Monocytes/macrophages committed to death by peroxynitrite nevertheless survive with a signaling response promoting Bad phosphorylation, as well as its cytosolic localization, via upstream activation of cytosolic phospholipase A2, 5-lipoxygenase, and protein kinase Cα. We now report evidence for an alternative mechanism converging in Bad phosphorylation when the expression/activity of the above enzymes are suppressed. Under these conditions, also associated with peroxynitrite-dependent severe inhibition of Akt, an additional Bad kinase, Bad dephosphorylation promoted its accumulation in the mitochondria and a prompt lethal response. PGE2 prevented toxicity via EP2 receptor-mediated protein kinase A-dependent Bad phosphorylation. This notion was established in U937 cells by the following criteria: 1) there was a strong correlation between survival and cAMP accumulation, both in the absence and presence of phosphodiesterase inhibitors; 2) direct activation of adenylyl cyclase afforded cytoprotection; and 3) PGE2 promoted loss of mitochondrial Bad and cytoprotection, mimicked by EP2 receptor agonists, and prevented by EP2 receptor antagonists or protein kinase A inhibitors. Finally, selected experiments performed in human monocytes/macrophages and in rat peritoneal macrophages indicated that the above cytoprotective pathway is a general response of cells belonging to the monocyte/macrophage lineage to both exogenous and endogenous peroxynitrite. The notion that two different pathways mediated by downstream products of arachidonic acid metabolism converge in Bad phosphorylation emphasizes the relevance of this strategy for the regulation of macrophage survival to peroxynitrite at the inflammatory sites.

Peroxynitrite, the coupling product of superoxide and NO, is produced by macrophages and other inflammatory cells in response to a variety of proinflammatory stimuli. This species is extremely reactive (1), promotes direct and indirect damage in different biomolecules (1, 2), and is heavily implicated in tissue damage associated with inflammatory conditions (3).

The above information is documented by an extensive literature, but much less is known about the effects of peroxynitrite in the perspective of peroxynitrite-producing cells. It is indeed reasonable to assume that these cells will be affected by peroxynitrite in the first place, when the oxidant is generated either within the cell or in its close vicinity. Our work performed in cells belonging to the monocyte/macrophage lineage provided evidence for an ingenious survival strategy based on the ability of these cells to respond to the oxidant with the triggering of signaling events that allow survival regardless of the damage accumulated (4, 5, 6, 7, 8, 9, 10, 11).

We found that these cells, as for many other cell types, are severely affected and committed to death by low concentrations of the oxidant. Unlike other cell types, however, monocytes/macrophages did not die since they were able to respond to molecules largely available at the inflammatory sites with a signaling response leading to survival.

Macrophages use arachidonic acid (ARA)4 (4, 5) and products of 5-lipoxygenase (5-LO) (9, 10, 11), as 5-hydroxyeicosatetraenoic acid (5-HETE), to promote downstream events associated with prevention of mitochondrial permeability transition (MPT)-dependent toxicity. We detected significant levels of Bad in the mitochondria of untreated cells and found that the above signaling leads to Bad phosphorylation, and thus to its cytosolic accumulation (7, 8). Phosphorylation inhibits binding of Bad to Bcl-2 or Bcl-xL and promotes its translocation to the cytosol, thus enabling Bcl-2 or Bcl-xL to exert its anti-MPT functions (12). Upstream inhibition of the survival signaling promotes the mitochondrial accumulation of Bad and the rapid onset of MPT-dependent toxicity taking place soon after the mitochondrial translocation of Bax (8). Bax can also dimerize with Bcl-2 or Bcl-xL, and it is provided with a well-defined pore-forming activity (13). Thus, the cytosolic accumulation of Bad creates optimal conditions for the anti-MPT functions of Bcl-2 and Bcl-xL.

In short, the survival strategy adopted by macrophages is simple, yet very effective: while committed to MPT-dependent toxicity, these cells nevertheless survive by promoting Bad phosphorylation via a pathway triggered by molecules they produce and/or are largely available in the extracellular inflammatory milieu.

In our experiments, phosphorylation of Bad was mediated by 5-HETE-dependent mitochondrial translocation of protein kinase Cα (PKCα) (6, 8, 9). Indeed, Bad phosphorylation may result from PKC-dependent pathways triggering a p90RSK-mediated phosphorylation at serine 112 (14), and this event obviously requires the mitochondrial localization of PKCα. An extensive literature documents that Bad phosphorylation is often mediated by protein kinase B (Akt), a serine-threonine protein kinase (15). It is difficult to establish whether this pathway is relevant in peroxynitrite signaling since few reports describe stimulation of Akt (16, 17), although others rather describe inhibition, most likely attributable to tyrosine nitration, in a variety of cell types (18, 19, 20, 21), including macrophages (22).

There is, however, a third pathway that may lead to Bad phosphorylation and survival in macrophages under inflammatory conditions. This pathway is dependent on phosphorylation mediated by protein kinase A (PKA), which is stimulated by receptor-mediated signaling driven by a variety of agonists, including PGs, that are largely released in the extracellular inflammatory milieu by both constitutive and inducible cyclooxygenases (23).

The present study provides results in line with the notion that Akt is severely inhibited by peroxynitrite, thereby ruling out the contribution of this pathway in the regulation of monocyte/macrophage survival. In remarkable contrast, PGs effectively signal survival through EP2 receptors by promoting Bad phosphorylation, as previously shown for the 5-LO-dependent pathway (7, 8, 9). Hence, ARA appears to be critical for macrophage survival to peroxynitrite in that it is a substrate for both 5-LO, directly (intracellular mechanism) promoting survival via PKCα-dependent Bad phosphorylation, and cyclooxygenases, thereby contributing to the definition of extracellular levels of PGs triggering PKA-dependent Bad phosphorylation.

