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Complex Genetic Response of Human Cells to Sublethal Levels of Pure Nitric Oxide¹

Department of Cancer Cell Biology and Division of Biological Sciences, Harvard School of Public Health, Boston, Massachusetts 02115

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 for possible defense genes and proteins induced upon treatment of cells with sublethal fluxes of pure NO. Two-dimensional gel analysis was performed for human embryonic lung fibroblasts (IMR-90) exposed for 90 min to pure NO at 280 nM/s, which revealed the reproducible induction of at least 12 proteins. Among these, a prominent polypeptide had M_r 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 after a 1-h exposure to NO at a constant flux of 280 nM/s. The mRNA encoding a tyrosine/threonine phosphatase (CL100/MKP-1) was also NO inducible (20 fold), whereas there was no increase in expression of the mRNA encoding manganese-containing superoxide dismutase. Induction of HO-1 mRNA was independent of the guanylate cyclase signaling pathway; addition of the analogue 8-bromo-cyclic GMP did not induce the HO-1 transcript, and the soluble guanylate cyclase inhibitor LY-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 2.5-fold induction in IMR-90 or HeLa cells exposed to NO. However, HO-1 mRNA was dramatically stabilized after exposure of IMR-90 cells to NO. Even a transient NO exposure produced elevated levels of HO-1 protein for 10 h, whereas continuous low-level NO treatment (35 nM/s) maintained elevated HO-1 mRNA expression for 8 h. These results reveal a complex mammalian response to NO that involves a new level of posttranscriptional control in response to this radical.

¹ This work was supported in part by Grant CA37831 (to B. D.) from the NIH. J. C. M. was supported by USPHS pre-doctoral training grant ES07155.

² To whom requests for reprints should be addressed, at Department of Cancer Cell Biology, Division of Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115-6021. Phone: (617) 432-3462; Fax: (617) 432-2590; E-mail: bdemple@hsph.harvard.edu.

Received 3/ 2/98. Accepted 6/ 3/98.

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