Departments of Biochemistry and Pathology, Center of Vascular Biology, Weill Medical College of Cornell University, New York, New York 10021
Submitted 10 April 2003 ; accepted in final form 22 January 2004
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ABSTRACT |
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reactive nitrogen species; prostaglandin H2 synthase; extracellular signal-regulated kinase; p38; cytosolic phospholipase A2
There is now strong evidence linking the NO· and arachidonic acid pathways. For instance, mice bearing a targeted deletion in the inducible NO synthase (iNOS) gene demonstrate reduced ability to synthesize prostaglandin E2 (PGE2) (42). Similarly, administration of NOS inhibitors to rats leads to a reduction in prostaglandin production in inflammatory lesions (56, 57). Furthermore, inflamed brain tissue from iNOS-knockout mice reveals decreased levels of PGE2 compared with wild-type mice (46, 54). In addition, arginine depletion can decrease prostaglandin synthesis in mouse macrophages stimulated with lipopolysaccharide (LPS) (54, 55). These observations indicate that NO· and/or NO-derived species can alter PGHS activity and eicosanoid production in vivo.
The chemistry of NO· under physiological conditions is complex, leading to the formation of various RNS that include NO·, nitrosonium ion (NO+), nitroxyl anion (NO), nitrite (NO2), nitrate (NO3), peroxynitrite (ONOO) (62) and nitrosoperoxocarbonate (ONOOCO2) (50). For this reason, the type of interaction between RNS and PGHS enzymes is highly dependent on the nature of the species under consideration. For example, ONOO, which arises from the reaction between NO· and superoxide radical anion (27), activates PGHS (34, 63), whereas NO· inhibits activation (63). The mechanism of ONOO-induced PGHS activation is likely similar to that of peroxide activation of PGHS, which involves oxidation of the heme moiety (15). ONOO has also been shown to cause Tyr nitration in PGHS in vascular smooth muscle cells (13) and in platelets (7). These results indicate that it is possible for a specific RNS such as ONOO to have both an inhibitory and activating effect.
Although RNS directly affect the activity of PGHS enzymes, the effect of RNS on the signaling pathways that lead to arachidonic acid release and increased PGHS activity has not been fully investigated. Arachidonic acid release from arachidonyl phospholipids is catalyzed by cytosolic phospholipase A2 (cPLA2) (9, 37). cPLA2 activation, which requires both phosphorylation and an increase in intracellular Ca2+ (25, 37), is achieved by several agonists, including growth factors, neurotransmitters, angiotensin II, vasopressin, LPS, colony-stimulating factor-1, and thrombin (32, 37, 38). cPLA2 is phosphorylated on Ser505 in vitro by mitogen-activated protein (MAP) kinases (MAPKs), including the extracellular signal-regulated kinases (ERKs) and p38 MAPK (32, 39, 45, 66), although the specific signaling mechanism varies in response to different agonists in different cell types. In addition, cPLA2 is phosphorylated on Ser727 by MAPK-interacting kinase I (MNK-I), or a closely related isoform (24), and on Ser515 by calcium/calmodulin-dependent protein kinase II (CaMKII) (44). When expressed in a baculovirus/Sf9 cell system, cPLA2 is phosphorylated on four serine residues (Ser437, Ser454, Ser505, and Ser727) (12). However, the signaling mechanisms by which RNS might activate cPLA2 are not yet fully elucidated.
The actions of RNS, such as ONOO, which is known to initiate lipid peroxidation (49), react with sulfhydryl groups (48), and cause oxidative damage via Tyr nitration (13, 28), are not limited to immediate chemical damage. ONOO has been found to activate calcium-dependent PKC, the ERKs, c-Jun NH2-terminal kinase (JNK), and p38 MAPK (3, 21, 29, 58). ONOO also affects cellular signaling by causing dimerization of the epidermal growth factor receptor (EGFR) in PC-12 cells, possibly through intermolecular dityrosine cross-linking (65), but may also occur independently of the EGFR receptor (71). It has been reported that ONOO causes the release of arachidonic acid from PC-12 cells (23) due to stimulation of a low-molecular-mass form of cPLA2 in mitochondria isolated from these cells (22). However, the specific role of ONOO in the signaling cascade leading to cPLA2 activation and arachidonic acid release in vascular smooth muscle cells has not yet been examined.
