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Biosynthesis of Phosphatidylcholine from a Phosphocholine Precursor Pool Derived from the Late Endosomal/Lysosomal Degradation of Sphingomyelin

2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês

10.1074/jbc.m101817200

1083-351X

Suzanne M. Jansen, J.E.M. Groener, Wim Bax, Anke Suter, Paul Säftig, Pentti Somerharju, Ben J. H. M. Poorthuis,

Lysosomal Storage Disorders Research

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [14C]phosphocholine derived from the lysosomal degradation of [14C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [14C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [14C]sphingomyelin-derived [14C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [3H]choline and sphingomyelin-derived [14C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts. Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [14C]phosphocholine derived from the lysosomal degradation of [14C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [14C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [14C]sphingomyelin-derived [14C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [3H]choline and sphingomyelin-derived [14C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts. phosphatidylcholine phosphocholine CTP:phosphocholine cytidylyltransferase sphingomyelin lysosomal acid phosphatase tartrate resistant acid phosphatase hexadecylphosphocholine fetal bovine serum apolipoprotein E low density lipoprotein Niemann-Pick A lysophosphatidylcholine high-performance thin-layer chromatography. De novo synthesis of phosphatidylcholine (PC)1 predominantly occurs via the CDP-choline pathway (1Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). Choline can enter the cell via the ATP-dependent choline transporter present in the cell membrane and is subsequently phosphorylated to phosphocholine (PCho) by the cytosolic enzyme choline kinase (EC 2.7.1.32). PC is then synthesized in two steps, catalyzed by the rate-limiting cytosolic and membrane-bound enzyme CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15) and the membrane-bound enzyme CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2). For some cell types, including fibroblasts, it has been reported that the steps of PC synthesis are tightly linked in a so-called metabolon (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 3Zlatkine P. Leroy C. Moll G. Le Grimellec C. Biochem. J. 1996; 315: 983-987Crossref PubMed Scopus (7) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). In these cell types only choline that entered the cells through the choline transporter was incorporated into PC. In contrast, PCho administered to cells by partial membrane permeabilization was not incorporated into PC (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar).A possible endogenous source of PCho and choline is provided by the lysosomal degradation of PC and sphingomyelin (SM) by acid sphingomyelinase (5Jansen S.M. Groener J.E.M. Poorthuis B.J.H.M. BBA (Biochim. Biophys. Acta) Libr. 1999; 1436: 363-369Crossref PubMed Scopus (6) Google Scholar). We hypothesized that if only choline, and not PCho, can enter the PC metabolon and be incorporated into PC in human fibroblasts, then PCho that is formed during the degradation of SM by acid sphingomyelinase in the lysosome must be degraded to choline to be incorporated into PC. Candidate enzymes for intralysosomal hydrolysis of PCho to choline are lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (TRAP) (6Waheed A. van Etten R.L. Gieselmann V. von Figura K. Biochem. Genet. 1985; 23: 309-319Crossref PubMed Scopus (40) Google Scholar, 7Waheed A. Gottschalk S. Hille A. Krentler C. Pohlmann R. Braulke T. Hauser H. Geuze H. von Figura K. EMBO J. 1988; 7: 2351-2358Crossref PubMed Scopus (85) Google Scholar, 8Clark S.A. Ambrose W.W. Anderson T.R. Terrell R.S. Toverud S.U. J. Bone Miner. Res. 1989; 4: 399-405Crossref PubMed Scopus (59) Google Scholar). Their in vivo substrates and functional roles are unclear. LAP is ubiquitously expressed, and TRAP is predominantly expressed in alveolar macrophages and osteoclasts (9Drexler H.G. Gignac S.M. Leukemia. 