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A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N 5 -(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate at concentrations from 10 to 1000 M as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.

Background: Nitric oxide (NO) has frequently been shown to display immunosuppressive activities. We describe here a molecular mechanism contributing to this effect. Materials and Methods: Murine T cell lymphoma EL4-6. 1 cells were activated with the physiological stimulus interleukin (IL)-13 to express IL-2 mRNA in the presence or absence of subtoxic concentrations of the physiological spontaneous NO donor S-nitrosocysteine (SNOC). Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyze the effects of NO on IL-2 mRNA expression and on the activity of the dominant regulating transcription factors Spi, EGR-1, and NFATc. Results: NO inhibits IL-13-induced IL-2 mRNA expression in EL4-6. 1 cells. The suppressive activity of NO was concentration dependent and found to be completely reversible. Importantly, NO at the concentrations used induced neither apoptosis nor necrosis. Dominant regulation of IL-2 gene expression is known to reside in the zinc finger transcription factors SpI or EGR-1 and in the non-zinc finger protein NFAT. NO abrogates the DNA binding activities of recombinant Spl and EGR-1. More importantly, gel shift assays also showed a lack of DNA binding of native Spl derived from NOtreated nuclear extracts and that from NO-treated viable lymphocytes. This effect is selective, as the DNA binding activity of recombinant NFATc was not affected by NO. Conclusion: Inactivation of zinc finger transcription factors by NO appears to be a molecular mechanism in the immunosuppressive activity of NO in mammals, thus contributing to NO-mediated inhibition of IL-2 gene expression after physiological stimuli. The exact understanding of the molecular mechanism leading to NOmediated, fully reversible suppression of immune reactions may lead to use of this naturally occuring tool as an aid in inflammatory diseases.

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