Nitric Oxide Down-regulates MKP-3 mRNA Levels

MAP kinase-dependent phosphorylation processes have been shown to interfere with the degradation of the antiapoptotic protein Bcl-2. The cytosolic MAP kinase phosphatase MAP kinase phosphatase-3 (MKP-3) induces apoptosis of endothelial cells in response to tumor necrosis factor α (TNFα) via dephosphorylation of the MAP kinase ERK1/2, leading to Bcl-2 proteolysis. Here we report that the endothelial cell survival factor nitric oxide (NO) down-regulated MKP-3 by destabilization of MKP-3 mRNA. This effect of NO was paralleled by a decrease in MKP-3 protein levels. Moreover, ERK1/2 was found to be protected against TNFα-induced dephosphorylation by coincubation of endothelial cells with the NO donor. Subsequently, both the decrease in Bcl-2 protein levels and the mitochondrial release of cytochrome c in response to TNFα were largely prevented by exogenous NO. In cells overexpressing MKP-3, no differences in phosphatase activity in the presence or absence of NO were found, excluding potential posttranslational modifications of MKP-3 protein by NO. These data demonstrate that upstream of theS-nitrosylation of caspase-3, NO exerts additional antiapoptotic effects in endothelial cells, which rely on the down-regulation of MKP-3 mRNA. MAP kinase-dependent phosphorylation processes have been shown to interfere with the degradation of the antiapoptotic protein Bcl-2. The cytosolic MAP kinase phosphatase MAP kinase phosphatase-3 (MKP-3) induces apoptosis of endothelial cells in response to tumor necrosis factor α (TNFα) via dephosphorylation of the MAP kinase ERK1/2, leading to Bcl-2 proteolysis. Here we report that the endothelial cell survival factor nitric oxide (NO) down-regulated MKP-3 by destabilization of MKP-3 mRNA. This effect of NO was paralleled by a decrease in MKP-3 protein levels. Moreover, ERK1/2 was found to be protected against TNFα-induced dephosphorylation by coincubation of endothelial cells with the NO donor. Subsequently, both the decrease in Bcl-2 protein levels and the mitochondrial release of cytochrome c in response to TNFα were largely prevented by exogenous NO. In cells overexpressing MKP-3, no differences in phosphatase activity in the presence or absence of NO were found, excluding potential posttranslational modifications of MKP-3 protein by NO. These data demonstrate that upstream of theS-nitrosylation of caspase-3, NO exerts additional antiapoptotic effects in endothelial cells, which rely on the down-regulation of MKP-3 mRNA. mitogen-activated kinase extracellular signal-regulated kinase nitric oxide MAP kinase phosphatase-3 sodium nitroprusside tumor necrosis factor α S-nitroso-n-acetylpenicillamine human umbilical vein endothelial cells p-nitrophenyl phosphate mitogen-activated protein kinase/extracellular signal-regulated kinase kinase analysis of variance 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (Z)-1-[N-(3-ammoniopropyl)- N-(n-propyl)amino]diazen-1-ium-1,2-diolate) Apoptosis is the enzymatically controlled form of cell death induced by stimulation of distinct cellular signal transduction pathways, as opposed to the lethal cell damage that is known as necrosis (1Cohen J.J. Immunol. Today. 1993; 14: 126-130Abstract Full Text PDF PubMed Scopus (1222) Google Scholar). In the past few years, several signaling systems have been identified that control apoptotic cell death (2Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5078) Google Scholar, 3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6104) Google Scholar, 4Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Whereas the caspase cascade executes the apoptotic pathway, MAP kinases1 are involved in modulating various regulatory pathways of the cell death machinery (5Karin M. Ann. N. Y. Acad. Sci. 1998; 851: 139-146Crossref PubMed Scopus (287) Google Scholar). Whereas the c-Jun N-terminal MAP kinase (stress-activated protein kinase) promotes apoptosis in various cell types (6Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1706) Google Scholar), the MAP kinase ERK1/2 exerts prosurvival functions (7Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5014) Google Scholar). Recently, ERK1/2 was shown to regulate protein levels of the antiapoptotic Bcl-2, thus linking ERK1/2 with the apoptotic signaling complex (8Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google Scholar). In detail, by maintaining Bcl-2 in its phosphorylated