Recognition of apoptotic cells by macrophages activates the peroxisome proliferator-activated receptor-gamma and attenuates the oxidative burst.

It is appreciated that phagocytosis of apoptotic cells (AC) is an immunological relevant process that shapes the pro- versus anti-inflammatory macrophage phenotype. It was our intention to study the respiratory burst, a prototype marker of macrophage activation, under the impact of AC. Following incubation of RAW264.7 macrophages with AC, we noticed attenuated production of reactive oxygen species (ROS) in response to PMA treatment, and observed a correlation between attenuated ROS formation and suppression of protein kinase Calpha (PKCalpha) activation. EMSA analysis demonstrated an immediate activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) following supplementation of AC to macrophages. In macrophages carrying a dominant-negative PPARgamma mutant, recognition of AC no longer suppressed PKCalpha activation, and the initial phase of ROS formation was largely restored. Interference with actin polymerization and transwell experiments suggest that recognition of AC by macrophages suffices to attenuate the early phase of ROS formation that is attributed to PPARgamma activation.

NF-kappaB and AP-1 activation by nitric oxide attenuated apoptotic cell death in RAW 264.7 macrophages.

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 microM) or with lipopolysaccharide, interferon-gamma, and NG-monomethyl-L-arginine (LPS/IFN-gamma/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-gamma/NMMA prestimulation activated nuclear factor (NF)-kappaB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-kappaB and activator protein-1 (AP-1). NF-kappaB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-kappaB site derived from the murine Cox-2 promoter, confirmed NF-kappaB activation after NO addition. An NF-kappaB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-gamma/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-kappaB activation, a low-level NO-elicited Cox-2 response required activation of both NF-kappaB and AP-1.

Protein thiol modification and apoptotic cell death as cGMP-independent nitric oxide (NO) signaling pathways.

Nitric oxide signaling is achieved through both cGMP-dependent and cGMP-independent mechanisms. The latter are exemplified by protein thiol modification followed by subsequent NAD(+)-dependent automodification of the glycolytic enzyme GAPDH, or by mechanisms inducing accumulation of the tumor suppressor gene p53 and causing apoptotic cell death. Both cGMP-independent actions are initiated using NO-releasing compounds and an active LPS/cytokine-inducible NO synthase. NO-synthase inhibitors block the release of NO and hinder downstream signaling mechanisms; they are therefore valuable pharmacological tools linking a defined cellular response to various NO actions. Signal transducing mechanisms elicited by NO can be studied using GAPDH as a representative example of NO-induced protein modification and are grouped as follows: --S-Nitrosylation reactions initiated by NO+ --NAD(+)-dependent, post-translational covalent automodification of GAPDH --Oxidative modification (thiol oxidation) and inhibition of GAPDH by NO-related agents, probably ONOO- GAPDH and several other protein targets may serve as molecular sensors of elevated NO concentrations and may transmit this message through posttranslational modification and oxidation-induced conformational changes as cGMP-independent NO signaling pathways. Toxicity of NO seems to be linked to both apoptosis and necrosis, depending on the chemistry of NO it undergoes in a given biological milieu. Toxicity manifests as a relative excess of NOx, metal-NO interactions, and ONOO- formation in relation to cellular defense systems. Although accumulation of the tumor-suppressor gene product p53 in response to NO opens a regulatory mechanism known to be involved in apoptotic cell death, cGMP-independent signaling pathways remain to be elucidated. As NO-dependent modification of GAPDH would imply down-regulation of glycolysis and concomitant energy production followed by cell death, our data so far do not support this assumption. In recent years, NO has proved to be a beneficial messenger with a potentially toxic activity. It will be challenging to investigate NO biochemistry in closer detail and to elucidate how NO targets biological systems, especially in relation to its pathophysiological role.

p53 accumulation in apoptotic macrophages is an energy demanding process that precedes cytochrome c release in response to nitric oxide.

