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• W2148052253 abstract "Carbon monoxide (CO), one of the end products of heme oxygenase activity,inhibits smooth muscle proliferation by decreasing ERK1/2 phosphorylation andcyclin D1 expression, a signaling pathway that is known to be modulated byreactive oxygen species (ROS) in airway smooth muscle cells (ASMCs). Twoimportant sources of ROS involved in cell signaling are the membrane NAD(P)Hoxidase and the mitochondrial respiratory chain. Thus, that CO could modulateredox signaling in ASMCs by interacting with the heme moiety of NAD(P)Hoxidase and/or the respiratory chain is a plausible hypothesis. Here we showthat a recently identified carbon monoxide-releasing molecule,[Ru(CO)3Cl2]2 (or CORM-2) 1) inhibits NAD(P)Hoxidase cytochrome b558 activity, 2) increases oxidantproduction by the mitochondria, and 3) inhibits ASMC proliferation andphosphorylation of the ERK1/2 mitogen-activated protein kinase and expressionof cyclin D1, two critical pathways involved in muscle proliferation. No sucheffects were observed with the negative control(Ru(Me2SO)4Cl2), which does not contain COgroups. Because both diphenylene iodinium or apocynin (inhibitors of NAD(P)Hoxidase) and rotenone (a molecule that increases mitochondrial ROS productionby blocking the respiratory chain) mimicked the effect of CORM-2 on cyclin D1expression and ASMC proliferation, the antiproliferative effect of CORM-2 isprobably related to inhibition of cytochromes on both NAD(P)H oxidase and therespiratory chain. The involvement of increased mitochondria-derived oxidantsis substantiated by the findings showing that the antioxidantN-acetylcysteine partially inhibited the effects of CORM-2. Thisstudy provides a new mechanism to explain redox signaling by CO. Carbon monoxide (CO), one of the end products of heme oxygenase activity,inhibits smooth muscle proliferation by decreasing ERK1/2 phosphorylation andcyclin D1 expression, a signaling pathway that is known to be modulated byreactive oxygen species (ROS) in airway smooth muscle cells (ASMCs). Twoimportant sources of ROS involved in cell signaling are the membrane NAD(P)Hoxidase and the mitochondrial respiratory chain. Thus, that CO could modulateredox signaling in ASMCs by interacting with the heme moiety of NAD(P)Hoxidase and/or the respiratory chain is a plausible hypothesis. Here we showthat a recently identified carbon monoxide-releasing molecule,[Ru(CO)3Cl2]2 (or CORM-2) 1) inhibits NAD(P)Hoxidase cytochrome b558 activity, 2) increases oxidantproduction by the mitochondria, and 3) inhibits ASMC proliferation andphosphorylation of the ERK1/2 mitogen-activated protein kinase and expressionof cyclin D1, two critical pathways involved in muscle proliferation. No sucheffects were observed with the negative control(Ru(Me2SO)4Cl2), which does not contain COgroups. Because both diphenylene iodinium or apocynin (inhibitors of NAD(P)Hoxidase) and rotenone (a molecule that increases mitochondrial ROS productionby blocking the respiratory chain) mimicked the effect of CORM-2 on cyclin D1expression and ASMC proliferation, the antiproliferative effect of CORM-2 isprobably related to inhibition of cytochromes on both NAD(P)H oxidase and therespiratory chain. The involvement of increased mitochondria-derived oxidantsis substantiated by the findings showing that the antioxidantN-acetylcysteine partially inhibited the effects of CORM-2. Thisstudy provides a new mechanism to explain redox signaling by CO. HO-1, 1The abbreviations used are: HO-1, heme