4-Hydroxynonenal (HNE) is an important product of lipid peroxidation that has been extensively studied as a mediator of the cytotoxic and genetic effects of oxidative stress. HNE adducts to protein have been detected in vitro and in vivo using monoclonal antibodies raised against certain HNE adducts. Among the proteins modified in vivo by HNE are the heat shock proteins, Hsp90 and Hsp70, and the chaperone, PDI. A complete inventory of HNE-modified proteins has not been recorded because of the difficulty in detecting adducts that are structurally distinct from those for which antibodies are available. We have synthesized azido and alkynyl derivatives of HNE that exhibit identical biological activities to HNE and which react with proteins to form similar types of adducts. The protein adducts formed from azido- or alkynyl-HNE can be conjugated to biotin using a modified Staudinger ligation or Click chemistry. This enables the recovery of all protein adducts regardless of their structure. Using this approach, we have detected a dose-responsive increase in protein adduction in colon cancer cells treated with HNE over a range of concentrations that induce changes in gene expression, necrosis, and apoptosis. The pattern of protein adduction helps to explain the changes in cell signaling induced by treatment of colon cancer ells with HNE.
Palabras clave: 4-Hydroxynonenal, Protein adducts, Mass spectrometry
Nitrolipids constitute a new class of lipid mediators acting on cell signaling that have been detected in biological fluids and tissues. Nitrated fatty acids can act by •NO-dependent and independent mechanisms affecting signal transduction pathways potentially important for inflammation, angiogenesis and atherogenesis. Atherosclerosis is a chronic immune-inflammatory disease in which the inhibition of inflammatory pathways can be beneficial. The anti-inflammatory role of nitrolinoleic acid (LNO2) has been shown in various in vitro and in vivo studies. We have shown by intravital microscopy assays in rat mesentery microcirculation that LNO2 diminished the migration of leukocytes in response to pro-inflammatory stimuli, as well as their rolling and adhesion to endothelium. Moreover, the expression of B2-integrin and ICAM-1 in leukocytes and endothelial cells isolated from microcirculation of Wistar rats was also inhibited by LNO2. Neovascularization has been suggested as acausative factor for atherosclerotic plaque growth and destabilization. In contrast to the inhibitory effects on inflammation, LNO2 stimulated angiogenesis as demonstrated by in vitro, ex vivo and in vivo assays. LNO2 enhanced human umbilical endothelial cells (HUVEC) migration, endothelial cell sprouting from murine aortic rings and angiogenesis in chick embryo chorioallantoic membrane. This pro-angiogenic action of LNO2 may have contributed to the absence of anti-atherogenic effects observed in the LDL receptor knock out mice. In this experimental model of atherosclerosis, LNO2 did not decrease the cross-sectional area of atherosclerotic lesions analyzed in the aortic root compared to linoleic acid. Thus, although nitrolinoleic acid has potent anti-inflammatory effects in rat microvasculature, it induces angiogenesis what could positively modulate atherogenesis in LDLr-/- mice. Further studies are necessary to elucidate the effects of LNO2 in other experimental models of atherosclerosis, as well as, to investigate the actions of different nitrated fatty acids on angiogenesis and atherogenesis. Financial Support: FAPESP, CAPES, CNPQ/Instituto do Milênio REDOXOMA
We are using a tandem MS approach to characterize esterified eicosanoids that form in activated immune cells. This approach has revealed a new class of 12/15-lipoxygenase (LOX) products that form in monocytes and macrophages following ionophore activation, that consist of specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines (PE-HETES). Using H218O water, the compounds were shown to form by direct oxidation of endogenous PE by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE, and increased significantly, in response to ionophore, collagen or convulxin. These products are retained in the cells, in contrast to free acids which are primarily secreted, their synthesis is regulated by PI3 kinase, PKC and Ca2+, and they are actively metabolized by the cells within 3 hr. Precursor scanning of platelet extracts for the major platelet derived prostanoid, thromboxane B2 (m/z 369.2) did not reveal PE-esters, indicating that this modification is restricted to the LOX pathway. We have now synthesized and chemically characterized one of these (18:0a/15-HETE-PE) and biological studies are underway in vitro and in vivo. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.
