Subject(s)
Allergy and Immunology/history , Anti-Infective Agents/immunology , Bacterial Infections/immunology , Leukocytes/immunology , Superoxides/immunology , Animals , Anti-Infective Agents/therapeutic use , Bacterial Infections/therapy , History, 20th Century , Humans , Oxidative Stress/immunology , Superoxides/therapeutic useABSTRACT
Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3zeta protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172) and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.
Subject(s)
14-3-3 Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Enzymologic , NADPH Oxidases/physiology , Adaptor Proteins, Signal Transducing , Cell Line, Tumor , Cell Membrane/metabolism , Cyclic AMP/metabolism , Cytosol/metabolism , Glutathione Synthase/metabolism , Humans , NADPH Oxidase 1 , NADPH Oxidases/metabolism , Phagocytes/metabolism , Phosphorylation , Reactive Oxygen Species , Time FactorsABSTRACT
OBJECTIVE: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation. METHODS: An approach was developed to delineate the cellular distribution of two selected oxidative enzymes, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidase (protein 67-kDa fraction). Immunolabeling with peroxidase substrate was utilized, which permits full delineation of the primary antibody in all microvascular structures of the mesentery. RESULTS: Xanthine oxidase is present in the endothelium of all segments of the microcirculation, in mast cells, and in parenchymal cells of the mesentery. NADPH oxidase can be detected in the endothelium, leukocytes, and mast cells and with lower levels in parenchymal cells. The mesentery of WKY and SHR has similar enzyme distributions with enhancements on the arteriolar and venular side of the microcirculation that coincide with the sites of enhanced free radical production recently reported. Immune label measurements under standardized conditions indicate that both enzymes are significantly enhanced in the SHR. Adrenalectomy, which serves to reduce the blood pressure and free radical production of the SHR to normotensive levels, leads to a reduction of NADPH and xanthine oxidase to normotensive levels, while supplementation of adrenalectomized SHR with dexamethasone significantly increases the oxidase expression in several parts of the microcirculation to levels above the WKY rats. CONCLUSION: The results indicate that enhanced expression of NADPH and xanthine oxidase in the SHR depends on an adrenal pathway that is detectable in the arteriolar and venular network at high and low pressure regions of the circulation.
Subject(s)
Hypertension/enzymology , Microcirculation/enzymology , NADPH Oxidases/metabolism , Xanthine Oxidase/metabolism , Adrenal Glands/physiology , Adrenalectomy , Animals , Dexamethasone/pharmacology , Free Radicals/metabolism , Leukocytes/enzymology , Male , Mesentery/blood supply , Mesentery/enzymology , Microcirculation/drug effects , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Tissue DistributionABSTRACT
The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.
Subject(s)
Down-Regulation , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Alanine/genetics , Animals , Cell Separation , Down-Regulation/genetics , Electrophoresis, Gel, Two-Dimensional , Enzyme Activation , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Mutagenesis, Site-Directed , Neutrophils/enzymology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphorylation , Protein Kinase C/metabolism , Rats , Serine/genetics , Sodium Dodecyl Sulfate/chemistry , Threonine/geneticsABSTRACT
NADPH oxidase is an enzyme that catalyzes the production of superoxide from oxygen and NADPH. It is a complex enzyme consisting of two membrane-bound components and three components in the cytosol, plus rac 1 or rac 2. Activation of the oxidase involves the phosphorylation of one of the cytosolic components. Recent crystallography data indicate that the tail of this cytosolic component lies in a groove between two Src homology 3 domains and, when phosphorylated, the tail leaves the groove and is replaced by the tail of one of the membrane-bound components. Chronic granulomatous disease is an inherited immune deficiency caused by the absence of one of the components of the oxidase. The most important recent advances in the field have been the crystallographic analysis of the oxidase and the use of antifungal agents in the prophylaxis of chronic granulomatous disease.
Subject(s)
Granulomatous Disease, Chronic/genetics , NADPH Oxidases/chemistry , Crystallography, X-Ray , Granulomatous Disease, Chronic/therapy , Humans , Models, Molecular , NADPH Oxidases/genetics , Protein Subunits/chemistry , Protein Subunits/genetics , src Homology DomainsABSTRACT
Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro. The steroids produced by cholesterol ozonolysis cause effects that are thought to be critical to the pathogenesis of atherosclerosis, including cytotoxicity, lipid-loading in macrophages, and deformation of the apolipoprotein B-100 secondary structure. We propose the trivial designation "atheronals" for this previously unrecognized class of steroids.