1994; 8: 359-368PubMed Google Scholar), suggesting a specific function of the latter enzyme in these cell types. Another possible route for lysosomal SM-derived PCho is transport across the lysosomal membrane (by an unknown transporter), followed by hydrolysis to choline and subsequent incorporation of choline into the PC synthesis metabolon. A candidate enzyme for the hydrolysis of cytosolic PCho is alkaline phosphatase. In human skin fibroblasts, alkaline phosphatase is predominantly located at the plasma membrane (10Fedde K.N. Whyte M.P. Am. J. Hum. Genet. 1990; 47: 767-775PubMed Google Scholar, 11Fedde K.N. Cole D.E.C. Whyte M.P. Am. J. Hum. Genet. 1990; 47: 776-783PubMed Google Scholar). Phosphoethanolamine and pyridoxal-5′-phosphate have been described as physiological substrates for this enzyme (10Fedde K.N. Whyte M.P. Am. J. Hum. Genet. 1990; 47: 767-775PubMed Google Scholar). We have investigated the possible role of LAP, TRAP, and alkaline phosphatase in the hydrolysis of PCho derived from the late endosomal/lysosomal degradation of SM.In this study we compared exogenous choline and late endosome/lysosome-derived PCho as substrates for PC synthesis. Hexadecylphosphocholine (HePC) was used as an inhibitor of PC synthesis (12Geilen C.C. Wieder T. Reutter W. J. Biol. Chem. 1991; 267: 6719-6724Abstract Full Text PDF Google Scholar, 13Detmar M. Geilen C.C. Wieder T. Orfanos C.E. Reutter W. J. Invest. Dermatol. 1994; 102: 490-494Abstract Full Text PDF PubMed Google Scholar, 14Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar, 15Posse de Chaves E. Vance D.E. Campenot R.B. Vance J.E. Biochem. J. 1995; 312: 411-417Crossref PubMed Scopus (37) Google Scholar, 16Boggs K. Rock C.O. Jackowski S. BBA (Biochim. Biophys. Acta) Libr. 1998; 1389: 1-12Crossref PubMed Scopus (60) Google Scholar). The results indicate that PCho derived from lysosomal SM is not degraded to choline but is incorporated into PC as such. We conclude that the PC synthesis metabolon either does not exist in fibroblasts or is not as inaccessible to PCho as indicated by previous studies.DISCUSSIONIn the present study we describe the incorporation of labeled PCho derived from the late endosomal/lysosomal degradation of SM into PC via the CDP-choline pathway of PC synthesis (1Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). We have shown that the uptake of [14C]SM·apoE was LDL receptor-mediated and that [14C]SM was exclusively metabolized in the late endosomal/lysosomal compartment, because NP-A fibroblasts, which are deficient in acid sphingomyelinase, did not metabolize [14C]SM. Also, in this cell line no evidence was found for the incorporation of SM-derived [14C]choline into PC, even after prolonged incubation. This finding excludes the involvement of the reverse reaction catalyzed by SM synthase (27van Helvoort A. van't Hoff W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar) in our experimental setup. In addition we have clearly shown that during the initial degradation of [14C]SM to [14C]PCho, [14C]PC synthesis was negligible, indicating a lag phase in the process of PC synthesis from SM-derived PCho.In these in situ experiments, we did not observe production of radiolabeled free choline from SM, indicating that SM-derived PCho was not hydrolyzed to choline prior to its incorporation into PC. Even under conditions of energy depletion, when resynthesis of PCho from choline would be inhibited, no radiolabeled free choline was detected. These results corroborate our previous in vitro studies with fibroblast homogenates at acidic pH, where we found PCho, and not choline, as an end product of PC degradation. PCho, which was predominantly generated by acid sphingomyelinase activity, was apparently not further degraded by acid phosphatases (5Jansen S.M. Groener J.E.M. Poorthuis B.J.H.M. BBA (Biochim. Biophys. Acta) Libr. 1999; 1436: 363-369Crossref PubMed Scopus (6) Google Scholar). Furthermore, in another