Arachidonic acid (AA) is the substrate for prostaglandin endoperoxide H synthase (PGHS) which is involved in eicosanoid metabolism. We recently reported a differential modulation of cyclooxygenase (COX) and peroxidase (POX) activities of PGHS-1 by peroxynitrite and nitric oxide (·NO) due to their reactions with AA radicals formed during PGHS catalytic cycle, redirecting AA-dependent cell signaling pathways. Nitrated lipids (nitroalkenes) have been detected in cell membranes and human plasma under oxidative/nitrative conditions. We have synthesized and characterized key isomers of nitroarachidonate AANO2) and demonstrated their ability to modulate inducible nitric oxide synthase (NOS2) expression during macrophage activation. Herein, we evaluate a) the modulatory role that AANO2 plays on PGHS-1 activity and b) the potential formation of AANO2 during COX catalytic cycle which would exert key anti-inflammatory actions. Chemically-synthesized AANO2 inhibited both COX (IC50~ 2 mM) and POX (Ki~ 130 mM). For COX-mediated AA nitration, peroxynitrite and/or ·NO were incubated at low oxygen tensions to favor their reactions with alkyl radicals, followed by Bligh and Dyer lipid extraction. Then, nitrated products were analyzed by LC/MS/MS and structures compared to the chemically-synthesized nitroalkenes. Next, the role of AANO2 on both PGHS-2 and NOS2 induction in activated macrophages was tested. In contrast to NOS2, AANO2 had no effect on PGHS-2 expression as determined by immunocytochemistry and western blot. Moreover, AANO2 treatment significantly decreased aortic lesion formation in a cholesterol-fed LDL receptor negative mice model of atherosclerosis. The involvement of the anti-inflammatory Nrf2 pathway in this process was suggested by following the induction of heme oxygenase 1 expression in macrophages and within the plaque. All together our data reveals that AANO2 is an enzymatically formed novel adaptative mediator of inflammatory responses.
Phase II enzymes are induced in response to acrolein, an environmental toxicant and component of tobacco smoke. While activation of Nrf2 and its translocation to the nucleus is common for almost all phase II enzymes, Nrf2 is not always involved and it needs to pair with another transcription factor to activate transcription. Thus, we proposed that signaling pathways vary among phase II genes. We used inhibitors of protein kinases, which are key enzymes in the signaling pathways to explore how five phase II genes, the catalytic and modulatory subunits of glutamate cystine ligase (GCLC and GCLM), NAD(P)H quinone oxidoreductase 1 and 2 (NQO1 and NQO2), and heme oxygenase-1 (HO-1), are affected in human bronchial epithelial cells with and without exposure to acrolein. HBE1 cells were pretreated with inhibitors that have been shown to be relatively specific for protein kinases before exposure to 20 μM acrolein.
The mRNA levels of GCLC, GCLM, NQO1, and HO-1 were determined with real-time PCR assay. We found that none of the three major MAP kinase pathways were involved in acrolein-induced phase 2 gene expression. In contrast, both RO-31-8220 and staurosporine, two inhibitors of PKCs, completely blocked acrolein-induced mRNA expression of NQO1 and the two GCL subunits but only partially abrogated the induction of HO-1 mRNA. The PKC family has 10 isozymes that are divided into three subfamilies, i.e., classical (α, βI, βII, and γ),
novel (δ, ε, η, and θ), and atypical PKC
(ζ and ι/l). The inhibitory effects of RO-31-8220 on phase 2 gene inductions seems not through inhibiting classical PKC, since other PKC inhibitors that inhibit classical PKCs, such as calphostin C, RO-31-8425, and RO-32-0432, had no any inhibitory effects on the phase 2 gene inductions. In addition, the inhibitory effect of RO-31-8220 on different PKCs is dose-dependent and at concentration (1 mM) that inhibits classical PKCs, it had no effect on phase 2 gene induction. Furthermore, calphostin C, a classical PKCs inhibitor, increased instead of inhibiting acrolein-induced phase 2 genes expression. As RO-31-82202 can only inhibit acrolein-induced phase II gene expression at concentrations higher than 2 mM and at this concentration RO-31-8220 could inhibit both novel and atypical PKCs, we investigated the possible role of novel PKCd and atypical
PKCζ and i in phase 2 genes induction. Rottlerin, a relatively specific inhibitor of PKCδ,
abrogated the induction of NQO1; and interestingly, rottlerin significantly increased the basal expression of HO-1 and enhanced the HO-1 induction by acrolein. The different effects of rottlerin on acrolein-caused NQO-1 and HO-1 induction once again suggest the difference of signaling pathways involved in phase 2 gene inductions by lipid peroxidation products. Considering the specificity of chemical inhibitors, further study with the use of siRNA of PKCδ will help to confirm the possible involvement of PKCδ in the observed effects of rottlerin.
