Nurit Hadad

The synergistic anti-inflammatory effects of lycopene, lutein, β-carotene, and carnosic acid combinations via redox-based inhibition of NF-κB signaling.

Inflammatory mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases; therefore much attention is focused on blunting their proinflammatory modes of action. The aims of the present research were to assess the effectiveness of combinations of carotenoids and phenolics, at concentrations that can be achieved in blood, to inhibit the release of inflammatory mediators from macrophages exposed to lipopolysaccharide (LPS) and to determine what the anti-inflammatory effect of the phytonutrient combinations was in an in vivo mouse model of peritonitis. Preincubation of mouse peritoneal macrophages with lycopene (1 μM) or Lyc-O-Mato (1 μM) and carnosic acid (2 μM), lutein (1 μM), and/or β-carotene (2 μM) 1h before addition of LPS for 24 h caused a synergistic inhibition of NO, prostaglandin E(2), and superoxide production derived from downregulation of iNOS, COX-2, and NADPH oxidase protein and mRNA expression and synergistic inhibition of TNFα secretion. We surmise that the anti-inflammatory action of the phytonutrient combinations used probably resides in their antioxidant properties, because they caused an immediate, efficient, and synergistic inhibition of LPS-induced internal superoxide production leading to a marked decrease in ERK and NF-κB activation. The anti-inflammatory effects of the selected phytonutrient combinations were also demonstrated in a mouse model of peritonitis: their supplementation in drinking water resulted in attenuation of neutrophil recruitment to the peritoneal cavity and in inhibition of inflammatory mediator production by peritoneal neutrophils and macrophages.

Nucleotide-binding sites in the voltage-dependent anion channel: Characterization and localization

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [α-32P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5′-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [α-32P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of ∼17- and ∼14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotide-binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.

Endothelial ICAM-1 Protein Induction Is Regulated by Cytosolic Phospholipase A2α via Both NF-κB and CREB Transcription Factors

The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A2α (cPLA2α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA2α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA2α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA2α (AS) that reduced cPLA2α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA2α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA2α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA2α activation, suggesting that NADPH oxidase acts upstream to cPLA2α. Attenuating cPLA2α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE2 that were essential for ICAM-1 upregulation. Inhibition of cPLA2α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser536 and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA2α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE2-mediated protein kinase A.

Reduction of cPLA2alpha overexpression: an efficient anti-inflammatory therapy for collagen-induced arthritis.

Cytosolic phospholipase A2α (cPLA2) plays an important role in the development of several inflammatory diseases. The aim of the present study is to determine whether inhibition of cPLA2 expression, using specific antisense oligonucleotides against cPLA2 (antisense), is efficient in reducing inflammation after its development. Two mouse models of inflammation were included in the study: thioglicolate peritonitis and collagen‐induced arthritis (CIA). The antisense was found to be specific and efficient in inhibiting cPLA2 expression and NADPH oxidase activity ex vivo in peritoneal phagocytes. Immunoblotting and immunohistochemistry analysis showed a significant elevation in cPLA2 expression in the inflamed joints of collagen‐induced arthritis mice localized in cell infiltrate, chondrocytes and the surrounding skin and skeletal muscle. Similarly, the cPLA2 metabolite, leukotriene B4, accumulated in the peritoneal cavity of mice with peritonitis. Inhibition of elevated cPLA2 expression after development of inflammation by intravenous administration of antisense resulted in a dramatic reduction in inflammation and a significant reduction in neutrophils recruitment to the site of inflammation in both mouse models of inflammation. Our results demonstrate the critical role of cPLA2 for the duration of inflammation and suggest that inhibition of cPLA2 expression by antisense oligonucleotides may serve as an efficient treatment of inflammatory diseases.

Regulatory role of cytosolic phospholipase A2α in NADPH oxidase activity and in inducible nitric oxide synthase induction by aggregated Aβ1–42 in microglia

In Alzheimers disease, extracellular deposits of amyloid β1–42 (Aβ1–42) may induce activation of microglial cells by releasing proinflammatory factors that contribute to the neurodegeneration process. Since the activation of cytosolic phospholipase A2α (cPLA2α) has been reported in inflammatory conditions, its role in primary rat microglial cell activated by aggregated Aβ1–42 was elucidated. The results of the present study show that activation of microglia by 5 μM aggregated Aβ1–42 (as evident by the amoeboid morphology and increased CD68 immunofluorescence reactivity) caused an immediate activation of cPLA2α, measured by its phosphorylated form and its specific activity, followed by a gradual elevation of its expression and activity during 24 h. Inhibition of cPLA2α expression and activity by the presence of 1 μM specific antisense resulted in a significant decrease in NADPH oxidase activity that releases superoxides, PGE2 formation, iNOS expression, and NO production, indicating a major role for cPLA2α in the regulation of these inflammatory processes. NADPH oxidase activity, which is under cPLA2α regulation, was found to upregulate cPLA2α and COX‐2 protein expression through the redox‐sensitive NFκB activation as evident by its phosphorylation on Ser‐536, resulting in increased PGE2 formation. The secreted PGE2 induced the synthesis of iNOS and the production of NO through the PKA‐CREB pathway. Taken together, our results suggest that the response of cPLA2α to aggregated Aβ1–42 is probably a key player in the oxidative stress present in AD, regulating potent oxidative agents: the production of superoxides by NADPH oxidase and NO formation by iNOS.

