Biochemical Properties of Mammalian Neutral Sphingomyelinase2 and Its Role in Sphingolipid Metabolism

Abstract

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg2+ and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 ± 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30–40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced ∼50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling. Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg2+ and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 ± 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30–40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced ∼50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling. Sphingolipid metabolites are now recognized as important components in signal transduction, not only in mammalian cells but also in yeast where they are implicated in heat stress responses. Ceramide, a major sphingolipid metabolite, has been shown to play important roles in apoptosis, cell cycle arrest, and differentiation (for recent reviews, see Refs. 1Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 2Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (284) Google Scholar, 3Pettus B. Chalfant C. Hannun Y.A. Biochim. Biophys. Acta. 2003; 1585: 114-125Crossref Scopus (689) Google Scholar). As a consequence of this diverse biology, the study and characterization of enzymes that regulate ceramide levels have become essential areas of study. Sphingomyelinases (SMases) 1The abbreviations used are: SMasessphingomyelinasesSMsphingomyelinA-SMaseacid sphingomyelinaseN-SMaseneutral sphingomyelinasePCphosphatidylcholinePAFplatelet-activating factorlyso-PAFlyso-platelet-activating factor1-alkyl-glycerol1-O-alkyl-2-lyso-sn-glycero-3-phosphocholinePSphosphatidylserinePLCphospholipase CMe2SOdimethyl sulfoxidePBSphosphate-buffered salineTNFtumor necrosis factor αMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideCMVcytomegalovirus are enzymes that cleave the phosphodiester linkage of sphingomyelin into ceramide and phosphocholine, and they are implicated in several pathways of signal transduction and cell regulation. Activation of acid sphingomyelinase (A-SMase) has been observed after treatment with UV-A radiation (4Zhang Y. Mattjus P. Schmid P.C. Dong Z. Zhong S. Ma W.Y. Brown R.E. Bode A.M. Schmid H.H. J. Biol. 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Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar), treatment with vitamin D (15Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar), and CD40 ligation (16Koppenhoefer U. Brenner B. Lang F. Gulbins E. FEBS Lett. 1997; 414: 444-448Crossref PubMed Scopus (29) Google Scholar). Recently, GW4869 was developed as an inhibitor for N-SMase, and it was shown to inhibit TNF-induced activation of N-SMase in MCF7 cells. GW4869 showed no inhibitory activity on other as and it showed activity the compared with the C. P. Y. H. E. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, GW4869 TNF-induced death and growth that N-SMase activation is an important for the of the induced by TNF C. P. Y. H. E. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). the N-SMase for GW4869 has not been sphingomyelin acid sphingomyelinase neutral sphingomyelinase factor lyso-platelet-activating factor phosphatidylserine phospholipase C tumor necrosis factor The results the to the gene(s) encoding N-SMase, and have been for this using a the N-SMase, a encoding a putative N-SMase was a S. H. S. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Recently, cloning on with mammalian nSMase1 S. K. M. M. U. S. A. 1998; PubMed Scopus Google Scholar) and nSMase2 K. S. G. U. S. A. 2000; PubMed Scopus Google Scholar), as putative of is on the ability of nSMase1 to SM in S. K. M. M. U. S. A. 1998; PubMed Scopus Google Scholar, H. Domae N. N. Hannun Y.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. F. J. 2002; PubMed Scopus Google Scholar, Y. K. F. Y. M. S. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar, F. M. J. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), the that it is to SM in cells S. K. M. M. U. S. A. 1998; PubMed Scopus Google Scholar, H. Domae N. N. Hannun Y.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In demonstrated in cells, nSMase1 as a C and not as a SMase H. Domae N. N. Hannun Y.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and this is by studies that the activity in SM and lyso-platelet-activating factor H. Domae N. N. Hannun Y.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. F. J. 2002; PubMed Scopus Google Scholar, Y. K. F. Y. M. S. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar, F. M. J. M. J. Biol. 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Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) was used as cells. and and The yeast a was of the mouse nSMase2 was by and The nSMase2 was by and the for the and the by the and and The of the was by an into yeast cells as C. M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and the of nSMase2 was induced by the cells in cells in and and A. with as C. M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). and cell by at for and the was at to the and the studies on and and substrate of nSMase2, by in in the of and at for The was at for and the was used for was using protein MCF7 cells in with serum at in a nSMase2 cloned into the was into MCF7 cells by using the H. S. Hannun Y.A. Obeid L.M. FASEB J. 2001; 15: PubMed Scopus Google Scholar). the of was to the and in and and cells the N-SMase activity in used as nSMase2 results with the for the by in and of and and for and used for the yeast MCF7 to of was used in the at the The was to with of at the was by the of of the by of of and of the was with of for The was as for the N-SMase that the The was as for the N-SMase that the no phosphatidylserine and of Cell was to of and in a of of at the by the of and J. PubMed Scopus Google Scholar) and by in The with and to at for The to was by to and the for The was as for the that the of was as H. T. Y. M. H. M. S. H. Domae N. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of protein by a for of The a and with by with a of for and of for The signal was by with to by the of and J. PubMed Scopus Google Scholar), and of used for Bielawska A. Hannun Y.A. 2000; PubMed Google Scholar). the measurement of the in with the of in and at for The by the of of by in and with and the to SM and the and the of was as Bielawska A. Hannun Y.A. 2000; PubMed Google Scholar). at in of growth The next the cells with in of growth for The cells with of and at the the was and the cells with of on in of and was with an of and with the and for at by the of and J. PubMed Scopus Google Scholar), and of used for and SM as by N. Salvayre R. Levade T. J. 1994; PubMed Scopus Google Scholar). in and by the of and J. PubMed Scopus Google Scholar). The was into and for ceramide and levels using the was using and to Bielawska A. Hannun Y.A. 2000; PubMed Google Scholar). labeling with cells with of in of for labeling with was by and cells with for the by the of and J. PubMed Scopus Google Scholar) and by in B. in a in of the of in was to well and at in for cells by the of of in to well and at The of the was by the at with a The of nSMase2 overexpression on cell growth by the as J. Hannun Y.A. Obeid L.M. J. Cell Biol. 1999; PubMed Scopus Google Scholar). Cell was by of as J. PubMed Scopus Google Scholar) with cells at in in and in the cells for in and with in the growth for The cells with and with of acid on for The acid in the in of and after with in a yeast overexpressing nSMase2 in the of GW4869 at the SM was as in the of the of The was and at with to the the of GW4869 In of at with the the of the In to that nSMase2 is a structural gene for neutral sphingomyelinase nSMase2 into the yeast in that for an endogenous inositol phosphosphingolipid phospholipase C as SMase activity and its SMase activity yeast H. Y. Luberto C. C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). As shown in overexpression of nSMase2 in the showed activity the vector-transfected cells. SMase activity was at compared with neutral a neutral activity on was in of and nSMase2-overexpressing cells, demonstrating that is not a substrate of this In the yeast inositol and not by the as and Y. A. cell the by at in the of the activity was in the and only of the activity was in the The of the nSMase2 was in the at by using of the acid of the nSMase2 that it putative In the of N-SMase activity in the of nSMase2-overexpressing cells was In the of in the the for SM and activity in of and nSMase2 ± ± ± ± ± ± in a N-SMase activity in the of nSMase2-overexpressing cells was dependent on and activity was at Mg2+ N-SMase activity at however, of not N-SMase activity nSMase2 activity was dependent on and in the the of was to The of nSMase2 on was only induced and activity In the of the activity was to that in the of as and acid only In to a inhibitor of the N-SMase, acts on using the yeast that of the with GW4869 inhibited activity by The was with at GW4869. In the was and only was observed at of on of the enzyme. As shown in the of GW4869 was dependent on the of in the the activity was at of was observed at GW4869. the of on GW4869 of the N-SMase activity to observed using N-SMase C. P. Y. H. E. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). nSMase2 is a in for GW4869. study the biochemical of nSMase2 in mammalian cells, MCF7 cells with the cloned in SMase activity in the cell of nSMase2 was by in the of compared with cells. The biochemical to observed with the The of not the activity The and for SM in the of and as with the study K. S. G. U. S. A. 2000; PubMed Scopus Google Scholar), nSMase2 SMase activity in mammalian cells. with nSMase1 showed that the SM as a substrate in it not in cells where it to primarily as a H. Domae N. N. Hannun Y.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to the overexpressed nSMase2 as a N-SMase in cells, the of SM and ceramide in the nSMase2 MCF7 cells was The of SM was 30–40% in nSMase2 cells compared with the The observed in SM to an increase in the levels of ceramide In and levels and in the levels of SM and ceramide in overexpressing cells suggest a role for this in the of the of nSMase2 on SM in cells, with nSMase2 cells with the of the increase in SM was in cells and was a decrease in the of SM in nSMase2 cells compared with that in cells the cells nSMase2 cells after of SM was in the nSMase2 was a decrease at of on the other was in the compared with the cells results suggest a of the on the of SM in the results observed with overexpression of nSMase2, demonstrating that these not to in the not the levels of other ceramide in nSMase2 compared with cells, with and the levels of compared and nSMase2 cells. The SM showed no in not has been shown that nSMase1 phospholipase C activity in and acts to in cells H. Domae N. N. Hannun Y.