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Originally published online as doi:10.1189/jlb.0406240 on August 2, 2006

Published online before print August 2, 2006
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(Journal of Leukocyte Biology. 2006;80:929-938.)
© 2006 by Society for Leukocyte Biology

5-Hydroxyeicosatetraenoic acid is a key intermediate of the arachidonate-dependent protective signaling in monocytes/macrophages exposed to peroxynitrite

Ilaria Tommasini, Andrea Guidarelli, Letizia Palomba, Liana Cerioni and Orazio Cantoni1

Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino "Carlo Bo," Urbino, Italy

1 Correspondence: Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino "Carlo Bo," Via S. Chiara, 27, Urbino (PU) 61029, Italy. E-mail: cantoni{at}uniurb.it


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ABSTRACT
 
Endogenous generation of arachidonic acid via selective activation of cytosolic phospholipase A2 has been implicated in the mechanism of monocytes/macrophage survival in the presence of peroxynitrite. In particular, the lipid messenger was shown to prevent the otherwise rapid onset of a mitochondrial permeability-transition (MPT)-dependent necrosis by causing the mitochondrial translocation of protein kinase C{alpha} (PKC{alpha}) and the ensuing cytosolic accumulation of the Bcl-2-antagonist of cell death (Bad), an event promoting the anti-MPT function of Bcl-2 (or Bcl-XL). Here, we show that the effects on PKC{alpha} are not mediated directly by arachidonate but rather, by downstream products of the enzyme 5-lipoxygenase (5-LO). Peroxynitrite elicited the nuclear membrane translocation of 5-LO and enhanced its enzymatic activity via a mechanism sensitive to low concentrations of inhibitors of 5-LO or the 5-LO-activating protein, as well as to genetic depletion of the latter enzyme. Inhibition of 5-LO activity was invariably associated with the cytosolic localization of PKC{alpha}, the mitochondrial accumulation of Bad, and a rapid MPT-dependent necrosis. All these events were prevented by nanomolar concentrations of the 5-LO product 5-hydroxyeicosatetraenoic acid.

Key Words: reactive nitrogen species • 5-lipoxygenase • mitochondria • cell survival


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INTRODUCTION
 
Cells belonging to the monocyte/macrophage lineage respond to numerous proinflammatory stimuli with the production of a variety of reactive species including peroxynitrite, the coupling product of nitric oxide and superoxide. It is not surprising that these cells are highly resistant to the above species, known to promote toxic effects in other cell types, thus causing extensive damage in inflamed tissues. Experimental work performed in our laboratory [1 , 2 ] suggested that monocytes/macrophages cope with peroxynitrite by using a defensive pathway involving activation of cytosolic phospholipase A2 (cPLA2). Hence, treatment with otherwise nontoxic levels of peroxynitrite caused the rapid onset of a mitochondrial permeability transition (MPT)-dependent necrosis in cells in which cPLA2 was pharmacologically inhibited or genetically depleted. This response was prevented by addition of exogenous arachidonic acid (AA) [3 ].

Subsequent studies identified protein kinase C{alpha} (PKC{alpha}) as a critical downstream target of AA [4 ]. In particular, PKC{alpha} responded to peroxynitrite with a cytosol to mitochondria translocation [5 ] causally linked to the cytosolic accumulation of Bcl2-antagonist of cell death (Bad) [6 ]. Inhibition of the survival signaling at the level of cPLA2 or PKC{alpha} was invariably associated with the mitochondrial accumulation of Bad and Bcl-2-antagonist x (Bax) and with a rapid MPT-dependent necrosis [5 , 6 ]. Thus, nontoxic concentrations of peroxynitrite nevertheless commit cells to MPT-dependent necrosis, which is, however, prevented by an AA-dependent survival signaling targeting Bad, a protein of the Bcl-2 family involved in the regulation of Bcl-2 (or Bcl-XL) activity. It is indeed well-established that proteins belonging to the Bcl-2 family can positively or negatively control MPT, at least in part through protein–protein interaction. Bcl-2 (or Bcl-XL) inhibits MPT [7 8 9 ], and its activity is lost on heterodimerization with Bad [10 , 11 ]. Consistently, we observed that U937 cells (which normally express high levels of Bcl-2), genetically depleted in Bcl-2, are killed by otherwise nontoxic concentrations of peroxynitrite, thus suggesting that Bcl-2 mediates a critical downstream response of the above survival signaling [6 ].

The present study addressed the question of whether the PKC{alpha}-dependent survival signaling cascade is triggered directly by AA itself or is rather mediated by its metabolites of the cyclooxygenase (COX)/lipoxygenase (LO) pathways. We found that nontoxic concentrations of peroxynitrite cause AA release associated with the activation of COX and LO. Only the second enzymatic activity, in particular, that mediated by 5-LO, was, however, implicated in the survival signaling pathway under investigation.