Human promonocytic tumor U937 cells were cultured as previously described (4). Human peripheral mononuclear cells were isolated by Ficoll gradient centrifugation and monocytes were purified by adherence on a plastic substrate in RPMI 1640 medium. Macrophages were obtained by culturing monocytes (1 × 106 cells/ml) for 8–10 days in RPMI 1640 medium. Macrophages were scraped with PBS/EDTA, centrifuged, and utilized for experiments. Rat peritoneal exudate macrophages were collected 4 days after intraperitoneal injection of 4% thioglycolate medium (Sigma-Aldrich) to 25- or 30-day-old Sprague-Dawley rats (Charles River Laboratories). The cells were plated on 35-mm culture dishes (Falcon, BD Labware) in DMEM supplemented with 2 mM glutamine, 25 mM HEPES, penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FBS (HyClone Laboratories) for 24 h. Nonadherent cells were removed with warm DMEM. At the treatment stage, rat peritoneal macrophages were between 4.5 and 5.0 × 105 cells/dish.

Peroxynitrite was synthesized by the reaction of nitrite with acidified H2O2, as described previously (24). For the different cell types and experiments, treatments were performed in 2 ml of prewarmed saline A (8.182 g/L NaCl, 0.372 g/L KCl, 0.336 g/L NaHCO3 and 0.9 g/L glucose) containing 4.5–5 × 105 cells. The same concentration of peroxynitrite (i.e., 100 μM) was used throughout the study. Arachidonyl trifluoromethyl ketone (AACOCF3), MK886, PGE2, ARA, butaprost, sulprostone, and Gö6850 were given to the cultures 3 min after peroxynitrite. Forskolin, KT5720, AH6809, and AH23848 were added to the cultures 30 min before peroxynitrite; 3-isobutyl-1-methylxanthine (IBMX) (5 or 30 min) and wortmannin (15 min) were also added to cultures before peroxynitrite. Formation of endogenous peroxynitrite was induced in rat peritoneal macrophages, as recently described (25). Briefly, the cells were treated with 10 μg/ml LPS (serotype 0127:B8; Sigma-Aldrich) plus 10 ng/ml IFN-γ (2 × 107 IU/mg of protein; R&D Systems, Space) and, after 2 h, also exposed to 2.5 μg/ml PMA (Sigma-Aldrich), with or without PGE2. Cytotoxicity, nitrite concentrations, or nitrotyrosine immunoreattivity was assessed after a further 9 h of growth. l-NAME, l-methionine, or apocynin was added to the cultures 10 min before addition of PMA.

Cytotoxicity was determined with the trypan blue exclusion assay. Briefly, an aliquot of the cell suspension was diluted 1/1 (v/v) with 0.4% trypan blue and the viable cells were counted with a hemocytometer.

Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to measure viability of rat peritoneal macrophages. MTT (25 μg/ml) was added directly to culture medium 1 h before the end of the incubation period. The medium was then removed and replaced with 1 ml of DMSO. Cellular MTT reductase activity was determined by measuring the absorbance of the DMSO extracts at 570 nm. Results are expressed as the percentage of MTT reducing activity of treated vs untreated cells.

The human AS-ON for cPLA2 (5′-GTA AGG ATC TAT AAA TGA CAT-3′), for FLAP (5′-TTC TTG ATC CAT GTT TGC TT-3′), and for PKCα (5′-GTT CTC GCT GGT GAG TTT CA-3′) were directed against the initiation site (cPLA2), the start codon of mRNA (FLAP), or against the 3′ region of the mRNA (PKCα). The NS-ON for cPLA2 (5′-AGT AGA TTG AAT AGA CAC TAT-3′), for FLAP (5′-CTT GTT TAA TCC CCT TTT GT-3′), and for PKCα (5′-TGG GCC GGG CAA TTT TTT TC-3′) were a random sequence of the AS bases. The ONs were phosphorothioate modified and synthesized by MWG Biotech. The phosphorothioate backbone provides resistance to exonuclease and increases the stability of the ON in serum and within the cell. U937 cells were transfected with the above ONs as follows: cells were washed twice with serum-free medium and seeded (1 × 106/ml) in serum-free RPMI 1640 for 6 h in the absence or presence of ONs (10 μM). A final concentration of 5% FBS was then added, the cells were cultured for an additional 24 h, and finally utilized for experiments. Using these conditions, we obtained good transfection efficiencies and thus avoided the use of transfection reagents, such as Lipofectamine. The effect of ON transfection on enzyme expression was tested by Western blot analysis (see below). Occasionally, cPLA2 or 5-LO activities were also assessed by measuring ARA release and leukotriene B4 formation, respectively, in response to peroxynitrite. The outcome of these experiments was in line with our previously published work (4, 9). We observed identical levels of ARA release in untreated cells, whereas there was a significant increase in ARA release only in the cells transfected with cPLA2 NS-ONs (typically 2-fold increase). Leukotriene B4 levels were also identical in untreated cells transfected with FLAP NS-ONs or AS-ONs. Peroxynitrite enhanced leukotriene B4 formation (typically a 1.5-fold increase) in cells transfected with the NS-ONs and had hardly any effect in the cells transfected with the AS-ONs.

After treatments, the cells were processed to obtain mitochondrial and cytosolic fractions, as described by Yu et al. (26), or the whole cell lysates, as described by Guidarelli et al. (6). Western blot analysis was next performed using Abs against cPLA2, FLAP, Akt, heat shock protein-60 (all obtained from Santa Cruz Biotechnology), pAkt, PKCα, Bad (all obtained from BD Transduction Laboratories), and actin (Sigma-Aldich). Details on Western blotting apparatus and conditions are reported elsewhere (6). Abs against Akt, actin, and heat shock protein-60 were used to assess the equal loading of the lanes.