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google as a substrate for In using as a substrate in the yeast In the activity on was that on SM In to N-SMase which as activity only in the of In of in the inhibited activity not activity was in nSMase2 yeast nSMase2 not activity in the of in mammalian cells, and cells with In of was into and was no in the levels of which of the nSMase2 not nSMase2 has substrate nSMase1 and to on SM as a substrate in cells. ceramide levels have been shown to cell cycle arrest, apoptosis, and/or cell differentiation (1Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 2Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (284) Google Scholar, 3Pettus B. Chalfant C. Hannun Y.A. Biochim. Biophys. Acta. 2003; 1585: 114-125Crossref Scopus (689) Google Scholar). the of in cellular sphingolipid with activity of nSMase2 in the growth of MCF7 nSMase2 compared with the cells. was observed a growth nSMase2 only to the of cells as by the which the of the to a and which the the cells, and cells to death of nSMase2 accelerated cell as is by cells with to apoptosis and its As shown in the overexpression of nSMase2 the of however, the of cells that the was and in nSMase2 and cells not results suggest that the overexpression of nSMase2 growth but not cell The growth was by the of into was observed that also by results a role of this in mediating growth through of it has been C. P. Y. H. E. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) that N-SMase at in to the generation of ceramide in to TNF in investigated nSMase2 by As shown in the stimulation of MCF7 with TNF for a ± increase in the N-SMase activity of nSMase2 compared with the cells. activation was observed in the cells in the of TNF ± however, the increase in activity was in the results suggest that nSMase2 is to TNF and that it is a enzyme. the of N-SMase has been for of on its and of cellular regulation has to pathways this C. Wiegmann K. D. R. M. F. Kronke M. J. 1995; 14: PubMed Scopus Google Scholar, S. N. Liu B. R. M. F. Hannun Y.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In this study that the cloned nSMase2 as a neutral sphingomyelinase in cells and that the protein SMase activity in and in cells. of the of the overexpression of nSMase2 on cell and that nSMase2 is a for the N-SMase GW4869. In the has focused on the N-SMase for its roles in mediating a variety of cellular processes including differentiation, cell cycle arrest, and programmed cell death (apoptosis) through the generation of ceramide (1Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 2Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (284) Google Scholar). in this several that that nSMase2 is a N-SMase and that it is involved in the regulation of SM in showed that the overexpressed nSMase2 has a substrate nSMase2 not activity nSMase2 is to regulate the of SM and ceramide in cells with a decrease in SM and increase in ceramide labeling showed that the nSMase2 accelerated of are the for which activity SM and as in and which also not the of SM in cells H. Domae N. N. Hannun Y.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of a role of nSMase2 in and was demonstrated with cellular that to in the of in to nSMase2 the N-SMase and the yeast showed on as activity B. K. Hannun Y.A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Y. Luberto C. C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). on the of acid in the for Y. S. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The results of this study that nSMase2 also is activated by and other and the of an in The on have for cellular activation of the enzyme. is in the however, nSMase2 was to in a of the K. S. G. U. S. A. 2000; PubMed Scopus Google Scholar). the other of that is not in other and this another for activation of studies on nSMase2 and regulation are have been for the generation of and the of sphingomyelin by the of that at T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (284) Google Scholar). The study of the has the of and the of the key enzymes on this A.H. E. R.T. 2001; PubMed Scopus Google Scholar, K. M. T. S. 2000; PubMed Scopus Google Scholar). the other have been to the neutral sphingomyelinase has been to inhibitory N-SMase F. M. T. K. T. J. 1999; PubMed Scopus Google Scholar, M. F. Y. S. H. S. R. T. J. 1999; PubMed Scopus Google Scholar). Recently, a for N-SMase another that and inhibitory activity on N-SMase, it showed no of acid SMase C. P. Y. H. E. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). the results show that GW4869 is to inhibit nSMase2 in and in a and with to observed for the nSMase2 the for GW4869 which as a to the roles of nSMase2 in ceramide As in ceramide levels have been shown to with apoptosis and cell cycle arrest, the growth of the MCF7 overexpressing nSMase2 showed decrease in growth after as the cells the phase. nSMase2 was as the protein Y. T. M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), which had been in a for involved in of nSMase2 play a role in The of a putative role of nSMase2 in cell cycle to and are to this studies C. P. Y. H. E. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Chem. 1999; PubMed Scopus Google Scholar, N. Medin J.A. 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J. 2002; PubMed Scopus Google Scholar) observed results in the cell where an not to play a role in the to results a role of nSMase1 in in the study the cellular activation of nSMase2 by TNF that this to TNF and that it is a In conclusion, these results suggest that nSMase2 is a sphingomyelinase that an important role in the regulation of SM and studies are to the role of this in ceramide generation and to the of its activation in to Moreover, the characterization of the of nSMase2 studies of its and the characterization of nSMase2 to of pathways by sphingolipid K. and S. for the mouse and nSMase2 for for on yeast and Bielawska for