status, ERK1/2 prevents Bcl-2 from ubiquitination, thereby inhibiting its degradation via the proteasome complex (8Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google Scholar, 9Breitschopf K. Haendeler J. Malchow P. Zeiher A.M. Mol. Cell. Biol. 2000; 20: 1886-1896Crossref PubMed Scopus (290) Google Scholar). Bcl-2 in turn prevents the mitochondrial release of cytochrome c (10Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4236) Google Scholar), an event that leads to formation of the apoptosome complex ultimately culminating in the activation of the executioner caspase-3 (11Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6150) Google Scholar). Besides the well established pro-apoptotic effect elicited by high concentrations of nitric oxide (NO), NO also exerts potent antiapoptotic effects in a variety of cells (12Mannick J.B. Asano K. Izumi K. Kieff E. Stamler J.S. Cell. 1994; 79: 1137-1146Abstract Full Text PDF PubMed Scopus (457) Google Scholar, 13Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. J. Exp. Med. 1997; 185: 601-608Crossref PubMed Scopus (782) Google Scholar). Several interactions of NO with the apoptotic signaling machinery have been postulated to explain the apoptosis inhibitory effects of NO. NO was shown to nitrosate not only the apoptosis executing enzyme caspase-3, where different apoptotic pathways converge (13Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. J. Exp. Med. 1997; 185: 601-608Crossref PubMed Scopus (782) Google Scholar, 14Mannick J.B. Miao X.Q. Stamler J.S. J. Biol. Chem. 1997; 272: 24125-24128Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), but also caspase-6, -7, and -8 (15Li J. Billiar T.R. Talanian R.V. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; 240: 419-424Crossref PubMed Scopus (472) Google Scholar, 16Dimmeler S. Zeiher A.M. Cell Death Differ. 1999; 6: 964-968Crossref PubMed Scopus (231) Google Scholar). Furthermore, NO has been implicated to inhibit caspase-dependent Bcl-2 cleavage and, consequently, the release of mitochondrial cytochrome c in MCF-7 hepatocytes and endothelial cells (17Kim Y.-M. Kim T.-H. Seol D.-W. Talanian R.V. Billiar T.R. J. Biol. Chem. 1998; 273: 31437-31441Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 18Suschek C.V. Krischel V. Bruch-Gerharz D. Berendji D. Krutmann J. Kroncke K.D. Kolb-Bachofen V. J. Biol. Chem. 1999; 274: 6130-6137Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 19Li J. Bombeck C.A. Yang S. Kim Y.-M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Importantly, NO was also reported to interact with p21ras and MAP kinase signaling (20Lander H.M. Jacovina A.T. Davis R.J. Tauras J.M. J. Biol. Chem. 1996; 271: 19705-19709Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). In Jurkat cells, NO was shown to activate the MAP kinases c-Jun N-terminal kinase and, though to a lesser extent, p38 and ERK1/2 byS-nitrosation of p21ras (21Lander H.M. Hajjar D.P. Hempstead B.L. Mirza U.A. Chait B.T. Campbell S. Quilliam L.A. J. Biol. Chem. 1997; 272: 4323-4326Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Here we address the effects of NO to interfere with the dephosphorylation of ERK1/2 as a potential target of the antiapoptotic capacity of NO in endothelial cells. We demonstrate that the down-regulation of the cytosolic MAP kinase phosphatase-3 (MKP-3) (22Muda M. Boschert U. Dickinson R. Martinou J.C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), which is known to dephosphorylate ERK1/2 (23Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (430) Google Scholar), maintains ERK1/2 active and, thus, inhibits the execution of apoptosis by preventing Bcl-2 degradation and mitochondrial release of cytochrome c. SNP, TNFα, and actinomycin D were obtained from Sigma; NG-monomethyl-l-arginine monoacetate and NOC-15 were from Alexis (Läufeling, Switzerland); and SNAP and 2′-amino-3′-methoxyflavone were from Biomol, Hamburg, Germany. Human umbilical vein endothelial cells (HUVEC; Cell Systems/Clonetics, Solingen, Germany; passage 2–4) were cultured in endothelial basal medium (Cell Cystems/Clonetics) supplemented with hydrocortisone (1 μg/ml), bovine brain extract (3 μg/ml), gentamicin (50 μg/ml), amphotericin B (50 μg/ml), epidermal growth factor (10 μg/ml), and 10% fetal calf serum (Life Technologies, Inc.) until the third passage. After detachment with trypsin, cells were grown in culture dishes for 18 h before experiments were performed. HUVEC