In this study, we found that arachidonic acid is released in vascular smooth muscle cells after challenge with ONOO. To investigate the hypothesis that ONOO stimulates the phosphorylation of signaling molecules leading to the activation of cPLA2, we examined the effect of ONOO on the p38 MAPK, CaMKII, and ERK signaling pathways. We believe our new findings define the contributions of specific signaling pathways linking ONOO to changes in eicosanoid metabolism in vascular cells.
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EXPERIMENTAL PROCEDURES |
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UV/Vis spectroscopy and handling of ONOO.
The concentration of ONOO was determined with a Perkin Elmer Lambda 20 spectrophotometer by measuring its absorbance at 302 nm ( = 1,670 M1·cm1). Matching quartz precision cells (Hellma, 1-cm path length; 1-ml or 100-µl volume) were used in our measurements. On receiving ONOO, the sample was thawed, its concentration was determined by UV/Vis spectroscopy, and it was divided into smaller aliquots, snap frozen, and stored at 80°C until required. During an experiment, an aliquot of ONOO was kept on dry ice until its addition was required, at which point it was thawed and added directly to the well. The remnants of the sample were immediately snap frozen on dry ice, and the concentration of the sample was determined after completion of the experiment. A comparison of the ONOO concentration before and after the experiment allowed us to determine the quality and consistency of ONOO used in the experiment. These protocols ensured reproducibility. Aliquots used in an experiment were not reused.
Smooth muscle cell culture.
Primary passage rat smooth muscle cells were isolated and characterized as described previously (47). Cells were plated either in six-well dishes (for arachidonic acid release or PGE2/6-keto-PGF1 measurements in the supernatant) or in 100-mm dishes (for the measurement of phosphorylated signaling molecules in the cell lysate) in DMEM supplemented with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) glutamine. All cells were incubated at 37°C in 5% CO2 in air. Cells were rendered quiescent for 34 days with serum-free medium (quiescent medium) containing insulin, transferrin, and selenium (ITS; Life Technologies) to suppress PGHS-2 expression (52). Nitrate/nitrite levels were measured with a colorimetric assay (Cayman Chemical) and were found to be nondetectable in the supernatant medium of quiescent cells. Cells between passages 6 and 15 were used in experiments. We did not see significant variation in our results related to passage number.
Treatment of smooth muscle cells. Quiescent rat smooth muscle cells were exposed to either quiescent medium or quiescent medium with added ONOO or arachidonic acid for 1 h at 37°C. In experiments requiring ONOO, ONOO from a stock solution (100200 mM) was added directly to the medium in the form of a single bolus while the dish was swirled (63). This immediate addition strategy was performed because ONOO has an extremely short half-life. In control experiments, ONOO was allowed to decompose (as determined spectrophotometrically) at room temperature for 23 days. In other control experiments, cells were treated with NaOH as vehicle control (data not shown). In experiments using the inhibitors (including SB-202190, U0126, KN-93, or BAPTA-AM and EGTA) cells were first pretreated with the inhibitor (1 h for SB-202190, KN-93, BAPTA-AM, and EGTA; 40 min for U0126) and then stimulated with ONOO (200 or 500 µM), or in some cases arachidonic acid (10 µM), for time points that ranged between 0 and 60 min, depending on the type of experiment being performed.
PGE2 and 6-keto-PGF1 measurements.
