Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death.

Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with sphingosine kinase, metabolizes ceramide to sphingosine-1-phosphate (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an "atypical apoptosis" characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.

Induction of stress-activated protein kinases/c-Jun N-terminal kinases by the p55 tumour necrosis factor receptor does not require sphingomyelinases.

Ceramide has been implicated in the activation of stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK). Binding of tumour necrosis factor (TNF) to its 55 kDa receptor (TR55) leads to the generation of ceramide through activation of either acid or neutral sphingomyelinase (A/N-SMase) as well as to potent activation of SAPK/JNK. We have examined a putative role of both N- and A-SMase in the TR55-dependent activation of SAPK/JNK. The analysis of TR55 deletion mutants expressed in 70Z/3 pre-B cells revealed that activation of SAPK/JNK occurs independently of N-SMase. Although both SAPK/JNK and A-SMase are activated by the death domain of TR55, pharmacological prevention of the TR55-dependent activation of A-SMase, or proteolytic degradation of A-SMase in 70Z/3 cells, did not impair SAPK/JNK activation, indicating that SAPK/JNK are not secondary to A-SMase. In addition, proteolytic degradation of A-SMase also did not affect SAPK/JNK activation by ultraviolet (UV-C) irradiation, arguing against a general role of A-SMase in stress-mediated responses. Furthermore, fibroblasts from Niemann-Pick A patients deficient in A-SMase did not show altered activation of SAPK/JNK in response to either TNF or UV-C. These results suggest that TR55 can activate SAPK/JNK without direct participation of sphingomyelinases or ceramide.