were exposed to constant laminar fluid flow by means of a cone and plate apparatus as described previously (24Fleming I. Bauersachs J. Fissthaler B. Busse R. Circ. Res. 1998; 82: 686-695Crossref PubMed Scopus (225) Google Scholar). COS-7 cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with glutamine (2 mm), penicillin-streptomycin, and 10% fetal calf serum. MKP-3 was amplified by polymerase chain reaction with oligonucleotides that were synthesized to containBamHI and EcoRV restriction sites and subsequently cloned into the respective sites of the pcDNA3.1-MycHis vector (InVitrogen, the Netherlands). Transient transfection of HUVEC was performed by incubation of 3.0 × 105 cells/6-cm well with 3 μg of plasmid as described previously (25Dimmeler S. Assmus B. Hermann C. Haendeler J. Zeiher A.M. Circ. Res. 1998; 83: 334-342Crossref PubMed Scopus (366) Google Scholar). To transiently transfect COS-7 cells, 7 μg of pcDNA3.1 plasmid containing the respective insert were employed using Superfect™ (Qiagen, Hilden, Germany). To determine ERK1/2 phosphorylation, HUVEC were lysed in buffer (20 mm Tris, 150 mmNaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton, 2, 5 mm sodium pyrophosphate, 1 mm glycerol phosphate, 1 mm Na3VO4, and 1 μm/ml leupeptin, pH 7, 4) for 15 min at 4 °C followed by centrifugation (20,000 × g, 15 min). Then, samples were run on a 11% SDS-polyacrylamide gel and blotted onto polyvinylidene fluoride membranes, and finally protein was probed using a phosphospecific antibody against p42/p44 (New England Biolabs). Western blot analysis of MKP-3 and Bcl-2 protein levels was performed by using an antibody directed against MKP-3 (kindly provided by Dr. Steve Arkinstall, Serono) and against Bcl-2 (Roche Molecular Biochemicals), respectively. To determine cytosolic cytochromec levels, the mitochondrial versus the cytosolic fraction was separated as described previously (26Walter D.H. Haendeler J. Galle J. Zeiher A.M. Dimmeler S. Circulation. 1998; 98: 1153-1157Crossref PubMed Scopus (146) Google Scholar). Western blot membranes were blocked with 5% milk powder, 1% fetal calf serum at room temperature for 1 h and probed with anti-cytochromec antibodies (PharMingen, San Diego, CA, 1:333 dilution). HUVEC or COS-7 cells transfected with the respective plasmid were lysed in 300 μl of buffer (1% Triton X-100, 0.32 m sucrose, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 2 mm dithiothreitol, 10 mmTris/HCl (pH 8)) for 15 min at 4 °C. After centrifugation (20,000 × g, 15 min), phosphatase activity of the supernatant was detected by colorimetric measurements of the hydrolysis of the phosphatase substrate pNPP (Sigma) at 405 nm. Total protein content was analyzed, and enzyme activity was calculated as ΔOD × mg protein−1 × s−1. Following the cotransfection of HUVEC with pcDNA3.1-lacZ (1 μg) and either pcDNA3.1-MKP-3 (2 μg) or the pcDNA3.1 control vector (2 μg), the transfected cells were identified by β-galactosidase staining. Viableversus dead stained cells were counted by two blinded investigators, and the results were expressed as dead/viable cells ×100. In addition, potential differences in cell death rate because of necrosis were excluded by measuring lactate dehydrogenase release. RNA was prepared according to Batt et al. (27Batt D.B. Carmichael G.G. Liu Z. Methods Mol. Biol. 1998; 86: 15-17PubMed Google Scholar), and 10 μg was loaded on 0.8% formamide-agarose gels. RNA was blotted on nylon membranes, and the blots were hybridized with a radioactively labeled full-length human MKP-3 probe and incubated for 24 h. Then the blots were washed (0.1% SDS, 0.2× SSC) and exposed to x-ray films. For preparation of nuclei, cells were detached with trypsin and lysed with Nonidet P-40. Nuclei (2 × 106) were separated by a 20.5% sucrose and incubated in the presence of and for min at °C to for the of mRNA. Then, RNA was as (27Batt D.B. Carmichael G.G. Liu Z. Methods Mol. Biol. 1998; 86: 15-17PubMed Google Scholar). To membranes, human full-length MKP-3 μg) or dehydrogenase (50 μg) were blotted onto nylon membranes using a blot were hybridized with the radioactively labeled for 24 h at washed (0.1% SDS, and exposed to x-ray films. are expressed as or as from at analysis was performed by To a potential of NO with apoptotic signal transduction the MAP kinase p42/p44 HUVEC were with TNFα in the presence or absence of the exogenous NO Then, phosphorylation of ERK1/2 was by