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MATERIALS AND METHODS
 
Chemicals
AA (sodium salt), nordihydroguaiaretic acid (NDGA), AA861, MK886, SC-560, and tetradecanoylphorbol acetate (TPA) as well as most of reagent-grade chemicals, were obtained from Sigma-Aldrich (Milan, Italy). 5-Hydroxyeicosatetraenoic acid (5-HETE), arachidonyl trifluoromethyl ketone (AACOCF3), and FK506 were obtained from Calbiochem (San Diego, CA). Cyclosporin A (CsA) was purchased from Sandoz A.G. (Bern, Switzerland). Rhodamine 123 was from Molecular Probes Europe (Leiden, The Netherlands). [3H] AA was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Cell culture and treatment conditions
U937 human myeloid leukemia cells were cultured in suspension in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS (Euroclone, Celbio Biotecnologie, Milan, Italy), penicillin (50 units/ml), and streptomycin (50 µg/ml; Euroclone) at 37°C in T-75 tissue-culture flasks (Corning, Corning, NY) gassed with an atmosphere of 95% air-5% CO2.

Human peripheral mononuclear cells were isolated by Ficoll gradient centrifugation, and monocytes were purified by adherence on a plastic substrate in RPMI. Macrophages were obtained as described previously [2 ].

Peroxynitrite was synthesized by the reaction of nitrite with acidified H2O2 as described previously [12 ] with minor modifications [13 ]. Treatments were performed in 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 2.5 x 105 cells/ml. Inhibitors of enzymes and/or AA/5-HETE were given to the cultures 3 min after peroxynitrite.

Cytotoxicity assay
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 (i.e., those excluding trypan blue) were counted with a hemocytometer.

Measurement of mitochondrial membrane potential
Cells were exposed for 15 min to 10 µM rhodamine 123 and then treated as detailed in the text. After treatments, the cells were washed, resuspended in 20 µl saline A, and stratified on a slide. Fluorescence images were captured with a BX-51 microscope (Olympus, Japan), equipped with a SPOT-RT camera unit (Diagnostic Instruments, Sterling Heights, MI). The excitation and emission wavelengths were 488 and 515 nm, respectively, with a 5-nm slit width for emission and excitation. 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 (Scion Corp., Frederick, MD). Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/ treatment condition/experiment.

Measurement of extracellular release of [3H] AA
The cells were labeled with [3H] AA (0.5 µCi/ml) and grown for 18 h. Prior to treatments, the cells were washed twice with a saline A, containing 1 mg/ml fatty acid-free BSA, and resuspended in a final volume of 1 ml saline A. After treatments, the cell suspension was centrifuged at 5000 g for 1.5 min, and 500 µl of the resulting supernatant was used for the determination of radioactivity in a Wallac 1409 liquid scintillation counter (Wallac, Turku, Finland).

Determination of LO or prostaglandin endoperoxide synthase (PGHS) activity
LO activity was measured spectrophotometrically by recording the formation of hydroperoxyeicosatetraenoic acid (HPETE) at 234 nm [14 ] and using a Cayman (Ann Arbor, MI) assay kit. COX activity of PGHS was measured polarographically [14 ] and is expressed as nmol O2 consumed/min/µg protein. Peroxidase activity of PGHS was assayed spectrophotometrically [14 ] and is expressed as mUnits/µg protein (1 Unit is the amount of enzyme causing a change in 590 nm absorbance of 1 Unit during 5 min incubation with the substrate). The protein concentration was determined using the Bio-Rad protein assay.

Transfection with cPLA2 or 5-LO-activating protein (FLAP) antisense (AS) and nonsense (NS) oligonucleotides (ONs)
The human AS-ON for cPLA2 (5'-GTA AGG ATC TAT AAA TGA CAT-3') and for FLAP (5'-TTC TTG ATC CAT GTT TGC TT-3') were directed against the initiation site [15 ] or the start codon of mRNA [16 ], respectively. The NS-ONs for cPLA2 (5'-AGT AGA TTG AAT AGA CAC TAT-3') and for FLAP (5'-CTT GTT TAA TCC CCT TTT GT-3') were a random sequence of the AS bases. The ONs were phosphorothioate-modified and synthesized by MWG Biotech (Florence, Italy). 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 described [3 ]. Treatments were performed 24 h after transfection. Using these conditions, we obtained a good transfection efficiency (see below) and thus, avoided the use of transfection agents, such as lipofectamine.

