidases are a major source of superoxide (O2−) in the cardiovascular system. The function of Nox4, a member of the Nox family of NADPH oxidases, in the heart is poorly understood. Objective: The goal of this study was to elucidate the role of Nox4 in mediating oxidative stress and growth/death in the heart. Methods and Results: Expression of Nox4 in the heart was increased in response to hypertrophic stimuli and aging. Neither transgenic mice with cardiac specific overexpression of Nox4 (Tg-Nox4) nor those with catalytically inactive Nox4 (Tg-Nox4-P437H) showed an obvious baseline cardiac phenotype at young ages. Tg-Nox4 gradually displayed decreased left ventricular (LV) function with enhanced O2− production in the heart, which was accompanied by increased apoptosis and fibrosis at 13 to 14 months of age. On the other hand, the level of oxidative stress was attenuated in Tg-Nox4-P437H. Although the size of cardiac myocytes was significantly greater in Tg-Nox4 than in nontransgenic, the LV weight/tibial length was not significantly altered in Tg-Nox4 mice. Overexpression of Nox4 in cultured cardiac myocytes induced apoptotic cell death but not hypertrophy. Nox4 is primarily localized in mitochondria and upregulation of Nox4 enhanced both rotenone- and diphenyleneiodonium-sensitive O2− production in mitochondria. Cysteine residues in mitochondrial proteins, including aconitase and NADH dehydrogenases, were oxidized and their activities decreased in Tg-Nox4. Conclusions: Upregulation of Nox4 by hypertrophic stimuli and aging induces oxidative stress, apoptosis and LV dysfunction, in part because of mitochondrial insufficiency caused by increased O2− production and consequent cysteine oxidation in mitochondrial proteins.

A redox-dependent pathway for regulating class II HDACs and cardiac hypertrophy.

Thioredoxin 1 (Trx1) facilitates the reduction of signaling molecules and transcription factors by cysteine thiol-disulfide exchange, thereby regulating cell growth and death. Here we studied the molecular mechanism by which Trx1 attenuates cardiac hypertrophy. Trx1 upregulates DnaJb5, a heat shock protein 40, and forms a multiple-protein complex with DnaJb5 and class II histone deacetylases (HDACs), master negative regulators of cardiac hypertrophy. Both Cys-274/Cys-276 in DnaJb5 and Cys-667/Cys-669 in HDAC4 are oxidized and form intramolecular disulfide bonds in response to reactive oxygen species (ROS)-generating hypertrophic stimuli, such as phenylephrine, whereas they are reduced by Trx1. Whereas reduction of Cys-274/Cys-276 in DnaJb5 is essential for interaction between DnaJb5 and HDAC4, reduction of Cys-667/Cys-669 in HDAC4 inhibits its nuclear export, independently of its phosphorylation status. Our study reveals a novel regulatory mechanism of cardiac hypertrophy through which the nucleocytoplasmic shuttling of class II HDACs is modulated by their redox modification in a Trx1-sensitive manner.

Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation

Protein–phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47phox, a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47phox, because protein kinase C treatment of the wild-type p47phox but not of a mutant protein with the S303/304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47phox nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions.

Modular domains mediating specific protein–protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67phox, which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40phox (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67phox, which abolishes its interaction with Cdc24p, confers on cells temperature‐ sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif‐containing region of p40phox, which restores the interaction with the altered Bem1p. This domain‐swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.

Solution structure of the PX domain, a target of the SH3 domain

The phox homology (PX) domain is a novel protein module containing a conserved proline-rich motif. We have shown that the PX domain isolated from the human p47phox protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 domain derived from the same protein. The solution structure of p47 PX has an α + β structure with a novel folding motif topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 domain. The proline-rich motif of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.

Postischemic Gene Transfer of Interleukin-10 Protects Against Both Focal and Global Brain Ischemia

Background—Gene therapy may be a promising approach for treatment of brain ischemia, although the efficiency of postischemic gene therapy is not established. Our goal in this study was to examine the effects of gene transfer of interleukin-10 (IL-10), an antiinflammatory cytokine, after induction of brain ischemia. Methods and Results—Brain ischemia was produced by either photochemical occlusion of distal middle cerebral artery for focal ischemia or bilateral carotid occlusion for global ischemia in spontaneously hypertensive rats. Adenoviral vectors encoding human IL-10 (AdIL10) or &bgr;-galactosidase (control) were injected into the lateral ventricle 90 or 60 minutes after focal or global ischemia. Five days after ischemia, IL-10, IL-1&bgr;, or tissue necrosis factor-&agr; in the cerebrospinal fluid, infarct volume, infiltrations of leukocytes/macrophages in the infarct area, or hippocampal neuronal damages were determined. The transduced IL-10 was released to the cerebrospinal fluid from the ventricular wall and increased to 7623±2965 pg/mL 5 days after AdIL10 transfection. Cerebral blood flow during ischemia was not different between treatments in either focal or global ischemia. Brain infarction of the AdIL10 group was significantly smaller and infiltrations of leukocytes and macrophages were fewer in the IL-10 treatment than control. Hippocampal neurons after global ischemia were more preserved, and the terminal deoxynucleotidyl transferase–mediated dUTP-biotin in situ nick end labeling–positive cells were diminished by the IL-10 gene transfer with attenuated IL-1&bgr; and augmented tissue necrosis factor-&agr;. Conclusions—Postischemic gene transfer of IL-10 into the lateral ventricle attenuated brain infarction and hippocampal damages, suggesting the promise for treatment of brain ischemia.

Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.

Regulation of myocardial growth and death by NADPH oxidase

The NADPH oxidases (Nox) are transmembrane proteins dedicated to producing reactive oxygen species (ROS), including superoxide and hydrogen peroxide, by transferring electrons from NAD(P)H to molecular oxygen. Nox2 and Nox4 are expressed in the heart and play an important role in mediating oxidative stress at baseline and under stress. Nox2 is primarily localized on the plasma membrane, whereas Nox4 is found primarily on intracellular membranes, on mitochondria, the endoplasmic reticulum or the nucleus. Although Nox2 plays an important role in mediating angiotensin II-induced cardiac hypertrophy, Nox4 mediates cardiac hypertrophy and heart failure in response to pressure overload. Expression of Nox4 is upregulated by hypertrophic stimuli, and Nox4 in mitochondria plays an essential role in mediating oxidative stress during pressure overload-induced cardiac hypertrophy. Upregulation of Nox4 induces oxidation of mitochondrial proteins, including aconitase, thereby causing mitochondrial dysfunction and myocardial cell death. On the other hand, Noxs also appear to mediate physiological functions, such as erythropoiesis and angiogenesis. In this review, we discuss the role of Noxs in mediating oxidative stress and both pathological and physiological functions of Noxs in the heart.