in vitro study the incubation of purified rat liver lysosomes with PC or PCho at acidic pH levels produced virtually no free choline (28Kunze H. BBA (Biochim. Biophys. Acta) Libr. 1993; 1169: 273-279PubMed Google Scholar).The possible role of acid phosphatases in the degradation of PCho in the present in situ study was investigated with embryonic fibroblasts from LAP and/or TRAP (double)-deficient mice. These mice have been developed to study the physiological role of the acid phosphatases (17Saftig P. Hartmann D. Lüllmann-Rauch R. Wolff J. Evers M. Köster A. Hetman M. von Figura K. Peters C. J. Biol. Chem. 1997; 272: 18628-18635Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Hayman A.R. Jones S.J. Boyde A. Foster D. Colledge W.H. Carlton M.B. Evans M.J. Cox T.M. Development. 1996; 122: 3151-3162Crossref PubMed Google Scholar).2 The natural substrates for these enzymes have not been elucidated. However, an early study reported that PCho was a poor substrate for a purified rat liver acid phosphatase in vitro (29Arsenis C. Touster O. J. Biol. Chem. 1968; 243: 5702-5708Abstract Full Text PDF PubMed Google Scholar). Our study shows that PCho is not an in situ substrate for acid phosphatases present in mouse fibroblasts.Because we have not found any indication that SM-derived PCho was hydrolyzed to free choline, it is likely that PCho is an end product of lysosomal degradation. However, most of the intracellular SM-derived PCho was present in the cytoplasm, not in the lysosomes, as was shown by digitonin-induced membrane permeabilization. This suggests that PCho must be transported out of the lysosomes by an as yet unknown transporter. Subsequently, it can serve as a substrate for PC synthesis. The experiments with alkaline phosphatase-deficient human fibroblasts indicate that PCho is not hydrolyzed in the cytosol by alkaline phosphatase. In conclusion, all results indicate that PCho is not hydrolyzed prior to its incorporation into PC.Our data point to a direct incorporation of cytosolic PCho, endogenously produced by the late endosomal/lysosomal breakdown of SM, into PC. This is not compatible with previous reports that concluded that in C6 rat glioma cells, C3H10T½ fibroblasts, and rat hepatocytes the intermediates of PC synthesis are channeled to the enzymes involved, defining a so-called PC synthesis metabolon (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). This conclusion was based on experiments in which PCho, which was delivered to the cytosol via partial membrane permeabilization, was not used for PC synthesis. However, our results show that PCho, which is derived from the lysosomal degradation of SM, is directly incorporated into PC via the CDP-choline pathway of PC synthesis. The latter is supported by the fact that HePC, a specific inhibitor of this pathway, inhibited the incorporation of both exogenous choline and endogenous PCho into PC. HePC has been shown by several groups to inhibit PC synthesis in different cell types (12Geilen C.C. Wieder T. Reutter W. J. Biol. Chem. 1991; 267: 6719-6724Abstract Full Text PDF Google Scholar, 13Detmar M. Geilen C.C. Wieder T. Orfanos C.E. Reutter W. J. Invest. Dermatol. 1994; 102: 490-494Abstract Full Text PDF PubMed Google Scholar, 14Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar, 15Posse de Chaves E. Vance D.E. Campenot R.B. Vance J.E. Biochem. J. 1995; 312: 411-417Crossref PubMed Scopus (37) Google Scholar, 16Boggs K. Rock C.O. Jackowski S. BBA (Biochim. Biophys. Acta) Libr. 1998; 1389: 1-12Crossref PubMed Scopus (60) Google Scholar), including human breast fibroblasts (30Wieder T. Zhang Z. Geilen C.C. Orfanos C.E. Giuliano A.E. Cabot M.C. Cancer Lett. 1996; 100: 71-79Crossref PubMed Scopus (23) Google Scholar). HePC inhibits CT, the rate-limiting enzyme of PC synthesis, which results in the accumulation of PCho. In those previous studies with HePC, the incorporation of exogenous choline into PC was investigated. In the present study with human skin fibroblasts, the inhibition of PC synthesis from exogenous radiolabeled choline