Isolation of the mitochondrial fraction
Cells were suspended in ice-cold, 5 mM HEPES (pH 7.5), 210 mM mannitol, 1 mM EGTA, 70 mM sucrose, and 110 µg/ml digitonin. The cells were disrupted in a glass homogenizer and centrifuged at 5000 g for 20 min at 4°C. The pellets were resuspended in the same buffer, homogenized again, and centrifuged at 2000 g for 5 min at 4°C. The supernatants (S1) were collected. The pellets were rehomogenized, centrifuged at 2000 g for 5 min at 4°C, and the resultant supernatants (S2) collected. S1 and S2 were pooled and centrifuged at 11,000 g for 10 min. The mitochondrial pellets were resuspended in lysis buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mM PMSF, 1 mM dithiothreitol) for 30 min on ice and then centrifuged at 15,000 g for 20 min. The supernatant was used as the soluble mitochondrial fraction.

Isolation of the nuclear and cytosolic fractions
Cells were swelled in an ice-cold hypotonic buffer (10 mM HEPES at pH 7.4, 5 mM MgCl2, 40 mM KCl, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) for 30 min, aspirated repeatedly through a 25-gauge needle (25 strokes), and finally, centrifuged at 200 g to pellet the nuclei. The resulting supernatant was centrifuged at 15,000 g for 30 min (cytosolic faction).

Preparation of the whole cell lysates
The cells were washed twice with PBS and incubated on ice for 1 h with lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM sodium vanadate, and 1 mM sodium fluoride, pH 8.0). Cells were then sonicated with a Sonicator Ultrasonic Liquid Processor XL (Heat System-Ultrasonics, Inc., Plainville, NY) and centrifuged at 21,500 g for 10 min at 4°C to remove detergent-insoluble material.

Western blot analysis
Subcellular fractions and whole cell lysates were subjected to denaturating electrophoresis in a 10% (cPLA2, 5-LO, and PKC{alpha}) or 12% (FLAP and Bad) polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The blots were blocked for 1 h at room temperature with 5% milk powder in Tris-buffered saline (140 mM NaCl, 50 mM Tris-HCl, pH 7.2) containing 0.06% Tween 20 and probed with a primary antibody against cPLA2, FLAP, PKC{alpha}, 5-LO, or Bad (1:500) overnight at 4°C. HRP-conjugate mAb (1:2000) was used for ECL detection.

Statistical analysis
Statistical analysis of the data for multiple comparisons was performed by ANOVA followed by a Dunnett’s test. For single comparison, the significance of differences between means was determined by Student’s t-test.


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RESULTS
 
LO inhibitors promote toxicity in U937 cells exposed to an otherwise nontoxic concentration of peroxynitrite
As we previously reported [1 , 2 ], a 10-min exposure to 100 µM peroxynitrite promotes activation of cPLA2, an event resulting in the release of the lipid messenger AA. The results illustrated in Table 1 confirm and extend these findings by showing that AA release is associated with a remarkable stimulation of the COX and peroxidase activities of PGHS as well as with the stimulation of LO activity, as measured by two independent assays.


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  		Table 1. The Effect of Various Inhibitors on the Activity of PLA2 and LO As Well As on the COX and Peroxidase Activities of PGHS in Cells Exposed to Peroxynitrite

To assess whether the cytoprotective signaling is triggered directly by AA or is mediated by products of AA metabolism, we measured the viability of cells exposed to a nontoxic concentration of peroxynitrite (100 µM) in combination with inhibitors selectively blunting COX or LO activities. We found that the COX-1 inhibitor SC-560 [17 ] abolishes COX and peroxidase activities of PGHS with no apparent effect on PLA2 or LO activities (Table 1) . SC-560 did not cause toxicity in the absence of additional treatments and also failed to promote toxicity (Fig. 1A ) under the same conditions of peroxynitrite exposure, in which toxicity is readily observed upon supplementation of a PLA2 inhibitor [1 ]. These results suggest that products of the COX pathway have hardly any effect on the survival signaling under investigation.


Figure 1
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  		Figure 1. LO inhibitors promote toxicity in cells exposed to an otherwise nontoxic concentration of peroxynitrite. The cells were exposed for 3 min to 0 (•) or 100 µM peroxynitrite ({circ}), centrifuged, and then postincubated for a further 57 min in fresh saline A containing increasing concentrations of SC-560 (A), NDGA (B), AA861 (C), or MK886 (D). Cytotoxicity was then determined using the trypan blue exclusion assay. Results represent the means ± SEM from three to five separate experiments. *, P < 0.01; **, P < 0.001, as compared with cells exposed to peroxynitrite alone (ANOVA followed by a Dunnett’s test).

