Thomas L. Leto

Genetic, biochemical, and clinical features of chronic granulomatous disease.

The reduced nicotinamide dinucleotide phosphate (NADPH) oxidase complex allows phagocytes to rapidly convert O2 to superoxide anion which then generates other antimicrobial reactive oxygen intermediates, such as H2O2, hydroxyl anion, and peroxynitrite anion. Chronic granulomatous disease (CGD) results from a defect in any of the 4 subunits of the NADPH oxidase and is characterized by recurrent life-threatening bacterial and fungal infections and abnormal tissue granuloma formation. Activation of the NADPH oxidase requires translocation of the cytosolic subunits p47phox (phagocyte oxidase), p67phox, and the low molecular weight GT-Pase Rac, to the membrane-bound flavocytochrome, a heterodimer composed of the heavy chain gp91phox and the light chain p22phox. This complex transfers electrons from NADPH on the cytoplasmic side to O2 on the vacuolar or extracellular side, thereby generating superoxide anion. Activation of the NADPH oxidase requires complex rearrangements between the protein subunits, which are in part mediated by noncovalent binding between src-homology 3 domains (SH3 domains) and proline-rich motifs. Outpatient management of CGD patients relies on the use of prophylactic antibiotics and interferon-gamma. When infection is suspected, aggressive effort to obtain culture material is required. Treatment of infections involves prolonged use of systemic antibiotics, surgical debridement when feasible, and, in severe infections, use of granulocyte transfusions. Mouse knockout models of CGD have been created in which to examine aspects of pathophysiology and therapy. Gene therapy and bone marrow transplantation trials in CGD patients are ongoing and show great promise.

Amyloid-β Induces Chemotaxis and Oxidant Stress by Acting at Formylpeptide Receptor 2, a G Protein-coupled Receptor Expressed in Phagocytes and Brain

Amyloid-β, the pathologic protein in Alzheimers disease, induces chemotaxis and production of reactive oxygen species in phagocytic cells, but mechanisms have not been fully defined. Here we provide three lines of evidence that the phagocyte G protein-coupled receptor (N-formylpeptide receptor 2 (FPR2)) mediates these amyloid-β-dependent functions in phagocytic cells. First, transfection of FPR2, but not related receptors, including the other known N-formylpeptide receptor FPR, reconstituted amyloid-β-dependent chemotaxis and calcium flux in HEK 293 cells. Second, amyloid-β induced both calcium flux and chemotaxis in mouse neutrophils (which express endogenous FPR2) with similar potency as in FPR2-transfected HEK 293 cells. This activity could be specifically desensitized in both cell types by preincubation with a specific FPR2 agonist, which desensitizes the receptor, or with pertussis toxin, which uncouples it from Gi-dependent signaling. Third, specific and reciprocal desensitization of superoxide production was observed whenN-formylpeptides and amyloid-β were used to sequentially stimulate neutrophils from FPR −/− mice, which express FPR2 normally. Potential biological relevance of these results to the neuroinflammation associated with Alzheimers disease was suggested by two additional findings: first, FPR2 mRNA could be detected by PCR in mouse brain; second, induction of FPR2 expression correlated with induction of calcium flux and chemotaxis by amyloid-β in the mouse microglial cell line N9. Further, in sequential stimulation experiments with N9 cells, N-formylpeptides and amyloid-β were able to reciprocally cross-desensitize each other. Amyloid-β was also a specific agonist at the human counterpart of FPR2, the FPR-like 1 receptor. These results suggest a unified signaling mechanism for linking amyloid-β to phagocyte chemotaxis and oxidant stress in the brain.

Genetic Demonstration of p47phox-Dependent Superoxide Anion Production in Murine Vascular Smooth Muscle Cells

Background—Previous investigations provide evidence that an enzyme related to the phagocyte NADPH oxidase produces superoxide in the blood vessel wall. These data, however, are confounded by observations that both NADPH and NADH serve as substrates for superoxide production in vascular cells. To clarify this issue, we compared the superoxide-generating capabilities of vascular smooth muscle cells (VSMCs) derived from wild-type (p47phox+/+;ph agocyte ox idase) mice with those from mice that lack p47phox (p47phox−/−; “knockout”), an essential component of the phagocyte NADPH oxidase. Methods and Results—VSMCs were derived from aortic explants harvested from p47phox+/+ or p47phox−/− mice. VSMCs from p47phox+/+ but not those from p47phox−/− mice produced superoxide after stimulation by phorbol myristate acetate. Consistent with this, p47phox was detected only in p47phox+/+ VSMCs. p47phox-transduced p47phox−/− but not enhanced green fluorescent protein-transduced p47phox−/− VSMCs generated significant levels of superoxide after stimulation by angiotensin II or platelet-derived growth factor-BB (PDGF-BB). Enhanced expression of recombinant p47phox in p47phox-transduced p47phox−/− cells correlated with superoxide production in these cells. Conclusions—These data provide direct functional proof that an oxidase requiring the p47phox component mediates superoxide release from VSMCs in the blood vessel wall in response to angiotensin II or PDGF-BB.

Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation

Dual oxidases (Duoxl and Duox2) are plasma membrane‐targeted hydrogen peroxide generators that support extracellular hemoperoxidases. Duox activator 2 (Duoxa2), initially described as an endoplasmic reticulum resident protein, functions as a maturation factor needed to deliver active Duox2 to the cell surface. However, less is known about the Duoxl/ Duoxal homologues. We identified four alternatively spliced Duoxal variants and explored their roles in Duox subcellular targeting and reconstitution. Duoxl and Duox2 are functionally rescued by Duoxa2 or the Duoxal variants that contain the third coding exon. All active maturation factors are cotransported to the cell surface when coexpressed with either Duoxl or Duox2, consistent with detection of endogenous Duoxal on apical plasma membranes of the airway epithelium. In contrast, the Duoxa proteins are retained in the endoplasmic reticulum when expressed without Duox. Duoxl/Duoxala and Duox2/Duoxa2 pairs produce the highest levels of hydrogen peroxide, as they undergo Golgi‐based carbohydrate modifications and form stable cell surface complexes. Cross‐functioning pairs that do not form stable complexes produce less hydrogen peroxide and leak superoxide. These findings suggest Duox activators not only promote Duox maturation, but they function as part of the hydrogen peroxide‐generating enzyme.—Morand, S., Ueyama, T., Tsujibe, S., Saito, N., Korzeniowska, A., Leto, T. L. Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation. FASEB J. 23, 1205–1218 (2009)

Nox4 involvement in TGF-beta and SMAD3-driven induction of the epithelial-to-mesenchymal transition and migration of breast epithelial cells

The epithelial-to-mesenchymal transition (EMT) is the development of increased cell plasticity that occurs normally during wound healing and embryonic development and can be coopted for cancer invasion and metastasis. TGF-beta induces EMT but the mechanism is unclear. Our studies suggest that Nox4, a member of the NADPH oxidase (Nox) family, is a source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. We found that TGF-beta induces Nox4 expression (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells. Conversely, cells expressing a dominant-negative form of Nox4 or Nox4-targeted shRNA showed significantly lower ROS production on TGF-beta treatment. Expression of a constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein expression, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3 dependent based on the effect of constitutively active SMAD3 increasing Nox4 promoter activity, whereas dominant-negative SMAD3 or SIS3, a SMAD3-specific inhibitor, had the opposite effect. Furthermore, Nox4 knockdown, dominant-negative Nox4 or SMAD3, or SIS3 blunted TGF-beta induced wound healing and cell migration, whereas cell proliferation was not affected. Our experiments further indicate that Nox4 plays a role in TGF-beta regulation of fibronectin mRNA expression, based on the effects of dominant-negative Nox4 in reducing fibronectin mRNA in TGF-beta-treated MDA-MB-231and MCF10A cells. Collectively, these data indicate that Nox4 contributes to NADPH oxidase-dependent ROS production that may be critical for the progression of the EMT in breast epithelial cells, and thereby has therapeutic implications.

Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells.

Src homology 3 (SH3) domains mediate specific protein‐protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain‐containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3‐mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151–284), spanning both SH3 domains, was required for flavocytochrome‐dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N‐terminal domain (residues 1–246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain‐mediated interaction involving the C‐termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.

P47(phox)-deficient NADPH oxidase defect in neutrophils of diabetic mouse strains, C57BL/6J-m db/db and db/+.

Deficiencies in neutrophil NADPH oxidase proteins have been demonstrated in humans with chronic granulomatous disease. However, no spontaneous mutation in murine NADPH oxidase has been reported. In this study we report that neutrophils from the diabetic mouse strains, C57BL/6J‐m heterozygous lean (leprdb/+ ) and homozygous obese (leprdb/db) mice produced no superoxide on stimulation. An absence of intact p47phox but not other oxidase proteins was observed in both mouse strains through the use of immunoblotting. Molecular analysis by reverse transcriptase‐polymerase chain reaction identified three abnormal p47phox mRNA transcripts. Sequencing of genomic DNA of p47phox revealed a point mutation at the –2 position of exon 8, which is consistent with aberrant splicing of the p47phox transcript. These results indicate that the C57BL/6J‐m db/db and db/+ mice are the first spontaneously derived murine model of NADPH oxidase deficiency involving a p47phox mutation. J. Leukoc. Biol. 67: 210–215; 2000.

Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor β-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress

ABSTRACT Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor β (TGF-β) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an ∼2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-β1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-β or the expression of dominant negative TGF-β receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-β-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease.

Specificity of p47phox SH3 domain interactions in NADPH oxidase assembly and activation.

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47phox, p67phox, and p22phox). p47phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22phox and cytosolic p67phox. To understand the specific roles of each SH3 domain of p47phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr167 or Tyr237-->Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47phox-p22phox interaction and plays a predominant role in oxidase activity and p47phox membrane assembly, while the second p47phox SH3 domain interacts with the NH2-terminal domain of p67phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.

Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.