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Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase
(1992)
Nitric oxide and nitric oxide-generating agents like 3-morpholinosydnonimine (SIN-1) stimulate the mono-ADP-ribosylation of a cytosolic, 39-kDa protein in various tissues. This protein was purified from human platelet cytosol by conventional and fast protein liquid chromatography techniques. N-terminal sequence analysis identified the isolated protein as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitric oxide stimulates the auto-ADP-ribosylation of GAPDH in a time and concentration-dependent manner with maximal effects after about 60 min. Associated with ADP-ribosylation is a loss of enzymatic activity. NAD(+)-free enzyme is not inhibited by SIN-1, indicating the absolute requirement of NAD+ as the substrate of the ADP-ribosylation reaction. Inhibition of the glycolytic enzyme GAPDH may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain.
Low concentrations of oxidized low density lipoprotein (OxLDL) are cytoprotective for phagocytes, although the underlying mechanisms remain unclear. We investigated signaling pathways used by OxLDL to attenuate apoptosis in monocytic cells. OxLDL at 25–50 μg/ml inhibited staurosporine-induced apoptosis in THP-1 cells and mouse peritoneal macrophages, and it was cytoprotective in human primary monocytes upon serum withdrawal. Attenuated cell demise was reversed by blocking extracellular signal-regulated kinase (ERK) signaling. Translocation of cytochrome c to the cytosol was attenuated by OxLDL, which again demanded ERK signaling. Analysis of Bcl-2 family proteins revealed phosphorylation of Bad at serine 112 as well as ERK-dependent inhibition of Mcl-1 degradation. Although the formation of reactive oxygen species (ROS) is an established signal generated by OxLDL, ROS scavengers did not interfere with cell protection by OxLDL. Thus, activation of the ERK signaling pathway by OxLDL is important to protect phagocytes from apoptosis.
Thioredoxin 1 and thioredoxin 2 have opposed regulatory functions on hypoxia-inducible factor-1α
(2007)
Hypoxia inducible factor 1 (HIF-1), a key regulator for adaptation to hypoxia, is composed of HIF-1alpha and HIF-1beta. In this study, we present evidence that overexpression of mitochondria-located thioredoxin 2 (Trx2) attenuated hypoxia-evoked HIF-1alpha accumulation, whereas cytosolic thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount. Transactivation of HIF-1 is decreased by overexpression of Trx2 but stimulated by Trx1. Inhibition of proteasomal degradation of HIF-1alpha in Trx2-overexpressing cells did not fully restore HIF-1alpha protein levels, while HIF-1alpha accumulation was enhanced in Trx1-overexpressing cells. Reporter assays showed that cap-dependent translation is increased by Trx1 and decreased by Trx2, whereas HIF-1alpha mRNA levels remained unaltered. These data suggest that thioredoxins affect the synthesis of HIF-1alpha. Trx1 facilitated synthesis of HIF-1alpha by activating Akt, p70S6K, and eIF-4E, known to control cap-dependent translation. In contrast, Trx2 attenuated activities of Akt, p70S6K, and eIF-4E and provoked an increase in mitochondrial reactive oxygen species production. MitoQ, a mitochondria specific antioxidant, reversed HIF-1alpha accumulation as well as Akt activation under hypoxia in Trx2 cells, supporting the notion of translation control mechanisms in affecting HIF-1alpha protein accumulation.
Introduction: Immune paralysis with massive T-cell apoptosis is a central pathogenic event during sepsis and correlates with septic patient mortality. Previous observations implied a crucial role of peroxisome proliferator-activated receptor gamma (PPARγ) during T-cell apoptosis.
Methods: To elucidate mechanisms of PPARγ-induced T-cell depletion, we used an endotoxin model as well as the caecal ligation and puncture sepsis model to imitate septic conditions in wild-type versus conditional PPARγ knockout (KO) mice.
Results: PPARγ KO mice showed a marked survival advantage compared with control mice. Their T cells were substantially protected against sepsis-induced death and showed a significantly higher expression of the pro-survival factor IL-2. Since PPARγ is described to repress nuclear factor of activated T cells (NFAT) transactivation and concomitant IL-2 expression, we propose inhibition of NFAT as the underlying mechanism allowing T-cell apoptosis. Corroborating our hypothesis, we observed up-regulation of the pro-apoptotic protein BIM and downregulation of the anti-apoptotic protein Bcl-2 in control mice, which are downstream effector proteins of IL-2 receptor signaling. Application of a neutralizing anti-IL-2 antibody reversed the pro-survival effect of PPARγ-deficient T cells and confirmed IL-2-dependent apoptosis during sepsis.
Conclusion: Apparently antagonizing PPARγ in T cells might improve their survival during sepsis, which concomitantly enhances defence mechanisms and possibly provokes an increased survival of septic patients.
Background: Microarray analysis still remains a powerful tool to identify new components of the transcriptosome and it has helped to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns, as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis. Result: We identified 188 genes that were significantly regulated by hypoxia, 81 genes were affected by nitric oxide, and 292 genes were induced by the co-treatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to hypoxia and/or nitric oxide treatments, but with different levels of expression. We observed that 166 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia regulated targets by NO. Conclusion: By eliminating the interference of steady state mRNA in gene expression profiling, we increased the sensitivity of mRNA analysis and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signalling.
We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.
Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.
Attenuated NOX2 expression impairs ROS production during the hypoinflammatory phase of sepsis
(2012)
Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased.
Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS.
Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis.
Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.