We therefore tested the effect of the LO inhibitor NDGA. As illustrated in Figure 1B , NDGA promoted toxicity in cells exposed to the otherwise nontoxic dose of peroxynitrite, and the maximal effects were observed at an extremely low concentration (0.08 µM). It should be noted that NDGA is considered poorly selective when used at micromolar concentrations but significantly more selective at the above concentrations [18 ]. Under the conditions used in toxicity studies, NDGA did not cause toxicity in the absence of additional treatments and abolished LO activity stimulated by peroxynitrite with no apparent effect on PLA2 or COX and peroxidase activities of PGHS (Table 1) . We next tested the effects of AA861 (Fig. 1C) , a selective 5-LO inhibitor [19 ], and MK886 (Fig. 1D) , a selective FLAP inhibitor [16 ]. Each of these inhibitors promoted peroxynitrite-dependent toxicity at low concentrations, devoid of intrinsic toxicity, and the maximal response was once again observed at levels causing LO inhibition with no effect on PLA2 or COX and peroxidase activities of PGHS (Table 1) . It is interesting that 0.08 µM NDGA, 1 µM MK886, or 1 µM AA861 also promoted an approximate 50% lethal response in human monocytes or macrophages exposed to a nontoxic dose of peroxynitrite (see Go Fig. 3 ).


Figure 2
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  		Figure 2. 5-HETE, or AA, prevents the lethal response mediated by an otherwise nontoxic concentration of peroxynitrite in cells in which cPLA2 is pharmacologically or genetically inhibited. cPLA2 AS-ON-transfected U937 cells (A) were exposed for 3 min to 100 µM peroxynitrite, centrifuged, and then postincubated for a further 57 min in fresh saline A in the absence or presence of increasing concentrations of AA (•) or 5-HETE ({circ}). Cells were then processed for the assessment of toxicity. The same concentration of peroxynitrite failed to cause loss of viability in cPLA2 NS-ON-transfected cells ({blacksquare}). The inset shows the level of cPLA2 protein expression in NS- and AS-ON-transfected cells. Under the same conditions, peroxynitrite ({blacksquare}) did not cause toxicity in nontransfected U937 cells (B), human monocytes (C), or macrophages (D). Toxicity was, however, observed upon addition of 50 µM AACOCF3, and this response was prevented by AA (•) as well as by 5-HETE ({circ}). AACOCF3, AA, or 5-HETE were given to the cells 3 min after peroxynitrite. Results represent the means ± SEM from four separate experiments. *, P < 0.01; **, P < 0.001, as compared with cells exposed to peroxynitrite alone (A) or associated (B–D) with AACOCF3 (ANOVA followed by a Dunnett’s test).


Figure 3
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  		Figure 3. 5-HETE, unlike AA, prevents the lethal response evoked by 5-LO inhibitors in cells exposed to an otherwise nontoxic concentration of peroxynitrite. Exposure (60 min) of U937 cells (A–C), human monocytes (D–F), or macrophages (G–I) to an otherwise nontoxic concentration of peroxynitrite (100 µM, {blacksquare}) promotes toxicity in the presence of NDGA (0.08 µM, A, D, and G), AA861 (1 µM, B, E, and H), or MK886 (1 µM, C, F, and I). This response is insensitive to AA (•) and abolished by 5-HETE ({circ}). LO-inhibitors, AA, or 5-HETE were given to the cells 3 min after the peroxynitrite. Results represent the means ± SEM from three separate experiments. *, P < 0.01; **, P < 0.001, as compared with cells exposed to peroxynitrite in the presence of each of the above LO inhibitors (ANOVA followed by a Dunnett’s test).

Further studies revealed that as previously observed using inhibitors of PLA2 [20 ], toxicity elicited by 100 µM peroxynitrite in U937 cells supplemented with NDGA, AA861, or MK886 was not associated with enhanced, delayed formation of H2O2 (not shown) and was mediated by MPT. Indeed, as indicated in Table 2 , cells were always rescued by CsA (0.5 µM), an inhibitor of MPT [21 ], but not by FK506 (1 µM), which shares with CsA the ability to inhibit calcineurin but fails to affect the formation of MPT pores [22 ]. In addition, toxicity was preceded by a CsA-sensitive decline in mitochondrial membrane potential, as measured by rhodamine 123 uptake (Table 2) , and opening of MPT pores, as measured by monitoring the changes in mitochondrial calcein fluorescence after quenching of the cytosolic signal with Co2+. These results were identical to those previously obtained with inhibitors of PLA2 [3 ] and are not shown here for the sake of brevity. Hence, cell death observed in the presence of inhibitors of the LO pathway is mediated by MPT, as it was observed under conditions in which the survival signaling was intercepted at the level of PLA2.


