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• W2037291567 abstract "Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult. Purified endothelial nitric-oxide synthase (eNOS) can generate superoxide (O⨪2) under special conditions but is only known to participate in cell signaling through NO. Here we show that eNOS regulates tumor necrosis factor α (TNFα) through a mechanism dependent on the production of O⨪2 and completely independent of NO. Expression of eNOS in transfected U937 cells increased phorbol 12-myristate 13-acetate-induced TNFα promoter activity and TNFα production.N ω-Methyl-l-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFα up-regulation. Likewise, Gln361eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFα. Similar to the effect of eNOS, a O⨪2 donor dose-dependently increased TNFα production in differentiated U937 cells. In contrast, cotransfection of superoxide dismutase with eNOS prevented TNFα up-regulation, as did partial deletion of the eNOS NADPH binding site, a mutation associated with loss of O⨪2 production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O⨪2, interrelated but often opposing free radical messengers. This arrangement has possible implications for atherosclerosis and septic shock where endothelial dysfunction results from imbalances in NO and O⨪2 production. Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult. Purified endothelial nitric-oxide synthase (eNOS) can generate superoxide (O⨪2) under special conditions but is only known to participate in cell signaling through NO. Here we show that eNOS regulates tumor necrosis factor α (TNFα) through a mechanism dependent on the production of O⨪2 and completely independent of NO. Expression of eNOS in transfected U937 cells increased phorbol 12-myristate 13-acetate-induced TNFα promoter activity and TNFα production.N ω-Methyl-l-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFα up-regulation. Likewise, Gln361eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFα. Similar to the effect of eNOS, a O⨪2 donor dose-dependently increased TNFα production in differentiated U937 cells. In contrast, cotransfection of superoxide dismutase with eNOS prevented TNFα up-regulation, as did partial deletion of the eNOS NADPH binding site, a mutation associated with loss of O⨪2 production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O⨪2, interrelated but often opposing free radical messengers. This arrangement has possible implications for atherosclerosis and septic shock where endothelial dysfunction results from imbalances in NO and O⨪2 production. endothelial nitric-oxide synthase tetrahydrobiopterin reactive oxygen species phorbol 12-myristate 13-acetate tumor necrosis factor α superoxide dismutase Nω-methyl-l-arginine Endothelial nitric-oxide synthase (eNOS)1 is a calcium-dependent NADPH oxidase that generates NO from oxygen and l-arginine (1.Janssens S.P. Shimouchi A. Quertermous T. Bloch D.B. Bloch K.D. J. Biol. Chem. 1992; 267: 14519-14522Abstract Full Text PDF PubMed Google Scholar, 2.Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar). It has a C-terminal reductase domain that binds NADPH, FAD, and FMN, and a N-terminal oxygenase domain that binds a heme moiety, tetrahydrobiopterin (BH4), and l-arginine (3.Chen P.F. Tsai A.L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). NO produced by eNOS regulates vascular tone through a cGMP-dependent signaling pathway (4.Waldman S.A. Murad F. J. Cardiovasc. Pharmacol. 1988; 12 (Suppl. 5): S115-S118Crossref PubMed Google Scholar). In addition, NO also has cGMP-independent effects within the vasculature, such as inhibition of leukocyte adhesion, that relies on its ability to inactivate or antagonize O⨪2 (5.Gaboury J. Woodman R.C. Granger D.N. Reinhardt P. Kubes P. Am. J. Physiol. 1993; 265: H862-H867PubMed Google Scholar, 6.Gaboury J.P. Anderson D.C. Kubes P. Am. J. Physiol. 1994; 266: H637-H642Crossref PubMed Google Scholar). Notably, in cell free systems, purified eNOS can be shown in the absence of BH4to generate small quantities of O⨪2 rather than NO (7.Xia Y. Tsai A.L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 8.Vasquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1229) Google Scholar). However, cell regulation directly attributable to eNOS production of O⨪2 has not been previously demonstrated. Superoxide (O⨪2) and other reactive oxygen species (ROS), conventionally viewed as cytotoxins, have recently been recognized as important signal transduction intermediates that regulate gene expression, cell differentiation, immune activation, and apoptosis (9.Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2314) Google Scholar, 10.Lo Y.Y. Cruz T.F. J. Biol. Chem. 1995; 270: 11727-11730Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 11.Ushio-Fukai M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 12.Lo Y.Y.C. Wong J.M.S. Cruz T.F. J. Biol. Chem. 1996; 271: 15703-15707Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 13.Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13524Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). In the vasculature, increased O⨪2 production disproportionate to NO synthesis has been associated with endothelial dysfunction, an early pathogenic event in atherosclerosis (14.Bouloumie A. Bauersachs J. Linz W. Scholkens B.A. Wiemer G. Fleming I. Busse R. Hypertension. 1997; 30: 934-941Crossref PubMed Scopus (284) Google Scholar, 15.Stroes E. Kastelein J. Cosentino F. Erkelens W. Wever R. Koomans H. Luscher T. Rabelink T. J. Clin. Invest. 1997; 99: 41-46Crossref PubMed Scopus (510) Google Scholar, 16.Pritchard Jr., K.A. Groszek L. Smalley D.M. Sessa W.C. Wu M. Villalon P. Wolin M.S. Stemerman M.B. Circ Res. 1995; 77: 510-518Crossref PubMed Scopus (450) Google Scholar, 17.Cosentino F. Katusic Z.S. Circulation. 1995; 91: 139-144Crossref PubMed Scopus (256) Google Scholar). Further, elevated O⨪2 production in the presence of NO has been linked to endothelial injury and organ damage in septic shock (18.Szabó C. Zingarelli B. Salzman A.L. Circ. Res. 1996; 78: 1051-1063Crossref PubMed Scopus (237) Google Scholar), possibly through the formation of peroxynitrite (ONOO−), a cytotoxic metabolite (19.Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 20.de la Monte S.M. Bloch K.D. Mol. Chem. Neuropathol. 1997; 30: 139-159Crossref PubMed Scopus (51) Google Scholar, 21.Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar, 22.Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (662) Google Scholar). However, the precise enzymatic origins of ROS in either signaling events or pathologic conditions have remained obscure (23.Wolin M.S. Microcirculation. 1996; 3: 1-17Crossref PubMed Scopus (117) Google Scholar, 24.Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1011) Google Scholar). ROS have many potential sources within cells including mitochondria, xanthine oxidase, cyclooxygenases, and NADPH oxidases, making it difficult to associate a specific enzyme with a corresponding ROS-related event (23.Wolin M.S. Microcirculation. 1996; 3: 1-17Crossref PubMed Scopus (117) Google Scholar, 24.Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1011) Google Scholar). We have previously described the expression of functional human eNOS in U937 cells using a pCEP4 vector (25.Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar). Human monoblastoid U937 cells lack NOS expression in either their naı̈ve or phorbol 12-myristate 13-acetate (PMA) differentiated states (26.Wang S. Yan L. Wesley R.A. Danner R.L. J. Biol. Chem. 1997; 272: 5959-5965Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). At resting levels of intracellular calcium, eNOS expression in transfected U937 cells does not result in NO release, but NO is rapidly produced upon exposure to calcium ionophore (25.Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar). The absence of soluble guanylate cyclase (26.Wang S. Yan L. Wesley R.A. Danner R.L. J. Biol. Chem. 1997; 272: 5959-5965Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), a pivotal target of NO-based signaling, and impaired synthesis of BH4 (27.Schoedon G. Troppmair J. Fontana A. Huber C. Curtius H.C. Niederwieser A. Eur. J. Biochem. 1987; 166: 303-310Crossref PubMed Scopus (117) Google Scholar), a cofactor whose deficiency is associated with O⨪2 production by eNOS in cell-free systems (7.Xia Y. Tsai A.L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar,8.Vasquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1229) Google Scholar), suggested that these cells might be useful for exploring NO-independent signaling by eNOS. A firefly luciferase reporter vector pGL2 (Promega) containing the full-length human tumor necrosis factor α (TNFα) promoter (pTNF-GL2) was used to assay for TNFα promoter activity. pTNF-GL2 (20 μg) and a vector (5 μg) for expression of secreted placental alkaline phosphatase (to control for transfection efficiency) were cotransfected into U937 cells with either the eNOS pCEP4 construct (20 μg) or the empty pCEP4 vector (20 μg) using electroporation (240 V, 960 microfarad). Then cells were differentiated with 100 nm PMA for 48 h, and luciferase and secreted placental alkaline phosphatase activity were measured (Promega and Tropix Inc). The human eNOS expression vector was constructed by using the eukaryotic expression vector pCEP4 (Invitrogen) (25.Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar). A Glu361 → Gln361 mutant eNOS cDNA was ligated into pCEP4 atHindIII/NotI sites to create Gln361eNOS. Human Cu/Zn superoxide dismutase (SOD) cDNA (American Type Culture Collection) was ligated into pCEP4 atHindIII/BamHI sites to generate the SOD expression vector. eNOS NADPH binding site deletion mutants, d(NADPH) eNOS and d(NADPH) Gln361eNOS, were constructed by digesting eNOS and Gln361eNOS with XhoI, thereby removing a 717-base pair fragment from eNOS containing the sequence for its NADPH-adenine binding site. All expression vectors were partially sequenced to confirm the correct sequence and orientation. U937 cells (American Type Culture Collection) were maintained in RPMI 1640 complete medium containing HEPES (25 mm), 10% fetal calf serum, l-glutamine (2 mm) and antibiotics. Empty pCEP4 (control vector) or expression vectors containing human eNOS, Gln361eNOS, Cu/Zn SOD, d(NADPH)eNOS, or d(NADPH)Gln361eNOS were transfected into cells by electroporation and then selected with hygromycin B (275 units/ml; Calbiochem), as described previously (25.Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar). After selection, transfectants (1 × 107 cells each) were suspended in 100 ml of RPMI 1640 complete medium and 100 nm PMA for 48 h. Adhered cells were removed by incubation with 1 mm EDTA in Hanks' balanced salt solution without Ca2+ and Mg2+ for 30 min at 37 °C. Cells were then washed with Hanks' balanced salt solution without Ca2+ and Mg2+, resuspended in RPMI 1640 complete medium, counted, tested for viability, and plated into 24-well plates at 5 × 105 cells/ml for 22 h. In one experiment, untransfected U937 cells were differentiated with PMA, as described above, and then incubated for 22 h with increasing doses of phenazine methosulfate, a O⨪2 donor (28.Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Cell-free supernatants were assayed for TNFα using an enzyme-linked immunosorbent assay (R & D Systems). NOS activity was determined by measuring the conversion of [14C]l-arginine to [14C]l-citrulline in total cell lysates from PMA-differentiated U937 cells, as reported previously (25.Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar). For the aconitase assay, differentiated cells incubated in RPMI complete medium for 22 h were removed by scraper, washed with ice-cold Hanks' balanced salt solution without Ca2+ and Mg2+, and disrupted using a microtip sonicator. Lysate supernatants were assayed for aconitase activity as described elsewhere (28.Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Data are shown as the means ± S.E., and differences were considered significant where p < 0.05. Paired comparisons were performed using t tests. Dose response effects were analyzed using a Sen-Theil estimate of slope followed by a one-sample t test. Three-way comparisons were made using t tests followed by Holm's multiple comparisons adjustment. Two-way analysis of variance followed by Fisher's least significant difference test was used for higher order comparisons. Differentiation of U937 cells with PMA (100 nm) induces development of monocytic characteristics and TNFα production (29.Hass R. Lonnemann G. Mannel D. Topley N. Hartmann A. Kohler L. Resch K. Goppelt-Strube M. Leuk. Res. 1991; 15: 327-339Crossref PubMed Scopus (43) Google Scholar). To determine the effect of eNOS expression on TNFα promoter activity, we used a luciferase reporter system in PMA-differentiated U937 cells. As shown in Fig.1 A, eNOS expression increased TNFα promoter activity as compared with control vector transfectants (p = 0.0004). Next we examined whether this increase in TNFα promoter activity resulted in increased TNFα protein production (Fig. 1 B). PMA-differentiated, eNOS-transfected U937 cells produced more TNFα compared with control vector-transfected cells (p = 0.01). However,N ω-methyl-l-arginine (l-NMA), an inhibitor that blocks the NOS activity of eNOS but not its NADPH oxidase activity (30.List B.M. Klosch B. Volker C. Gorren A.C. Sessa W.C. Werner E.R. Kukovetz W.R. Schmidt K. Mayer B. Biochem. J. 1997; 323: 159-165Crossref PubMed Scopus (139) Google Scholar), did not prevent eNOS enhancement of TNFα production. These findings suggested that eNOS produced a signaling molecule other than NO that up-regulated TNFα production and raised the possibility that this alternative messenger was related to the NADPH oxidase activity of eNOS. To further eliminate the possibility that NO was responsible for TNFα up-regulation by eNOS, we tested the mutant Gln361eNOS, which