Activation of the Endothelial Nitric-oxide Synthase by Tumor Necrosis Factor-α

Abstract

Cell death via apoptosis induced by tumor necrosis factor-␣ (TNF-␣) 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-␣-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-␣-induced apoptosis. This protection was mediated by the generation of NO. TNF-␣ 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-␣ controls its own apoptotic program.

Regulation and measurement of oxidative stress in apoptosis

“…The authors found that TNFα activated eNOS through ceramide formation. Generation of NO in this system was anti-apoptotic (Bulotta et al, 2001). These conflicting results highlight the dual effects that RNI and indeed ROS can have in different cells.…”

Ceramide: A common pathway for atherosclerosis?

“…Zhang et al directly measured NO levels in the intact endothelium of coronary arteries by using realtime fluorescence microscopy and found NO levels were reduced in ceramide-treated vessels [70]. Cell culture studies have shown that ceramide reduces the release of bioactive NO in human umbilical vein endothelial cells [73,74]. Overwhelming evidence demonstrates that ceramide-induced oxidative stress may be the major reason for its effects on the reduction of NO bioavailability and endothelial dysfunction.…”

Recent advances in the immunobiology of ceramide

“…NOS III activation can involve ceramide generation by either acid or neutral sphinomyelinases, stimulated either by basic fibroblast growth factor (bFGF) in CHO-K1 cells (Goldkorn et al, 1998). It can also involve tumor necrosis factor-α (TNF-α) in HeLa cell clones transfected with NOS III under a tetracycline-responsive element (Barsacchi et al, 2003;Bulotta et al, 2001). NOS III activation by TNF-α requires stimulation of the phosphatidylinositol 3 kinase (PI3K)/Akt pathway while bFGF activated NOS III is independent of PI3/Akt activation.…”