oxygenase; ASMC, airway smoothmuscle cell; CO, carbon monoxide; CORM, carbon monoxide-releasing molecule;DCFH-DA, 2,7-dichlorofluorescein diacetate; DPI, diphenylene iodinium; ERK,extracellular signal-regulated kinase; NAC, N-acetylcysteine; NOS,nitric-oxide synthase; ROS, reactive oxygen species;[Ru(CO3)Cl2]2,tricarbonyldichlororuthenium(II) dimer; PDGF, platelet-derived growth factor;CM-H2XRos, chloromethyl dihydro-X-rosamine; Pipes,1,4-piperazinediethanesulfonic acid. the limitingstep enzyme in heme degradation, is strongly involved in the control of smoothmuscle proliferation (1Durante W. J. Cell.Physiol. 2003; 195: 373-382Crossref PubMed Scopus (151) Google Scholar). Wehave recently reported that bilirubin, one of the products of heme breakdownby HO-1, modulates redox signaling pathways of human ASMCs resulting ininhibition of cell proliferation(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Other studies have shownthat CO, another metabolite of HO activity, could also have anantiproliferative effect on vascular(3Peyton K.J. Reyna S.V. Chapman G.B. Ensenat D. Liu X.M. Wang H. Schafer A.I. Durante W. Blood. 2002; 99: 4443-4448Crossref PubMed Scopus (144) Google Scholar, 4Morita T. Mitsialis S.A. Koike H. Liu Y. Kourembanas S. J. Biol. Chem. 1997; 272: 32804-32809Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 5Stanford S.J. Walters M.J. Hislop A.A. Haworth S.G. Evans T.W. Mann B.E. Motterlini R. Mitchell J.A. Eur. J. Pharmacol. 2003; 473: 135-141Crossref PubMed Scopus (36) Google Scholar)and bronchial smooth muscle tissues(6Song R. Mahidhara R.S. Liu F. Ning W. Otterbein L.E. Choi A.M.K. Am. J. Respir. Cell Mol.Biol. 2002; 27: 603-610Crossref PubMed Scopus (114) Google Scholar). This effect has also beenobserved in models in vivo where CO protected against restenosis ofcarotid arteries following balloon injury(7Otterbein L.E. Zuckerbraun B.S. Haga M. Liu F. Song R. Usheva A. Stachulak C. Bodyak N. Smith R.N. Csizmadia E. Tyagi S. Akamatsu Y. Flavell R.J. Billiar T.R. Tzeng E. Bach F.H. Choi A.M. Soares M.P. Nat. Med. 2003; 9: 183-190Crossref PubMed Scopus (447) Google Scholar) and hypoxic pulmonaryhypertension (8Christou H. Morita T. Hsieh C.M. Koike H. Arkonac B. Perrella M.A. Kourembanas S. Circ.Res. 2000; 86: 1224-1229Crossref PubMed Scopus (196) Google Scholar). Themechanisms by which CO exerts its antiproliferative effects in ASMCs appear torely on inhibition of ERK1/2 phosphorylation and cyclin D1 expression(6Song R. Mahidhara R.S. Liu F. Ning W. Otterbein L.E. Choi A.M.K. Am. J. Respir. Cell Mol.Biol. 2002; 27: 603-610Crossref PubMed Scopus (114) Google Scholar). The involvement of ERKpathway inhibition by CO has been described in other cellular models(9Choi B.M. Pae H.O. Kim Y.M. Chung H.T. Hepatology. 2003; 37: 810-823Crossref PubMed Scopus (91) Google Scholar), but the precise mechanismexplaining this interaction remains unknown. Interestingly the specificpathway responsible for cyclin D1 expression is sensitive to oxidants(10Brar S.S. Kennedy T.P. Whorton A.R. Murphy T.M. Chitano P.C. Hoidal J.R. J. Biol.Chem. 1999; 274: 20017-20026Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In view of thesefindings, the existence of a potential “CO sensor” that regulatesASMC proliferation cannot be excluded a priori and represents achallenging hypothesis to explore further. Two important sources of oxidants involved in the control of cellproliferation are the NAD(P)H oxidase(11Brar S.S. Kennedy T.P. Sturrock A.B. Huecksteadt T.P. Quinn M.T. Murphy T.M. Chitano P. Hoidal J.R. Am. J. Physiol. 