Involvement of Acid β-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

Abstract

Activation of protein kinase C (PKC) promotes the salvage pathway of ceramide formation, and acid sphingomyelinase has been implicated, in part, in providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007) J. Biol. Chem. 282, 11549–11561). In the present study, we examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKCδ, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKCδ is an upstream regulator of this pathway. Activation of protein kinase C (PKC) promotes the salvage pathway of ceramide formation, and acid sphingomyelinase has been implicated, in part, in providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007) J. Biol. Chem. 282, 11549–11561). In the present study, we examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKCδ, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKCδ is an upstream regulator of this pathway. Sphingolipids are abundant components of cellular membranes, many of which are emerging as bioactive lipid mediators thought to play crucial roles in cellular responses (1Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1489) Google Scholar, 2Futerman A.H. Riezman H. Trends Cell Biol. 2005; 15: 312-318Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Ceramide, a central sphingolipid, serves as the main precursor for various sphingolipids, including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of formation of ceramide has been demonstrated through the action of three major pathways: the de novo pathway (3Smith E.R. Merrill Jr., A.H. J. Biol. Chem. 1995; 270: 18749-18758Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 4Linn S.C. Kim H.S. Keane E.M. Andras L.M. Wang E. Merrill Jr., A.H. Biochem. Soc. Trans. 2001; 29: 831-835Crossref PubMed Scopus (0) Google Scholar), the sphingomyelinase pathway (5Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar), and the salvage pathway (6Tettamanti G. Bassi R. Viani P. Riboni L. Biochimie. 2003; 85: 423-437Crossref PubMed Scopus (115) Google Scholar, 7Kitatani K. Idkowiak-Baldys J. Hannun Y.A. Cell Signal. 2008; 20: 1010-1018Crossref PubMed Scopus (406) Google Scholar, 8Riboni L. Bassi R. Conti M. Tettamanti G. FEBS Lett. 1993; 322: 257-260Crossref PubMed Scopus (36) Google Scholar). The latter plays an important role in constitutive sphingolipid turnover by salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that serve as sphingolipid backbones for ceramide and dihydroceramide as well as all complex sphingolipids (Fig. 1A). Metabolically, ceramide is also formed from degradation of glycosphingolipids (Fig. 1A) usually in acidic compartments, the lysosomes and/or late endosomes (9Kolter T. Sandhoff K. Annu. Rev. Cell Dev. Biol. 2005; 21: 81-103Crossref PubMed Scopus (346) Google Scholar). The stepwise hydrolysis of complex glycosphingolipids eventually results in the formation of glucosylceramide, which in turn is converted to ceramide by the action of acid β-glucosidase 1 (GBA1) 2The abbreviations used are: GBA1, acid β-glycosidase 1; C16-ceramide, N-palmitoyl-d-erythro-sphingosine; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, 4β-phorbol 12-myristate 13-acetate; SCR, scrambled sequence; siRNA, small interference RNA; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). 2The abbreviations used are: GBA1, acid β-glycosidase 1; C16-ceramide, N-palmitoyl-d-erythro-sphingosine; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, 4β-phorbol 12-myristate 13-acetate; SCR, scrambled sequence; siRNA, small interference RNA; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). (9Kolter T. Sandhoff K. Annu. Rev. Cell Dev. Biol. 2005; 21: 81-103Crossref PubMed Scopus (346) Google Scholar, 10Dinur T. Osiecki K.M. Legler G. Gatt S. Desnick R.J. Grabowski G.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1660-1664Crossref PubMed Scopus (64) Google Scholar). Severe defects in GBA1 activity cause Gaucher disease, which is associated with aberrant accumulation of the lipid substrates (10Dinur T. Osiecki K.M. Legler G. Gatt S. Desnick R.J. Grabowski G.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1660-1664Crossref PubMed Scopus (64) Google Scholar, 11Jmoudiak M. Futerman A.H. Br. J. Haematol. 2005; 129: 178-188Crossref PubMed Scopus (225) Google Scholar, 12Grabowski G.A. Horowitz M. Baillieres Clin. Haematol. 1997; 10: 635-656Abstract Full Text PDF PubMed Scopus (133) Google Scholar, 13Kolter T. Sandhoff K. Biochim. Biophys. Acta. 2006; 1758: 2057-2079Crossref PubMed Scopus (279) Google Scholar, 14Futerman A.H. van Meer G. Nat. Rev. Mol. Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (620) Google Scholar). On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also form ceramide (15Quintern L.E. Schuchman E.H. Levran O. Suchi M. Ferlinz K. Reinke H. Sandhoff K. Desnick R.J. EMBO J. 1989; 8: 2469-2473Crossref PubMed Scopus (136) Google Scholar, 16Schuchman E.H. Suchi M. Takahashi T. Sandhoff K. Desnick R.J. J. Biol. Chem. 1991; 266: 8531-8539Abstract Full Text PDF PubMed Google Scholar). Either process results in the generation of lysosomal ceramide that can then be deacylated by acid ceramidase (17Bernardo K. Hurwitz R. Zenk T. Desnick R.J. Ferlinz K. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1995; 270: 11098-11102Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), releasing sphingosine that may escape the lysosome (18Riboni L. Bassi R. Caminiti A. Prinetti A. Viani P. Tettamanti G. Ann. N. Y. Acad. Sci. 1998; 845: 46-56Crossref PubMed Scopus (14) Google Scholar). The released sphingosine may become a substrate for either sphingosine kinases or ceramide synthases, forming sphingosine 1-phosphate or ceramide, respectively (3Smith E.R. Merrill Jr., A.H. J. Biol. Chem. 1995; 270: 18749-18758Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 19Riboni L. Prinetti A. Bassi R. Tettamanti G. FEBS Lett. 1994; 352: 323-326Crossref PubMed Scopus (25) Google Scholar, 20Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 21Gillard B.K. Clement R.G. Marcus D.M. Glycobiology. 1998; 8: 885-890Crossref PubMed Scopus (83) Google Scholar). In a related line of investigation, our studies (20Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Zeidan Y.H. Hannun Y.A. J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 23Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) have begun to implicate protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol ester (PMA) was shown to stimulate the salvage pathway resulting in increases in ceramide. All the induced ceramide was inhibited by pretreatment with a ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating acute activation of the de novo pathway and establishing a role for ceramide synthesis (20Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Moreover, labeling studies also implicated the salvage pathway because PMA induced turnover of steady state-labeled sphingolipids but did not affect de novo labeled ceramide in pulse-chase experiments. Moreover, PKCδ, among PKC isoforms, was identified as an upstream molecule for the activation of acid sphingomyelinase in the salvage pathway (22Zeidan Y.H. Hannun Y.A. J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Interestingly, the PKCδ isoform induced the phosphorylation of acid sphingomyelinase at serine 508, leading to its activation and consequent formation of ceramide. The activation of acid sphingomyelinase appeared to contribute to ∼50% of the salvage pathway-induced increase in ceramide (28Ziulkoski A.L. Zimmer A.R. Guma F.C. Biochem. Biophys. Res. Commun. 2001; 281: 971-975Crossref PubMed Scopus (11) Google Scholar) (also, see Fig. 4C). This raised the possibility that distinct routes of ceramide metabolism may account for the remainder of ceramide generation. In this study, we investigated glucocerebrosidase GBA1 as a candidate for one of the other routes accounting for PKC-regulated salvage pathway of ceramide formation. Materials—Peroxidase-conjugated antibodies for rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). Enhanced chemiluminescence kit was from Thermo Scientific. NBD-C12-ceramide was from Avanti Polar Lipids. pSPORT GBA1 expression vectors were from ATCC. Cell Culture—MCF-7 cells were grown in RPMI 1640 supplemented with l-glutamine and 10% fetal bovine serum, respectively. Cells were maintained at less than 80% confluence under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, at 37 °C). Transfection with Small Interfering RNA (siRNA)—Cells (1 × 105 cells/60-mm dish) were transfected with 5 nm double-stranded siRNAs for scrambled sequence (SCR), GBA1, or PKCδ using Oligofectamine (Invitrogen) according to the manufacturer's instructions. After 48 h, transfection reagents were washed out, and cells were stimulated with 100 nm PMA in RPMI 1640 medium supplemented with 10% fetal bovine serum. Sequences