Intracellular cAMP levels were determined in cell extracts (0.1 M HCl) by an ELISA kit (Cayman Chemical).

Nitrite production was estimated using the Griess reaction. Briefly, 800 μl of samples was mixed with 60 μl sulfanilamide (12.5 mM) and 60 μl HCl (6 M). After 5 min, 60 μl N-(1-naphthyl)-ethylene diamine was added to the mixture and the absorbance was read at 540 nm using a Uvicon 923 spectrophotometer. Nitrite concentration was calculated from a standard curve of NaNO2 in the medium.

Nitrotyrosine-containing proteins were detected using an immunocytochemical technique following the manufacturer’s instructions. Briefly, rat peritoneal macrophages were seeded on polylysine-coated coverslips and treated as indicated above. The coverslips were then removed from the dishes, washed, and the cells fixed for 1 min with ethanol/acetic acid (95/5, v/v). These preparations were finally rinsed with PBS and blocked in PBS-containing BSA (2%, w/v). Rabbit polyclonal anti-nitrotyrosine (5 μg/ml in PBS containing 2% BSA) was used as a primary Ab. After 18 h at 4°C, the cell monolayers were washed and exposed to FITC-conjugated secondary Ab for 2 h in the dark. Stained cells were analyzed using a BX-51 fluorescence microscope (Olympus Italia) equipped with a SPOT-RT camera unit (Diagnostic Instruments, Delta Sistemi). The excitation and emission wavelengths were 495 and 515 nm, respectively. Images were collected with exposure times of 100–400 ms, digitally acquired, and processed for fluorescence determination at the single cell level on a personal computer using Scion Image software. Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells per treatment condition per experiment.

Statistical analysis of the data for multiple comparisons was performed by ANOVA followed by a Dunnett’s test. For comparison between two groups, Student’s unpaired t test was used.

Previous work from our laboratory showed that peroxynitrite-dependent activation of cPLA2, associated with ARA metabolism via the 5-LO pathway, promotes a cascade of events leading to cytoprotection (4, 5, 6, 7, 8, 9, 10, 11). Under these conditions, 5-LO translocated to the nuclear membrane, an event critical for interaction with FLAP and 5-HETE formation (27, 28), and indeed was readily detected after peroxynitrite exposure (9). Thus, consistently with these findings, down-regulation of cPLA2 or FLAP expression with specific AS-ONs (Fig. 1,A, inset) promoted toxicity after exposure to otherwise nontoxic concentrations of peroxynitrite (100 μM, Fig. 1,A). Cells transfected with the respective NS-ONs did not show appreciable changes vs the nontransfected cells in terms of protein expression (Fig. 1,A, inset) or susceptibility to peroxynitrite (not shown). Pharmacological inhibitors of cPLA2 (AACOCF3) or FLAP (MK886), under conditions inhibiting ARA release and 5-HETE formation (Refs. 4, 9 and Materials and Methods), respectively, promoted toxicity in nontransfected cells (Fig. 1 B) and, with similar outcomes, in cells transfected with NS-ONs (not shown).

FIGURE 1.

PGE2 prevents the lethal response mediated by an otherwise nontoxic concentration of peroxynitrite in cells supplemented with either AS-ONs or pharmacological inhibitors of cPLA2 and FLAP. A, cPLA2 and FLAP AS-ON-transfected cells were exposed for 3 min to peroxynitrite (100 μM), for an additional 57 min to increasing concentration of PGE2, and finally analyzed with the trypan blue exclusion assay. Also shown is the effect of peroxynitrite alone in cells transfected with cPLA2 NS-ONs (similar results were obtained in cells transfected with FLAP NS-ONs). The inset shows the levels of cPLA2 and FLAP expression in nontransfected cells as well as in NS-ON- and AS-ON-transfected cells. B, Nontransfected cells were treated as indicated above with the further addition of either AACOCF3 (50 μM) or MK886 (1 μM). Also shown is the effect of peroxynitrite alone. C, Cells, with or without prior addition of wortmannin (300 nM), were treated for 60 min with peroxynitrite, AACOCF3, and PGE2 (3 μM) as indicated above (see Materials and Methods for details) and processed for the assessment of viability. D, Cells were exposed to wortmannin and/or peroxynitrite for 10 min, lysed, and finally processed for Western blot analysis using Ab against pAkt and Akt. Blots shown in D are representative of three separate experiments. Results represent the means ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells exposed to peroxynitrite alone (A and C) or associated with AACOCF3 or MK886 (B) (ANOVA followed by Dunnett’s test).

FIGURE 1.

PGE2 prevents the lethal response mediated by an otherwise nontoxic concentration of peroxynitrite in cells supplemented with either AS-ONs or pharmacological inhibitors of cPLA2 and FLAP. A, cPLA2 and FLAP AS-ON-transfected cells were exposed for 3 min to peroxynitrite (100 μM), for an additional 57 min to increasing concentration of PGE2, and finally analyzed with the trypan blue exclusion assay. Also shown is the effect of peroxynitrite alone in cells transfected with cPLA2 NS-ONs (similar results were obtained in cells transfected with FLAP NS-ONs). The inset shows the levels of cPLA2 and FLAP expression in nontransfected cells as well as in NS-ON- and AS-ON-transfected cells. B, Nontransfected cells were treated as indicated above with the further addition of either AACOCF3 (50 μM) or MK886 (1 μM). Also shown is the effect of peroxynitrite alone. C, Cells, with or without prior addition of wortmannin (300 nM), were treated for 60 min with peroxynitrite, AACOCF3, and PGE2 (3 μM) as indicated above (see Materials and Methods for details) and processed for the assessment of viability. D, Cells were exposed to wortmannin and/or peroxynitrite for 10 min, lysed, and finally processed for Western blot analysis using Ab against pAkt and Akt. Blots shown in D are representative of three separate experiments. Results represent the means ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells exposed to peroxynitrite alone (A and C) or associated with AACOCF3 or MK886 (B) (ANOVA followed by Dunnett’s test).

