Activation of the endothelial nitric-oxide synthase by tumor necrosis factor-alpha. A novel feedback mechanism regulating cell death.
Cell death via apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) 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-alpha-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-alpha-induced apoptosis. This protection was mediated by the generation of NO. TNF-alpha 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-alpha controls its own apoptotic program.
Bulotta
S
S
Faculty of Pharmacy, University of Catanzaro Magna Graecia, 88021 Roccelletta di Borgia, Italy.
Barsacchi
R
R
Rotiroti
D
D
Borgese
N
N
Clementi
E
E
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D006152:Cyclic GMP / Q000096:biosynthesis; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005246:Feedback; D006367:HeLa Cells; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000187:drug effects* / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D009025:Morpholines; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C033110:RV 538; D000255:Adenosine Triphosphate; D003513:Cycloheximide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester