Cell death via apoptosis induced by tumor necrosis factor-α (TNF-α) plays an important role in many physiological and pathological conditions. The signal transduction pathway activated by this cytokine is known to be regulated by several intracellular messengers. In particular, in many systems nitric oxide (NO) has been shown to protect cells from TNF-α-induced apoptosis. However, whether NO can be generated by the cytokine to down-regulate its own apoptotic program has never been studied. We have addressed this question in HeLa Tet-off cell clones stably transfected with the endothelial NO synthase under a tetracycline-responsive promoter. Endothelial NO synthase, induced about 100-fold in these cells by removal of the antibiotic, retained the characteristics of the native enzyme of endothelial cells, both in terms of intracellular localization and functional activity. Expression of the endothelial NO synthase was sufficient to protect from TNF-α-induced apoptosis. This protection was mediated by the generation of NO. TNF-α itself stimulated endothelial NO synthase activity to generate NO through a pathway involving its lipid messenger, ceramide. Our results identify a novel mechanism of regulation of a signal transduction pathway activated by death receptors and suggest that NO may constitute a built-in mechanism by which TNF-α controls its own apoptotic program.
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