Overexpression of CuZn superoxide dismutase protects RAW 264.7 macrophages against nitric oxide cytotoxicity

Abstract

Initiation of nitric oxide (NO.)-mediated apoptotic cell death in RAW 264.7 macrophages is associated with up-regulation of mitochondrial manganese superoxide dismutase (MnSOD; SOD2) and down-regulation of cytosolic copper zinc superoxide dismutase (CuZnSOD; SOD1) at their individual mRNA and protein levels. To evaluate the decreased CuZnSOD expression and the initiation of apoptosis we stably transfected macrophages to overexpress human CuZnSOD. Individual clones revealed a 2-fold increase in CuZnSOD activity. Expression of a functional and thus protective CuZnSOD was verified by attenuated superoxide (O2(.)-)-mediated apoptotic as well as necrotic cell death. In this study we showed that SOD-overexpressing macrophages (R-SOD1-12) were also protected against NO.-initiated programmed cell death. Protection was substantial towards NO. derived from exogenously added NO donors or when NO. was generated by inducible NO synthase activation, and was evident at the level of p53 accumulation, caspase activation and DNA fragmentation. Stimulation of parent and SOD-overexpressing cells with a combination of lipopolysaccharide and murine interferon gamma produced equivalent amounts of nitrite/nitrate, which ruled out attenuated inducible NO. synthase activity during protection. Because protection by a O2(.)--scavenging system during NO. -intoxication implies a role of NO. and O2(.)- in the progression of cell damage, we used uric acid to delineate the role of peroxynitrite during NO.-elicited apoptosis. The peroxynitrite scavenger uric acid left S-nitrosoglutathione or spermine-NO-elicited apoptosis unaltered, blocking only 3-morpholinosydnonimine-mediated cell death. As a result we exclude peroxynitrite from contributing, to any major extent, to NO. -mediated apoptosis. Therefore protection observed with CuZnSOD overexpression is unlikely to stem from interference with peroxynitrite formation and/or action. Unequivocally, the down-regulation of CuZnSOD is associated with NO. cytotoxicity, whereas CuZnSOD overexpression protects macrophages from apoptosis.

Cu/Zn Superoxide Dismutase Plays Important Role in Immune Response

“…Because macrophages produce both superoxide and NO, they may be particularly vulnerable to oxidant injury by this mechanism. The important role of SOD-1 was demonstrated when overexpression of SOD-1 in RAW 264.7 macrophages protected them against NO cytotoxicity (64). As expected, SOD-1-overexpressing cells coped with the higher levels of oxygen radicals generated by oxidative stress (65), paraquat (18), DSF (6), thiram (7), and NO cytotoxicity (64), thereby preventing the initiation of apoptotic or other arrest-signaling pathways.…”

p53 accumulation in apoptotic macrophages is an energy demanding process that precedes cytochrome c release in response to nitric oxide

“…All other chemicals were of the highest grade of purity and commercially available, or as speci®ed (Messmer et al, 1998;Brockhaus and BruÈ ne, 1999).…”

“…Activity of caspase-3 was determined as previously described (Brockhaus and BruÈ ne, 1999). Substrate cleavage and accumulation of AMC was followed¯uorometrically.…”

“…Western blots of p53, Bcl-x L , and PARP Cells were lysed and extracts were prepared for Western blot analysis as previously described (Brockhaus and BruÈ ne, 1999). Twenty-®ve microgram protein (for detection of Bcl-x L ) or 100 mg protein (for detection of p53 and PARP) were mixed with the same volume of 26 sample bu er (125 mM Tris/HCl, 2% SDS, 10% glycerin, 1 mM DTT, 0.002% bromophenol blue, pH 6.9) and boiled for 10 min.…”

Nitric Oxide, Mitochondria, and Cell Death

“…NO may stimulate ROS production in cells, possibly by inhibition of catalase, depletion of glutathione, and inhibition of the respiratory chain. Preventing NO-induced ROS can block NO-induced p53 activation and apoptosis (68). How p53 induces apoptosis is not clear, but the mechanisms may involve transcription-dependent and -independent processes: a) p53-mediated activation of expression of several pro-apoptotic proteins (such as Bax, Noxa, etc.)…”