Subject(s)
Arteriosclerosis/metabolism , Carotid Arteries/metabolism , Cholestanes/metabolism , Cholesterol/metabolism , Norsteroids/metabolism , Ozone/metabolism , Sterols/metabolism , Cholestanes/blood , Cholestanes/pharmacology , Dimethyl Sulfoxide/pharmacology , Endarterectomy, Carotid , Foam Cells/drug effects , Foam Cells/physiology , Humans , Hydrazones/metabolism , Indigo Carmine/metabolism , Inflammation , Leukocytes/metabolism , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/pharmacology , Norsteroids/blood , Norsteroids/pharmacology , Oxidation-Reduction , Singlet Oxygen/metabolism , Sterols/blood , Sterols/pharmacology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O(2)(-) at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47(PHOX) occurs during the activation of the enzyme in intact cells. p47(PHOX) carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47(PHOX) is required for the activation of the oxidase. Earlier studies with both intact cells and a kinase-dependent, cell-free system have suggested that protein kinase C can phosphorylate those serines of p47(PHOX) whose phosphorylation is necessary for its activity. Work with inhibitors suggested that a phosphatidylinositol 3-kinase-dependent pathway also can activate the oxidase. Phosphorylation of p47(PHOX) by Akt (protein kinase B), whose activation depends on phosphatidylinositol 3-kinase, could be the final step in such a pathway. We now find that Akt activates the oxidase in vitro by phosphorylating serines S304 and S328 of p47(PHOX). These results suggest that Akt could participate in the activation of the leukocyte NADPH oxidase.
Subject(s)
NADPH Oxidases/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Base Sequence , DNA Primers , Enzyme Activation , Peptide Mapping , Phosphoproteins/chemistry , Phosphorylation , Proto-Oncogene Proteins c-aktABSTRACT
Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H(2)O(3)) and ozone (O(3)) from singlet oxygen ((1)O(2)(*)) and water. Given that neutrophils have the potential both to produce (1)O(2)(*) and to bind antibodies, we considered that these cells could be a biological source of O(3). We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O(3). This process is independent of surface antibody concentration down to 50% of the resting concentration, suggesting that surface IgG is highly efficient at intercepting the neutrophil-generated (1)O(2)(*). Vinylbenzoic acid, an orthogonal probe for ozone detection, is oxidized by activated neutrophils to 4-carboxybenzaldehyde in a manner analogous to that obtained for its oxidation by ozone in solution. This discovery of the production of such a powerful oxidant in a biological context raises questions about not only the capacity of O(3) to kill invading microorganisms but also its role in amplification of the inflammatory response by signaling and gene activation.
Subject(s)
Antibodies/metabolism , Isatin/analogs & derivatives , Neutrophils/immunology , Neutrophils/metabolism , Ozone/metabolism , Animals , Catalase/metabolism , Humans , In Vitro Techniques , Indigo Carmine/metabolism , Isatin/metabolism , Molecular Probes/metabolism , Oxidation-Reduction , Styrenes/metabolismABSTRACT
The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O2- (superoxide) at the expense of NADPH. The O2- then dismutes to H2O2, which serves to oxidize Cl- to HOCl, a potent microbicidal agent that is used by leukocytes to kill invading microorganisms. This oxidation is catalyzed by myeloperoxidase. O2 is also used to make other microbicidal oxidants, some in reactions with nitric oxide. The oxidase itself is highly complex, consisting of four unique subunits and Rac2. In the resting cell, two of the subunits, p22PHOX and gp91PHOX, are located in the membrane, and the other two, p47PHOX and p67PHOX, are in the cytosol. The electron-carrying components of the oxidase are located in gp91PHOX; the NADPH binding site is generally regarded to be in gp91PHOX as well, but there is some evidence that it may be in p67PHOX. When the oxidase is activated, p47PHOX is phosphorylated at specific sites, and the cytosolic components plus Rac2 migrate to the membrane to assemble the active oxidase.
Subject(s)
Leukocytes/enzymology , NADPH Oxidases/physiology , Nitric Oxide/physiology , Humans , Oxidation-ReductionABSTRACT
Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H2O2 production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.
Subject(s)
Antibodies, Catalytic/metabolism , Arthus Reaction/immunology , Escherichia coli/immunology , Inflammation/immunology , Neutrophils/metabolism , Ozone/metabolism , Animals , Antibodies, Catalytic/immunology , Arthus Reaction/metabolism , Blood Bactericidal Activity , Catalase/metabolism , Catalysis , Hematoporphyrins/metabolism , Humans , Hydrogen Peroxide/metabolism , Indigo Carmine/metabolism , Inflammation/metabolism , Mice , Neutrophil Activation , Neutrophils/immunology , Oxidation-Reduction , Rabbits , Rats , Rats, Sprague-Dawley , Respiratory Burst , Singlet Oxygen/metabolism , Ultraviolet Rays , Water/metabolismABSTRACT
Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3'-phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3'-phosphoinositides in vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3'-phosphoinositides in vivo. We show that the C2A domain of JFC1 is the only domain present in this protein that localizes to the plasma membrane in living cells. Moreover, the C2A domain of JFC1 binds 3'-phosphoinositides in vitro with similar specificity as that described for full-length JFC1, suggesting that the domain mediates the specific membrane localization of the full-length protein. Furthermore, the C2A domain of JFC1 colocalized with the pleckstrin homology domain of Akt in vivo, and both the JFC1 C2A domain and the full-length JFC1 dissociated from the membrane in the presence of PI 3-kinase specific inhibitors. We also show that the association of the C2A domain to the membrane is modulated by calcium. From these results we analyze possible mechanisms for the role of JFC1 in cellular trafficking.
