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• W2002404304 abstract "The production of nitric oxide (NO) by inducible NO synthase (iNOS) regulates many aspects of physiology and pathology. The expression of iNOS needs to be tightly regulated to balance the broad ranging properties of NO. We have investigated the feedback regulation of cytokine-induced iNOS expression by NO in human cells. The pharmacological inhibition of iNOS activity reduced iNOS protein levels in response to cytokine stimulation in a human epithelial cell line (A549 cells) as well as in primary human astrocytes and bronchial epithelial cells. The addition of exogenous NO using a NO donor prevented the reduction in iNOS levels caused by blockade of iNOS activity. Examination of signaling pathways affected by iNOS indicated that NO S-nitrosylated Ras. Transfection of cells with a S-nitrosylation-resistant Ras mutant reduced iNOS protein levels, indicating a role for this Ras modification in the amplification of iNOS levels. Further, the induction of iNOS protein levels correlated with the late activation of the phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR) pathways, and inhibition of these signaling molecules reduced iNOS levels. Altogether, our findings reveal a previously unknown regulatory pathway that amplifies iNOS expression in human cells.Background: Feedback regulatory processes controlling iNOS expression are incompletely defined.Results: Induction of iNOS expression was attenuated by 1) inhibition of iNOS activity, 2) prevention of Ras S-nitrosylation, and 3) inhibition of PI3K and mTOR activity.Conclusion: iNOS-derived NO amplifies iNOS expression through S-nitrosylation of Ras and activation of PI3K and mTOR.Significance: We have defined a previously unrecognized positive feedback pathway that amplifies human iNOS expression. The production of nitric oxide (NO) by inducible NO synthase (iNOS) regulates many aspects of physiology and pathology. The expression of iNOS needs to be tightly regulated to balance the broad ranging properties of NO. We have investigated the feedback regulation of cytokine-induced iNOS expression by NO in human cells. The pharmacological inhibition of iNOS activity reduced iNOS protein levels in response to cytokine stimulation in a human epithelial cell line (A549 cells) as well as in primary human astrocytes and bronchial epithelial cells. The addition of exogenous NO using a NO donor prevented the reduction in iNOS levels caused by blockade of iNOS activity. Examination of signaling pathways affected by iNOS indicated that NO S-nitrosylated Ras. Transfection of cells with a S-nitrosylation-resistant Ras mutant reduced iNOS protein levels, indicating a role for this Ras modification in the amplification of iNOS levels. Further, the induction of iNOS protein levels correlated with the late activation of the phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR) pathways, and inhibition of these signaling molecules reduced iNOS levels. Altogether, our findings reveal a previously unknown regulatory pathway that amplifies iNOS expression in human cells. Background: Feedback regulatory processes controlling iNOS expression are incompletely defined. Results: Induction of iNOS expression was attenuated by 1) inhibition of iNOS activity, 2) prevention of Ras S-nitrosylation, and 3) inhibition of PI3K and mTOR activity. Conclusion: iNOS-derived NO amplifies iNOS expression through S-nitrosylation of Ras and activation of PI3K and mTOR. Significance: We have defined a previously unrecognized positive feedback pathway that amplifies human iNOS expression. Nitric oxide (NO) is a bioactive gas involved in many aspects of physiology and pathology (1Moncada S. Higgs E.A. The discovery of nitric oxide and its role in vascular biology.Br. J. Pharmacol. 2006; 147: S193-S201Crossref PubMed Scopus (662) Google Scholar). It is produced from the conversion of l-arginine to l-citrulline and NO by a family of nitric-oxide synthases (NOSs), of which there are three isoforms: neuronal NOS (nNOS), 3The abbreviations used are: nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; CM, cytokine mixture; l-NAME, l-NG-nitroarginine methyl ester; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of rapamycin complex 2; ODQ, 1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one; sGC, soluble guanylate cyclase; EGFR, epidermal growth factor receptor. inducible NOS (iNOS), and endothelial NOS (eNOS). nNOS and eNOS are expressed constitutively in neurons and endothelial cells, respectively, and iNOS is induced in several cell types (2Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S. H Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.Nature. 1991; 351: 714-718Crossref PubMed Scopus (2167) Google Scholar, 3Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar, 4Nishida K. Harrison D.G. Navas J.P. Fisher A.A. Dockery S.P. Uematsu M. Nerem R.M. Alexander R.W. Murphy T.J. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.J. Clin. Invest. 1992; 90: 2092-2096Crossref PubMed Scopus (614) Google Scholar, 5Marsden P.A. Schappert K.T. Chen H.S. Flowers M. Sundell C.L. Wilcox J.N. Lamas S. Michel T. Molecular cloning and characterization of human endothelial nitric oxide synthase.FEBS Lett. 1992; 307: 287-293Crossref PubMed Scopus (418) Google Scholar, 6Xie Q.W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages.Science. 1992; 256: 225-228Crossref PubMed Scopus (1738) Google Scholar, 7Lowenstein C.J. Glatt C.S. Bredt D.S. Snyder S.H. Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 6711-6715Crossref PubMed Scopus (621) Google Scholar). The induction of iNOS is involved in the regulation of immunity, the clearance of pathogens, and the pathogenesis of several conditions (8Bogdan C. Röllinghoff M. Diefenbach A. The role of nitric oxide in innate immunity.Immunol. Rev. 2000; 173: 17-26Crossref PubMed Scopus (516) Google Scholar, 9Nathan C. Is iNOS beginning to smoke?.Cell. 2011; 147: 257-258Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Cytokines are one of the main inducers of iNOS expression. In mice, the proinflammatory cytokines TNF, IL-1, and IFNγ act together to induce very high levels of iNOS in many cell types (10Stuehr D.J. Cho H.J. Kwon N.S. Weise M.F. Nathan C.F. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 7773-7777Crossref PubMed Scopus (728) Google Scholar). Pathogen-derived molecules, such as lipopolysaccharide, also stimulate iNOS expression (11Xie Q.W. Whisnant R. Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon γ and bacterial lipopolysaccharide.J. Exp. Med. 1993; 177: 1779-1784Crossref PubMed Scopus (1028) Google Scholar). In contrast to mice, expression of iNOS in humans is relatively restricted to certain cell types, and controversy exists regarding differences between the regulation of human and mouse expression of this enzyme (12Schneemann M. Schoedon G. Species differences in macrophage NO production are important.Nat. Immunol. 2002; 3: 102Crossref PubMed Scopus (94) Google Scholar). iNOS expression has been documented in macrophages from patients with Mycobacterium tuberculosis infection, although neither bacterial components nor the combination of TNF, IL-1, and IFNγ induces substantial levels of iNOS expression in cultured human macrophages (13Nicholson S. Bonecini-Almeida Mda G. Lapa e Silva J.R. Nathan C. Xie Q.W. Mumford R. Weidner J.R. Calaycay J. Geng J. Boechat N. Linhares C. Rom W. Ho J.L. Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.J. Exp. Med. 1996; 183: 2293-2302Crossref PubMed Scopus (400) Google Scholar, 14Schneemann M. Schoedon G. Hofer S. Blau N. Guerrero L. Schaffner A. Nitric oxide synthase is not a constituent of the antimicrobial armature of human mononuclear phagocytes.J. Infect. Dis. 1993; 167: 1358-1363Crossref PubMed Scopus (317) Google Scholar, 15Padgett E.L. Pruett S.B. Evaluation of nitrite production by human monocyte-derived macrophages.Biochem. Biophys. Res. Commun. 