Close modal

The results illustrated in Fig. 1, A and B, also indicate that addition of PGE2 after peroxynitrite promotes cytoprotection under conditions in which cytotoxicity would arise as a consequence of inhibition of the survival signaling, at the level of either cPLA2 or 5-LO. Interestingly, lower concentrations of PGE2 were required for maximal protection against inhibition of lipoxygenase activity. This observation implies a contribution of endogenous PGs in that more ARA becomes available for the cyclooxygenases upon inhibition of lipoxygenases.

These results are consistent with a signaling initiated by extracellular PGs, which may cooperate with the previously described intracellular signaling driven by 5-LO products (9) to promote Bad phosphorylation. A contribution of the Akt pathway, however, is unlikely (Fig. 1, C and D) since peroxynitrite suppressed Akt phosphorylation as effectively as wortmannin, a PI3K inhibitor. Additionally, wortmannin did not promote toxicity in cells exposed to peroxynitrite (Fig. 1,C), as previously observed with the inhibitors of either cPLA2 or 5-LO (Fig. 1 B).

The above results indicate that PGE2 rescues U937 cells committed to death by peroxynitrite via upstream inhibition of the survival signaling. We therefore asked the question of whether the downstream events of this signaling were the same as those previously identified downstream to 5-LO activation, as the mitochondrial translocation of PKCα causally linked to the cytosolic accumulation of Bad (7, 8, 9).

As previously shown (8, 9), peroxynitrite indeed promoted the mitochondrial accumulation of PKCα, and this response was suppressed by AACOCF3 (Fig. 2,A). The observation that the effect of the PLA2 inhibitor was sensitive to ARA, but insensitive to PGE2, is therefore consistent with the notion that the survival response evoked by PGE2 does not involve PKCα. This notion is further supported by the observation that PGE2, unlike ARA, prevented toxicity mediated by peroxynitrite and Gö6850, an inhibitor of PKC (Fig. 2,B). Additionally, down-regulation of PKCα expression with specific AS-ONs (Fig. 2,C, inset) promoted toxicity after exposure to peroxynitrite via an ARA-insensitive albeit PGE2-sensitive mechanism (Fig. 2,C). Finally, cells transfected with the NS-ONs did not show reduced PKCα expression (Fig. 2 C) and were resistant to peroxynitrite (not shown) (6).

FIGURE 2.

PGE2 signals survival via a PKCα-independent mechanism. A, Cells were treated for 10 min with peroxynitrite, AACOCF3, ARA (0.1 μM), and PGE2, as indicated in the figure (see Materials and Methods for details). Western blot analysis for PKCα was performed in the mitochondrial fraction. B, Cells were exposed for 60 min to peroxynitrite, Gö6850 (3 μM), ARA, PGE2, and butaprost (50 μM), as indicated, and finally analyzed with the trypan blue exclusion assay. C, PKCα AS-ON-transfected cells were treated as indicated in B (with the exception of Gö6850, not employed in these experiments) and analyzed with the trypan blue exclusion assay. The inset shows the level of PKCα expression in nontransfected cells and in cells transfected with PKCα NS-ONs or AS-ONs. Blots shown in A and C are representative of three separate experiments. Results represent the means ± SEM from three separate experiments. ∗, p < 0.01 as compared with cells exposed to peroxynitrite (B) or to untreated cells (C) (ANOVA followed by Dunnett’s test). The relative amount of PKCα was quantified by densitometric analysis and expressed as OD integration.

FIGURE 2.

PGE2 signals survival via a PKCα-independent mechanism. A, Cells were treated for 10 min with peroxynitrite, AACOCF3, ARA (0.1 μM), and PGE2, as indicated in the figure (see Materials and Methods for details). Western blot analysis for PKCα was performed in the mitochondrial fraction. B, Cells were exposed for 60 min to peroxynitrite, Gö6850 (3 μM), ARA, PGE2, and butaprost (50 μM), as indicated, and finally analyzed with the trypan blue exclusion assay. C, PKCα AS-ON-transfected cells were treated as indicated in B (with the exception of Gö6850, not employed in these experiments) and analyzed with the trypan blue exclusion assay. The inset shows the level of PKCα expression in nontransfected cells and in cells transfected with PKCα NS-ONs or AS-ONs. Blots shown in A and C are representative of three separate experiments. Results represent the means ± SEM from three separate experiments. ∗, p < 0.01 as compared with cells exposed to peroxynitrite (B) or to untreated cells (C) (ANOVA followed by Dunnett’s test). The relative amount of PKCα was quantified by densitometric analysis and expressed as OD integration.

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The next question addressed was whether Bad is a target of the PGE2-dependent survival signaling. The results illustrated in Fig. 3,A indicate that significant levels of Bad are located in the mitochondria of untreated U937 cells and that peroxynitrite promotes the mitochondria to cytosol translocation of the protein, sensitive to the PKC inhibitor Gö6850, which indeed caused a remarkable mitochondrial Bad accumulation. This observation is in keeping with our previous findings indicating that, under the above conditions, Bad phosphorylation and cytosolic accumulation require activation and mitochondrial translocation of PKCα (7, 8). Interestingly, the process of mitochondrial accumulation of Bad mediated by peroxynitrite in PKC-inhibited cells was sensitive to PGE2 (Fig. 3 A).

FIGURE 3.

PGE2 signaling leads to the cytosolic localization of Bad. Cells were treated for 10 min, as indicated in the figure (see Materials and Methods for details). KT5720 or AH6809 were used at 3 or 50 μM, respectively. After treatments, mitochondrial and cytosolic fractions were isolated and processed for Western blot analysis using an Ab against Bad. Blots shown are representative of three separate experiments. The relative amount of Bad was quantified by densitometric analysis and expressed as OD integration.