Quiescent smooth muscle cells in six-well dishes were exposed to ONOO (100, 200, and 500 µM) for 1 h (or for time points between 0 and 60 min), and the supernatant medium was assayed for either PGE2 or 6-keto-PGF1
formation with enzyme immunoassay assay kits (Amersham Pharmacia). Quiescent smooth muscle cells in six-well dishes were also exposed to 100500 µM nitrite, S-nitroso-N-acetyl-penicillamine (SNAP), 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), and SIN-1. Total protein in the cell lysate was determined by using either the Lowry or the modified Lowry method (41). After exposure of the cells to 100, 200, and 500 µM ONOO, nitrate/nitrite levels in the supernatant medium were found to be 114 ± 7, 222 ± 4, and 473 ± 10 µM nitrate/nitrite, respectively, which closely reflects the expected concentration of the decomposition products of ONOO.
Western blotting.
After treatments, cells were washed with PBS and lysed in buffers, the composition of which varied depending on which phosphorylated signaling molecule was under investigation. The modified lysis buffer used for detecting the phospho-specific forms of p38, MEK1/2, ERK1/2, cPLA2, Raf-1, and MKK3/6 comprised (in mM) 20 Tris·HCl pH 7.5, 1 EDTA, 1 EGTA, 150 NaCl, 2.5 sodium pyrophosphate, 1 -glycerolphosphate, 1 sodium vanadate, and 1 phenylmethylsulfonyl fluoride (PMSF) with 1% Triton X-100 and 1 µg/ml leupeptin. The modified lysis buffer used for detecting the phospho-specific form of CaMKII comprised (in mM) 20 Tris·HCl pH 8, 2 EDTA, 2 EGTA, 10 sodium phosphate, 2 dithiothreitol, 25 benzamidine, and 1 PMSF with 25 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, and 10 µg/ml aprotinin. The cells were scraped, transferred to an Eppendorf tube, vortexed, and put on ice for 30 min. The lysates were clarified by centrifugation (10,000 g at 4°C) for 10 min. The protein sample (2550 µg) was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked for 90 min at 25°C with 5% nonfat milk in a PBS-1% Tween buffer. The membrane was then incubated overnight at 4°C with an appropriate dilution of the phospho-specific antibody, followed by incubation for 75 min with a 1:2,000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody. The immunoblot signal was visualized through enhanced chemiluminescence.
Determination of arachidonic acid release. Confluent rat smooth muscle cells in six-well dishes were labeled for 24 h with [3H]arachidonic acid (0.51 µCi/ml) in quiescent DMEM. The cells were washed three times with quiescent DMEM containing 0.2% fatty acid-free bovine serum albumin (BSA) and once in PBS to remove all unincorporated [3H]arachidonic acid. Approximately 8090% of the added [3H]arachidonic acid was incorporated into the cells. The cells were treated with and without inhibitors and stimulated with ONOO as described above. Before ONOO addition, 100 µl of the medium was removed and counted for the presence of 3H label. After ONOO treatment, another 100 µl of the medium was removed and counted. The difference obtained served as the measure of arachidonic acid release.
Miscellaneous. Cell viability was assessed by in situ staining with Trypan blue, and cell number was determined by hemocytometer. At 500 µM ONOO, cell viability experiments demonstrated no significant cell death (data not shown). ONOO becomes toxic under our experimental conditions at levels of 1 mM and higher. Unless otherwise stated, data are reported as averages ± SE, with significant differences determined by a single-factor ANOVA or by Student's t-test.
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RESULTS |
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DISCUSSION |
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In this study, we have examined the specific role of ONOO in initiating cellular signaling cascades leading to the activation of cPLA2 and the eventual release of arachidonic acid in smooth muscle cells, which has not yet been characterized. We investigated pathways that have shown precedence in cPLA2 activation: p38, CaMKII, and the ERKs (32, 39, 44, 45, 66). We observed phosphorylation of MEK1/2, ERK1/2, p38, and cPLA2 in smooth muscle cells after incubation with ONOO in both a time- and concentration-dependent manner (Figs. 2 and 3). Using SIN-1, a molecule that generates both NO· and superoxide anion on decomposition, we demonstrated that in situ generation of ONOO activates the same signaling molecules as added ONOO (Fig. 2). The ONOO concentrations used are consistent with those previously reported by others to activate MAPKs (29) and the EGFR (65). Although the ONOO concentrations are high, it is necessary to consider that ONOO decomposition occurs with a half-life of 1 s at pH 7.4. This suggests that 500 µM ONOO decays to 2 µM in <10 s, which is within the range of physiological concentrations of ONOO (16). Thus only a small fraction of the total ONOO concentration added comes into contact with the cell (72). In addition, given the reactivity of ONOO with multiple targets in the cell (13, 28, 48, 49), it is not possible to define the local concentration of ONOO required to initiate the signaling molecules that we have studied.