2002; 282: L782-L795Crossref PubMed Scopus (138) Google Scholar,12Page K. Li J. Hodge J.A. Liu P.T. Vanden Hoeck T.L. Becker L.B. Pestell R.G. Rosner M.R. Hershenson M.B. J. Biol. Chem. 1999; 274: 22065-22071Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and the mitochondrialrespiratory chain(13Deshpande S.S. Angkeow P. Huang J. Ozaki M. Irani K. FASEB J. 2000; 14: 1705-1714Crossref PubMed Scopus (205) Google Scholar, 14Carriere A. Fernandez Y. Rigoulet M. Penicaud L. Casteilla L. FEBS Lett. 2003; 550: 163-167Crossref PubMed Scopus (96) Google Scholar, 15Li N. Ragheb K. Lawler G. Sturgis J. Rajwa B. Melendez J.A. Robinson J.P. J. Biol.Chem. 2003; 278: 8516-8525Abstract Full Text Full Text PDF PubMed Scopus (1020) Google Scholar, 16Horimoto M. Fulop P. Derdak Z. Wands J.R. Baffy G. Hepatology. 2004; 39: 386-392Crossref PubMed Scopus (97) Google Scholar).NAD(P)H oxidase is made of an assembly of different proteins:gp91phox and p22phox (whichheterodimerize to form the cytochrome b558) andp67phox, p47phox, and Rac1 or -2subunits. Gp91phox, the catalytic moiety of the oxidase,is a plasma membrane-associated flavohemoprotein containing one flavin-adeninedinucleotide and two hemes that catalyzes the NAD(P)H-dependent reduction ofoxygen to form superoxide(17Babior B. Lambeth J. Nauseef W. Arch. Biochem. Biophys. 2002; 397: 342-344Crossref PubMed Scopus (712) Google Scholar). Activation of NAD(P)Hoxidase has been associated with an increased proliferative response(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,11Brar S.S. Kennedy T.P. Sturrock A.B. Huecksteadt T.P. Quinn M.T. Murphy T.M. Chitano P. Hoidal J.R. Am. J. Physiol. 2002; 282: L782-L795Crossref PubMed Scopus (138) Google Scholar). The mitochondrial respiratory chain is composed of different cytochromesaccounting for 85–90% of the O2 consumed in the cell.Approximately 1–3% of this O2 is incompletely metabolized anddiverted into superoxide anion(18Boveris A. Chance B. Biochem.J. 1973; 134: 707-716Crossref PubMed Scopus (2112) Google Scholar). Inhibition of electrontransfer in the mitochondrial respiratory chain is associated with asignificant increase in the production of superoxide anion and hydrogenperoxide and a decreased cell proliferation(14Carriere A. Fernandez Y. Rigoulet M. Penicaud L. Casteilla L. FEBS Lett. 2003; 550: 163-167Crossref PubMed Scopus (96) Google Scholar,16Horimoto M. Fulop P. Derdak Z. Wands J.R. Baffy G. Hepatology. 2004; 39: 386-392Crossref PubMed Scopus (97) Google Scholar). It must be noted thatthis is in contrast with the mitogenic effect of NAD(P)H oxidase-derivedsuperoxide anion. Although there is no clear explanation for the discrepancybetween the effects of mitochondria- and NAD(P)H oxidase-derived ROS on cellproliferation, most evidence suggests that the intensity and subcellularorigins of ROS may be crucial in their effects on cell fate. Because CO has a high affinity for heme groups (for a review, see Ref.19Piantadosi C.A. Antioxid. RedoxSignal. 2002; 4: 259-270Crossref PubMed Scopus (171) Google Scholar), we hypothesized that COcould behave as a modulator of redox signals by interacting with heme andinhibiting cytochrome b558 of the NAD(P)H oxidase and/orcytochromes of the respiratory chain, consequently affecting redox-mediatedcell proliferation. Recently a group of transition metal carbonyls have been characterized ascarbon monoxide-releasing molecules (CORMs) to liberate CO in biologicalsystems providing a useful tool in research to examine the mechanism by whichCO exerts its pharmacological