has a single amino acid substitution from Glu to Gln at position 361 in the l-arginine binding site of wild type eNOS. This mutation abolishes the ability of eNOS to produce NO, but NADPH oxidase activity remains intact (31.Chen P.F. Tsai A.L. Berka V. Wu K.K. J. Biol. Chem. 1997; 272: 6114-6118Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Expression of Gln361eNOS (Fig. 1 C) up-regulated TNFα production compared with control vector cells (p = 0.01) and had an effect similar to that of wild type eNOS (p = 0.5). Fig.1 D shows that wild type eNOS and Gln361eNOS were both expressed by their respective transfectants. Further, PMA differentiation is shown to increase expression of eNOS genes incorporated into the pCEP4 vector as described previously (25.Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar). Empty vector transfectants did not express eNOS either before or after PMA differentiation. Thus, two separate experiments, one using a NOS inhibitor (Fig. 1 B) and the other a mutant eNOS unable to produce NO (Fig. 1 C), demonstrate that up-regulation of TNFα by eNOS was independent of NO. Next, phenazine methosulfate, a O⨪2 donor (28.Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), was shown to increase TNFα production in a dose-dependent manner (Fig.2 A; p = 0.001). This result was consistent with the possibility that ROS generated by eNOS could play a role in TNFα production. To further test whether O⨪2 released by eNOS might be responsible for TNFα up-regulation, Cu/Zn SOD was cotransfected into U937 cells. In PMA-differentiated transfectants (Fig. 2 B), SOD expression was found to totally abolish TNFα up-regulation by eNOS (p = 0.007). eNOS expression was not affected by the coexpression of Cu/Zn SOD (Fig. 2 C). These data suggest that eNOS-induced TNFα production required the production of O⨪2or a closely related metabolite. To investigate the role of NADPH oxidase activity in TNFα up-regulation, the adenine binding site for NADPH was deleted from eNOS. Two NADPH binding site deletion mutants, d(NADPH)eNOS and d(NADPH)Gln361eNOS, were constructed, one from wild type eNOS and the other from Gln361eNOS. Again we found that wild type eNOS expression (Fig.3 A) increased TNFα production compared with control vector (p = 0.002). However, neither d(NADPH)eNOS nor d(NADPH)Gln361eNOS expression significantly altered TNFα production (p = 0.4). This result indicates that the NADPH oxidase activity of eNOS, which requires an intact NADPH recognition site, was necessary for TNFα up-regulation in PMA-differentiated U937 cells. Fig.3 B demonstrates that eNOS and its NADPH binding site deletion mutants were all expressed in the PMA-differentiated cells used for these experiments. Finally, NOS activity and O2 production were measured by l-arginine to l-citrulline conversion (25.Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar) and by aconitase assay (28.Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar,32.Fridovich I. J. Biol. Chem. 1997; 272: 18515-18517Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar), respectively, to determine which enzymatic function corresponded to the ability of transfectants to up-regulate TNFα. The presence of NOS activity in cells transfected with wild type human eNOS or with one of the eNOS mutants did not correlate with the capacity to up-regulate TNFα (Fig. 4 A). Only wild type eNOS transfectants were found to have NOS activity compared with control cells (p = 0.001). Although the Gln361eNOS mutant also up-regulated TNFα (Fig.1 C), it was completely devoid of NOS activity (Fig.4 A; compared with control p = 0.3). Next, suppression of aconitase activity was used as a measure of intracellular O⨪2 production (28.Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 32.Fridovich I. J. Biol. Chem. 1997; 272: 18515-18517Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar). Aconitase activity in both wild type eNOS transfectants and Gln361eNOS mutants was lower than that of control vector transfectants (p< 0.0003 for both), indicating the presence of increased O⨪2production (Fig. 4 B). In contrast, both d(NADPH)eNOS and d(NADPH)Gln361eNOS transfected U937 cells, which do not produce increased amounts of TNFα (Fig. 3 A), had aconitase activity similar to that measured in control vector transfectants (Fig.4 B; p = 0.1, for both). This shows, as expected, that the NADPH binding site mutants of eNOS did not produce increased amounts of O⨪2. Notably, l-NMA, a NOS inhibitor, did not change the aconitase activity pattern of eNOS transfectants (p = 0.8), demonstrating that decreases in aconitase activity in eNOS transfectants relative to control vector were not related to NO production (Fig. 4 C) (19.Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar). Further, Fig. 4 D shows that eNOS and its respective mutants were expressed in the PMA-differentiated U937 cells used in these experiments. Thus, the ability of various eNOS transfectants to up-regulate TNFα corresponded to their capacity to generate O⨪2 but not NO. Many cell types including endothelial cells, fibroblasts, hepatocytes, and vascular smooth muscle cells have been shown to utilize ROS as second messengers (9.Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2314) Google Scholar, 10.Lo Y.Y. Cruz T.F. J. Biol. Chem. 1995; 270: 11727-11730Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 11.Ushio-Fukai M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 12.Lo Y.Y.C. Wong J.M.S. Cruz T.F. J. Biol. Chem. 1996; 271: 15703-15707Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 23.Wolin M.S. Microcirculation. 1996; 3: 1-17Crossref PubMed Scopus (117) Google Scholar, 24.Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1011) Google Scholar). These ROS-dependent signal transduction pathways have been shown to regulate important cellular functions such as growth, differentiation, gene expression, and apoptosis. However, even as the signaling mechanisms distal to ROS release are becoming increasingly well defined, identification of the precise enzymatic sources for ROS involved in specific signal transduction events has been relatively elusive (23.Wolin M.S. Microcirculation. 1996; 3: 1-17Crossref PubMed Scopus (117) Google Scholar, 24.Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1011) Google Scholar). The results presented here demonstrate that eNOS may be a source of O⨪2within cells and that eNOS-generated ROS can participate in the modulation of inflammatory responses. eNOS has close homology with cytochrome P-450 reductases (1.Janssens S.P. Shimouchi A. Quertermous T. Bloch D.B. Bloch K.D. J. Biol. Chem. 1992; 267: 14519-14522Abstract Full Text PDF PubMed Google Scholar, 2.Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar) and can generate small amounts of O⨪2 in cell-free systems deficient inl-arginine and BH4 (7.Xia Y. Tsai A.L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 8.Vasquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1229) Google Scholar). In intact cells andin vivo, the availability of substrate and cofactors might be expected to keep this alternative electron shunting pathway inactive. However, several investigations (14.Bouloumie A. Bauersachs J. Linz W. Scholkens B.A. Wiemer G. Fleming I. Busse R. Hypertension. 1997; 30: 934-941Crossref PubMed Scopus (284) Google Scholar, 15.Stroes E. Kastelein J. Cosentino F. Erkelens W. Wever R. Koomans H. Luscher T. Rabelink T. J. Clin. Invest. 1997; 99: 41-46Crossref PubMed Scopus (510) Google Scholar, 16.Pritchard Jr., K.A. Groszek L. Smalley D.M. Sessa W.C. Wu M. Villalon P. Wolin M.S. Stemerman M.B. Circ Res. 1995; 77: 510-518Crossref PubMed Scopus (450) Google Scholar, 17.Cosentino F. Katusic Z.S. Circulation. 1995; 91: 139-144Crossref PubMed Scopus (256) Google Scholar) using human endothelial cell cultures, canine coronary arteries, or aortic rings from rats and rabbits are consistent with the possibility that eNOS might be capable of releasing oxygen intermediates under certain pathological conditions. Notably, the infusion of BH4 into patients with hypercholesterolemia was shown to restore endothelial-dependent vascular responses (15.Stroes E. Kastelein J. Cosentino F. Erkelens W. Wever R. Koomans H. Luscher T. Rabelink T. J. Clin. Invest. 1997; 99: 41-46Crossref PubMed Scopus (510) Google Scholar), supporting the concept that eNOS dysfunction with impaired NO production and possibly increased O⨪2 formation might occur in vivo (16.Pritchard Jr., K.A. Groszek L. Smalley D.M. Sessa W.C. Wu M. Villalon P. Wolin M.S. Stemerman M.B. Circ Res. 1995; 77: 510-518Crossref PubMed Scopus (450) Google Scholar). The capacity of a single enzyme to produce both NO and O⨪2 may have important implications for atherosclerosis (14.Bouloumie A. Bauersachs J. Linz W. Scholkens B.A. Wiemer G. Fleming I. Busse R. Hypertension. 1997; 30: 934-941Crossref PubMed Scopus (284) Google Scholar, 15.Stroes E. Kastelein J. Cosentino F. Erkelens W. Wever R. Koomans H. Luscher T. Rabelink T. J. Clin. Invest. 1997; 99: 41-46Crossref PubMed Scopus (510) Google Scholar, 16.Pritchard Jr., K.A. Groszek L. Smalley D.M. Sessa W.C. Wu M. Villalon P. Wolin M.S. Stemerman M.B. Circ Res. 1995; 77: 510-518Crossref PubMed Scopus (450) Google Scholar, 17.Cosentino F. Katusic Z.S. Circulation. 