by HePC and the subsequent accumulation of PCho were confirmed. HePC also inhibited PC synthesis from lysosomal SM-derived PCho in these cells, suggesting that this PCho, like exogenous choline, entered the CDP-choline pathway of PC synthesis that is regulated by CT. The fact that the molecular species of PC synthesized from exogenous [3H]choline and of PC synthesized from late endosomal/lysosomal [14C]PCho were nearly identical indicates a common diacylglycerol precursor pool. This supports the idea that [14C]SM-derived PCho enters the normal PC biosynthetic pathway, rather than incorporating to PC via another route.In conclusion, our results clearly indicate that in human skin fibroblasts, PCho that is produced by acid sphingomyelinase activity is transported intact from the late endosomal/lysosomal compartment to the cytoplasm and then enters the CDP-choline pathway of PC synthesis. This means that if the proposed PC synthesis metabolon exists, it is not as impermeable to intermediates of PC synthesis as indicated by previous studies (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). De novo synthesis of phosphatidylcholine (PC)1 predominantly occurs via the CDP-choline pathway (1Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). Choline can enter the cell via the ATP-dependent choline transporter present in the cell membrane and is subsequently phosphorylated to phosphocholine (PCho) by the cytosolic enzyme choline kinase (EC 2.7.1.32). PC is then synthesized in two steps, catalyzed by the rate-limiting cytosolic and membrane-bound enzyme CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15) and the membrane-bound enzyme CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2). For some cell types, including fibroblasts, it has been reported that the steps of PC synthesis are tightly linked in a so-called metabolon (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 3Zlatkine P. Leroy C. Moll G. Le Grimellec C. Biochem. J. 1996; 315: 983-987Crossref PubMed Scopus (7) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). In these cell types only choline that entered the cells through the choline transporter was incorporated into PC. In contrast, PCho administered to cells by partial membrane permeabilization was not incorporated into PC (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). A possible endogenous source of PCho and choline is provided by the lysosomal degradation of PC and sphingomyelin (SM) by acid sphingomyelinase (5Jansen S.M. Groener J.E.M. Poorthuis B.J.H.M. BBA (Biochim. Biophys. Acta) Libr. 1999; 1436: 363-369Crossref PubMed Scopus (6) Google Scholar). We hypothesized that if only choline, and not PCho, can enter the PC metabolon and be incorporated into PC in human fibroblasts, then PCho that is formed during the degradation of SM by acid sphingomyelinase in the lysosome must be degraded to choline to be incorporated into PC. Candidate enzymes for intralysosomal hydrolysis of PCho to choline are lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (TRAP) (6Waheed A. van Etten R.L. Gieselmann V. von Figura K. Biochem. Genet. 1985; 23: 309-319Crossref PubMed Scopus (40) Google Scholar, 7Waheed A. Gottschalk S. Hille A. Krentler C. Pohlmann R. Braulke T. Hauser H. Geuze H. von Figura K. EMBO J. 1988; 7: 2351-2358Crossref PubMed Scopus (85) Google Scholar, 8Clark S.A. Ambrose W.W. Anderson T.R. Terrell R.S. Toverud S.U. J. Bone Miner. Res. 1989; 4: 399-405Crossref PubMed Scopus (59) Google Scholar). Their in vivo substrates and functional roles are unclear. LAP is ubiquitously expressed, and TRAP is predominantly expressed in alveolar macrophages and osteoclasts (9Drexler H.G. Gignac S.M. Leukemia. 1994; 8: 359-368PubMed Google Scholar), suggesting a specific function of the latter enzyme in these cell types. Another possible route for lysosomal SM-derived PCho is transport across the lysosomal membrane (by an unknown transporter), followed by hydrolysis to choline and subsequent incorporation of choline into the PC synthesis metabolon. A candidate enzyme for the hydrolysis of cytosolic PCho is alkaline phosphatase. In human skin fibroblasts, alkaline phosphatase is predominantly located at the plasma membrane (10Fedde K.N. Whyte M.P. Am. J. Hum. Genet. 