Apoptosis in response to stress signals activates effector caspases known to be regulated by the release of cytochrome c (Cyt c) from mitochondria and the subsequent ATP-dependent activation of the death regulator apoptotic protease-activating factor 1 (Apaf-1). Experiments were carried out to determine whether the release of Cyt c is evoked by NO. in RAW 264.7 macrophages and to position signaling components relative to mitochondria. S-nitrosoglutathione and spermine-NO caused a fast p53 accumulation, followed by Bcl-xL downregulation, Cyt c release, and caspase activation. These alterations were absent in p53 antisense expressing macrophages (R delta p53asn-11). In Bcl-2 overexpressing cells (Rbcl2-14) Cyt c relocation and caspase activation were abrogated although p53 accumulation remained intact. The use of caspase inhibitors revealed Cyt c release and decreased Bcl-xL expression to be caspase independent. ATP-depleted cells showed a shift from apoptosis towards necrosis and no p53 accumulation or caspase activation upon NO. addition. Conclusively, NO.-mediated apoptosis in macrophages is entirely controlled by the mitochondrial pathway with the implication that Cyt c relocation demands p53 accumulation. Moreover, pulse-chase-experiments in combination with the ATP-depletion protocol identified p53 accumulation and stabilization as an energy requiring process. This allowed to dissect two ATP-dependent steps, one is in association with Apaf-1 formation, while the other resides in p53 accumulation.

Etoposide and cisplatin induced apoptosis in activated RAW 264.7 macrophages is attenuated by cAMP-induced gene expression.

Previous observations suggest expression of cyclooxygenase-2 to convey macrophage protection towards apoptotic cell death. We reasoned prostaglandin formation and in turn a cAMP increase as the underlying protective principle. Here we report that exposure of macrophages to lipopolysaccharide/interferon-gamma or lipophilic cAMP analogs such as dibutyryl-cAMP or 8-bromo-cAMP for 15 h attenuated DNA fragmentation and accumulation of the tumor suppressor p53 in response to the chemotherapeutic agents cisplatin and etoposide, compared to cells that received chemotherapeutic agents only. In contrast, a 1 h lasting preexposure period revealed no protection. The demand for a long incubation period with cAMP-derivates implied cAMP-mediated gene activation as the underlying principle. Therefore, we treated cells with oligonucleotides containing a cAMP-response element (CRE) binding site. Using this decoy-approach we scavaged activated cAMP response element binding protein prior to its promoter activating ability. Incubating macrophages with decoy, but not with control oligonucleotides, reduced cAMP evoked protection and simultaneously restored p53 accumulation in response to chemotherapeutic agents. Our studies demonstrate that cAMP-initiated gene activation regulates the sensitivity towards DNA damaging agents via inhibition of a p53 dependent pathway.

Up-regulation of Bcl-2 by redox signals in glomerular mesangial cells.

The mediators nitric oxide (NO) and superoxide (O2-) are known to regulate cell death and survival. In mesangial cells (MC), NO induced apoptosis and in higher concentrations necrosis. Intriguingly, cogeneration of NO and O2- in a balanced ratio promoted cell protection. Under these conditions, we noticed the accumulation of the anti-apoptotic protein Bcl-2. Its up-regulation is based on an increase in mRNA and protein level. To investigate whether oxidative stress elicits Bcl-2 expression in general, we further used the chemically unrelated oxidative agents diamide and butyl hydroperoxide. Both stimulated mRNA and protein up-regulation of Bcl-2. But in contrast to diamide, butyl hydroperoxide evoked apoptosis and necrosis despite Bcl-2 accumulation. As diamide was non-toxic, we used diamide as a Bcl-2 activator to protect MC against a subsequent toxic dose of NO. We conclude that redox changes promote Bcl-2 up-regulation that may confer cellular protection towards apoptosis.

Nitric oxide (NO): an effector of apoptosis.

It is appreciated that the production of nitric oxide (NO) from L-arginine metabolism is an essential determinate of the innate immune system, important for nonspecific host defense, as well as tumor and pathogen killing. Cytotoxicity as a result of a substantial NO-formation is established to initiate apoptosis, characterized by upregulation of the tumor suppressor p53, changes in the expression of pro- and anti-apoptotic Bcl-2 family members, cytochrome c relocation, activation of caspases, chromatin condensation, and DNA fragmentation. Proof for the involvement of NO was demonstrated by blocking adverse effects by NO-synthase inhibition. However, NO-toxicity is not a constant value and NO may achieve cell protection as well. In part this is understood by transcription and translation of protective proteins, such as cyclooxygenase-2. Alternatively, protection may result as a consequence of a diffusion controlled NO/O2- (superoxide) interaction that redirects the apoptotic initiating activity of NO towards protection. NO is endowed with the unique ability to initiate and to block apoptosis, depending on multiple variables that exist to be elucidated. The crosstalk between cell destructive and protective signaling pathways under the modulatory influence of NO will determine the impact of NO in apoptotic cell death and survival.