1992; 186: 775-781Crossref PubMed Scopus (108) Google Scholar). Also, evaluation of human iNOS gene promoter activity in transgenic mice expressing human iNOS promoter enhanced GFP has shown extensive species-specific distinctions in expression patterns of iNOS under both basal and inflammatory settings (16Yu Z. Xia X. Kone B.C. Expression profile of a human inducible nitric oxide synthase promoter reporter in transgenic mice during endotoxemia.Am. J. Physiol. Renal. Physiol. 2005; 288: F214-F220Crossref PubMed Scopus (14) Google Scholar). These findings highlight the importance of defining mechanisms regulating iNOS expression specifically in human cells to provide insight into human physiology and pathology. Inducible expression of iNOS has been defined in only a handful of primary human cell types such as astrocytes, hepatocytes, and bronchial epithelial cells (17Lee S.C. Brosnan C.F. Cytokine regulation of iNOS expression in human glial cells.Methods. 1996; 10: 31-37Crossref PubMed Scopus (22) Google Scholar, 18Nussler A.K. Di Silvio M. Billiar T.R. Hoffman R.A. Geller D.A. Selby R. Madariaga J. Simmons R.L. Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin.J. Exp. Med. 1992; 176: 261-264Crossref PubMed Scopus (382) Google Scholar). Certain human cell lines have also been instrumental in defining the role of signaling pathways in the regulation of human iNOS expression. The cytokines TNF, IL-1, and IFNγ act in synergy to induce human iNOS gene expression through the coordinated activation of NF-κB, JAK-STAT1, ERK1/2, and p38 MAPK pathways (19Taylor B.S. de Vera M.E. Ganster R.W. Wang Q. Shapiro R.A. Morris Jr., S.M. Billiar T.R. Geller D.A. Multiple NF-κB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene.J. Biol. Chem. 1998; 273: 15148-15156Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 20Ganster R.W. Taylor B.S. Shao L. Geller D.A. Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 8638-8643Crossref PubMed Scopus (279) Google Scholar, 21Poljakovic M. Nygren J.M. Persson K. Signalling pathways regulating inducible nitric oxide synthase expression in human kidney epithelial cells.Eur. J. Pharmacol. 2003; 469: 21-28Crossref PubMed Scopus (35) Google Scholar, 22Kristof A.S. Marks-Konczalik J. Moss J. Mitogen-activated protein kinases mediate activator protein-1-dependent human inducible nitric-oxide synthase promoter activation.J. Biol. Chem. 2001; 276: 8445-8452Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In contrast, phosphatidylinositol 3-kinase (PI3K) inhibits cytokine-induced human iNOS gene promoter activity, although the effect of this signaling pathway on post-transcriptional regulation of iNOS levels has not been examined (23Kristof A.S. Fielhaber J. Triantafillopoulos A. Nemoto S. Moss J. Phosphatidylinositol 3-kinase-dependent suppression of the human inducible nitric-oxide synthase promoter is mediated by FKHRL1.J. Biol. Chem. 2006; 281: 23958-23968Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Activation of Ras is also involved in the induction of iNOS in some cell types, although how Ras interacts with other signaling pathways in this setting is incompletely defined (24Pahan K. Liu X. McKinney M.J. Wood C. Sheikh F.G. Raymond J.R. Expression of a dominant-negative mutant of p21ras inhibits induction of nitric oxide synthase and activation of nuclear factor-κB in primary astrocytes.J. Neurochem. 2000; 74: 2288-2295Crossref PubMed Scopus (83) Google Scholar). The cellular effects of NO are mediated largely by the activation of soluble guanylate cyclase (sGC) and by protein S-nitrosylation. Activation of sGC by NO initiates cGMP signaling pathways by converting GTP to cGMP (25Tennyson A.G. Lippard S.J. Generation, translocation, and action of nitric oxide in living systems.Chem. Biol. 2011; 18: 1211-1220Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). NO also directly modifies proteins by S-nitrosylation, which is a post-translational modification that involves the covalent addition of a nitroso moiety onto cysteines to form S-nitrosocysteine. This type of protein modification regulates the enzymatic properties of a large number of proteins and has recently been suggested to have similar biological significance as protein phosphorylation (26Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1722) Google