activities(20Motterlini R. Clark J.E. Foresti R. Sarathchandra P. Mann B.E. Green C.J. Circ. Res. 2002; 90: e17-e24Crossref PubMed Google Scholar, 21Motterlini R. Mann B.E. Johnson T.R. Clark J.E. Foresti R. Green C.J. Curr. Pharm.Des. 2003; 9: 2525-2539Crossref PubMed Scopus (231) Google Scholar, 22Clark J.E. Naughton P. Shurey S. Green C.J. Johnson T.R. Mann B.E. Foresti R. Motterlini R. Circ. Res. 2003; 93: e2-e8Crossref PubMed Google Scholar, 23Foresti R. Hammad J. Clark J.E. Johnson T.R. Mann B.E. Friebe A. Green C.J. Motterlini R. Br. J. Pharmacol. 2004; 142: 453-460Crossref PubMed Scopus (269) Google Scholar).Therefore, in this study we investigated whether CO, administered using CORM-2([Ru(CO)3Cl2]2), could modulate PDGF-inducedhuman ASMC proliferation and examined whether this effect involvesheme-dependent ROS-producing pathways such as the NAD(P)H oxidase and themitochondrial respiratory chain. Reagents—[Ru(CO)3Cl2]2(CORM-2) was obtained from Aldrich.Ru(Me2SO)4Cl2, the negative control forCORM-2, was synthesized as described previously(20Motterlini R. Clark J.E. Foresti R. Sarathchandra P. Mann B.E. Green C.J. Circ. Res. 2002; 90: e17-e24Crossref PubMed Google Scholar).[methyl-3H]Thymidine was purchased from PerkinElmer LifeSciences, and PDGF-AB was from R&D Systems (Abingdon, UK). Apocynin(acetovanillone) was from Acros Organices (Geel, Belgium). DCFH-DA andCM-H2XRos (MitoTracker® Red) were from Molecular Probes(Eugene, OR). Polyclonal anti-p42/44 (phosphorylated and non-phosphorylated)antibody was purchased from New England Biolabs (Ozyme,Saint-Quentin-en-Yvelines, France), monoclonal anti-HO-1 and anti-nitric-oxidesynthase type 1 (NOS1) antibodies were from StressGen, and anti-cyclin D1antibody was from Santa Cruz Biotechnology (Tebu, Le-Perray-en-Yvelines,France). Culture media, supplements, and fetal calf serum were fromInvitrogen. Tissue culture plasticware was supplied by Costar Corp.(Cambridge, MA). Reagents for Western blotting were from Bio-Rad. Otherreagents were from Sigma. Human Airway Smooth Muscle Cell Isolation and Culture—Primary cultures of human bronchial smooth muscle were established as alreadydescribed (2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,24Berger P. Perng D.W. Thabrew H. Compton S.J. Cairns J.A. McEuen A.R. Marthan R. Tunon De Lara J.M. Walls A.F. J. Appl. Physiol. 2001; 91: 1372-1379Crossref PubMed Scopus (158) Google Scholar). Briefly human bronchiwere obtained from lung resection for cancer of six different patients anddissected from the surrounding parenchyma. Then the epithelium was removed,and bundles of smooth muscle were dissected under binocular microscope. Smoothmuscle was cut into 1-mm square pieces, termed explants, and incubated in6-well plates with Dulbecco's modified Eagle's medium, 10% heat inactivatedserum, and antibiotics in a humidified atmosphere of 5% CO2, 95%air at 37 °C as published previously(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Cultures from passages 3to 5 were used for experiments. In all of the experiments CORM-2,Ru(Me2SO)4Cl2, and other molecules (DPI,apocynin, or rotenone) were added to the medium 30 min before PDGF. [3H]Thymidine Incorporation—Cell proliferationwas assessed by measurement of [methyl-3H]thymidineincorporation as described previously(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Briefly 70% confluentcells were seeded in 24-well plates and serum-deprived for 24 h beforestimulation with 50 ng/ml PDGF-AB for another 24h. As stated before, CORM-2,Ru(Me2SO)4Cl2, DPI, apocynin, or rotenonewere added