The dual role of S-nitrosoglutathione (GSNO) during thymocyte apoptosis.

Nitric oxide (NO) is known to regulate redox-sensitive signalling pathways in physiology and pathophysiology. Depending on its concentration, the NO-releasing compound S-nitrosoglutathione (GSNO) causes negative and positive regulation of thymocyte apoptosis. At levels below 0.6 mM, GSNO produces deoxyribonucleic acid (DNA) laddering, which is inhibited by activation of protein kinase C (PKC), cycloheximide treatment, and calcium chelation. Higher concentrations of the NO donor (1-2 mM) suppress thymocyte apoptosis initiated by the classical agonist dexamethasone. Inhibition of apoptosis by NO is analogous to the action of the thiol-blocking compound N-ethylmaleimide (NEM) and the glutathione-S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB). Inhibition of apoptosis results from thiol modification of critical proteins in response to NO treatment. Depending on the concentration, GSNO can be involved either in toxic or in protective signalling in thymocyte biology.

Modulation of inducible nitric oxide synthase in RINm5F cells.

The rat insulinoma beta-cell line RINm5F, which shares some homology with pancreatic islets, was used to study nitric oxide synthase induction. Nitric oxide is involved during beta-cell destruction and possibly in propagation of insulin-dependent diabetes mellitus. The cytokine interleukin-1 (IL-1) turned out to be the ultimate inducer, whereas tumour necrosis factor-alpha (TNF) and unexpectedly the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate; 10 nM) synergistically promoted nitrite accumulation. Besides employing TPA directly, the synergistic effect of TNF could be traced back to protein kinase C activation since protein kinase C inhibitors (IC50 value for staurosporine: 4 nM) potently suppressed nitrite production in the case of IL-1/TNF administration. Further experiments using anti-TNF antibodies aimed to an autocrine loop following IL-1 addition to RINm5F cells, possibly involved in nitrite generation. Moreover, the nitric oxide synthase inductive IL-1 signal was antagonized by lipophilic cAMP analogues. Our results for nitrite accumulation in RINm5F cells point to activating protein kinase C and inhibitory protein kinase A signalling pathways.

Wortmannin interferes with thrombin-evoked secondary calcium redistribution in human platelets.

Wortmannin has previously been reported to inhibit calcium entry in thrombin-stimulated human platelets. We extend these findings by demonstrating that the redistribution of calcium from intracellular stores features two separate, consecutive phases the second of which is selectively abolished by wortmannin. The primary release of calcium from Ins 1,4,5 P3-sensitive stores remains unaffected. Hence, wortmannin is interfering with regulation of any secondary, sustained calcium accumulation in the cytosolic compartment of activated platelets, originating either from intracellular stores or from calcium entry. We assume that wortmannin blocks a common step in receptor-dependent regulation of calcium entry. We assume that wortmannin blocks a common step in receptor-dependent regulation of calcium entry and intracellular calcium circulation.

Transcription factors p53 and HIF-1alpha as targets of nitric oxide.

It is widely recognized that the production of nitric oxide (NO) from L-arginine metabolism is an essential determinate of diverse signalling cascades throughout the body, with a major impact during nonspecific host defence. Biological actions of NO and derived species comprise physiological as well as pathological entities, with an impressive and steadily growing number of signalling pathways and/or protein targets being involved. It is now appreciated that NO not only acts as an effector molecule but also as an autocrine as well as paracrine modulator of rapid and delayed cellular responses. Among multiple targets the tumour suppressor p53 and the hypoxia inducible factor-1alpha (HIF-1alpha) emerged. Accumulation of p53 in response to NO delivery may account for an interference in cell cycle progression and/or initiation of apoptosis that is found in close correlation with inducible NO synthase (NOS) expression. Quite similarly, accumulation of HIF-1alpha not only occurs during hypoxia, but also under conditions of NO delivery, thus mimicking a situation of reduced oxygen availability. Interestingly, p53 and HIF-1alpha share regulatory elements that cause protein stabilization in part as a result of impaired ubiquitin-evoked protein degradation. Here, we summarize current knowledge on the impact of NO on p53- and HIF-1alpha-stabilization and we will discuss pathophysiological consequences. These examples may help to shape and refine current concepts of NO action with an emphasis on transcription factor regulation.

