Involvement of de NovoCeramide Biosynthesis in Tumor Necrosis Factor-α/Cycloheximide-induced Cerebral Endothelial Cell Death

Jan Xu,Chen-Hsiung Yeh,Shawei Chen,Luming He,Stefano L Sensi,Lorella M.T Canzoniero,Dennis W Choi,Chung Y Hsu

doi:10.1074/jbc.273.26.16521

Jan Xu, Chen-Hsiung Yeh + Show 6 more

Open Access

https://doi.org/10.1074/jbc.273.26.16521

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Abstract

Cytokines, including tumor necrosis factor-alpha (TNF-alpha), may elicit cytotoxic response through the sphingomyelin-ceramide signal transduction pathway by activation of sphingomyelinases and the subsequent release of ceramide: the universal lipid second messenger. Treatment of bovine cerebral endothelial cells (BCECs) with TNF-alpha for 16 h followed by cycloheximide (CHX) for 6 h resulted in an increase in ceramide accumulation, DNA fragmentation, and cell death. Application of a cell permeable ceramide analogue C2 ceramide, but not the biologically inactive C2 dihydroceramide, also induced DNA laddering and BCEC death in a concentration- and time-dependent manner. TNF-alpha/CHX-mediated ceramide production apparently is not a result of sphingomyelin hydrolysis because sphingomyelin content does not decrease in this death paradigm. In addition, an acidic sphingomyelinase inhibitor, desipramine, had no effect on TNF-alpha/CHX-induced cell death. However, addition of fumonisin B1, a selective ceramide synthase inhibitor, attenuated TNF-alpha/CHX-induced intracellular ceramide elevation and BCEC death. Together, these findings suggest that ceramide plays at least a partial role in this paradigm of BCEC death. Our results show, for the first time, that ceramide derived from de novo synthesis is an alternative mechanism to sphingomyelin hydrolysis in the BCEC death process initiated by TNF-alpha/CHX.

Highlights

Cytokines are key mediators in inflammatory and immune disorders of the central nervous system injury [1,2,3,4,5]
It is noteworthy that signs of cell death (e.g. lactate dehydrogenase (LDH) release) induced by TNF-␣/CHX were preceded by DNA fragmentation, which began as early as 1 h after CHX incubation and persisted to reach a maximum at 8 h
Ceramide Synthase Inhibitor Attenuated TNF-␣/CHX-induced bovine cerebral endothelial cells (BCECs) Death—Because the previous results revealed that ceramide generation by TNF-␣/CHX treatment was not from sphingomyelin hydrolysis, we considered the possibility of de novo synthesis of ceramide as an alternative mechanism

Summary

Introduction

Cytokines are key mediators in inflammatory and immune disorders of the central nervous system injury [1,2,3,4,5]. We report here that sequential TNF-␣ and CHX treatment induces a progressive elevation of intracellular ceramide level, which is followed by DNA fragmentation and cell death in BCECs. More importantly, ceramide in this BCEC death paradigm is derived from de novo synthesis, but not the conventional pathway, namely the hydrolysis of sphingomyelin. Our findings provide evidence for a crucial role of ceramide in the TNF-␣/CHX-induced apoptotic process and indicate that de novo ceramide synthesis is a unique signaling mechanism in this cell death paradigm in BCECs involving TNF-␣.

Results

Conclusion