to the medium 30 min before PDGF. Thymidine (4 μCi/ml) was addedfor the last 18 h of stimulation. The amount of incorporated thymidine wasmeasured by scintillation counting. Results were obtained in quadruplicate andexpressed in cpm. Because CORM-2 and DPI were diluted in Me2SO, asimilar concentration of Me2SO was added to the media for all ofthe experimental conditions in which these molecules were not utilized. Cellular Toxicity and Viability—Cellular toxicity wasassessed by cell count and trypan blue exclusion test, and cell viability wasdetermined by quantification of lactate dehydrogenase released into themedium. Apoptosis—Flow cytometric determination of apoptosis wasperformed using propidium iodide incorporation. Untreated and treated cellswere collected after 24 h by trypsinization and centrifugation for 10 min at1400 rpm at 4 °C. Cells were then resuspended in ice-coldphosphate-buffered saline, pelleted by centrifugation, and fixed by ice-cold70% ethanol overnight. After another centrifugation, cells were resuspended in100 μl of RNase A (180 μg/ml in phosphate-buffered saline) and incubatedat room temperature for 30 min. Then 200 μl of propidium iodide were addedto the suspension (final concentration, 50 μg/ml), and a subsequentincubation at room temperature for 15 min followed. Samples were placed in iceto stop the reaction and analyzed within 1 h by flow cytometry. Results areexpressed as the percentage of apoptotic cells. Extracellular Superoxide Anion Production by ASMCs: Cytochrome cReduction Assay—Ferricytochrome c reduction was measuredas described previously (25Thabut G. El-Benna J. Samb A. Corda S. Megret J. Leseche G. Vicaut E. Aubier M. Boczkowski J. J. Biol. Chem. 2002; 277: 22814-22821Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).Briefly cells were cultured in 6-well plates. Before addition of PDGF, themedium was replaced with Hank's balanced salt solution without phenol red andincubated in 1 ml of the same buffer with and without 300 units/ml superoxidedismutase. Subsequently ferricytochrome c was added at a finalconcentration of 1 mg/ml to the reaction buffer solution followed by additionof PDGF. After 1 h, the buffer was removed, and absorbance at 550 nm wasmeasured immediately. Superoxide anion production was calculated from thedifferences in the absorbances between samples with and without superoxidedismutase using an extinction coefficient of 21.1mm–1 cm–1 for reducedferricytochrome c. ASMC Cytochrome b558 Spectra Analysis—Thespectrum of cytochrome b558 was analyzed as describedpreviously (25Thabut G. El-Benna J. Samb A. Corda S. Megret J. Leseche G. Vicaut E. Aubier M. Boczkowski J. J. Biol. Chem. 2002; 277: 22814-22821Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Briefly cellswere lysed in phosphate-buffered saline in the presence of 2% Triton X-100 at4 °C for 10 min. They were then treated with 10 μm CORM-2 orRu(Me2SO)4Cl2. Then the difference betweenthe reduced and oxidized spectrum was recorded with a dual beam scanningspectrophotometer (Uvikon). The base-line (oxidized) spectrum was measured at400–600 nm, and then a few grains of sodium dithionite were added to thesample cuvette, and a new spectrum was recorded. The subtraction betweenspectra was performed automatically. Membranes were prepared as described by Dang and co-workers(26Dang P.M. Dewas C. Gaudry M. Fay M. Pedruzzi E. Gougerot-Pocidalo M.A. El Benna J. J.Biol. Chem. 1999; 274: 20704-20708Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly 1 ×107 cells/ml were sonicated for 3 × 5 s on ice