Lucia Rossetti Lopes

Regulation of NAD(P)H Oxidase by Associated Protein Disulfide Isomerase in Vascular Smooth Muscle Cells

NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22phox, Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22phox, Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.

Effect of fatty acids on leukocyte function

Fatty acids have various effects on immune and inflammatory responses, acting as intracellular and intercellular mediators. Polyunsaturated fatty acids (PUFAs) of the omega-3 family have overall suppressive effects, inhibiting lymphocyte proliferation, antibody and cytokine production, adhesion molecule expression, natural killer cell activity and triggering cell death. The omega-6 PUFAs have both inhibitory and stimulatory effects. The most studied of these is arachidonic acid that can be oxidized to eicosanoids, such as prostaglandins, leukotrienes and thromboxanes, all of which are potent mediators of inflammation. Nevertheless, it has been found that many of the effects of PUFA on immune and inflammatory responses are not dependent on eicosanoid generation. Fatty acids have also been found to modulate phagocytosis, reactive oxygen species production, cytokine production and leukocyte migration, also interfering with antigen presentation by macrophages. The importance of fatty acids in immune function has been corroborated by many clinical trials in which patients show improvement when submitted to fatty acid supplementation. Several mechanisms have been proposed to explain fatty acid modulation of immune response, such as changes in membrane fluidity and signal transduction pathways, regulation of gene transcription, protein acylation, and calcium release. In this review, evidence is presented to support the proposition that changes in cell metabolism also play an important role in the effect of fatty acids on leukocyte functioning, as fatty acids regulate glucose and glutamine metabolism and mitochondrial depolarization.

Updating the effects of fatty acids on skeletal muscle

In this review we updated the fatty acid (FA) effects on skeletal muscle metabolism. Abnormal FA availability induces insulin resistance and accounts for several of its symptoms and complications. Efforts to understand the pathogenesis of insulin resistance are focused on disordered lipid metabolism and consequently its effect on insulin signaling pathway. We reviewed herein the FA effects on metabolism, signaling, regulation of gene expression and oxidative stress in insulin resistance. The elevated IMTG content has been associated with increased intracellular content of diacylglycerol (DAG), ceramides and long‐chain acyl‐coenzyme A (LCA‐CoA). This condition has been shown to promote insulin resistance by interfering with phosphorylation of proteins of the insulin pathway including insulin receptor substrate‐1/2 (IRS), phosphatidylinositol‐3‐kinase, (PI3‐kinase) and protein kinase C. Although the molecular mechanism is not completely understood, elevated reactive oxygen (ROS) and nitrogen species (RNS) are involved in this process. Elevated ROS/RNS activates nuclear factor‐kappaB (NFkB), which promotes the transcription of proinflammatory tumoral necrosis factor alpha (TNFα), decreasing the insulin response. Therefore, oxidative stress induced by elevated FA availability may constitute one of the major causes of insulin resistance in skeletal muscle. J. Cell. Physiol. 217: 1–12, 2008.

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2.

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O(2)(•-)) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O(2)(•-) production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.

Novel Role of Protein Disulfide Isomerase in the Regulation of NADPH Oxidase Activity: Pathophysiological Implications in Vascular Diseases

Vascular cell NADPH oxidase complexes are key sources of signaling reactive oxygen species (ROS) and contribute to disease pathophysiology. However, mechanisms that fine-tune oxidase-mediated ROS generation are incompletely understood. Besides known regulatory subunits, upstream mediators and scaffold platforms reportedly control and localize ROS generation. Some evidence suggest that thiol redox processes may coordinate oxidase regulation. We hypothesized that thiol oxidoreductases are involved in this process. We focused on protein disulfide isomerase (PDI), a ubiquitous dithiol disulfide oxidoreductase chaperone from the endoplasmic reticulum, given PDIs unique versatile role as oxidase/isomerase. PDI is also involved in protein traffic and can translocate to the cell surface, where it participates in cell adhesion and nitric oxide internalization. We recently provided evidence that PDI exerts functionally relevant regulation of NADPH oxidase activity in vascular smooth muscle and endothelial cells, in a thiol redox-dependent manner. Loss-of-function experiments indicate that PDI supports angiotensin II-mediated ROS generation and Akt phosphorylation. In addition, PDI displays confocal co-localization and co-immunoprecipitates with oxidase subunits, indicating close association. The mechanisms of such interaction are yet obscure, but may involve subunit assembling stabilization, assistance with traffic, and subunit disposal. These data may clarify an integrative view of oxidase activation in disease conditions, including stress responses.

Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox‐dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref‐A, an ER‐Golgi‐disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild‐type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss‐ or gain‐of‐function experiments indicated that PMA‐driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi.

Protein disulfide isomerase redox-dependent association with p47phox: evidence for an organizer role in leukocyte NADPH oxidase activation

Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI‐mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell‐free system, PDI inhibitors scrRNase (100 μg/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by ∼40%), whereas PDIred diminished (by ∼60%) superoxide generation. No change occurred after incubation with PDI serine‐mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by ∼70%. Thus, oxidized PDI supports, whereas reduced PDI down‐regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol‐mediated interaction with p47phox in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47phox. PDI‐p47phox association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47phox in cytosol. Incubation of purified PDI (>80% reduced) and p47phox in vitro promoted their arachidonate‐dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re)cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox‐dependent enzyme complex organizer.

Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src–Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10−7 mol/L, 0–120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase–derived ROS and c-Src–dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs.

Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O⨪2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47 PHOX , a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O⨪2 production, we now show that p47 PHOX phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67 PHOX , Rac2, and phosphorylated p47 PHOX , suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O⨪2 production than activation by protein kinase C-phosphorylated p47 PHOX , indicating that a system that employs protein kinase C-phosphorylated p47 PHOX as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.

Endoplasmic Reticulum Stress and Nox-Mediated Reactive Oxygen Species Signaling in the Peripheral Vasculature: Potential Role in Hypertension

SIGNIFICANCE Reactive oxygen species (ROS) are produced during normal endoplasmic reticulum (ER) metabolism. There is accumulating evidence showing that under stress conditions such as ER stress, ROS production is increased via enzymes of the NADPH oxidase (Nox) family, especially via the Nox2 and Nox4 isoforms, which are involved in the regulation of blood pressure. Hypertension is a major contributor to cardiovascular and renal disease, and it has a complex pathophysiology involving the heart, kidney, brain, vessels, and immune system. ER stress activates the unfolded protein response (UPR) signaling pathway that has prosurvival and proapoptotic components. RECENT ADVANCES Here, we summarize the evidence regarding the association of Nox enzymes and ER stress, and its potential contribution in the setting of hypertension, including the role of other conditions that can lead to hypertension (e.g., insulin resistance and diabetes). CRITICAL ISSUES A better understanding of this association is currently of great interest, as it will provide further insights into the cellular mechanisms that can drive the ER stress-induced adaptive versus maladaptive pathways linked to hypertension and other cardiovascular conditions. More needs to be learnt about the precise signaling regulation of Nox(es) and ER stress in the cardiovascular system. FUTURE DIRECTIONS The development of specific approaches that target individual Nox isoforms and the UPR signaling pathway may be important for the achievement of therapeutic efficacy in hypertension.