of siRNAs for SCR, PKCδ, and acid sphingomyelinase are shown in previous studies (22Zeidan Y.H. Hannun Y.A. J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 23Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and sequences of GBA1 siRNAs are shown in Fig. 3A. Transfection with Human GBA1 Expression Vector—MCF-7 cells (1 × 105 cells/35-mm dish) were transfected with the indicated amounts of pSPORT GBA1 vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 24 h, transfection reagents were washed out, and cells were stimulated with 100 nm PMA for 12 h in RPMI 1640 supplemented with 10% fetal bovine serum. Western Blotting—Cells were washed three times with PBS and then lysed using Laemmli buffer. The protein samples (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis (4–20% gradient gels). Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with PBS, 0.1% Tween 20 (PBS-T) containing 5% nonfat dried milk, washed with PBS-T, and incubated with rat polyclonal antibodies for GBA1 (1 to 1,500) in PBS-T containing 5% nonfat dried The were washed with PBS-T and incubated with with in PBS-T containing 5% nonfat dried was using chemiluminescence of were transfected with the indicated siRNAs and then After the cells were and in a acid and the protein samples (20 μg) were incubated with at 37 for in the buffer. were and by using as the a of by acid was determined. of (1 × 105 cells/35-mm dish) were incubated with at 37 for 12 The labeled cells were in RPMI 1640 containing 10% fetal bovine and for 12 The cells were stimulated with 100 nm PMA for the indicated were J. Biochem. PubMed Scopus Google Scholar) and by using as the The to ceramide, of glucosylceramide and or sphingomyelin was and the by of were by treatment with GBA1 not Moreover, of and by also demonstrated that 95% of Thus, in MCF-7 are thought to be of by of ceramide in lipid was by as in Bielawski J. Hannun Y.A. A. 2006; PubMed Scopus Google Scholar). were by the of were as the of with various and was by or Glucosylceramide is a central for complex glycosphingolipids, and degradation of glucosylceramide is crucial for forming lysosomal ceramide and its sphingosine. The latter is for ceramide in a ceramide (Fig. 1A). previous observations indicated that activation of PKC by PMA in MCF-7 cells accumulation of by the salvage pathway in which ceramide including contribute to of recycled sphingosine (20Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). shown in Fig. PMA induced accumulation of of of which was to accumulation of of ceramide synthase by fumonisin attenuated of the ceramide by acid sphingomyelinase appeared to contribute to of the (Fig. important to whether GBA1, a lysosomal in the salvage pathway of ceramide formation. the of GBA1, glucosylceramide was at by MCF-7 cells labeled with were to whether PKC activation induced the degradation of glucosylceramide. with cells declined but significantly after PMA treatment to 1 h (Fig. This degradation of glucosylceramide from of acid glucocerebrosidase of GBA1 in formation of ceramide through the salvage siRNAs were to the of all three sequences and Fig. was shown in Fig. treatment of MCF-7 cells with three distinct GBA1 siRNAs the activity of acid glucocerebrosidase with treatment with with the activity of acid protein expression of GBA1 was decreased with GBA1 (Fig. that GBA1 of the acid glucocerebrosidase Thus, all sequences of GBA1 were to significantly of GBA1 expression levels as well as acid glucocerebrosidase Next, we the of GBA1 PMA-induced formation of ceramide. of the siRNAs for GBA1 levels of with GBA1 by three siRNAs and significantly inhibited PMA-induced generation of by ∼50% to treatment (Fig. to other ceramide GBA1 levels of ceramide and and decreased levels of (Fig. In to GBA1, of acid sphingomyelinase also inhibited PMA increases of by ∼50% (Fig. 4C). This is with our previous observations (22Zeidan Y.H. Hannun Y.A. J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). GBA1 is to also contribute to of of acid sphingomyelinase after PMA In a MCF-7 cells were transfected with GBA1 expression expression of GBA1 significantly increased the levels of ceramide and PMA-induced formation of ceramide (Fig. These results to the that degradation of glucosylceramide to and PKC-regulated ceramide levels. whether GBA1 activity to the degradation of glucosylceramide, of GBA1 were by glucosylceramide (Fig. or the of (Fig. GBA1 PMA-induced formation of