Although it has been established that ONOO stimulates both ERK and p38 MAPK in PC-12 cells (29), until this study it was not clear which MAPK is responsible for the ONOO-mediated activation of cPLA2 in vascular smooth muscle cells. To determine which MAPK is responsible for the ONOO-mediated activation of cPLA2, we used specific inhibitors aimed at blocking either MEK1/2 or p38 MAPK. The highly specific MEK1/2 inhibitor U0126 served to prevent ERK1/2 phosphorylation (14), whereas SB-202190 specifically inhibited p38 MAPK (36). On incubation with the MEK-1/2 inhibitor U0126, ONOO-induced arachidonic acid release and cPLA2 phosphorylation were significantly reduced to control levels, indicating full inhibition (Fig. 6). Upstream activation of ERK1/2 and MEK1/2 likely involves Raf-1, which is the main effector recruited by GTP-bound Ras (2). In these studies, we have demonstrated that Raf-1 is also phosphorylated after ONOO incubation. These findings suggest its involvement in the activation of the ERK-MAPK pathway. The initial signal leading to the activation of the Raf-1/MEK/ERK pathway, however, remains unknown. ONOO is known to permeate the membrane (1), where it could cause chemical modification of specific molecules that, in turn, initiate phosphorylation of signaling molecules. Use of the p38 inhibitor SB-202190 also led to a significant reduction in ONOO-induced arachidonic acid release, although only partial reduction was attained, indicating incomplete involvement of the p38 MAPK pathway (Fig. 4). p38 MAPK phosphorylation is mediated by the upstream signaling kinases MKK6/MKK3, which were also phosphorylated after ONOO incubation. p38 MAPK is known to participate in signal transduction initiated by cellular stress (10, 68), and thus it is reasonable to assume that ONOO, a molecule that is capable of inducing oxidative reactions, mediates the activation of p38 MAPK. The fact that complete inhibition of arachidonic acid release was achieved with the MEK1/2 inhibitor U0126 suggests that the p38 MAPK pathway is initiated by metabolites downstream of MEK1/2. This finding is supported by the fact that ONOO-induced p38 phosphorylation was inhibited by U0126 (Fig. 7A). Here, it was also demonstrated that the addition of arachidonic acid activates p38 in smooth muscle cells, a process that is independent of the MEK/ERK pathway (Fig. 7B). Recently, it was shown that cPLA2-dependent arachidonic acid metabolites contribute to norepinephrine-induced activation of p38 MAPK in vascular smooth muscle cells (30). In particular, the arachidonic acid metabolites of lipoxygenase, 5(S)-hydroxyeicosatetraenoic acid (HETE), 12(S)-HETE, and 15(S)-HETE, are known to activate p38 MAPK (30, 51, 67). The PGHS metabolite, PGE2, is also known to cause p38 MAPK activation (18). Thus formation of arachidonic acid metabolites may cause a positive feedback mechanism in the cPLA2-catalyzed arachidonic acid process. Taken in total, our findings indicate that the major ONOO-driven pathway in smooth muscle cells leading to arachidonic acid release and subsequent eicosanoid production is the MEK/ERK pathway and that initiation of the p38 MAPK pathway is dependent on metabolites generated from the MEK/ERK pathway.
