The International Journal of Biochemistry & Cell Biology 40 (2008) 2660–2670 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel Glutathione and copper, zinc superoxide dismutase are modulated by overexpression of neuronal nitric oxide synthase Sara Baldelli a,1 , Katia Aquilano a,1 , Giuseppe Rotilio a,b , Maria R. Ciriolo a,b,∗ a Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy b Research Center IRCCS San Raffaele La Pisana, Via dei Bonacolsi snc, 00163 Rome, Italy article info Article history: Received 8 April 2008 Received in revised form 19 May 2008 Accepted 20 May 2008 Available online 28 May 2008 Keywords: nNOS overexpression SOD1 down-regulation GSH Sp1 AP1 abstract This study examines the effects of neuronal nitric oxide overexpression (nNOS) in neuronal and non-neuronal cell lines. The up-regulation of nNOS causes an increase in the intracellu- lar concentration of glutathione (GSH) that was mandatory for counteracting NO-mediated cytotoxicity. Indeed, inhibition of GSH synthesis by buthionine sulfoximine (BSO) signif- icantly enhances NO toxicity. nNOS increase also mediates a down-regulation of copper, zinc superoxide dismutase (SOD1) in terms of mRNA production, protein and activity lev- els. The nNOS inhibitor (7-Ni), while restores the GSH content, does not recover the SOD1 level, suggesting that NO is not directly involved in SOD1 modulation. SOD1 reduction is most probably due to an increased DNA binding capacity of AP-1, which seems to play a negative role in the capacity of Sp1 to bind to the sod1 gene promoter. Actually, this study demonstrates that nNOS directly interacts with Sp1, both in the cytosol as well as in the nucleus, forming a stable heterocomplex that could have an important physiological role in the modulation of Sp1 activity. © 2008 Elsevier Ltd. All rights reserved. 1. Introduction Nitric oxide (NO) is a gaseous messenger molecule with pleiotropic action and is physiologically synthesized by a Abbreviations: nNOS cells, cells transiently transfected with nNOS cDNA cloned into the pME18S vector; mock cells, cells transfected with the pME18S empty vector; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; ONOO - , peroxynitrite; O2 •- , superoxide radical; SOD1, superoxide dismutase; GSH, reduced glutathione; GSNO, S-nitrosoglutathione; ALS, amyotrophic lateral sclerosis; GSSG, oxidized GSH; ROS, reactive oxygen species; RNS, reactive nitrogen species; PBS, phosphate-buffered saline; DTT, dithiothreitol; NBT, nitro blue tetrazolium; 7-Ni, 7-nitroindazole; MG132, carbobenzoxy-l-leucyl-l-leucyl-l-leucinal; l-NAME, N G -nitro-l- arginine-methyl-ester; l-NMA, l-N  -methylarginine; carboxy PTIO, 2-(4- carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt; EMSA, electrophoretic mobility shift assay; NO2Tyr, nitrotyrosine; BrdU, 5-bromo-2 ′ -deoxyuridine. ∗ Corresponding author at: Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy. Tel.: +39 0672594369; fax: +39 0672594311. E-mail address: ciriolo@bio.uniroma2.it (M.R. Ciriolo). 1 These authors equally contributed to this work. class of enzymes called NO synthases (NOS). There are three isoforms of NOS: neuronal NOS (nNOS, also known as NOS1), inducible NOS (iNOS, or NOS2) and endothelial NOS (eNOS or NOS3). iNOS expression is induced by inflam- matory cytokines, endotoxin, hypoxia and oxidative stress, whereas nNOS and eNOS are constitutive enzymes whose activity is dependent on cytosolic Ca 2+ . However, several evidences demonstrate that the expression of the nos1 gene can be regulated by a large variety of physiological and pathological stimuli (Dawson et al., 1998; Forstermann et al., 1998; Aquilano et al., 2007) that ultimately lead to nitrosative stress. Copper, zinc superoxide dismutase (SOD1) may rep- resent a protective factor against NO toxicity since by scavenging the radical superoxide (O 2 •- ) prevents the formation of ONOO - , a highly reactive oxidant that might constitute the primary pathogenic event leading to nitrosative damage of cellular macromolecules (Rotilio et al., 2002; Ciriolo et al., 2000). In the absence of its co- factors nNOS can also produce O 2 •- at the level of the oxygenase domain, leading to its inactivation (Daff et al., 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.05.013