FIGURE 3.

PGE2 signaling leads to the cytosolic localization of Bad. Cells were treated for 10 min, as indicated in the figure (see Materials and Methods for details). KT5720 or AH6809 were used at 3 or 50 μM, respectively. After treatments, mitochondrial and cytosolic fractions were isolated and processed for Western blot analysis using an Ab against Bad. Blots shown are representative of three separate experiments. The relative amount of Bad was quantified by densitometric analysis and expressed as OD integration.

Close modal

These results collectively indicate that PGE2 signals survival via a PKCα-independent mechanism nevertheless leading to the cytosolic accumulation of Bad.

The results illustrated in Fig. 4,A indicate that concentrations of PGE2 >3 μM (10 min) are required to significantly enhance U937 cell cAMP. The observation that a 5-min exposure to phosphodiesterase inhibitor (IBMX) promotes a leftward shift of the above curve leading to appreciable cAMP levels after exposure to <3 μM PGE2, however, provides an indication of transient increases of the second messenger also under these conditions. Cells were next treated with peroxynitrite/AACOCF3, manipulated to enhance cAMP as detailed above, and assayed for survival. The results shown in Fig. 4, A and B, collectively provide evidence for a good correlation between these parameters, a notion further supported by the observation that longer exposure (30 min) to IBMX promotes even larger increases in cAMP (Fig. 4,A) and virtually afforded complete cytoprotection in the absence of PGE2. As a final note, the effects of PGE2 are mimicked by forskolin, an activator of adenylyl cyclase (Fig. 4 C).

FIGURE 4.

PGE2 signals survival via cAMP. A, Cells, with or without prior exposure to IBMX (300 μM), were treated for 10 min with increasing concentration of PGE2 and processed for the assessment of cAMP levels. B and C, Cells were treated for 60 min as indicated in the figure (see Materials and Methods for details) and analyzed with the trypan blue exclusion assay. Results represent the mean ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells treated in the absence of PGE2 (A and B) or forskolin (C) (ANOVA followed by Dunnett’s test).

FIGURE 4.

PGE2 signals survival via cAMP. A, Cells, with or without prior exposure to IBMX (300 μM), were treated for 10 min with increasing concentration of PGE2 and processed for the assessment of cAMP levels. B and C, Cells were treated for 60 min as indicated in the figure (see Materials and Methods for details) and analyzed with the trypan blue exclusion assay. Results represent the mean ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells treated in the absence of PGE2 (A and B) or forskolin (C) (ANOVA followed by Dunnett’s test).

Close modal

Additional experiments revealed that the effects of PGE2 are both insensitive to wortmannin (Fig. 1,C) and abolished by the PKA inhibitor KT5720 (Fig. 5,A). The specificity of the latter response is emphasized by the observation that KT5720 promoted a concentration-dependent lethal response in cells committed to death by peroxynitrite/AACOCF3 and rescued with PGE2, whereas PKA inhibition had no significant effect on cytoprotection offered by ARA. Additionally, KT5720 neither promoted toxicity in the presence of peroxynitrite/absence of other treatments nor affected the lethal response mediated by peroxynitrite/AACOCF3. Finally, KT5720 also caused toxicity in cells committed to death by peroxynitrite/Gö6850 but rescued with PGE2 (not shown) and under the same conditions promoted a remarkable mitochondrial Bad accumulation (Fig. 3 B).

FIGURE 5.

PGE2 signals survival via EP2 receptor-dependent PKA activation. Cells were treated for 60 min as indicated in the figure (see Materials and Methods for details) and analyzed with the trypan blue exclusion assay. Results represent the means ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells treated in the absence of KT5720 (A), AH6809 (C), butaprost (D), or as compared with cells exposed to peroxynitrite alone (B). (ANOVA followed by Dunnett’s test).

FIGURE 5.

PGE2 signals survival via EP2 receptor-dependent PKA activation. Cells were treated for 60 min as indicated in the figure (see Materials and Methods for details) and analyzed with the trypan blue exclusion assay. Results represent the means ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells treated in the absence of KT5720 (A), AH6809 (C), butaprost (D), or as compared with cells exposed to peroxynitrite alone (B). (ANOVA followed by Dunnett’s test).

Close modal

Collectively, these results further emphasize the involvement of cAMP in the PGE2-dependent cytoprotective signaling and imply a role for PKA.

The results illustrated above demonstrate a role for cAMP in PGE2-induced cytoprotection, thereby ruling out the involvement of EP1 receptors, resulting in the elevation of intracellular free Ca2+ (29) and EP3 receptors, decreasing cAMP (30). This notion is also confirmed by the observed inability of sulprostone, an EP1–EP3 agonist, to promote cytoprotection after exposure to peroxynitrite/AACOCF3 (Table I). Additionally, sulprostone promoted loss of viability in cells exposed to peroxynitrite alone. EP2 and EP4 receptors, stimulating adenylyl cyclase activity via a Gs protein (31), are therefore likely candidates for the PGE2-dependent cytoprotective mechanism. The results illustrated in Fig. 5,B would suggest the selective involvement of EP2 receptors since the cytoprotective effects of PGE2, while insensitive to the EP4 antagonist AH23848, were lost upon exposure to the EP2 antagonist AH6809. The specificity of the effects of AH6809 was established by using the same approach adopted for the PKA inhibitor KT5720. AH6809 promoted a concentration-dependent lethal response in cells committed to death by peroxynitrite/AACOCF3 and rescued with PGE2, whereas the EP2 receptor antagonist had no significant effect on cytoprotection offered by ARA (Fig. 5,C). Additionally, AH6809 failed to promote toxicity in the absence of other treatments and did not affect the lethal response mediated by peroxynitrite/AACOCF3. Importantly, loss of mitochondrial Bad associated with exposure to PGE2 was also inhibited by AH6809 (Fig. 3 B).