Western blot analysis using a phosphospecific shown in stimulation of endothelial cells with the TNFα in a dephosphorylation of ERK1/2, as described previously (8Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google Scholar). In the exogenous NO ERK1/2 dephosphorylation by TNFα at exogenous NO with ERK1/2 dephosphorylation in response to ERK1/2 dephosphorylation is known to be a for degradation of the antiapoptotic protein Bcl-2, the of exogenous NO on Bcl-2 protein degradation was For Bcl-2 levels to TNFα in the presence or absence of the NO or SNAP were by Western 1 B that the degradation of Bcl-2 protein stimulation with TNFα is largely prevented by coincubation with or To the of the effect of NO on ERK1/2 phosphorylation and Bcl-2 protein levels, the release of cytochrome c from in cells was the of NO. protein were from endothelial cells with TNFα in the presence or absence of to the cytosolic fraction from the in 1 the release of cytochrome c from in response to TNFα is by coincubation with These data an inhibitory of NO in TNFα-induced apoptosis signaling upstream of the by maintaining ERK1/2 The of ERK1/2 dephosphorylation Bcl-2 against degradation and, inhibits the mitochondrial release of NO is known to regulate byS-nitrosation of (13Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. J. Exp. Med. 1997; 185: 601-608Crossref PubMed Scopus (782) Google Scholar, J.S. M.E. J. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The MAP kinase phosphatase MKP-3, which ERK1/2, an at its the of which was shown to MKP-3 phosphatase activity (23Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (430) Google Scholar). we NO exerts an inhibitory effect on the phosphatase activity of MKP-3 on a posttranslational by of the activity of HUVEC and COS-7 cell overexpressing MKP-3 was in the presence and absence of NO by of a pNPP hydrolysis The phosphatase activity in HUVEC overexpressing MKP-3 was not by an coincubation with in of the activity in control cells with no by a in incubation with the NO NOC-15 of COS-7 cell MKP-3 protein of control NO not to MKP-3 phosphatase activity on a posttranslational Furthermore, we the effect of NO on the signal event by MKP-3, the release of cytochrome c from In cells, the release of cytochrome c into the induced by MKP-3 was not by exogenous NO 2 that NO not interfere with MKP-3 we a regulatory effect of NO on MKP-3 MKP-3 levels were incubation of HUVEC with the NO or SNAP for 2, and h. shown in MKP-3 is down-regulated in the presence of the NO results were obtained using the NO SNAP not The down-regulation of MKP-3 was on the protein as by Western blot analysis 3 To the effect of endothelial cells were exposed to which the endothelial NO R.M. Circ. Res. 1996; 79: PubMed Scopus Google Scholar, D. J. Y. K. A. Nature. 1999; PubMed Scopus Google Scholar, S. B. I. Hermann C. Busse R. Zeiher A.M. Nature. 1999; PubMed Scopus Google Scholar). of human endothelial cells to constant laminar flow induced a decrease in MKP-3 levels, as shown in 3 D. effect was paralleled by a in MKP-3 protein levels the to constant laminar which was prevented by the NO NG-monomethyl-l-arginine monoacetate not To the by which NO MKP-3 MKP-3 was by shown in 4 the rate of MKP-3 was not by the NO MKP-3 was by incubation of HUVEC with actinomycin D. the of MKP-3 4 that NO MKP-3 mRNA. To a of MKP-3 for TNFα-induced ERK1/2 MKP-3 levels were stimulation of endothelial cell with shown in TNFα induced a in MKP-3 levels, which was largely by NO. NO from exogenous as well as from MKP-3 levels in endothelial of NO on MKP-3 rate and of MKP-3 MKP-3 and dehydrogenase from incubation of HUVEC in the absence or presence of for 2 h were by the of MKP-3 in with dehydrogenase not MKP-3 in the absence or presence of (20 was detected by blot analysis the of actinomycin D of experiments are of TNFα-induced of MKP-3 by exogenous NO. MKP-3 levels stimulation of HUVEC with TNFα (50 for h in the absence and presence of (10 as with 18 RNA MKP-3 was to cell death in HUVEC by ERK1/2 (8Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google K. Haendeler J. Malchow P. Zeiher A.M. Mol. Cell. Biol. 2000; 20: 1886-1896Crossref PubMed Scopus (290) Google Scholar). we the of NO on cell death by Death rate of HUVEC was by of MKP-3 with control cells that were transfected with the pcDNA3.1 vector with no insert and cells were exposed to levels of NO provided by the NO SNP, cell death by MKP-3 was and Furthermore, to NO release also cell death control levels This effect was by the of the of NO NG-monomethyl-l-arginine monoacetate death signaling in HUVEC induced by MKP-3, that not for the of MKP-3 is to exogenous as well as to NO. to a potential of NO on the kinase kinase we the effects of NO on cell death by MKP-3 of with shown in NO was of cell death by MKP-3 in the presence of the NO a of endothelial cell survival S. Zeiher A.M. Cell Death Differ. 