1990; 47: 767-775PubMed Google Scholar, 11Fedde K.N. Cole D.E.C. Whyte M.P. Am. J. Hum. Genet. 1990; 47: 776-783PubMed Google Scholar). Phosphoethanolamine and pyridoxal-5′-phosphate have been described as physiological substrates for this enzyme (10Fedde K.N. Whyte M.P. Am. J. Hum. Genet. 1990; 47: 767-775PubMed Google Scholar). We have investigated the possible role of LAP, TRAP, and alkaline phosphatase in the hydrolysis of PCho derived from the late endosomal/lysosomal degradation of SM. In this study we compared exogenous choline and late endosome/lysosome-derived PCho as substrates for PC synthesis. Hexadecylphosphocholine (HePC) was used as an inhibitor of PC synthesis (12Geilen C.C. Wieder T. Reutter W. J. Biol. Chem. 1991; 267: 6719-6724Abstract Full Text PDF Google Scholar, 13Detmar M. Geilen C.C. Wieder T. Orfanos C.E. Reutter W. J. Invest. Dermatol. 1994; 102: 490-494Abstract Full Text PDF PubMed Google Scholar, 14Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar, 15Posse de Chaves E. Vance D.E. Campenot R.B. Vance J.E. Biochem. J. 1995; 312: 411-417Crossref PubMed Scopus (37) Google Scholar, 16Boggs K. Rock C.O. Jackowski S. BBA (Biochim. Biophys. Acta) Libr. 1998; 1389: 1-12Crossref PubMed Scopus (60) Google Scholar). The results indicate that PCho derived from lysosomal SM is not degraded to choline but is incorporated into PC as such. We conclude that the PC synthesis metabolon either does not exist in fibroblasts or is not as inaccessible to PCho as indicated by previous studies. DISCUSSIONIn the present study we describe the incorporation of labeled PCho derived from the late endosomal/lysosomal degradation of SM into PC via the CDP-choline pathway of PC synthesis (1Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). We have shown that the uptake of [14C]SM·apoE was LDL receptor-mediated and that [14C]SM was exclusively metabolized in the late endosomal/lysosomal compartment, because NP-A fibroblasts, which are deficient in acid sphingomyelinase, did not metabolize [14C]SM. Also, in this cell line no evidence was found for the incorporation of SM-derived [14C]choline into PC, even after prolonged incubation. This finding excludes the involvement of the reverse reaction catalyzed by SM synthase (27van Helvoort A. van't Hoff W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar) in our experimental setup. In addition we have clearly shown that during the initial degradation of [14C]SM to [14C]PCho, [14C]PC synthesis was negligible, indicating a lag phase in the process of PC synthesis from SM-derived PCho.In these in situ experiments, we did not observe production of radiolabeled free choline from SM, indicating that SM-derived PCho was not hydrolyzed to choline prior to its incorporation into PC. Even under conditions of energy depletion, when resynthesis of PCho from choline would be inhibited, no radiolabeled free choline was detected. These results corroborate our previous in vitro studies with fibroblast homogenates at acidic pH, where we found PCho, and not choline, as an end product of PC degradation. PCho, which was predominantly generated by acid sphingomyelinase activity, was apparently not further degraded by acid phosphatases (5Jansen S.M. Groener J.E.M. Poorthuis B.J.H.M. BBA (Biochim. Biophys. Acta) Libr. 1999; 1436: 363-369Crossref PubMed Scopus (6) Google Scholar). Furthermore, in another in vitro study the incubation of purified rat liver lysosomes with PC or PCho at acidic pH levels produced virtually no free choline (28Kunze H. BBA (Biochim. Biophys. Acta) Libr. 1993; 1169: 273-279PubMed Google Scholar).The possible role of acid phosphatases in the degradation of PCho in the present in situ study was investigated with embryonic fibroblasts from LAP and/or TRAP (double)-deficient mice. These mice have been developed to study the physiological role of the acid phosphatases (17Saftig P. Hartmann D. Lüllmann-Rauch R. Wolff J. Evers M. Köster A. Hetman M. von Figura K. Peters C. J. Biol. Chem. 1997; 272: 18628-18635Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Hayman A.R. Jones S.J. Boyde A. Foster D. Colledge W.H. Carlton M.B. Evans M.J. Cox T.M. Development. 