in 1 ml oflysis buffer (10 mm Pipes, pH 7.3, 3 mmMgCl2, 100 mm KCl, 5 mm NaCl) supplementedwith 0.5 mm phenylmethylsulfonyl fluoride, 1 mm EGTA, 10μg/ml leupeptin, and 10 μg/ml pepstatin. Non-lysed cells, nuclei, andparticulates were discarded by centrifugation at 10,000 × g for10 min at 4 °C. Cytosolic and light membrane fractions were separated bycentrifugation at 150,000 × g on a 15–34% (w/w) sucrosegradient for 30 min at 4 °C. Membranes were collected from the interface,and then pellets were used for determination of the spectrum. NAD(P)H Oxidase Assay by Lucigenin-enhanced Chemiluminescence in ASMCMembranes—Semiconfluent cells were serum-deprived for 24 h beforestimulation with 50 ng/ml PDGF-AB for another 24 h. Then membranes wereprepared as described above. Membranes were treated with 10 μmDPI or 10 μm CORM-2 orRu(Me2SO)4Cl2. The lucigenin assay wasperformed in Pipes buffer(26Dang P.M. Dewas C. Gaudry M. Fay M. Pedruzzi E. Gougerot-Pocidalo M.A. El Benna J. J.Biol. Chem. 1999; 274: 20704-20708Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), 50 μmlucigenin, and 100 μm NAD(P)H. Chemiluminescence (cpm) wasmeasured in a luminometer (Berthold LB935) for 10 min. Results were expressedas cpm/10 min/cell number. Isolation of Human Neutrophils and Superoxide Anion ProductionAssay—Venous blood was collected from healthy adult volunteers, andneutrophils were isolated by dextran sedimentation and density gradientcentrifugation as described previously(26Dang P.M. Dewas C. Gaudry M. Fay M. Pedruzzi E. Gougerot-Pocidalo M.A. El Benna J. J.Biol. Chem. 1999; 274: 20704-20708Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Erythrocytes wereremoved by lysis with hypotonic solutions. Following isolation, cells wereresuspended in the appropriate medium such as Hank's balanced salt solution. Acell count was performed, and cell viability was determined using the trypanblue exclusion method. Following isolation, cells were resuspended in Hank's balanced saltsolution at a concentration of 2 million/ml. Superoxide production wasdetermined by measuring superoxide dismutase-inhibitable cytochrome creduction (26Dang P.M. Dewas C. Gaudry M. Fay M. Pedruzzi E. Gougerot-Pocidalo M.A. El Benna J. J.Biol. Chem. 1999; 274: 20704-20708Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The cellsuspension (1 ml) was placed in a cuvette with 1 ml of Hank's balanced saltsolution containing 160 μm cytochrome c. The cuvettewas placed in the thermostated chamber of the spectrophotometer (BeckmanDU640) and allowed to stabilize at 37 °C. After a base line was recorded,cells were stimulated with 100 ng/ml phorbol myristate acetate. The referencecuvette contained 5 units of superoxide dismutase in addition to the mixture.Changes in absorbance at 550 nm were measured over a 10-min period. Resultswere calculated as nanomoles of superoxide produced/2 million cells/10 min fortotal superoxide production using a molar extinction coefficient of 21.1mm–1 cm–1. Intracellular ROS Production: DCFH-DA Oxidation—Cells werecultured in 96-well plates as described previously(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). DCFH-DA (10μm final concentration in Me2SO) was added 1 h beforestimulation; then the medium was removed, cells were washed twice withphosphate-buffered saline, fresh medium was added, and cells were stimulatedwith 50 ng/ml PDGF. Fluorescence was measured immediately after PDGF additionat 480–555 nm every 7 min during a 45-min period using a multiwellfluorescence plate reader. Intracellular ROS (especially hydroxyl