Finally, we considered the involvement of PI3K. LY294002 had no any effect on acrolein-induced NQO1, but it decreased induction of HO-1 by about 50%. It also had no effect on expression of the two GCL genes. In contrast, wortmannin, another inhibitor of PI3K, increased the basal expression of HO-1 and its induction by acrolein. The opposing effects of these two supposed PI3K inhibitors on HO-1 expression suggests that PI3K-independent factors may be involved in these effects. Further study with siRNA of PI3K will confirm whether PI3K is involved in acrolein-caused phase 2 genes induction.
Nitrated fatty acids (NO2-FA) are endogenous derivatives that exert signaling actions through different mechanisms that include serving as ligands for peroxisome proliferator-activated receptor (PPAR) activation and electrophilic reactivity. PPARs are nuclear receptors that regulate metabolism and inflammation, with endogenous ligands for this receptor presently not well defined. Activation of PPARs by ligand binding induces conformational changes that facilitate co-repressor release and co-activator recruitment. Ligand-specific co-regulator protein responses induce unique patterns of receptor-dependent gene expression and clinical outcomes. PPARg activation by thiazolidinediones (TZDs) increase insulin sensitivity, suppress chronic inflammatory processes and is one approach for treating diabetes. NO2-FA are potent PPARg ligands that appear to induce unique differentiated cell responses compared with TZDs, motivating more detailed studies of PPARg activation by NO2-FA. Mass spectrometric analysis of PPARg ligand binding domain (LBD) incubated with NO2-FA revealed covalent adduction of Cys285 by electrophilic NO2-FA. This nitroalkylation is fast, concentration-dependent and detectable by MS analysis using NO2-FA at PPARg-LBD mol ratios of 1:60. When NO2-FAs were derivatized to allyl esters, no nitroalkylation was detectable. The identity of the modified tryptic peptide containing Cys285 was confirmed by comparison with nitroalkylated synthetic LBD peptide. Further MS analysis also detected in vivo modification of PPARg by NO2-FA, with a limit of detection of 100 amol on column. Real time binding of NO2-FA to PPARg measured via surface plasmon resonance showed reduced Cys285 was essential for initial docking of NO2-FA and subsequent LBD nitroalkylation, with the carboxylic acid promoting interactions with positively charged residues in the ligand binding pocket. Time-resolved Förster resonance energy transfer (TR-FRET) showed NO2-FA induced unique patterns of coregulator protein interactions compared with TZDs. Importantly, TRAP220/DRIP2, a key mediator of triglyceride accumulation in adipocytes, was poorly recruited by PPARg upon NO2-FA activation. In concordance, less triglyceride accumulation with NO2-FA was found during adipocyte differentiation when compared to TZDs. In summary, activation of PPARg by NO2-FA is biologically-relevant, specific, involves covalent reaction with Cys285 and is a potential therapeutic strategy for treating inflammation and metabolic disorders.
Background: Previously we and others have proposed that nitrate tolerance as well as cross-tolerance is likely due to increased production of reactive oxygen species (ROS) leading to an inhibition of the nitroglycerin (GTN)-metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH-2) as well as to decreased nitric oxide (NO) bioavailability and/or impaired NO signaling. Importantly, pentaerithrityl tetranitrate (PETN) treatment has been shown to induce no tolerance and to possess antioxidant properties in vitro and in vivo. Methods: Wistar rats were chronically infused with PETN (10.5µg/kg/min for 3d) or GTN (6.6µg/kg/min for 3d). Blood pressure (BP) was assessed by telemetry-assisted catheter method, endothelial function was assessed by isometric tension recording, protein expression by Western blot and mRNA levels by RT-PCR analysis. ROS were measured by chemiluminescence, ALDH-2 activity was determined by HPLC and heme oxygenase-1 activity by plasma bilirubin levels. Results: Chronic PETN treatment, in contrast to GTN, did not induce nitrate tolerance or cross-tolerance. The protein and mRNA expression of the antioxidant enzymes heme oxygenase-1 (HO-1) and ferritin was increased (1,6 and 1,5-fold, respectively) in response to PETN but not GTN infusion (p<0,05). In contrast to GTN therapy, NO signalling, ROS formation and the activity of the GTN/PETN metabolizing enzyme ALDH-2 were not significantly modified upon chronic PETN treatment. PETN and GTN caused a small decrease in BP on the first day of infusion which was still present on the 6th day of PETN infusion but resulted in an increased BP for GTN on day 6. The inhibitor of HO-1 expression apigenin induced a tolerance-like impaired PETN potency in PETN in vivo treated rats whereas the HO-1 inducer hemin dramatically improved tolerance in GTN in vivo infused rats. Conclusions: In vivo PETN-treatment in Wistar rats induces neither nitrate tolerance nor cross-tolerance. These beneficial effects may be explained, at least in part, by induction of the antioxidant enzyme HO-1 and ferritin, and by the lack of stimulation of ROS production by PETN and are not shared by GTN.