1996; 122: 3151-3162Crossref PubMed Google Scholar).2 The natural substrates for these enzymes have not been elucidated. However, an early study reported that PCho was a poor substrate for a purified rat liver acid phosphatase in vitro (29Arsenis C. Touster O. J. Biol. Chem. 1968; 243: 5702-5708Abstract Full Text PDF PubMed Google Scholar). Our study shows that PCho is not an in situ substrate for acid phosphatases present in mouse fibroblasts.Because we have not found any indication that SM-derived PCho was hydrolyzed to free choline, it is likely that PCho is an end product of lysosomal degradation. However, most of the intracellular SM-derived PCho was present in the cytoplasm, not in the lysosomes, as was shown by digitonin-induced membrane permeabilization. This suggests that PCho must be transported out of the lysosomes by an as yet unknown transporter. Subsequently, it can serve as a substrate for PC synthesis. The experiments with alkaline phosphatase-deficient human fibroblasts indicate that PCho is not hydrolyzed in the cytosol by alkaline phosphatase. In conclusion, all results indicate that PCho is not hydrolyzed prior to its incorporation into PC.Our data point to a direct incorporation of cytosolic PCho, endogenously produced by the late endosomal/lysosomal breakdown of SM, into PC. This is not compatible with previous reports that concluded that in C6 rat glioma cells, C3H10T½ fibroblasts, and rat hepatocytes the intermediates of PC synthesis are channeled to the enzymes involved, defining a so-called PC synthesis metabolon (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). This conclusion was based on experiments in which PCho, which was delivered to the cytosol via partial membrane permeabilization, was not used for PC synthesis. However, our results show that PCho, which is derived from the lysosomal degradation of SM, is directly incorporated into PC via the CDP-choline pathway of PC synthesis. The latter is supported by the fact that HePC, a specific inhibitor of this pathway, inhibited the incorporation of both exogenous choline and endogenous PCho into PC. HePC has been shown by several groups to inhibit PC synthesis in different cell types (12Geilen C.C. Wieder T. Reutter W. J. Biol. Chem. 1991; 267: 6719-6724Abstract Full Text PDF Google Scholar, 13Detmar M. Geilen C.C. Wieder T. Orfanos C.E. Reutter W. J. Invest. Dermatol. 1994; 102: 490-494Abstract Full Text PDF PubMed Google Scholar, 14Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar, 15Posse de Chaves E. Vance D.E. Campenot R.B. Vance J.E. Biochem. J. 1995; 312: 411-417Crossref PubMed Scopus (37) Google Scholar, 16Boggs K. Rock C.O. Jackowski S. BBA (Biochim. Biophys. Acta) Libr. 1998; 1389: 1-12Crossref PubMed Scopus (60) Google Scholar), including human breast fibroblasts (30Wieder T. Zhang Z. Geilen C.C. Orfanos C.E. Giuliano A.E. Cabot M.C. Cancer Lett. 1996; 100: 71-79Crossref PubMed Scopus (23) Google Scholar). HePC inhibits CT, the rate-limiting enzyme of PC synthesis, which results in the accumulation of PCho. In those previous studies with HePC, the incorporation of exogenous choline into PC was investigated. In the present study with human skin fibroblasts, the inhibition of PC synthesis from exogenous radiolabeled choline by HePC and the subsequent accumulation of PCho were confirmed. HePC also inhibited PC synthesis from lysosomal SM-derived PCho in these cells, suggesting that this PCho, like exogenous choline, entered the CDP-choline pathway of PC synthesis that is regulated by CT. The fact that the molecular species of PC synthesized from exogenous [3H]choline and of PC synthesized from late endosomal/lysosomal [14C]PCho were nearly identical indicates a common diacylglycerol