radical) orhydrogen peroxide oxidize dichlorodihydrofluorescein, yielding the fluorescentproduct dichlorodifluorescein(25Thabut G. El-Benna J. Samb A. Corda S. Megret J. Leseche G. Vicaut E. Aubier M. Boczkowski J. J. Biol. Chem. 2002; 277: 22814-22821Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar,27Carter W. Narayanan P. Robinson J. J. Leukoc. Biol. 1994; 55: 253-258Crossref PubMed Google Scholar). Mitochondrial ROS Production—To assess whether the increasein intracellular ROS production would originate from mitochondria, we used thespecific fluorescent probe MitoTracker CM-H2XRos (500 nmfinal concentration in Me2SO, 30 min before PDGF), which becomesfluorescent upon oxidation(28Young T.A. Cunningham C.C. Bailey S.M. Arch. Biochem. Biophys. 2002; 405: 65-72Crossref PubMed Scopus (111) Google Scholar). The protocol was similarto that used for DCFH-DA except that only one measurement was takenimmediately after addition of PDGF. The fluorescence was measured at578–599 nm. Immunoblotting for HO-1, NOS1, Phosphorylated ERK1/2, andCyclin D1—Western blot analysis for HO-1, NOS1, phosphorylatedERK1/2, and cyclin D1 was performed as described previously(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,29Taille C. Foresti R. Lanone S. Zedda C. Green C. Aubier M. Motterlini R. Boczkowski J. Am. J.Respir. Crit. Care Med. 2001; 163: 753-761Crossref PubMed Scopus (64) Google Scholar). For HO-1 and NOS1expression, cells were treated with increasing concentrations of CORM-2 orRu(Me2SO)4Cl2 for 24 h and then scraped inlysis buffer. Fifty micrograms of proteins were loaded in each well. Anti-HO-1and -NOS1 monoclonal primary antibodies (1:1000 dilution) was applied for 1 h,whereas anti-cyclin D1 antibody (1:200 dilution) was incubated overnight. Formeasurements of ERK1/2 phosphorylation, cells were treated with differentconcentrations of CORM-2 for different times, then stimulated with 50 ng/mlPDGF for 10 min, and finally scraped in lysis buffer containing phosphataseinhibitors (2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A primarypolyclonal antibody was used at 1:2000 dilution. Using the same blots, theexpression of the housekeeping protein β-actin or total ERK1/2 wasevaluated using monoclonal antibodies. Optical densities were measured with aPerfect Image 2.01 image analysis system (Iconix, Courtaboeuf, France). Theresults were expressed as the ratio of the expression of HO-1, NOS1,phospho-ERK, or cyclin D1 over that of β-actin or total ERK1/2. Statistical Analysis—Values are given as the means ±S.E. The data were analyzed by one-way analysis of variance or non-parametrictests when appropriate. The significance for all statistics was accepted atp < 0.05. CORM-2 Inhibits PDGF-induced ASMC Proliferation—Toinvestigate how CO influences cell proliferation, we analyzed the effect ofCORM-2 on PDGF-induced ASMC proliferation(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We found that PDGF induceda 4-fold increase in ASMC proliferation. Treatment of cells with CORM-2 (10μm) 30 min before stimulation with PDGF reduced cellproliferation by about 50%. In contrast, the negative control(Ru(Me2SO)4Cl2, 10 μm), whichdoes not contain CO groups, did not modify the proliferative response(Fig. 1), confirming that COliberated from CORM-2 is directly involved in the observed effect. Thisconfirms that CORM-2 has effects similar to CO administrated as a gas in ASMCs(6Song R. Mahidhara R.S. Liu F. Ning W. Otterbein L.E. Choi A.M.K. Am. J. Respir. Cell Mol.Biol. 2002; 27: 603-610Crossref PubMed Scopus (114) Google