Table I.

EP1 and EP3 receptors are not involved in the survival signaling mediated by PGE2

TreatmentaTrypan Blue-Negative Cells (% of Control)
Without SulprostoneWith Sulprostone
Untreated 98.3 ± 0.9 92.0 ± 1.0 
Peroxynitrite 94.8 ± 0.8 75.8 ± 7.4b 
+AACOCF3 53.5 ± 4.1 51.7 ± 6.0 
TreatmentaTrypan Blue-Negative Cells (% of Control)
Without SulprostoneWith Sulprostone
Untreated 98.3 ± 0.9 92.0 ± 1.0 
Peroxynitrite 94.8 ± 0.8 75.8 ± 7.4b 
+AACOCF3 53.5 ± 4.1 51.7 ± 6.0 
a

Cells were treated 3 min with peroxynitrite (100 μM) and for an additional 57 min with AACOCF3 (50 μM) with or without sulprostone (50 μM). The number of viable cells was then counted with the trypan blue exclusion assay. Results represent the means ± SEM from three separate experiments.

b

p < 0.01 compared to cells treated without sulprostone (unpaired Student’s t test).

The involvement of EP2 receptors in the PGE2-dependent survival signaling is also emphasized by the observation that butaprost, an EP2 receptor agonist, prevents toxicity mediated by peroxynitrite associated with either AACOCF3 (Fig. 5,D) or Gö6850 (Fig. 2,B). Additionally, butaprost promoted survival in cells transfected with PKCα AS-ONs after exposure to the sole peroxynitrite (Fig. 2,C). Butaprost also mimicked the effects of PGE2 on the mitochondrial accumulation of Bad promoted by peroxynitrite/Gö6850 (Fig. 3 B).

Collectively, the above results indicate that PGE2 signals U937 cell survival through EP2 receptors, thereby triggering PKA-dependent inactivation of Bad.

Our previous work showed that the cPLA2/5-LO/PKCα-dependent survival signaling is not restricted to U937 cells but, rather, is a general response of cells belonging to the monocyte/macrophage lineage (6, 9, 10). The results illustrated in Fig. 6 strongly suggest that the same is true for the PGE2-dependent survival signaling described in this study. Indeed, PGE2 was protective for both monocytes (Fig. 6,A) and macrophages (Fig. 6,B) exposed to an otherwise nontoxic dose of peroxynitrite and AACOCF3. Additionally, the survival signaling promoted by PGE2 was sensitive to either KT5720 or AH6809 and mimicked by butaprost in both monocytes (Fig. 6,C) and macrophages (Fig. 6 D).

FIGURE 6.

PGE2 signals survival also in human monocytes and macrophages. Human monocytes (A and C) purified by adherence on a plastic substrate and macrophages (B and D) obtained from monocytes grown for 10 days were treated (60 min) as indicated in the figure (see Materials and Methods for details) and analyzed with the trypan blue exclusion assay. Results represent the means ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells exposed to peroxynitrite associated with AACOCF3 (A and B) or to peroxynitrite alone (C and D) (ANOVA followed by Dunnett’s test).

FIGURE 6.

PGE2 signals survival also in human monocytes and macrophages. Human monocytes (A and C) purified by adherence on a plastic substrate and macrophages (B and D) obtained from monocytes grown for 10 days were treated (60 min) as indicated in the figure (see Materials and Methods for details) and analyzed with the trypan blue exclusion assay. Results represent the means ± SEM from three to five separate experiments. ∗∗, p < 0.01; ∗, p < 0.05 as compared with cells exposed to peroxynitrite associated with AACOCF3 (A and B) or to peroxynitrite alone (C and D) (ANOVA followed by Dunnett’s test).

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We therefore conclude that the PGE2-dependent survival signaling identified in U937 cells is also operative in human monocytes and macrophages.

Having established that PGE2 affords cytoprotection against exogenous peroxynitrite in a human tumor pro-monocytic cell line as well as in human monocytes/macrophages, we sought to determine whether the same protective effects are mediated against endogenous peroxynitrite. The above cell types, however, do not produce significant amounts of the oxidant in response to proinflammatory stimuli, and we therefore employed a recently published strategy, involving the sequential addition of LPS/IFN-γ and PMA, to achieve significant levels of peroxynitrite formation in activated rat peritoneal macrophages (25). As documented in Fig. 7, this treatment (9 h) indeed promoted a l-NAME- (1 mM, a NO synthase inhibitor), l-methionine- (20 mM, a peroxynitrite scavenger), and apocyanin- (10 μM, a NADPH oxidase inhibitor) sensitive loss of MTT-reductase activity (Fig. 7,A, an index of cytotoxicity) and induction of nitrotyrosine immunoreactivity (Fig. 7,C, an index of peroxynitrite formation). The proinflammatory stimuli also promoted nitrite accumulation (Fig. 7,B, an index of NO formation), and this response was prevented only by l-NAME. These results provide evidence for the specificity of the effects of the inhibitors employed and lead to the univocal conclusion that endogenous peroxynitrite mediates rat macrophage toxicity in response to the above proinflammatory stimuli. Also note that in the absence of PMA, LPS/IFN-γ promoted formation of NO (Fig. 7,B) but failed to generate peroxynitrite (Fig. 7,C) and toxicity (Fig. 7 A).

FIGURE 7.