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  		Table 2. Effects of CsA or FK506 on Mitochondrial Membrane Potential and Survival of Cells Exposed to Peroxynitrite and Different LO Inhibitors

Taken together, these results indicate that stimulation of cPLA2 activity mediated by peroxynitrite is followed by activation of COX and LO. These results also suggest the involvement of products of the 5-LO pathway in the cytoprotective signaling, preventing peroxynitrite-dependent MPT and toxicity.

Involvement of 5-HETE in the protective signaling preventing monocyte/macrophage cell death induced by peroxynitrite
To provide additional indication for an involvement of 5-LO in the AA-dependent cytoprotective signaling, experiments were performed using U937 cells in which cPLA2 expression was reduced (61.5%, inset to Fig. 2A ) by previous exposure to cPLA2 AS-ONs. As shown in Figure 2A , approximately 50% of these cells were killed by a concentration of peroxynitrite (100 µM), which does not promote toxicity in nontransfected cells (Fig. 1) or in cells transfected with cPLA2 NS-ONs. It is interesting that toxicity was prevented by AA (the PLA2 product) as well as by 5-HETE, the 5-LO product. It is important to note that as we reported previously [20 ], transfection with the AS-ONs abolishes AA release mediated by peroxynitrite (not shown).

The outcome of experiments using nontransfected cells, in which PLA2 activity was, however, suppressed by a pharmacological inhibitor (AACOCF3, 50 µM), is consistent with the findings above, as toxicity mediated by the otherwise nontoxic dose of peroxynitrite was also suppressed by AA or 5-HETE (Fig. 2B) . It is interesting that cytoprotection was also observed in human monocytes (Fig. 2C) or macrophages (Fig. 2D) exposed to peroxynitrite and AACOCF3, but the minimal dose of the lipid messengers required for full survival was in both circumstances significantly greater than that necessary to rescue U937 cells.

Finally, the results illustrated in Figure 3 indicate that 5-HETE, unlike AA, also prevented toxicity mediated by peroxynitrite in cells supplemented with 0.08 µM NDGA (Fig. 3A 3D and 3G) , 1 µM MK886 (Fig. 3B 3E and 3H) , or 1 µM AA861 (Fig. 3C 3F and 3I) . Once again, a greater concentration of the lipid messenger was required for human monocytes (Fig. 3D 3E 3F) and macrophages (Fig. 3G 3H 3I) than for U937 cells (Fig. 3A 3B 3C) .

Taken together, the results presented above provide strong experimental evidence for an involvement of 5-HETE downstream to AA in the protective signaling, preventing MPT-dependent necrosis induced by peroxynitrite in monocytes/macrophages.

Genetic depletion of FLAP promotes a 5-HETE-sensitive, AA-insensitive, lethal response in U937 cells exposed to otherwise nontoxic concentrations of peroxynitrite
The results above indicate an involvement of 5-HETE in the cytoprotective signaling under investigation and imply that 5-LO translocates to the nuclear membrane, where FLAP is located. This event was indeed found to take place after addition of peroxynitrite, and supplementation of AA861 prevented this response (Fig. 4A ). To provide additional information documenting the involvement of 5-LO/FLAP in the survival signaling, we performed experiments using cells with reduced FLAP expression. U937 cells were transfected with phosphorothioate-modified AS-ONs (10 µM) directed against the start codon of human FLAP mRNA [16 ], and complementary NS-ONs were used as a negative control. Western blot analyses using a mAb against the human FLAP showed that the level of the protein was indeed significantly lower (55% decrease) in cells treated with the AS-ONs compared with cells treated with the NS-ONs (Fig. 4B) . Furthermore, peroxynitrite did not cause significant stimulation of LO activity in FLAP down-regulated cells (Fig. 4C) . A remarkable increase in LO activity was observed in cells transfected with the NS-ONs. The expression of FLAP was identical in nontransfected cells or NS-ON-transfected cells (Fig. 4B) , and identical results were obtained in toxicity studies (Fig. 4D) . The FLAP NS-ON-transfected cells were resistant to peroxynitrite, and toxicity was observed after addition of MK886. 5-HETE, unlike AA, prevented toxicity. In contrast with these results, peroxynitrite promoted toxicity in the FLAP AS-ON-transfected cells, and this response was not enhanced further by MK886. 5-HETE, unlike AA, afforded cytoprotection under both conditions.


Figure 4
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  		Figure 4. Transfection with FLAP AS-ONs promotes toxicity in cells exposed to an otherwise nontoxic concentration of peroxynitrite. (A) Cells were treated for 3 min with 0 or 100 µM peroxynitrite, centrifuged, and then postincubated for a further 7 min in fresh saline A in the absence or presence of 1 µM AA861, as indicated in the figure. Western blot analysis for 5-LO was performed on the cytosolic and nuclear fractions. The blots were then washed and reprobed for lamin B and actin proteins. The blots shown are representative of three separate experiments with similar outcomes. (B) The amount of FLAP protein was determined by Western blot analysis in nontransfected cells as well as in cells transfected with FLAP NS- and AS-ONs. The blot shown is representative of three separate experiments. (C) FLAP NS- or AS-ON-transfected cells were exposed for 10 min to 0 or 100 µM peroxynitrite, and LO activity was determined in the cell extracts, as indicated in Materials and Methods. Results represent the means ± SEM from three separate experiments. *, P < 0.001, as compared with untreated cells (unpaired Student’s t-test). (D) FLAP NS- or AS-ON-transfected cells were exposed for 3 min to 100 µM peroxynitrite, centrifuged, and then postincubated for a further 57 min in fresh saline A in the absence or presence of 1 µM MK886 alone or associated with 0.1 µM AA or 0.1 µM 5-HETE. Cytotoxicity was then determined. Results represent the means ± SEMfrom three to five separate experiments. *, P < 0.001, as compared with untreated cells (unpaired Student’s t-test).

Taken together, these studies document the involvement of 5-LO in the protective signaling, preventing MPT-dependent necrosis induced by peroxynitrite.

5-HETE promotes the mitochondrial translocation of PKC{alpha}
The results above indicate an involvement of 5-LO metabolites (e.g., 5-HETE) downstream to AA in the cytoprotective signaling under investigation. The observation that TPA (a PKC activator) prevents toxicity in cells exposed to an otherwise nontoxic concentration of peroxynitrite and a FLAP inhibitor, or a LO inhibitor, suggests that the effects of the 5-LO metabolites are upstream to PKC (Fig. 5A ). Consistently, the results illustrated in Figure 5B indicate that the mitochondrial translocation of PKC{alpha} elicited by peroxynitrite is prevented by AACOCF3 via a mechanism sensitive to AA and 5-HETE. Similar results were obtained using MK886 in the place of AACOCF3, but under these conditions, the mitochondrial translocation of PKC{alpha} was only observed after addition of 5-HETE.


Figure 5
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  		Figure 5. 5-HETE promotes the mitochondrial translocation of PKC{alpha} and the cytosolic localization of Bad. (A) Cells were exposed for 3 min to 0 or 100 µM peroxynitrite, centrifuged, and then postincubated for a further 57 min in fresh saline A in the absence or presence of 1 µM AA861 or 1 µM MK886. Toxicity observed in the presence of the inhibitors was prevented by 100 ng/ml TPA (given to the cells 5 min prior to peroxynitrite). Results represent the means ± SEM from four separate experiments. *, P < 0.001, as compared with untreated cells (unpaired Student’s t-test). (B and C) Cells were treated for 3 min with peroxynitrite, centrifuged, and then postincubated for a further 7 min in fresh saline A in the absence or presence of various additions, as indicated in the figure. Western blot analysis for PKC{alpha}, or Bad, was performed in the mitochondrial fractions. The blots were then washed and reprobed for heat shock protein 60 kDa (HSP-60). The blots shown are representative of three separate experiments with similar outcomes.

We previously showed that the accumulation of PKC{alpha} in the mitochondrial fraction is causally linked to the mitochondria to cytosol translocation of Bad [6 ]. This event was indeed observed after addition of peroxynitrite, and MK886 not only prevented this response but actually caused an increase of mitochondrial Bad (Fig. 5C) . 5-HETE, unlike AA, abolished the effects of the FLAP inhibitor. Similar results were obtained using AA861 in the place of MK886 (not shown). The observation that pharmacological inhibition of 5-LO is rapidly followed by cell death is therefore readily explained by the significant accumulation of Bad in the mitochondria. We previously showed that this condition is associated with the loss of the anti-MPT activity of Bcl-2 [6 ] with the mitochondrial translocation of Bax [5 ] and with a rapid MPT-dependent necrosis [6 ].

Collectively, the results above indicate that 5-HETE is involved in the mitochondrial translocation of PKC{alpha}, a critical event leading to the cytosolic localization of Bad.


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DISCUSSION
 
Previous studies from our laboratory indicate that nontoxic concentrations of peroxynitrite nevertheless commit U937 cells to a MPT-dependent toxicity, however, prevented by the parallel activation of a survival pathway in which cPLA2-released AA is critically involved [1 2 3 ]. The downstream event subsequently identified was represented by the mitochondrial translocation of PKC{alpha} [5 ] causally linked to the cytosolic translocation of the fraction of Bad, which is normally detected in the mitochondria of untreated cells [6 ].