Involvement of CaMKII in ONOO-mediated activation of ERK1/2 was previously reported in PC-12 cells (29). Here, we demonstrate that CaMKII is also activated by ONOO in rat smooth muscle cells (Fig. 5). CaMKII requires Ca2+ and calmodulin (CaM) for activation, and, once activated, CaMKII autophosphorylates at Thr286, which allows phosphorylation of target substrates (17, 61). Phosphorylation of Thr286 results in partial activation of the kinase, even in the absence of Ca2+/CaM (61). The CaMKII-specific inhibitor KN-93 (40) can prevent CaMKII autophosphorylation. Although norepinephrine-induced activation of CaMKII leads to cPLA2 phosphorylation on Ser515 (but not on Ser505 or Ser727) with concomitant arachidonic acid release (44), this was not observed in our studies using ONOO as an agonist. Our studies indicate that full-length cPLA2 is activated with phosphorylation occurring at Ser505.
In addition to phosphorylation, cPLA2 activation also requires a source of Ca2+ (25, 37). In this regard, ONOO exposure is known to cause increased cytosolic Ca2+, which depends on the presence of extracellular Ca2+ (3). Coupled with the role of ONOO in cellular signaling, we thus examined the role of Ca2+ as a necessary requirement for the activation of cPLA2. Ca2+ chelation was found to prevent the release of arachidonic acid after ONOO addition (Fig. 8). These results implicate a role for the calcium-dependent form of PLA2, which requires Ca2+ for the translocation of cPLA2 from the cytosol to membranes where phospholipids are present (70). It could be argued that the increased permeability of Ca2+ is due to membrane damage by ONOO. However, the fact that maximal cPLA2 activation and prostaglandin production occurred 2030 min after ONOOaddition and the fact that this process was prevented by specific inhibitors of cell signaling molecules indicate that cell damage is unlikely to be the cause.
Finally, the role of ONOO in cellular signaling has implications in the context of inflammation and, in particular, vascular disease. Atherosclerotic lesions have been shown to contain iNOS, which is capable of generating both NO· and superoxide (69) and may promote the activity of ONOO (8). The rate of ONOO production is three times faster than the rate by which superoxide is scavenged by superoxide dismutase (4), indicating that ONOO formation is favorable in a pathophysiological setting. Prolonged exposure to ONOO leads to Tyr nitration that will inhibit PGI2 synthase (59, 7375) and PGHS (13), thus limiting the production of PGI2 (64). Indeed, nitrated PGHS-1 has been observed in human atherosclerotic lesions, although the mechanism of its formation remains unknown (13). Although the direct effects of ONOO in the pathology of vascular disease remain a question, it is apparent that ONOO mediates the production of eicosanoids via the activation of ERK in vascular smooth muscle cells. In this regard, it is interesting to note that elevated levels of activated ERK1/2 have been found in rabbit atherosclerotic lesions compared with control tissue (26). It is possible that the localization of iNOS in the vicinity of an atherosclerotic lesion elevates ONOO levels, thereby activating the ERK family of MAPKs. Thus, although the actions of ONOO are considered deleterious on an acute chemical basis, ONOO may provide the cell with a means to combat some of the clinical manifestations of inflammation through specific eicosanoids before Tyr nitration becomes prominent. Increased PGHS-2 expression in atherosclerosis (60) can contribute to the increased eicosanoid production that is observed in atherosclerotic patients (6, 19). However, the fact that iNOS gene removal causes a reduction in PGE2 synthesis in mice (42) argues for a role of RNS in this process. It is intriguing to consider that ONOO-induced signaling pathways (in addition to ONOO activation of PGHS) may be contributing to increased eicosanoid output in atherosclerotic patients. Our results indicate that studies investigating the role of the NO· pathway on eicosanoid production need to consider the contribution of signaling pathways initiated by RNS. These findings may provide evidence for a new role of ONOO during vascular disease.
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GRANTS |
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ACKNOWLEDGMENTS |
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M. J. Resnick and R. Lindenbaum are currently medical students at the University of Pennsylvania School of Medicine and the Sackler Institute of Graduate Biomedical Sciences at New York University School of Medicine, respectively.
Present address of D. Mittar: Dept. of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143 005, India.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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