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Complex genetic response of human cells to sublethal levels of pure nitric oxide
1Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115-6021, USA.
Abstract:
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 approximately 280 nM/s, which revealed the reproducible induction of at least 12 proteins. Among these, a prominent polypeptide had Mr approximately 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 approximately 280 nM/s. The mRNA encoding a tyrosine/threonine phosphatase (CL100/MKP-1) was also NO inducible (approximately 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 < or = 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 > or = 10 h, whereas continuous low-level NO treatment (35 nM/s) maintained elevated HO-1 mRNA expression for > or = 8 h. These results reveal a complex mammalian response to NO that involves a new level of posttranscriptional control in response to this radical.
Insights
Nitric oxide (NO) induces defense genes in mammalian cells, notably heme oxygenase-1 (HO-1). This induction involves post-transcriptional stabilization of HO-1 mRNA, revealing a novel NO response mechanism.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Biology
Background:
- Nitric oxide (NO) is a crucial signaling molecule in mammals, involved in cell communication and immune responses.
- Mammalian cells face varying NO levels, necessitating defense mechanisms against potential damage.
- Understanding NO-induced gene expression is key to deciphering cellular defense strategies.
Purpose of the Study:
- To identify defense genes and proteins induced by sublethal nitric oxide (NO) exposure in mammalian cells.
- To investigate the molecular mechanisms underlying NO-mediated gene induction, focusing on heme oxygenase-1 (HO-1).
- To explore the role of signaling pathways and mRNA stability in the cellular response to NO.
Main Methods:
- Human embryonic lung fibroblasts (IMR-90) and HeLa cells were treated with controlled fluxes of pure NO.
- Two-dimensional gel electrophoresis was used to analyze protein expression changes.
- Northern blot analysis quantified mRNA levels of specific genes, including HO-1, CL100/MKP-1, and manganese superoxide dismutase.
- Luciferase reporter assays assessed transcriptional regulation.
- mRNA stability assays were performed to evaluate post-transcriptional control.
Main Results:
- Sublethal NO exposure induced at least 12 proteins, including a prominent 32 kDa polypeptide identified as heme oxygenase-1 (HO-1).
- NO treatment significantly upregulated HO-1 mRNA (up to 70-fold) and CL100/MKP-1 mRNA (up to 20-fold), but not manganese superoxide dismutase mRNA.
- HO-1 induction was dose-dependent and time-sensitive, peaking 3-5 hours post-exposure.
- Transcriptional activation of HO-1 was modest (<2.5-fold), but NO exposure dramatically stabilized HO-1 mRNA, prolonging protein expression.
- HO-1 induction was independent of the guanylate cyclase signaling pathway.
Conclusions:
- Mammalian cells possess inducible defense mechanisms against sublethal nitric oxide (NO) fluxes.
- Heme oxygenase-1 (HO-1) is a key NO-inducible defense protein.
- NO-mediated HO-1 expression is primarily regulated at the post-transcriptional level through mRNA stabilization.
- This study reveals a novel aspect of NO signaling involving post-transcriptional control of gene expression in mammalian cells.