by (Fig. In GBA1 induced a 10% increase in with PMA which of complex sphingolipids to glucosylceramide after PMA treatment (Fig. also be that GBA1 treatment did not PMA-induced hydrolysis of (Fig. These not of GBA1 acid sphingomyelinase but also of GBA1 in a of the PMA-induced formation of ceramide. Reciprocally, the by also in after PMA cells failed to degradation of PMA treatment (Fig. These indicate that PMA promotes degradation of glucosylceramide by ceramide formed from glucosylceramide is converted to sphingosine that can then serve as a source for ceramide. we that PMA induced of the sphingosine levels (20Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this study, GBA1 significantly the of sphingosine after PMA treatment to a of ∼50% (Fig. These indicate that PKC activation by PMA promotes GBA1 degradation of glucosylceramide to sphingosine as an source of ceramide formation from the salvage pathway. PMA treatment activation of and novel PKCs the salvage pathway of ceramide formation acid sphingomyelinase or of PKCδ isoform the protein expression of other PKC and blocked PMA-induced formation of ceramide, PKCδ was implicated as a in the salvage pathway (22Zeidan Y.H. Hannun Y.A. J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). whether PKCδ GBA1 of PKCδ sphingolipid were examined (Fig. Silencing PKCδ significantly the degradation of glucosylceramide in PMA treatment in cells induced an increase in that to 5% of all and the of this was to the of the of of sphingomyelin and glucosylceramide PMA-induced in levels of sphingolipids including ceramide and sphingomyelin failed to in These that PKCδ is the PKC that the generation of ceramide in the salvage pathway. The from this for the that GBA1 plays an important role in the regulated formation of ceramide through the sphingosine salvage pathway. PKCδ was identified as the regulator in the salvage pathway of ceramide formation. These novel of GBA1 also contribute to the of the and to its in a of cellular Activation of PKCs by PMA promotes the salvage pathway of ceramide formation in which GBA1 to sphingosine salvage by lysosomal ceramide. lysosomal GBA1 hydrolyzes glucosylceramide to generate lysosomal ceramide. acid ceramidase lysosomal ceramide to sphingosine that is recycled to form ceramide through the action of ceramide The major to from this is the of a novel role for GBA1 as a for the formation of ceramide. This is by the of of GBA1 attenuated PMA-induced formation of C16-ceramide, which is the major ceramide formed from the salvage pathway in MCF-7 cells (Fig. of GBA1 PMA-induced ceramide formation (Fig. PMA treatment significantly decreased levels of glucosylceramide, and this was by the of GBA1 and and GBA1 PMA-induced formation of sphingosine (Fig. This is also by our studies (20Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) that PMA-induced ceramide was inhibited by fumonisin (Fig. and not by myriocin, thus of the de novo pathway and of sphingosine in the formation of ceramide. This was by results with of which also attenuated PMA-induced ceramide accumulation K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). GBA1 is suggested to lysosomal for of sphingosine in the salvage pathway of ceramide formation. The results from this and previous studies (22Zeidan Y.H. Hannun Y.A. J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) also implicate PKCδ as the major isoform involved in the of PMA the salvage pathway. Thus, of PKCδ was shown to all the ceramide formed in to PMA, and in this PMA-induced in ceramide, glucosylceramide, and sphingomyelin (Fig. Thus, PKCδ upstream of GBA1 and acid sphingomyelinase (22Zeidan Y.H. Hannun Y.A. J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) in the salvage pathway. This of PKCδ and is of because PKCδ has as the major isoform of PKC involved in responses and responses also associated with ceramide M. M. J. T. T. M. J. Clin. PubMed Scopus Google Scholar, 2003; 8: PubMed Scopus Google Scholar). The results that GBA1 appeared to contribute ∼50% of the induced ceramide (Fig. the levels of glucosylceramide are significantly less than of sphingomyelin. This both the through the acid sphingomyelinase and GBA1 and the that GBA1 all lysosomal glycosphingolipids are eventually converted to Activation of the salvage pathway the hydrolysis of both sphingomyelin and glucosylceramide complex (6Tettamanti G. 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