Sphingosine 1-Phosphate Inhibits Activation of Caspases that Cleave Poly(ADP-ribose) Polymerase and Lamins during Fas- and Ceramide-mediated Apoptosis in Jurkat T Lymphocytes

Abstract

Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800–803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7. Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800–803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7. Physiologic cell death occurs through an evolutionary conserved suicide process, termed apoptosis, which plays a considerable role in early development and homeostasis of adult tissues (1Vaux D.L. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2239-2244Crossref PubMed Scopus (913) Google Scholar). Ceramide has recently emerged as a critical component of apoptosis (2Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1631) Google Scholar, 3Hannun Y. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1510) Google Scholar). A variety of stress stimuli, such as tumor necrosis factor α (TNFα), 1The abbreviations used are: TNFα, tumor necrosis factor α; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin; DEVD, Asp-Glu-Val-Asp; ICE, interleukin1β-converting enzyme; PARP, enzyme poly(ADP-ribose) polymerase; PKC, protein kinase C; SPP, sphingosine 1-phosphate; TPA, 12-O-tetradecanoylphorbol-13-acetate; PBS, phsophate-buffered saline; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Fas ligand, growth factor withdrawal, anticancer drugs, oxidative stress, heat shock, ionizing radiation, and ultraviolet light increase cellular ceramide which, in turn, is capable of inducing apoptosis (3Hannun Y. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1510) Google Scholar, 4Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Crossref PubMed Scopus (475) Google Scholar). This apoptotic effect is blocked by addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol, both activators of protein kinase C (PKC) (2Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1631) Google Scholar, 5Jarvis W.D. Fornari Jr., F.A. Browning J.L. Gewirtz D.A. Kolesnick R.N. Grant S. J. Biol. Chem. 1994; 269: 31685-31692Abstract Full Text PDF PubMed Google Scholar), suggesting that PKC activation counteracts ceramide-mediated apoptosis. Activation of PKC in diverse cell types stimulates sphingosine kinase activity resulting in intracellular accumulation of sphingosine 1-phosp