Ceramide induces apoptosis via CPP32 activation

Abstract

Although both ceramide and interleukin-ll] convert-apoptosis [15][16][17][18]. However, ICE itself might not be a true mammalian Ced-3 counterpart because apoptosis proceeds aling enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report most normally in ICE-deficient mice [19,20]. So far at least here that cell-permeable ceramide induced cleavage and activaseven mammalian Ced-3 homologues have been identified, tion of CPP32, a Ced-3/ICE-like protease, but not ICE. , especially the death domain, but not Apoptosis is a well-regulated process of cell death that is TNFRII(p75), raising the possibility that ceramide and ICEimportant in events such as embryogenesis, cell growth conlike proteases exist in the same signaling pathway. We theretrol and lymphocyte repertoire formation. Recent investigafore addressed this question in the present study and demontions have revealed that there are several key pathways in strated that CPP32 acts downstream of ceramide and its actiapoptotic signal transduction, including the sphingomyelinvation is required for ceramide-induced apoptosis. ceramide pathway [1,2] and the pathway involving interleukin-l~ converting enzyme (ICE)/Ced-3 family proteases [3].2. Materials and methods Ceramide is a newly identified lipid second messenger that is generated through the hydrolysis of sphingomyelin by 2.1. Reagents and cell lines sphingomyelinases (SMase) [1,2]. Signals from cell surface re-N-Acetyl ceramide (C2-ceramide) was purchased from Wako (Osaceptors such as Fas (CD95/Apol) [4][5][6][7] and tumor necrosis ka), C2-dihydroceramide from Calbiochem and dioctanoylglycerol factor receptor (TNFR) [8][9][10][11] activate SMase and generate from Sigma, and dissolved in ethanol. The final ethanol concentration in the culture medium was always less than 0.1%. Mouse IgM anticeramide, even in the cell-free system [12]. Synthetic cellhuman Fas monoclonal antibody (CH-11) was purchased from Medpermeable ceramide can mimic such apoptosis. This cytotoxic ical Biological Laboratories (Nagoya) and anti-CPP32 monoclonal effect is specific because structural analogues such as dihydroantibody from Transduction Laboratories. Tetrapeptide inhibitors ceramide do not induce apoptosis [13,14]. These data suggest for CPP32 (Ac-DEVD-CHO) and ICE (Ac-YVAD-CHO), and tetrathat ceramide is a physiological mediator of Fas-and TNFRpeptide substrates for CPP32 (Ac-DEVD-AMC) and ICE (Ac-YVAD-AMC) [22,27] were purchased from Peptide Industries (Osamediated apoptosis, ka) and dissolved in DMSO. The human T cell line, Jurkat, was ICE family proteases, which are mammalian Ced-3 homomaintained in RPMI 1640 medium containing 10% fetal calf serum logues, have also been implicated in Fas-and TNF-mediated (FCS). An anti-Fas-resistant (FR) subline of Jurkat was established by culturing wild-type Jurkat cells in the presence of 100 ng/ml antiFas (CH-11) for 1 month. *Corresponding author. Fax: (81) (3) 5803-0131.2.2. Immunoblotting of CPP32 **Research fellow o...

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