In principle, activation of PKC{alpha} might take place as a consequence of direct activation mediated by AA, as previously observed in other systems [23 , 24 ] or via an indirect mechanism involving the action of metabolites of the LO or COX pathways [25 26 27 28 ]. The results presented in this study indicate that AA is not directly responsible for the triggering of the above events; rather, the cytoprotective signaling appears to be initiated by metabolites of the 5-LO pathway.

The first clue in this direction was given by carefully controlled inhibitor studies showing that treatments blunting LO activity, in the absence of detectable effects on PLA2 or COX and peroxidase activities of PGHS (Table 1) , promote toxicity in cells exposed to otherwise nontoxic concentrations of peroxynitrite (Fig. 1) . However, toxicity was not observed under conditions of COX inhibition in the absence of effects on PLA2 or LO activities.

It is important to note that as we previously observed under conditions in which the survival signaling was inhibited at the level of cPLA2 [1 , 20 ] or PKC{alpha} [4 ], toxicity caused by the above treatments was mediated by MPT. We observed loss of mitochondrial membrane potential (Table 2) , loss of mitochondrial calcein (not shown), and toxicity (Table 2) sensitive to the MPT inhibitor CsA. It was also interesting to observe that the effects of a general inhibitor of LO, NDGA, were mimicked by two inhibitors selectively blunting 5-LO activity, directly (AA861) [19 ] or indirectly (MK886) [16 ]. The 5-LO enzyme, localized in the cytosol or in the nuclear eucromatin, translocates to the nuclear membrane upon cell activation [29 ] and promotes the transformation of AA into 5-HETE; this reaction requires an 18-kDa membrane-bound protein, FLAP [30 ], sensitive to inhibition by MK886 [16 ].

The results above are therefore consistent with the possibility that downstream products of 5-LO are critically involved in the survival response under investigation. The following lines of evidence put more weight on this notion: 1) Peroxynitrite indeed stimulates 5-LO activity. Two distinct biochemical assays provided experimental evidence for the involvement of a LO activity sensitive to general LO inhibitors as well as to AA861 or MK886 (Table 1) . In addition, peroxynitrite failed to stimulate LO activity in cells genetically depleted in FLAP (Fig. 4C) . Finally, peroxynitrite caused the translocation of 5-LO to the nuclear envelope, and this response was sensitive to AA861 (Fig. 4A) . 2) Toxicity, mediated by an otherwise nontoxic dose of peroxynitrite in cells in which the activity of cPLA2 was pharmacologically inhibited or genetically depleted, is prevented by exogenous AA as well as by 5-HETE (Fig. 2) . These results indicate that inhibition of the survival signaling at the cPLA2 level can be circumvented by the addition of the cPLA2 product (e.g., AA) as well as of the 5-LO product, 5-HETE. Hence, the latter is a likely intermediate of the survival signaling operating downstream to AA. 3) Toxicity, observed in cells supplemented with nonselective LO inhibitors, or with selective inhibitors of 5-LO or FLAP, was insensitive to AA but prevented by 5-HETE (Fig. 3) . Likewise, 5-HETE prevented toxicity in cells genetically depleted in FLAP, whereas AA had hardly any effect (Fig. 4D) . These results indicate that the effects of the above inhibitors on peroxynitrite toxicity are specifically mediated by their ability to intercept the survival signaling upstream to 5-HETE formation and downstream to AA release (i.e., via inhibition of 5-LO).

5-HETE is therefore a likely trigger of the AA-dependent mitochondrial translocation of PKC{alpha} in cells exposed to a nontoxic, but nevertheless MPT-committing, peroxynitrite concentration. The observation that toxicity arising after 5-LO inhibition is sensitive to TPA, a potent PKC activator, is consistent with this notion (Fig. 5A) . In addition, prevention of the peroxynitrite-dependent mitochondrial translocation of PKC{alpha} by upstream inhibition of the survival signaling was achieved via inhibition of PLA2, sensitive to exogenous AA or 5-HETE, or FLAP, sensitive to 5-HETE but insensitive to AA (Fig. 5B) .