1999; 6: 964-968Crossref PubMed Scopus (231) Google I. Busse R. J. Mol. Cell. 1999; Full Text PDF PubMed Scopus Google Scholar). interactions of NO with signal transduction have been described to explain the prosurvival effects of levels of NO as by the endothelial NO In we the of the MAP kinase phosphatase MKP-3 as a potential target of the effect of NO in endothelial cells. We demonstrate that NO MKP-3 and, with the TNFα-induced dephosphorylation of the MAP kinase Subsequently, NO prevents Bcl-2 degradation and the release of cytochrome c from which results in the of endothelial cells from ERK1/2 is an established in the antiapoptotic (7Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5014) Google Scholar). The has previously been reported to against apoptosis induced by growth factor P. Mol. Cell. Biol. 1999; PubMed Scopus Google I. M.E. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of the was shown to the Bcl-2 in the of from which the of cells from apoptosis V. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In endothelial cells, phosphorylation was to Bcl-2 and its degradation (8Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google Scholar, 9Breitschopf K. Haendeler J. Malchow P. Zeiher A.M. Mol. Cell. Biol. 2000; 20: 1886-1896Crossref PubMed Scopus (290) Google Scholar). In with the prosurvival of phosphorylated ERK1/2, its by MAP kinase phosphatase signaling in response to cellular was shown to (8Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google Scholar, 9Breitschopf K. Haendeler J. Malchow P. Zeiher A.M. Mol. Cell. Biol. 2000; 20: 1886-1896Crossref PubMed Scopus (290) Google Scholar, M. W. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the of ERK1/2 activation via the of MAP kinase phosphatase an additional in cellular NO is of with apoptosis signaling in The variety of interactions of NO with signaling the of Bcl-2 in B A.M. S. A. C. J. 1995; PubMed Scopus Google and the of caspase activity byS-nitrosation of the (13Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. J. Exp. Med. 1997; 185: 601-608Crossref PubMed Scopus (782) Google Scholar, J. Billiar T.R. Talanian R.V. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; 240: 419-424Crossref PubMed Scopus (472) Google Scholar, B. Breitschopf K. Haendeler J. Zeiher A.M. A. Dimmeler S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google as well as by a Y.-M. Talanian R.V. Billiar T.R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). we report the down-regulation of MKP-3 levels and, thus, the of ERK1/2 phosphorylation by NO as a that to the effects of NO in endothelial cells. data demonstrate that NO exerts an inhibitory effect upstream of the of the signaling cascade by inhibiting of These the of NO to apoptosis signaling to a of as with the antiapoptotic by the of the caspase-3 Moreover, by ERK1/2 phosphorylation via down-regulation of MKP-3, NO that activation of the and by ERK1/2 in to the prosurvival of Bcl-2 levels. the of MKP-3 protein levels by NO is by of MKP-3 as opposed to the posttranslational of caspase The down-regulation of MKP-3 by NO was of the rate as shown by experiments but is by a destabilization of MKP-3 mRNA. of has previously been The of NO in endothelial cells was shown to the of by which is known to rely on the of J. Cell. 1996; PubMed Scopus Google Scholar). Moreover, was to be by of a protein D.B. K.D. J. 1997; PubMed Scopus Google Scholar). the the of MKP-3 by NO the of a protein is the of the NO In we demonstrate that stimulation with TNFα, levels of NO ERK1/2 phosphorylation via down-regulation of MKP-3 levels, thereby constant phosphorylation of the ERK1/2 target Bcl-2, which prevents the degradation of Bcl-2 and, the release of cytochrome c from the antiapoptotic of the effects of NO from and of cytochrome c release and posttranslational modifications of protein as well as the of to control apoptotic signaling We Dr. M. Camps and Dr. Steve Arkinstall for with the antibody and and for