precursor pool. This supports the idea that [14C]SM-derived PCho enters the normal PC biosynthetic pathway, rather than incorporating to PC via another route.In conclusion, our results clearly indicate that in human skin fibroblasts, PCho that is produced by acid sphingomyelinase activity is transported intact from the late endosomal/lysosomal compartment to the cytoplasm and then enters the CDP-choline pathway of PC synthesis. This means that if the proposed PC synthesis metabolon exists, it is not as impermeable to intermediates of PC synthesis as indicated by previous studies (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). In the present study we describe the incorporation of labeled PCho derived from the late endosomal/lysosomal degradation of SM into PC via the CDP-choline pathway of PC synthesis (1Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). We have shown that the uptake of [14C]SM·apoE was LDL receptor-mediated and that [14C]SM was exclusively metabolized in the late endosomal/lysosomal compartment, because NP-A fibroblasts, which are deficient in acid sphingomyelinase, did not metabolize [14C]SM. Also, in this cell line no evidence was found for the incorporation of SM-derived [14C]choline into PC, even after prolonged incubation. This finding excludes the involvement of the reverse reaction catalyzed by SM synthase (27van Helvoort A. van't Hoff W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar) in our experimental setup. In addition we have clearly shown that during the initial degradation of [14C]SM to [14C]PCho, [14C]PC synthesis was negligible, indicating a lag phase in the process of PC synthesis from SM-derived PCho. In these in situ experiments, we did not observe production of radiolabeled free choline from SM, indicating that SM-derived PCho was not hydrolyzed to choline prior to its incorporation into PC. Even under conditions of energy depletion, when resynthesis of PCho from choline would be inhibited, no radiolabeled free choline was detected. These results corroborate our previous in vitro studies with fibroblast homogenates at acidic pH, where we found PCho, and not choline, as an end product of PC degradation. PCho, which was predominantly generated by acid sphingomyelinase activity, was apparently not further degraded by acid phosphatases (5Jansen S.M. Groener J.E.M. Poorthuis B.J.H.M. BBA (Biochim. Biophys. Acta) Libr. 1999; 1436: 363-369Crossref PubMed Scopus (6) Google Scholar). Furthermore, in another in vitro study the incubation of purified rat liver lysosomes with PC or PCho at acidic pH levels produced virtually no free choline (28Kunze H. BBA (Biochim. Biophys. Acta) Libr. 1993; 1169: 273-279PubMed Google Scholar). The possible role of acid phosphatases in the degradation of PCho in the present in situ study was investigated with embryonic fibroblasts from LAP and/or TRAP (double)-deficient mice. These mice have been developed to study the physiological role of the acid phosphatases (17Saftig P. Hartmann D. Lüllmann-Rauch R. Wolff J. Evers M. Köster A. Hetman M. von Figura K. Peters C. J. Biol. Chem. 1997; 272: 18628-18635Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Hayman A.R. Jones S.J. Boyde A. Foster D. Colledge W.H. Carlton M.B. Evans M.J. Cox T.M. Development. 1996; 122: 3151-3162Crossref PubMed Google Scholar).2 The natural substrates for these enzymes have not been elucidated. However, an early study reported that PCho was a poor substrate for a purified rat liver acid phosphatase in vitro (29Arsenis C. Touster O. J. Biol. Chem. 1968; 243: 5702-5708Abstract Full Text PDF PubMed Google Scholar). Our study shows that PCho is not an in situ substrate for acid phosphatases present in mouse fibroblasts. Because we have not found any indication that SM-derived PCho was hydrolyzed to free choline, it is likely that PCho is an end product of lysosomal degradation. However, most of the intracellular SM-derived PCho was present in the cytoplasm, not in the lysosomes, as was shown by digitonin-induced membrane permeabilization. This