Scholar). Lander et al. (27Lander H.M. Ogiste J.S. Pearce S.F. Levi R. Novogrodsky A. Nitric oxide-stimulated guanine nucleotide exchange on p21ras.J. Biol. Chem. 1995; 270: 7017-7020Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 28Lander H.M. Hajjar D.P. Hempstead B.L. Mirza U.A. Chait B.T. Campbell S. Quilliam L.A. A molecular redox switch on p21ras: structural basis for the nitric oxide-p21ras interaction.J. Biol. Chem. 1997; 272: 4323-4326Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar) have shown that Ras is S-nitrosylated on cysteine 118 and that this leads to Ras activation. However, very little is known about how this modification of Ras affects cell biology. The mammalian target of rapamycin (mTOR) is a 289-kDa serine threonine protein kinase that regulates cell size, proliferation, survival, and metabolism (29Powell J.D. Delgoffe G.M. The mammalian target of rapamycin: linking T cell differentiation, function, and metabolism.Immunity. 2010; 33: 301-311Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). In mammalian cells, mTOR forms two complexes, mTOR complex 1 (mTORC1) and mTORC2. Activation of mTORC1 supports cell growth by increasing protein translation and lipid biosynthesis through activation of p70 S6 kinase and the translation initiation factor eIF-4E BP1. mTORC2 promotes proliferation and survival, at least in part, by inhibiting activity of the transcription factors Foxo1 and Foxo3a (30Laplante M. Sabatini D.M. mTOR signaling at a glance.J. Cell Sci. 2009; 122: 3589-3594Crossref PubMed Scopus (1642) Google Scholar). The Akt and mTOR pathways are tightly linked as Akt can activate mTORC1 and this kinase is activated downstream of mTORC2 (31Inoki K. Li Y. Zhu T. Wu J. Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling.Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2392) Google Scholar, 32Sarbassov D.D. Ali S.M. Sengupta S. Sheen J.H. Hsu P.P. Bagley A.F. Markhard A.L. Sabatini D.M. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Mol. Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2178) Google Scholar). iNOS expression is tightly controlled by feedback mechanisms that fine-tune its expression. The nature of NO-mediated feedback regulation of iNOS expression is cell- and context-dependent. NO, either provided exogenously by NO donors or synthesized endogenously, has been reported to act as part of a negative feedback loop to down-regulate iNOS expression (33Griscavage J.M. Rogers N.E. Sherman M.P. Ignarro L.J. Inducible nitric oxide synthase from a rat alveolar macrophage cell line is inhibited by nitric oxide.J. Immunol. 1993; 151: 6329-6337Crossref PubMed Google Scholar, 34Hinz B. Brune K. Pahl A. Nitric oxide inhibits inducible nitric oxide synthase mRNA expression in RAW 264.7 macrophages.Biochem. Biophys. Res. Commun. 2000; 271: 353-357Crossref PubMed Scopus (46) Google Scholar). iNOS-derived NO can also inhibit iNOS enzymatic function through post-translational modifications (35Albakri Q.A. Stuehr D.J. Intracellular assembly of inducible NO synthase is limited by nitric oxide-mediated changes in heme insertion and availability.J. Biol. Chem. 1996; 271: 5414-5421Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 36Galijasevic S. Saed G.M. Diamond M.P. Abu-Soud H.M. Myeloperoxidase up-regulates the catalytic activity of inducible nitric oxide synthase by preventing nitric oxide feedback inhibition.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14766-14771Crossref PubMed Scopus (72) Google Scholar). In contrast to negative regulation, NO positively regulates iNOS expression in certain rodent cell types and in human cells, but the mechanisms by which this positive feedback occurs is poorly understood (37Mühl H. Pfeilschifter J. Amplification of nitric oxide synthase expression by nitric oxide in interleukin 1β-stimulated rat mesangial cells.J. Clin. Invest. 1995; 95: 1941-1946Crossref PubMed Scopus (80) Google Scholar, 38Boese M. Busse R. Mülsch A. Schini-Kerth V. Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP.Br. J. Pharmacol. 