PGE2 prevents the lethal response mediated by endogenous peroxynitrite in rat peritoneal macrophages exposed to proinflammatory stimuli. Cells were treated (9 h) as indicated in the figure (see Materials and Methods for details) and analyzed for viability (A), nitrite concentration (B), or nitrotyrosine immunoreactivity (C). In particular, PMA, and eventually PGE2 (2.5 μM), was given to the cultures 2 h after LPS/IFN-γ, whereas l-NAME (1 mM), l-methionine (20 mM), or apocynine (10 μM) was given 10 min before PMA. Results represent the means ± SEM from three to five separate experiments. ∗, p < 0.01 or #, p < 0.01 as compared with untreated or LPS/IFN-γ/PMA-treated cells, respectively (ANOVA followed by Dunnett’s test).

FIGURE 7.

PGE2 prevents the lethal response mediated by endogenous peroxynitrite in rat peritoneal macrophages exposed to proinflammatory stimuli. Cells were treated (9 h) as indicated in the figure (see Materials and Methods for details) and analyzed for viability (A), nitrite concentration (B), or nitrotyrosine immunoreactivity (C). In particular, PMA, and eventually PGE2 (2.5 μM), was given to the cultures 2 h after LPS/IFN-γ, whereas l-NAME (1 mM), l-methionine (20 mM), or apocynine (10 μM) was given 10 min before PMA. Results represent the means ± SEM from three to five separate experiments. ∗, p < 0.01 or #, p < 0.01 as compared with untreated or LPS/IFN-γ/PMA-treated cells, respectively (ANOVA followed by Dunnett’s test).

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It was interesting to observe that PGE2, given 2 h after LPS/IFN-γ(i.e., at the time of PMA addition) prevented loss of MTT staining (Fig. 7,A) under the same conditions in which hardly any effect was detected on nitrite release (Fig. 7,B) or nitrotyrosine immunoreactivity (Fig. 7 C). Hence, macrophage deletion was prevented via a mechanism downstream to peroxynitrite formation.

We therefore conclude that PGE2 also promotes survival in rat peritoneal macrophages otherwise committed to death by endogenous peroxynitrite.

The present work extends our previous findings on the mechanism whereby monocytes/macrophages survive to peroxynitrite, a reactive species extensively produced by these and other cell types in response to proinflammatory stimuli. The use of inflammatory products to promote survival is clearly an ingenious strategy for cells producing and sensing peroxynitrite at the inflammatory sites. Monocytes/macrophages indeed survive with a signaling response involving the sequential activation of cPLA2 (4, 5, 6) and 5-LO (9, 10, 11), thereby promoting the mitochondrial translocation of PKCα (8), an event associated with the cytosolic accumulation of Bad and Bax (7, 8, 9). Thus, this signaling optimizing the anti-MPT functions of Bcl-2/Bcl-xL (12) promotes monocyte/macrophage survival even under harsh conditions of peroxynitrite exposure, regardless of the damage accumulated by the cells (4, 5). Obviously these cells may also use exogenous ARA, which can freely cross the plasma membrane, to support the 5-LO-dependent pathway leading to Bad phosphorylation.

The present study describes an additional ARA-dependent pathway leading to Bad phosphorylation in, and survival of, monocytes/macrophages otherwise driven to death via inhibition of the 5-LO-dependent signaling. This pathway is triggered by PGs, as PGE2, via EP2 receptor activation leading to PKA-dependent Bad phosphorylation. Hence, different pathways may converge in eliciting Bad phosphorylation in macrophages at the inflammatory sites, since sustained ARA release mediated by different PLA2 isoforms (32) may stimulate both the 5-LO/PKCα- and the PGE2/PKA-dependent signaling pathways. Inflammation is indeed associated with enhanced PG formation, due to both constitutive and inducible cyclooxygenases (23). Additionally, each of the two different pathways may successfully promote Bad phosphorylation and survival when the other is suppressed. Finally, the PI3K/Akt pathway, regulating Bad phosphorylation and promoting cytoprotection in response to an array of survival factors in different toxicity paradigms, is clearly not involved in the regulation of monocyte/macrophage survival to peroxynitrite. Exposure to nontoxic concentrations of peroxynitrite, but promoting toxicity in cells in which cPLA2 or FLAP was either knocked down (Fig. 1,A) or inhibited pharmacologically (Fig. 1,B), was indeed associated with suppression of Akt phosphorylation (Fig. 1,D). Additionally, wortmannin failed to promote toxicity in the presence of peroxynitrite but nevertheless suppressed Akt phosphorylation alone or in combination with the oxidant (Fig. 1, C and D).

Toxicity observed under the above conditions was not only sensitive to supplementation of downstream products of the inhibited steps (e.g., ARA in cPLA2-inhibited cells and 5-HETE in both cPLA2- and FLAP-inhibited cells), as previously documented (4, 5, 9), but was also suppressed by micromolar concentrations of PGE2 (Fig. 1, A and B). The requirement of lower concentrations of PGs to rescue 5-LO-inhibited cells implies the contribution of enforced formation of endogenous PGs in the PGE2-dependent survival signaling.

The cytoprotective effects of PGE2 were not mediated by activation of the Akt pathway, as expected (Fig. 1,C), and were not associated with the mitochondrial translocation of PKCα (Fig. 2,A), critically involved in Bad phosphorylation promoted via the 5-LO-dependent signaling (6, 7, 8, 9). More generally, we can rule out the involvement of PKCα and other PKC isoforms, since PGE2 afforded protection also in cells in which the 5-LO-dependent signaling was intercepted downstream with a general PKC inhibitor (Fig. 2,B) or via down-regulated PKCα expression (Fig. 2 C).

The protective effects of PGE2 were, however, associated with the triggering of events promoting Bad phosphorylation, and its cytosolic accumulation (Fig. 3 A), as normally occurs is response to peroxynitrite via the 5-LO/PKCα-dependent signaling (6, 7, 8, 9). Hence, PGE2 must promote Bad phosphorylation via activation of a Bad kinase different from Akt or PKC.