Scholar) and is a useful tool forstudying the mechanisms of action of CO. It must be noted that no celltoxicity, as assessed by lactate dehydrogenase measurement in the medium andtrypan blue exclusion test, was evident at the concentrations of CORM-2 used.Cell toxicity of the compound was only observed at 100 μm (datanot shown). To further confirm that the effect of CORM-2 was mediated by CO releasedfrom the compound, we performed additional experiments in the presence ofmyoglobin, a CO scavenger. Indeed we observed that myoglobin (10μm) effectively reversed the antiproliferative effect of CORM-2(Fig. 1). Because HO-1 is known to inhibit smooth cell proliferation by itself(2Taille C. Almolki A. Benhamed M. Zedda C. Megret J. Berger P. Leseche G. Fadel E. Yamaguchi T. Marthan R. Aubier M. Boczkowski J. J. Biol. Chem. 2003; 278: 27160-27168Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and because heavy metalscan induce HO-1 expression(30Maines M.D. Annu. Rev. Pharmacol.Toxicol. 1997; 17: 517-554Crossref Scopus (2218) Google Scholar), we assessed whether theruthenium-containing CORM-2 or its negative control could induce HO-1expression per se at the time when proliferation was measured.However, we found no change in HO-1 protein expression after 24-h incubationof cells in the presence of 1 and 10 μm CORM-2(Fig. 2). We also investigatedwhether CORM-2 influenced the expression of NOS1 because this enzyme has beenshown to modulate ASMC proliferation(31Patel H.J. Belvisi M.G. Donnelly L.E. Yacoub M.H. Chung K.F. Mitchell J.A. FASEBJ. 1999; 13: 1810-1816Crossref PubMed Scopus (58) Google Scholar). We found no change inNOSI protein expression after 24-h incubation of cells in the presence ofCORM-2 (data not shown). Inhibition of NAD(P)H Oxidase by CORM-2 Contributes to the Attenuationof Cell Proliferation—As stated in the Introduction, NAD(P)Hoxidase-derived superoxide anions are involved in ASMC proliferation(11Brar S.S. Kennedy T.P. Sturrock A.B. Huecksteadt T.P. Quinn M.T. Murphy T.M. Chitano P. Hoidal J.R. Am. J. Physiol. 2002; 282: L782-L795Crossref PubMed Scopus (138) Google Scholar,32Page K. Li J. Corbit K.C. Rumilla K.M. Soh J.W. Weinstein I.B. Albanese C. Pestell R.G. Rosner M.R. Hershenson M.B. Am. J. Respir. Cell Mol.Biol. 2002; 27: 204-213Crossref PubMed Scopus (36) Google Scholar). We first confirmed thesefindings by using DPI (10 μm), an inhibitor of flavin-containingenzymes such as NAD(P)H oxidase(33Hancock J.T. White J.I. Jones O.T. Silver I.A. Free Radic. Biol. Med. 1991; 11: 25-29Crossref PubMed Scopus (22) Google Scholar). As expected, DPIsignificantly reduced PDGF-induced cell proliferation(Fig. 1). It must be noted,however, that DPI could inhibit all flavo-containing enzymes such as NOS andrespiratory chain complex I and could also increase ROS production(34Riganti C. Gazzano E. Polimeni M. Costamagna C. Bosia A. Ghigo D. J. Biol. Chem. 2004; 279: 47726-47731Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). We think that theseeffects of DPI are unlikely under our experimental conditions because we havepreviously shown that the same concentration of DPI (10 μm) usedin the present study decreased intracellular ROS production in ASMCs(25Thabut G. El-Benna J. Samb A. Corda S. Megret J. Leseche G. V" @default.
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• W2148052253 title "Mitochondrial Respiratory Chain and NAD(P)H Oxidase Are Targets for theAntiproliferative Effect of Carbon Monoxide in Human Airway SmoothMuscle" @default.
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