COMPLEX GENETIC RESPONSE OF HUMAN-CELLS TO SUBLETHAL LEVELS OF PURE NITRIC-OXIDE

NO is a biologically generated free radical that serves diverse roles in mammalian cell signaling and immune-mediated cell killing. Because mammalian cells might be exposed to varying levels of NO, we tested fo r possible defense genes and proteins induced upon treatment of cells with sublethal fluxes of pure NO. Two-dimensional gel analysis was per formed for human embryonic lung fibroblasts (IMR-90) exposed for 90 mi n to pure NO at similar to 280 nM/s, which revealed the reproducible i nduction of at least 12 proteins. Among these, a prominent polypeptide had M-r similar to 32,000, similar to the well-known oxidative stress protein heme oxygenase-1 (HO-1). Northern blot analysis of IMR-90 and HeLa cells demonstrated the NO-mediated induction of HO-1 mRNA up to 70-fold over the levels in untreated cells. HO-1 induction depended on the NO dose and subsequent expression time and was maximal 3-5 h afte r a 1-h exposure to NO at a constant flux of similar to 280 nM/s. The mRNA encoding a tyrosine/threonine phosphatase (CL100MKP-1) was also N O inducible (similar to 20 fold), whereas there was no increase in exp ression of the mRNA encoding manganese-containing superoxide dismutase , Induction of HO-1 mRNA was independent of the guanylate cyclase sign aling pathway; addition of the analogue 8-bromo-cyclic GMP did not ind uce the HO-1 transcript, and the soluble guanylate cyclase inhibitor L Y-83583 did not block HO-1 induction by NO in IMR-90 cells. Luciferase reporter constructs containing up to 4.7 kb of DNA upstream of the HO -1 transcription start site showed less than or equal to 2.5-fold indu ction in IMR-90 or HeLa cells exposed to NO. However, HO-1 mRNA was dr amatically stabilized after exposure of IMR-90 cells to NO. Even a tra nsient NO exposure produced elevated levels of HO-1 protein for greate r than or equal to 10 h, whereas continuous low-level NO treatment (35 nM/s) maintained elevated HO-1 mRNA expression for greater than or eq ual to 8 h. These results reveal a complex mammalian response to NO th at involves a new level of posttranscriptional control in response to this radical.