Adaptation to the oxidative stress conditions is of great importance for life. Under such conditions, thioredoxin-1 (TRX-1) plays an important role as a cellular redox regulator. Previous observations consistently reported the occurrence of nuclear translocation of TRX-1 in mammalian cells exposed to reactive oxygen species and other oxidants. Recent findings by our group (BBRC 348: 1254; 2006) found a correlation between this migration and the activation of the p21Ras-ERK1/2 MAP kinases signaling pathway when cells were stimulated with NO donors. High levels of NO, characterizing a condition of nitrosative stress, promoted TRX-1 nuclear translocation. The present work aims to describe the interaction between TRX-1 and ERK1/2 MAP kinases and its consequences on TRX-1 nuclear migration under conditions of nitrosative and oxidative stresses. Western blot analysis revealed a co-migration of TRX-1 and phospho-ERK1/2 to the nucleus in HeLa cells exposed to 500 µM SNAP, a NO donor. In addition, TRX-1 did not migrate to the nucleus in HeLa cells over-expressing an ERK cytoplasmic anchor (PEA-15) and exposed to 1 mM H2O2. This mutant over-expressed TXNIP gene and further studies will be employed into its promoter sequence to acknowledge the Transcription Factors that are associated with this event. In conclusion, our findings strongly suggest that stress-mediated TRX-1 nuclear migration is associated with the activation of the ERK1/2 MAP kinases and TXNIP gene expression. Acknowledgments: Financial support was provided by FAPESP and CNPq/Milênio.
Palabras clave: Thioredoxin, oxidative/nitrosative stresses, protein interaction
Hydrogen sulfide induces a sustained rise of cytosolic calcium in cerebellar granule neurons through activation of L-type voltage-dependent calcium channels and can elicit glutamate-mediated excitotoxic cell death.
Garcia-Bereguiain, M.A. 1(*); Samhan-Arias, A.K. 1; Gutierrez-Merino, C. 1
1 - Depto. Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Extremadura | (*) Spain
The high endogenous levels of hydrogen sulfide (H2S), 10-160 µM, measured in human, rat and bovine brains have led to the suggestion that H2S can function as an endogenous neuromodulator. Cerebellar granule neurons (CGN) in culture express cystathionine â-synthase, the major enzyme producing H2S in brain, although H2S levels in the culture dish are below our detection limit (i.e. <5 µM) due to its rapid diffusion to the atmosphere. Here, we show that H2S concentration can be maintained in cell cultures within the range reported for rat brain by the application of repetitive pulses of sodium hydrogen sulfide. Less than two hours exposure to H2S concentrations within 50 and 120 ìM, i.e. within the upper segment of the reported physiological range of H2S in rat brain, produces a large shift of the intracellular calcium homeostasis in CGN in culture leading to a large and sustained increase of cytosolic calcium concentration. Only one hour exposure to H2S concentrations within 100 and 300 ìM raises intracellular calcium to the neurotoxic range, ensuing nearly 50% cell death after 2 hours. L-type Ca2+ channels antagonists nimodipine and nifedipine block both the H2S-induced rise of cytosolic calcium and cell death. The N-methyl-D-aspartate receptor antagonists (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid afford a nearly complete protection against H2S-induced CGN death and largely attenuated the rise of cytosolic calcium. The rise of cytosolic calcium produced by one hour exposure to H2S concentrations within 100 and 300 ìM leads to glutamate-induced excitotoxic CGN death. We conclude that H2S is a major modulator of calcium homeostasis in cerebellar granule neurons as it induces activation of Ca2+ entry through L-type Ca2+ channels, thereby stimulating the neuronal activity. Noteworthy, H2S concentrations displaying significant neurotoxicity for CGN in culture are less than twice the highest concentrations reported for H2S in brain. Work supported by Grants SAF2003-08275 of the Spanish Ministerio de Educacion y Ciencia and 3PR05A078 of the Junta de Extremadura.