1996; 119: 707-715Crossref PubMed Scopus (28) Google Scholar, 39Pérez-Sala D. Cernuda-Morollón E. Díaz-Cazorla M. Rodríguez-Pascual F. Lamas S. Posttranscriptional regulation of human iNOS by the NO/cGMP pathway.Am. J. Physiol. Renal Physiol. 2001; 280: F466-F473Crossref PubMed Google Scholar, 40Sheffler L.A. Wink D.A. Melillo G. Cox G.W. Exogenous nitric oxide regulates IFN-γ plus lipopolysaccharide-induced nitric oxide synthase expression in mouse macrophages.J. Immunol. 1995; 155: 886-894PubMed Google Scholar). We showed previously that iNOS-derived NO augments human allogeneic immune responses (41Choy J.C. Wang Y. Tellides G. Pober J.S. Induction of inducible NO synthase in bystander human T cells increases allogeneic responses in the vasculature.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1313-1318Crossref PubMed Scopus (32) Google Scholar, 42Choy J.C. Pober J.S. Generation of NO by bystander human CD8 T cells augments allogeneic responses by inhibiting cytokine deprivation-induced cell death.Am. J. Transplant. 2009; 9: 2281-2291Crossref PubMed Scopus (7) Google Scholar) and, in the current study, we define a positive feedback mechanism by which iNOS-derived NO amplifies iNOS protein expression in human cells. Pharmacological inhibition of iNOS activity attenuated iNOS protein expression, and the addition of exogenous NO was sufficient to rescue iNOS levels. Potentiation of iNOS expression by NO was due to S-nitrosylation of Ras, which increased iNOS levels through supporting the sustained activation of Akt. This NO-mediated augmentation of iNOS protein levels also involved activation of the mTOR pathway, as demonstrated by the attenuation of iNOS protein levels by rapamycin. Altogether, our findings highlight an important regulatory process controlling the expression of iNOS in human cells and identify a novel role of Ras S-nitrosylation in this process. Human lung epithelial cells (A549) were purchased from ATCC and cultured in F-12K medium containing 10% FBS at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Primary human astrocytes were purchased from Lonza, cultured as above in ABM medium (Lonza, Basel, Switzerland) supplemented with the AGM SingleQuot Kit (Lonza), and used between passages 4 and 12 for experiments. Primary bronchial epithelial cells were also purchased from Lonza, cultured in BEGM with the associated supplements, and used between passages 2 and 6. RAW 264.7 cells were cultured in DMEM + 10% FCS. All experiments were performed at least three times. For all experiments using A549 cells, confluent cells were incubated in basal medium in the absence of serum for 24 h prior to cytokine treatment. Primary cells were not serum-starved. To induce iNOS, cells were treated with a cytokine mixture (CM) of recombinant human IL-1α (PeproTech, Rocky Hill, NJ), recombinant human TNFα (PeproTech), and recombinant human IFNγ (Invitrogen) for the indicated times. iNOS activity was inhibited by incubating cells with 1400W (50 μm unless indicated otherwise; Sigma-Aldrich) or l-NAME (Cayman Chemicals, Ann Arbor, MI) 1 h prior to cytokine stimulation. Nitric oxide was added using the slow release NO donor NOC-18 (Calbiochem). Inhibition of signaling pathways was achieved by incubating cells with the pharmacological inhibitors FPT inhibitor II (100 μm; Calbiochem), LY294002 (50 μm; Calbiochem), Akt inhibitor VII (50 μm, Calbiochem), PD98059 (50 μm; Cayman Chemicals), epidermal growth factor receptor (EGFR) inhibitor (20 μm; Calbiochem), and PP2 (20 μm; Calbiochem). sGC was inhibited using 1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one (ODQ; Cayman Chemicals), and the mTOR pathway was inhibited with rapamycin (50 nm; LC Laboratories, Woburn, MA). Cells were lysed as described previously (43von Rossum A. Krall R. Escalante N.K. Choy J.C. Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIPS expression.J. Biol. Chem. 2011; 286: 21137-21144Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Briefly, cells were washed two times with Dulbecco's phosphate-buffered saline and then harvested in 1× radioimmune precipitation buffer (1% (v/v) Triton X-100, 1%(w/v) sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 50 mm Tris·HCl, 2 mm EDTA supplemented with fresh 1 mm sodium orthovanadate, 1 mm PMSF, 50 mm NaF, 10 mm β-glycerophosphate) using a rubber scraper. The lysate was then centrifuged at 17,000 × g for 10 min to remove insoluble material. Immunoblot was performed as described previously (43von Rossum A. Krall R. Escalante N.K. Choy J.C. Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIPS expression.J. Biol. Chem. 2011; 286: 21137-21144Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Primary antibodies used were: polyclonal anti-iNOS (sc-651; Santa Cruz Biotechnology), monoclonal anti-phospho-Thr-308-Akt (2965S; Cell Signaling Technology), monoclonal anti-phospho-ERK1/2 (4370S; Cell Signaling Technology), monoclonal anti-phospho-p70 S6 kinase (9234S; Cell Signaling Technology), monoclonal anti-p70 S6 kinase (2708S; Cell Signaling Technology), monoclonal anti-β-actin (A1978, Sigma-Aldrich), polyclonal anti-Ras (3965S, Cell Signaling Technology), and monoclonal anti-Ras (OP23, Calbiochem). Total RNA was isolated from cells using an RNeasy mini kit as per the manufacturer's instructions (Qiagen), and TaqMan quantitative RT-PCR was performed as described (44Escalante N.K. von Rossum A. Lee M. Choy J.C. CD155 on human vascular endothelial cells attenuates the acquisition of effector functions in CD8 T cells.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 1177-1184Crossref PubMed Scopus (24) Google Scholar) using validated primer/probe sets for iNOS and GAPDH (Applied Biosystems). Data were acquired on an ABI 7900HT fast real-time PCR system (Applied Biosystems). NO production was measured by quantifying the accumulation of nitrite in culture supernatants as described previously (41Choy J.C. Wang Y. Tellides G. Pober J.S. Induction of inducible NO synthase in bystander human T cells increases allogeneic responses in the vasculature.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1313-1318Crossref PubMed Scopus (32) Google Scholar) using a NO-specific chemiluminescence analyzer (Sievers, Boulder, CO). A549 cells were harvested by trypsinization, and 1 × 106 cells were transfected with 6 μg of wild type or 6 μg of C118S Ras plasmid using an Amaxa nucleofector as per the manufacturer's instructions (Lonza) and as described previously (45Liu A.C. Lee M. McManus B.M. Choy J.C. Induction of endothelial nitric oxide synthase expression by IL-17 in human vascular endothelial cells: implications for vascular remodeling in transplant vasculopathy.J. Immunol. 2012; 188: 1544-1550Crossref PubMed Scopus (20) Google Scholar). The wild-type (WT) and C118S Ras plasmids were kindly provided by Dr. Kenneth Tang (Cornell University). Following transfection, cells were rested in 6-well tissue culture plates for 24 h and then used for experiments. The biotin switch method to measure S-nitrosylation was performed using the S-nitrosylation protein detection assay kit (Cayman Chemicals) as per the manufacturer's instructions. Following the biotin switch assay, Ras was immunoprecipitated using a monoclonal Ras antibody (Cell Signaling Technology), and the immunoprecipitate was immunoblotted for biotin using an HRP-conjugated monoclonal biotin antibody (Cell Signaling Technology) and a rabbit polyclonal Ras antibody (EMD Millipore). Immunoblot images were quantified by densitometry using the National Institutes of Health ImageJ software, and protein expression data were normalized to β-actin levels. Unless otherwise indicated, data are expressed as mean ± S.E. obtained in at least three independent experiments. Statistical comparisons were made with a Student's t test or with analysis of variance followed by Tukey's honestly significant difference test. Statistically significant differences were defined as p < 0.05. To study the impact of iNOS-derived NO on the feedback regulation of human iNOS expression, A549 cells were stimulated with CM composed of TNFα (10 ng/ml), IL-1α (10 ng/ml), and IFNγ (50 ng/ml). This stimulus is known to optimally induce the expression of iNOS (19Taylor B.S. de Vera M.E. Ganster