Subject(s)
Membrane Proteins/metabolism , Phosphatidylinositols/metabolism , 3T3 Cells , Animals , Base Sequence , Binding Sites , Calcium/metabolism , Cell Membrane/metabolism , DNA Primers , Membrane Proteins/chemistry , Mice , Phosphatidylinositol 3-Kinases/metabolism , PhosphorylationABSTRACT
The human promoter region of JFC1, a phosphatidylinositol 3,4,5-trisphosphate binding ATPase, was isolated by amplification of a 549 bp region upstream of the jfc1 gene by the use of a double-PCR system. By primer extension analysis we mapped the transcription initiation site at nucleotide -321 relative to the translation start site. Putative regulatory elements were identified in the jfc1 TATA-less promoter, including three consensus sites for nuclear factor-kappaB (NF-kappaB). We analysed the three putative NF-kappaB binding sites by gel retardation and supershift assays. Each of the putative NF-kappaB sites interacted specifically with recombinant NF-kappaB p50, and the complexes co-migrated with those formed by the NF-kappaB consensus sequence and p50. An antibody to p50 generated a supershifted complex for these NF-kappaB sites. These sites formed specific complexes with nuclear proteins from tumour necrosis factor alpha (TNFalpha)-treated WEHI 231 cells, which were supershifted with antibodies against p50 and p65. The jfc1 promoter was transcriptionally active in various cell lines, as determined by luciferase reporter assays following transfection with a jfc1 promoter luciferase vector. Co-transfection with NF-kappaB expression vectors or stimulation with TNFalpha resulted in significant transactivation of the jfc1 promoter construct, although transactivation of a mutated jfc1 promoter was negligible. The expression of a dominant negative IkappaB (inhibitor kappaB) decreased basal jfc1 promoter activity. The cell lines PC-3, LNCaP and DU-145, but not Epstein-Barr virus-transformed lymphocytes, showed a dramatic increase in the expression of JFC1 after treatment with TNFalpha, suggesting that transcriptional activation of JFC1 by the TNFalpha/NF-kappaB pathway is significant in prostate carcinoma cell lines.
Subject(s)
Membrane Proteins/genetics , NF-kappa B/physiology , Prostatic Neoplasms/metabolism , Transcriptional Activation/physiology , Tumor Necrosis Factor-alpha/physiology , Up-Regulation/physiology , Base Sequence , Blotting, Western , Cloning, Molecular , DNA , Humans , Male , Molecular Sequence Data , Promoter Regions, Genetic , Prostatic Neoplasms/pathology , Recombinant Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H(2)O(2) potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(2)O(2) response. It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.
Subject(s)
Endothelial Growth Factors/metabolism , Hydrogen Peroxide/pharmacology , Keratinocytes/metabolism , Lymphokines/metabolism , Oxidants/pharmacology , Skin Physiological Phenomena , Wound Healing , Animals , Cell Line , Collagen/metabolism , Gene Deletion , Gene Transfer Techniques , Green Fluorescent Proteins , Humans , Immunoblotting , Luciferases/metabolism , Luminescent Proteins/metabolism , Male , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , NADPH Oxidases/metabolism , Neovascularization, Pathologic , Oxidation-Reduction , Phosphotyrosine/metabolism , Plasmids/metabolism , Precipitin Tests , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleases/metabolism , Skin/pathology , Time Factors , Transcriptional Activation , Transfection , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , rac1 GTP-Binding Protein/metabolismABSTRACT
The leukocyte NADPH oxidase is regulated chiefly by phosphorylation of the serines of p47(PHOX), one of its cytosolic subunits. Its activity is also regulated, however, by the four cysteines of the same subunit, as indicated by the replacement of those cysteines by alanines.
Subject(s)
Cysteine/metabolism , Gene Expression Regulation, Enzymologic , Leukocytes/enzymology , NADPH Oxidases/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Serine/metabolism , Alanine/metabolism , Cell-Free System , Cytosol/enzymology , Humans , Lymphocytes/metabolism , Mutation , Oxidation-Reduction , PhosphorylationABSTRACT
Activation of the phagocyte NADPH oxidase complex requires assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac with the membrane-bound cytochrome b558. We recently established a direct interaction between p67PHOX and cytochrome b558. In the present study, we show that removal of the C-terminal domain of p67PHOX increased its binding to cytochrome b558. Whereas phosphorylated p40PHOX alone did not bind to cytochrome b558, phosphorylated p47PHOX did, and, moreover, it allowed the binding of p40PHOX to the cytochrome. Furthermore, both increased the binding of p67PHOX) to the cytochrome. Phosphorylated p47PHOX thus appears to increase the binding of p67(PHOX) to cytochrome b558 by serving as an adapter, bringing p67PHOX into proximity with cytochrome b558, whereas phosphorylated p40(PHOX) may increase the binding by inducing a conformational change that allows p67PHOX to interact fully with cytochrome b558.