Modulation of nitric oxide-evoked apoptosis by the p53-downstream target p21(WAF1/CIP1).

When produced in excess, the inflammatory mediator nitric oxide (NO) attenuates cell-cycle progression at the G1 phase in tight correlation with p21(WAF1/CIP1) expression, provokes accumulation of the tumor suppressor p53, and initiates apoptosis/necrosis as judged on cell accumulation in the sub-G1 phase. To verify the role of p21(WAF1/CIP1) in modulating cell-cycle arrest vs. apoptosis, we transfected stably antisense p21(WAF1/CIP1)-encoding plasmids. Following NO exposure, accumulation of p21(WAF1/CIP1), but not p53, was largely attenuated in antisense p21(WAF1/CIP1) transfectants. Moreover, the G1 cell-cycle arrest was abrogated, and cells were sensitized toward apoptosis compared with parent macrophages. In contrast, antisense elimination of p53 attenuated p53 as well as p21(WAF1/CIP1) expression, abolished the G1 cell-cycle arrest, and prevented apoptosis. We conclude that p21(WAF1/CIP1) is a downstream target of p53 in macrophages that modulate the sensitivity toward the immune-modulator NO.

Protein thiol modification of glyceraldehyde-3-phosphate dehydrogenase and caspase-3 by nitric oxide.

The regulation of enzyme activity function is a major factor in the cellular response to a changing environment. One mechanism of enzyme activity regulation includes post-translational protein thiol modification by nitric oxide (NO) or its redox species. Major routs used by NO to modify cysteine residues of proteins include S-nitrosation, oxidation, mixed disulfide formation with glutathione, and the covalent attachment of nucleotide cofactors, i.e NAD(+)/NADH. Critical thiol centers serve as recognition sites for NO, thus channeling the NO signal through post-translational modifications and oxidation into cellular functions. Here, we summarize current knowledge on active site thiol modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and caspase-3 by nitric oxide. Although very different in their cellular function, both enzymes contain highly reactive cysteines which represent sensitive targets for NO. Our studies are supportive of a potential role of S-nitrosation and mixed disulfide formation as a general signaling mechanism that allows sensing of nitrosative stress. At the same time, modification of GAPDH and caspase-3 by NO show the diversity of mechanisms (S-nitrosation versus oxidations) that we are confronted with as a result of NO delivery, especially comparing in vitro studies with cellular systems. In the future it will be challenging to dissect how nitrosative and oxidative signaling mechanisms overlap and how intracellular communication systems allow their activation in a selective way.

Calcium mobilization in human platelets by receptor agonists and calcium-ATPase inhibitors.

Inhibitors of the endoplasmic reticulum Ca(2+)-ATPase like thapsigargin (TG) and 2,5-di (tert-butyl)-1,4-benzohydroquinone (tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 microM) mobilized 100% of sequestered Ca2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 microM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway (Ki approximately 7 microM). Therefore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca(2+)-ATPase inhibitors require the self-amplification mechanism of endogenously formed thromboxane A2 to cause a similar response. We conclude that the calcium store sensitive to Ca(2+)-ATPase inhibitors is a subset of the receptor sensitive calcium pool.

Nitric oxide preferentially stimulates auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase compared to alcohol or lactate dehydrogenase.

Recently we demonstrated that the radical nitric oxide (NO) stimulates the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) resulting in enzyme inhibition. To further characterize this auto-ADP-ribosylation reaction we studied alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) for comparison. Whereas auto-ADP-ribosylation of ADH was stimulated to a minor extent by the NO-liberating agent 3-morpholinosydnonimine (SIN-1), LDH was unaffected. The susceptibility of dehydrogenases towards auto-ADP-ribosylation correlated with the potency of NO to decrease enzyme activity. Again, GAPDH was much more sensitive compared to ADH, whereas LDH again was unaffected. Interestingly, the efficiency of the SH-alkylating agent N-ethylmaleimide (NEM) to inhibit the enzymatic activity of the chosen dehydrogenases correlates with the sensitivity of dehydrogenases towards NO. These studies demonstrate the requirement of a reactive SH-group besides the NAD+ binding site as a prerequisite for NO-stimulated auto-ADP-ribosylation reactions. Furthermore, we establish that under physiological conditions and among the dehydrogenases tested, only GAPDH is a potential target for this post-translational protein modification mechanism.

Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation.

The toxic reactive aldehyde lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) is thought to be a major contributor to oxidant stress-mediated cell injury. HNE induced apoptosis in RAW 264.7 murine macrophage cells in a dose-dependent manner within 6-8 h after exposure. Expression of the antiapoptotic protein Bcl-2 in stably transfected RAW 264.7 cells prevented HNE-induced internucleosomal DNA fragmentation and apoptosis, and these cells resume growth after a temporary (24-48 h) growth delay. While parental RAW 264.7 cells released mitochondrial cytochrome c within 3 h after HNE exposure, expression of Bcl-2 prevented cytochrome c release. In control cells, p53 protein levels peaked at 6-9 h after HNE exposure and then declined, while in Bcl-2 expressing cells, p53 levels were maximal at 6-9 h and remained elevated up to 96 h. Expression of SV40 large T-antigen, which forms a stable complex with p53 protein, via stable transfection-blocked transactivation of the p53-regulated gene p21(WAF1/CIP1), but did not affect induction of apoptosis by HNE, suggesting that p53 function is not important in HNE-induced apoptosis. These results suggest that cytochrome c release, but not p53 accumulation, plays an essential role in HNE-induced apoptosis in RAW 264.7 cells.

Nitric oxide and superoxide inhibit platelet-derived growth factor receptor phosphotyrosine phosphatases.

Platelet derived growth factor receptor (PDGFR) became tyrosine autophosphorylated in rat mesangial cells shortly after platelet derived growth factor (PDGF) ligation in a tyrosine kinase inhibitor (tyrphostin AG 1296) sensitive manner. Ligand-independent, massive tyrosine PDGFR phosphorylation was achieved by diverse NO releasing compounds. Phosphorylation was slow compared to PDGF, revealed a concentration- and time-dependency, and was not mimicked by lipophilic cyclic-GMP analogues. Interleukin-1 beta/cAMP activated mesangial cells released NO and in turn showed PDGFR phosphorylation. A NO-synthase involvement was assured by L-NG-nitroarginine methyl ester inhibition. PDGFR phosphorylation was also achieved by the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. NO- and O2(.-)-evoked PGDFR phosphorylation was N-acetylcysteine reversible. Cell free dephosphorylation assays revealed PDGFR dephosphorylation by tyrosine phosphatases. Receptor dephosphorylation by cytosolic phosphatases was completed within 30 min and was sensitive to the readdition of NO donors or orthovanadate. In addition, phosphatase activity determined in a direct dephosphorylation assay using the substrate para-nitrophenyl phosphate was attenuated by NO or vanadate. We conclude that cytosolic protein tyrosine phosphatases are targeted by exogenously supplied or endogenously generated NO in mesangial cells. Radical (NO. or O2.-) formation shifts the phosphorylation--dephosphorylation equilibrium towards phosphorylation, thus integrating redox-mediated responses into established signal transducing pathways.

p53 expression in nitric oxide-induced apoptosis.

Nitric oxide (NO) is a diffusible messenger involved in several patho-physiological processes including immune-mediated cytotoxicity and neural cell killing. NO or the products of its redox chemistry can cause DNA damage and activate subsequent lethal reactions including energy depletion and cell necrosis. However, regardless of whether it is endogenously produced in response to cytokines, or generated by chemical breakdown of donor molecules, NO can also induce apoptosis in different systems. Here, we report that NO generation in response to a cytokine induced NO-synthase or by NO donors stimulates the expression of the tumor suppressor gene, p53, in RAW 264.7 macrophages or pancreatic RINm5F cells prior to apoptosis. NO-synthase inhibitors such as NG-monomethyl-L-arginine prevent the inducible NO generation as well as p53 expression and apoptosis. Since p53 expression is linked to apoptosis in some cells exposed to DNA damaging agents, we suggest that NO-induced apoptosis in these cell systems is the consequence of DNA damage and subsequent expression of this tumor suppressor gene.