R.W. Wang Q. Shapiro R.A. Morris Jr., S.M. Billiar T.R. Geller D.A. Multiple NF-κB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene.J. Biol. Chem. 1998; 273: 15148-15156Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The induction of iNOS expression was evident in CM-treated cells as early as 3–4 h after stimulation and continued to increase until 6 h after stimulation, which was the last time point examined (Fig. 1A). CM also induced the production of NO that was concomitant with the induction of iNOS expression. NO levels were significantly increased at 4 h after stimulation and continued to increase thereafter (Fig. 1B). Also, NO production in response to CM was completely prevented by the iNOS inhibitor 1400W (data not shown). The role of iNOS activity in regulating iNOS protein levels was studied by treating cells with the iNOS inhibitor 1400W or the pan-NOS inhibitor l-NAME followed by examination of iNOS protein expression by immunoblot 6 h later. The inhibition of iNOS activity using either inhibitor significantly reduced CM-induced iNOS protein levels (Fig. 1C). To confirm that NO produced by iNOS was involved in the augmentation of iNOS protein levels, the NO donor NOC-18 was added to cells at the same time as iNOS inhibition with 1400W. The addition of exogenous NO in this manner prevented the reduction in iNOS expression caused by pharmacological inhibition, indicating that iNOS-derived NO stimulates a positive feedback pathway that amplifies iNOS protein levels (Fig. 1D). Interestingly, inhibition of iNOS activity did not affect iNOS mRNA levels, indicating a specific effect of NO in the amplification of protein levels through a post-transcriptional mechanism (Fig. 1E). Other studies have shown in mouse cells that iNOS-derived NO can inhibit iNOS expression (33Griscavage J.M. Rogers N.E. Sherman M.P. Ignarro L.J. Inducible nitric oxide synthase from a rat alveolar macrophage cell line is inhibited by nitric oxide.J. Immunol. 1993; 151: 6329-6337Crossref PubMed Google Scholar, 34Hinz B. Brune K. Pahl A. Nitric oxide inhibits inducible nitric oxide synthase mRNA expression in RAW 264.7 macrophages.Biochem. Biophys. Res. Commun. 2000; 271: 353-357Crossref PubMed Scopus (46) Google Scholar). It is possible that NO has pleiotropic effects on cell signaling and that the effect of iNOS-derived NO may be different in humans and mice (12Schneemann M. Schoedon G. Species differences in macrophage NO production are important.Nat. Immunol. 2002; 3: 102Crossref PubMed Scopus (94) Google Scholar). As such, the effect of iNOS activity on the feedback regulation of iNOS expression in a mouse cell line was examined. Stimulation of RAW 264.7 cells with LPS + IFNγ induced iNOS expression and, in contrast to our observation in human cells but consistent with previous studies examining mouse macrophages (33Griscavage J.M. Rogers N.E. Sherman M.P. Ignarro L.J. Inducible nitric oxide synthase from a rat alveolar macrophage cell line is inhibited by nitric oxide.J. Immunol. 1993; 151: 6329-6337Crossref PubMed Google Scholar, 34Hinz B. Brune K. Pahl A. Nitric oxide inhibits inducible nitric oxide synthase mRNA expression in RAW 264.7 macrophages.Biochem. Biophys. Res. Commun. 2000; 271: 353-357Crossref PubMed Scopus (46) Google Scholar), inhibition of iNOS activity with 1400W increased iNOS levels (Fig. 1F). The role of iNOS activity in regulating iNOS protein levels was also examined in primary cells. Primary human astrocytes stimulated with CM express iNOS with maximal levels apparent at 24 h after stimulation (Fig. 2A). Consistent with our findings in A549 cells, inhibition of iNOS activity with 1400W dose-depen" @default.
• W2002404304 created "2016-06-24" @default.
• W2002404304 creator A5030664166 @default.
• W2002404304 creator A5091758810 @default.
• W2002404304 date "2013-05-01" @default.
• W2002404304 modified "2023-09-29" @default.
• W2002404304 title "Positive Feedback Regulation of Human Inducible Nitric-oxide Synthase Expression by Ras Protein S-Nitrosylation" @default.
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