Collectively, these results are in keeping with those obtained in toxicity studies and provide a clear-cut indication that 5-HETE is critically involved in the peroxynitrite-dependent mitochondrial translocation of PKC{alpha}. It is not surprising, and in keeping with our previous findings [5 , 6 ], that all of the above manipulations preventing the mitochondrial translocation of PKC{alpha} were associated with a remarkable mitochondrial accumulation of Bad (Fig. 5C) preceding the rapid onset of a MPT-dependent necrosis (Figs. 1 2 3 , Table 2 ). It should be also noted that as we recently showed [6 ], peroxynitrite causes the cytosolic translocation of the fraction of Bad, normally residing in the mitochondrial compartment. We reproducibly identified a significant amount of Bad in the mitochondria of untreated U937 cells [6 ], and the loss of this fraction of Bad appears to be critical for prevention of MPT in cells committed to this event by peroxynitrite [6 ]. The mitochondrial loss of Bad is expected to promote survival, as these conditions allow the full expression of the anti-MPT activity of Bcl-2 and Bcl-XL. In particular, we previously showed that upstream inhibition of the survival signaling is associated with the loss of the anti-MPT activity of Bcl-2 [6 ], with the mitochondrial translocation of Bax [5 ] and with a rapid MPT-dependent necrosis [6 ].

The results above indicate that activation of 5-LO is a likely intermediate step, downstream to AA and upstream to PKC{alpha}, preventing toxicity in U937 cells exposed to peroxynitrite. It is important, however, that critical experiments using human monocytes or macrophages produced results identical to those obtained in U937 cells (Figs. 2 and 3) . These findings, along with others [4 5 6 ], provide strong evidence suggesting that cPLA2, 5-LO, and PKC{alpha} are sequentially involved in the signaling leading to the mitochondrial loss of Bad and prevention of MPT-dependent toxicity, also in human monocytes and macrophages exposed to peroxynitrite.

As we previously observed [2 ], monocytes and macrophages committed to death by PLA2 inhibitors and otherwise nontoxic concentrations of peroxynitrite require significantly greater concentrations of AA (i.e., 3 µM) than U937 cells (i.e., 0.1 µM) for survival (Fig. 2C and 2D) . A need for the same, greater concentrations of AA was also observed in DMSO-differentiated U937 cells [31 ]. We now report that much greater concentrations of 5-HETE are required to rescue monocytes/macrophages than U937 cells supplemented with inhibitors of PLA2 or LO after exposure to otherwise nontoxic concentrations of peroxynitrite. The reasons for the observed differences are not readily explained and therefore, are currently under investigation.

In conclusion, the results presented in this study fill an important gap left by our previous work about the cytoprotective signaling triggered by AA in monocytes/macrophages exposed to peroxynitrite. It appears that AA does not directly cause the mitochondrial translocation of PKC{alpha}; rather, this event is mediated by 5-HETE and/or other downstream products of the 5-LO pathway. The results above, along with other results previously published on this topic [1 2 3 4 5 6 ], lead us to propose that cells belonging to the monocyte/macrophage lineage cope with peroxynitrite, according to the mechanism outlined in Figure 6 . It is interesting to note that inflammatory cells, although committed to MPT-dependent toxicity by a reactive species they produce (i.e., peroxynitrite), nevertheless, survive in response to products largely available at the inflammatory sites (i.e., AA and 5-HETE) with a simple, yet effective, survival mechanism. AA and 5-HETE are sequentially involved in a key step, causing the mitochondrial translocation of PKC{alpha}, a critical event leading to the cytosolic localization of Bad. These conditions, by preventing the heterodimerization of Bad with Bcl-2 or Bcl-XL, allow the full expression of the anti-MPT function of the latter two proteins. Thus, as we previously noted for bona fide inhibitors of PLA2 [1 , 2 , 20 ] or for agents indirectly leading to PLA2 inhibition (i.e., glucocorticoids) [32 ], pharmacological inhibitors of LO are likely to cause an anti-inflammatory effect, not only by inhibiting the formation of species causing tissue damage but also by selectively promoting death of inflammatory cells at the inflammatory sites.


Figure 6
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  		Figure 6. Survival signaling preventing toxicity in monocytes/macrophages committed to MPT by peroxynitrite. An otherwise nontoxic concentration of peroxynitrite nevertheless commits cells to MPT-dependent necrosis, however, prevented by a survival signaling triggered by cPLA2-released AA. The results presented in this study indicate that the previously identified, downstream events (i.e., the mitochondrial translocation of PKC{alpha} and the ensuing cytosolic accumulation of Bad) are not mediated directly by AA but rather, by some 5-LO metabolite, presumably 5-HETE. These conditions, by preventing the heterodimerization of Bad with Bcl-2, or Bcl-XL, allow the full expression of the anti-MPT function of the latter two proteins. Inhibition of survival signaling at the level of cPLA2, 5-LO, or PKC{alpha} was invariably associated with the mitochondrial accumulation of Bad and Bax and with a rapid MPT-dependent necrosis.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Italian Association for Cancer Research (Associazione Italiana Ricerca sul Cancro) to O. C.

Received April 3, 2006; revised May 26, 2006; accepted June 14, 2006.


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