suggests that PCho must be transported out of the lysosomes by an as yet unknown transporter. Subsequently, it can serve as a substrate for PC synthesis. The experiments with alkaline phosphatase-deficient human fibroblasts indicate that PCho is not hydrolyzed in the cytosol by alkaline phosphatase. In conclusion, all results indicate that PCho is not hydrolyzed prior to its incorporation into PC. Our data point to a direct incorporation of cytosolic PCho, endogenously produced by the late endosomal/lysosomal breakdown of SM, into PC. This is not compatible with previous reports that concluded that in C6 rat glioma cells, C3H10T½ fibroblasts, and rat hepatocytes the intermediates of PC synthesis are channeled to the enzymes involved, defining a so-called PC synthesis metabolon (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). This conclusion was based on experiments in which PCho, which was delivered to the cytosol via partial membrane permeabilization, was not used for PC synthesis. However, our results show that PCho, which is derived from the lysosomal degradation of SM, is directly incorporated into PC via the CDP-choline pathway of PC synthesis. The latter is supported by the fact that HePC, a specific inhibitor of this pathway, inhibited the incorporation of both exogenous choline and endogenous PCho into PC. HePC has been shown by several groups to inhibit PC synthesis in different cell types (12Geilen C.C. Wieder T. Reutter W. J. Biol. Chem. 1991; 267: 6719-6724Abstract Full Text PDF Google Scholar, 13Detmar M. Geilen C.C. Wieder T. Orfanos C.E. Reutter W. J. Invest. Dermatol. 1994; 102: 490-494Abstract Full Text PDF PubMed Google Scholar, 14Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar, 15Posse de Chaves E. Vance D.E. Campenot R.B. Vance J.E. Biochem. J. 1995; 312: 411-417Crossref PubMed Scopus (37) Google Scholar, 16Boggs K. Rock C.O. Jackowski S. BBA (Biochim. Biophys. Acta) Libr. 1998; 1389: 1-12Crossref PubMed Scopus (60) Google Scholar), including human breast fibroblasts (30Wieder T. Zhang Z. Geilen C.C. Orfanos C.E. Giuliano A.E. Cabot M.C. Cancer Lett. 1996; 100: 71-79Crossref PubMed Scopus (23) Google Scholar). HePC inhibits CT, the rate-limiting enzyme of PC synthesis, which results in the accumulation of PCho. In those previous studies with HePC, the incorporation of exogenous choline into PC was investigated. In the present study with human skin fibroblasts, the inhibition of PC synthesis from exogenous radiolabeled choline by HePC and the subsequent accumulation of PCho were confirmed. HePC also inhibited PC synthesis from lysosomal SM-derived PCho in these cells, suggesting that this PCho, like exogenous choline, entered the CDP-choline pathway of PC synthesis that is regulated by CT. The fact that the molecular species of PC synthesized from exogenous [3H]choline and of PC synthesized from late endosomal/lysosomal [14C]PCho were nearly identical indicates a common diacylglycerol precursor pool. This supports the idea that [14C]SM-derived PCho enters the normal PC biosynthetic pathway, rather than incorporating to PC via another route. In conclusion, our results clearly indicate that in human skin fibroblasts, PCho that is produced by acid sphingomyelinase activity is transported intact from the late endosomal/lysosomal compartment to the cytoplasm and then enters the CDP-choline pathway of PC synthesis. This means that if the proposed PC synthesis metabolon exists, it is not as impermeable to intermediates of PC synthesis as indicated by previous studies (2George T.P. Morash S.C. Cook H.W. Byers D.M. Palmer F.B. St C. Spence M.W. Biochim. Biophys. Acta. 1989; 1004: 283-291Crossref PubMed Scopus (67) Google Scholar, 4Bladergroen B.A. Geelen M.J.H. Reddy A.C.P. Declercq P.E. van Golde L.M.G. Biochem. J. 1998; 334: 511-517Crossref PubMed Scopus (17) Google Scholar). We thank Drs. L. M. G. van Golde and M. Houweling (Laboratory of Veterinary Biochemistry, Utrecht University, Utrecht, The Netherlands) for helpful discussions and critically reading the manuscript.