Induction of Cytosolic Phospholipase A2α Is Required for Adipose Neutrophil Infiltration and Hepatic Insulin Resistance Early in the Course of High-Fat Feeding

In established obesity, inflammation and macrophage recruitment likely contribute to the development of insulin resistance. In the current study, we set out to explore whether adipose tissue infiltration by neutrophils that occurs early (3 days) after initiating a high-fat diet (HFD) could contribute to the early occurrence of hepatic insulin resistance and to determine the role of cytosolic phospholipase A2α (cPLA2α) in this process. The 3-day HFD caused a significant upregulation of cPLA2α in periepididymal fat and in the liver. A specific antisense oligonucleotide (AS) effectively prevented cPLA2α induction, neutrophil infiltration into adipose tissue (likely involving MIP-2), and protected against 3-day HFD–induced impairment in hepatic insulin signaling and glucose over-production from pyruvate. To sort out the role of adipose neutrophil infiltration independent of cPLA2α induction in the liver, mice were injected intraperitoneally with anti–intracellular adhesion molecule-1 (ICAM-1) antibodies. This effectively prevented neutrophil infiltration without affecting cPLA2α or MIP-2, but like AS, prevented impairment in hepatic insulin signaling, the enhanced pyruvate-to-glucose flux, and the impaired insulin-mediated suppression of hepatic glucose production (assessed by clamp), which were induced by the 3-day HFD. Adipose tissue secretion of tumor necrosis factor-α (TNF-α) was increased by the 3-day HFD, but not if mice were treated with AS or ICAM-1 antibodies. Moreover, systemic TNF-α neutralization prevented 3-day HFD–induced hepatic insulin resistance, suggesting its mediatory role. We propose that an acute, cPLA2α-dependent, neutrophil-dominated inflammatory response of adipose tissue contributes to hepatic insulin resistance and glucose overproduction in the early adaptation to high-fat feeding.

Cytosolic Phospholipase A2α Is Targeted to the p47phox-PX Domain of the Assembled NADPH Oxidase via a Novel Binding Site in Its C2 Domain

We have previously demonstrated a physical interaction between cytosolic phospholipase A2α (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A(2)α dependent PGE(2)via both PKA and PKB pathways.

Cytosolic phospholipase A(2)α (cPLA(2)α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA(2)α which coincided with a significant increase in cell proliferation. The inhibition of cPLA(2)α activity by pyrrophenone or by antisense oligonucleotide against cPLA(2)α (AS) or inhibition of prostaglandin E(2) (PGE(2)) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE(2). The secreted PGE(2) activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE(2). But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE(2). AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA(2)α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA(2)α-dependent PGE(2) production. PGE(2)via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.

Cytosolic phospholipase A2α upregulation mediates apoptotic neuronal death induced by aggregated amyloid-β peptide1–42

Increased cytosolic phospholipase A2α (cPLA2α) immunoreactivity and transcript were observed in Alzheimers disease (AD) brain associated with amyloid deposits. Thus, the present study examined whether cPLA2α upregulation participate in cortical neuron damage induced by aggregated Aβ1-42 and determined its role in the signaling events leading to damage, using an antisense technology. Exposure of primary cortical neurons to 1μM aggregated Aβ1-42 for 24h induced up-regulation and activation of cPLA2α and apoptotic cell death of about 30% as detected by: cell count, MTT reduction, caspases-3 and -8 activation, DAPI and TUNEL staining, that were prevented by inhibition of cPLA2α up-regulation and activity in the presence of antisense against cPLA2α (AS). cPLA2α was rapidly activated upon addition of aggregated Aβ1-42, as determined by its phosphorylated form on serine 505, and this activity was dependent on NADPH oxidase activity. NOX2- and NOX4-NADPH oxidase upregulation at 24h of aggregated Aβ1-42 exposure was not affected by the presence of AS, but superoxide production was reduced, probably due to NOX2 inhibition. cPLA2α upregulation led to activation of neutral sphingomyelinase (N-SMase) as its activity was inhibited in the presence of AS, and could be restored by addition of arachidonic acid. Addition of ceramide analog induced caspase-8 activation leading to caspase-3 activation and apoptotic neuronal death. In conclusion, our results suggest that cPLA2α activity plays a crucial role in the signaling cascade leading to apoptotic neuronal death by aggregated Aβ1-42 probably via activation of N-SMase, ceramide production and caspases-3 and -8.

Direct Effect of Human Immunodeficiency Virus Protease Inhibitors on Neutrophil Function and Apoptosis via Calpain Inhibition

ABSTRACT Impairment of neutrophil functions and high levels of apoptotic neutrophils have been reported in human immunodeficiency virus (HIV) patients. The aim of the present study was to investigate the direct in vitro effects of the different HIV protease inhibitors (PIs) on neutrophil functions and apoptosis and to explore their mechanisms of action. The effects of nelfinavir (NFV), saquinavir (SQV), lopinavir (LPV), ritonavir (RTV), and amprenavir (APV) in the range of 5 to 100 μg/ml on neutrophil function, apoptosis, and μ-calpain activity were studied. The neutrophil functions studied included superoxide production stimulated by 5 ng/ml phorbol myristate acetate, 5 × 10−7 M N-formyl-methionyl-leucyl-phenylalanine, and 1 mg/ml opsonized zymosan; specific chemotaxis; random migration; and phagocytosis. Apoptosis was determined by DNA fragmentation, fluorescein isothiocyanate-annexin V binding, and nuclear morphology. All three neutrophil functions, as well as apoptosis, were similarly affected by the PIs. SQV and NFV caused marked inhibition and LPV and RTV caused moderate inhibition, while APV had a minor effect. μ-Calpain activity was not affected by the PIs in neutrophil lysate but was inhibited after its translocation to the membranes after cell stimulation. SQV, which was the most potent inhibitor of neutrophil functions and apoptosis, caused significant inhibition of calpain activity, while APV had no effect. The similar patterns of inhibition of neutrophil functions and apoptosis by the PIs, which coincided with inhibition of calpain activity, suggest the involvement of calpain activity in the regulation of these processes.