1995; 91: 139-144Crossref PubMed Scopus (256) Google Scholar) and other illnesses such as Alzheimer's disease (20.de la Monte S.M. Bloch K.D. Mol. Chem. Neuropathol. 1997; 30: 139-159Crossref PubMed Scopus (51) Google Scholar, 21.Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar) and septic shock (18.Szabó C. Zingarelli B. Salzman A.L. Circ. Res. 1996; 78: 1051-1063Crossref PubMed Scopus (237) Google Scholar) in which an altered balance between these two free radicals might lead to tissue injury. Direct chemical interaction of NO and O⨪2produces peroxynitrite (ONOO−), a highly toxic metabolite that can damage macromolecules and injure cells (19.Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 22.Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (662) Google Scholar). In addition to the possibility of toxic effects, eNOS-generated O⨪2 and other ROS molecules derived from it, in particular H2O2, are likely to participate in signal transduction events and the regulation of cell function. In our eNOS transfected U937 cells, the molecular identity of the ROS that up-regulates TNFα was not precisely determined. Although Cu/Zn SOD efficiently converts O⨪2 to H2O2 and effectively blocked eNOS-related increases in TNFα production, effector molecules other than O⨪2 may contribute to this response. Manna et al. (13.Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13524Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar) have shown that redox-dependent activation of NF-κB probably reflects the total cellular balance of a number of prooxidants and antioxidants. Further, Manna et al. suggest that induction of manganese SOD by TNFα and ROS-dependent NF-κB activation may function as a negative feedback loop (13.Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13524Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 33.Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Crossref PubMed Scopus (212) Google Scholar). Therefore, ROS up-regulation of TNFα in our system may lead to manganese SOD induction, which might attenuate further proinflammatory responses and thus reduce injury caused by eNOS-generated O⨪2. Notably, vessel shear stress has been shown to increase endothelial cell expression of both Cu/Zn SOD and eNOS (34.Dimmeler S. Hermann C. Galle J. Zeiher A.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 656-664Crossref PubMed Scopus (267) Google Scholar). Co-regulation of these genes might serve to reduce the prooxidant potential of eNOS and to thereby maximally suppress oxidant-triggered apoptosis (34.Dimmeler S. Hermann C. Galle J. Zeiher A.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 656-664Crossref PubMed Scopus (267) Google Scholar). In the current investigation, we have shown that eNOS can generate small amounts of O⨪2 in intact cells. This eNOS-derived O⨪2 participated in cell signaling events that regulated the production of TNFα, a pivotal mediator of inflammation. Depending on the cell-type and triggering event investigated, ROS have been shown to transduce signals through the activation of tyrosine kinases, mitogen-activated protein kinases, and c-Jun N-terminal kinases and ultimately to alter promoter activity by increasing the DNA binding of transcription factors such as NFκB and AP-1 (9.Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2314) Google Scholar, 10.Lo Y.Y. Cruz T.F. J. Biol. Chem. 1995; 270: 11727-11730Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 11.Ushio-Fukai M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 12.Lo Y.Y.C. Wong J.M.S. Cruz T.F. J. Biol. Chem. 1996; 271: 15703-15707Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 13.Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13524Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). In recent experiments, we have found in U937 cells that eNOS-based O⨪2signaling activates p44/42 mitogen-activated protein kinase (35.Wang W. Wang S. Wesley R. Danner R. FASEB J. 1999; 13 (abstr.) 132Google Scholar). Therefore, eNOS may occupy a signal bifurcation point controlling the production of two interrelated but distinct free radical messengers. Determining whether this arrangement exists in endothelium and, if so, how it is regulated might lead to an understanding of its possible benefits, given the potential danger it poses for the formation of ONOO−. We thank Kenneth D. Bloch for providing us with human eNOS cDNA and Pei-Feng Chen for providing human Gln361 eNOS cDNA. We also thank Martha Vaughan, Joel Moss, and Torren Finkel for review and discussion of our manuscript." @default.
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