The biological actions of PGE2 are mediated by four distinct pharmacological classes of G protein-coupled receptors, EP1–EP4 (31). EP1 is not widely distributed and enhances phosphoinositol turnover, thereby resulting in the elevation of intracellular free Ca2+ (29, 31). EP2 receptors are more widespread and increase intracellular cAMP via adenylyl cyclase activation (31). An opposite response is mediated by EP3 receptors, the most ubiquitous EP receptors, inhibiting adenylyl cyclase activity through Gi proteins (30, 31). Finally, EP4 receptors, like EP2, also enhance adenylyl cyclase activity (31). However, the effects mediated by EP2 and EP4 receptors are primarily through PKA- and PI3K-dependent pathways, respectively (33, 34).

The above information strongly suggests that PGE2 promotes U937 cell survival via EP2 receptor signaling and that, under these conditions, PKA mediates Bad phosphorylation. PKA may indeed induce Bad phosphorylation on Ser112 and, more importantly, in Ser155, an event promoting the dissociation of Bad from Bcl-2/Bcl-xL and its interaction with the 14-3-3 protein (35, 36). This effect may contribute to the anti-apoptotic effects of cAMP-elevating agents and more generally result in prevention of MPT associated with either necrotic or apoptotic death. MPT is indeed critically involved in the lethal response mediated by peroxynitrite after inhibition of the 5-LO-dependent survival signaling (7, 9).

The above mechanism of PGE2-mediated cytoprotection via EP2 receptor-dependent PKA signaling was demonstrated by the following criteria. First, PGE2 promoted cAMP accumulation, and this response was significantly enhanced via phosphodiesterase inhibition (Fig. 4,A). There was a good correlation between cAMP accumulation and survival (Fig. 4, A and B), and indeed adenylyl cyclase activators reproduced cytoprotection mediated by PGE2 (Fig. 4,C). Finally, PGE2 promoted loss of mitochondrial Bad and cytoprotection, mimicked by EP2 receptor agonists and prevented by EP2 receptor antagonists or PKA inhibitors (Figs. 2, 3, and 5).

These results therefore provide compelling evidence for an alternative mechanism, triggered by PGE2, promoting the cytosolic accumulation of Bad and survival to peroxynitrite under conditions in which the 5-LO-dependent survival pathway is inhibited. It was therefore interesting to obtain results in line with these findings when human monocytes/macrophages were used in the place of U937 cells (Fig. 6). The outcome of our studies implies that the above cytoprotective pathway is a general response of cells belonging to the monocyte/macrophage lineage.

To further establish the biological relevance of our findings we investigated whether PGE2 affords cytoprotection also against endogenous peroxynitrite. For this purpose, we could not use human monocytes/macrophages, which ex vivo respond to proinflammatory stimuli with poor inducible NO synthase expression, despite the recognition of the occurrence of this event in vivo in a variety of human diseases involving inflammation (37). We therefore employed a toxicity paradigm known to promote peroxynitrite-dependent toxicity (25) involving sequential exposure of rat peritoneal macrophages to LPS/IFN-γ and PMA. Under the same conditions in which survival was mediated by different treatments preventing peroxynitrite formation (i.e., via inhibition of NO or NADPH reductase activities) or scavenging of peroxynitrite (i.e., using l-methionine), PGE2 was found to afford cytoprotection via a mechanism downstream to peroxynitrite formation (Fig. 7).

Hence, PGE2 signals macrophage survival against both endogenous and exogenous peroxynitrite. The notion that monocytes/macrophages cope with peroxynitrite with the triggering of two different pathways mediated by downstream products of ARA metabolism (Fig. 8) indicates that the survival strategy of these cells is based on their ability to signal survival in response to products largely available under inflammatory conditions. The notion that both pathways converge in Bad phosphorylation indicates that this strategy is of great advantage for the survival of cells that survive and perform energy-demanding functions at the inflammatory sites, regardless of the damage accumulated in response to a large variety of reactive species, including peroxynitrite.

FIGURE 8.

Mechanism(s) whereby cells belonging to monocyte/macrophage lineage cope with peroxynitrite. Monocytes and macrophages survive to peroxynitrite, a reactive species extensively produced by these cells in response to pro-inflammatory stimuli, via activation of two ARA-dependent pathways leading to Bad phosphorylation and to the ensuing cytosolic accumulation. ARA metabolism via the 5-LO pathway (gray arrows) promotes PKCα-dependent Bad phosphorylation. An alternative signaling converging in Bad phosphorylation (black arrows) is driven by PGs, as PGE2, via an EP2 receptor-mediated PKA-dependent mechanism.

FIGURE 8.

Mechanism(s) whereby cells belonging to monocyte/macrophage lineage cope with peroxynitrite. Monocytes and macrophages survive to peroxynitrite, a reactive species extensively produced by these cells in response to pro-inflammatory stimuli, via activation of two ARA-dependent pathways leading to Bad phosphorylation and to the ensuing cytosolic accumulation. ARA metabolism via the 5-LO pathway (gray arrows) promotes PKCα-dependent Bad phosphorylation. An alternative signaling converging in Bad phosphorylation (black arrows) is driven by PGs, as PGE2, via an EP2 receptor-mediated PKA-dependent mechanism.

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The authors have no financial conflicts of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (to O.C.).

4

Abbreviations used in this paper: ARA, arachidonic acid; AACOCF3, arachidonyl trifluoromethyl ketone; AS-ON, antisense oligonucleotide; cPLA2, cytosolic phospholipase A2; FLAP, 5-LO-activating protein; 5-HETE, 5-hydroxyeicosatetraenoic acid; IBMX, 3-isobutyl-1-methylxanthine; l-NAME, NG-nitro-l-arginine methyl ester; 5-LO, 5-lipoxygenase; MPT, mitochondrial permeability transition; NS-ON, nonsense oligonucleotide; ON, oligonucleotide; PKA, protein kinase A; PKCα, protein kinase Cα.

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