Role of Multiple Drug Resistance Protein 1 in Neutral but Not Acidic Glycosphingolipid Biosynthesis

Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246–6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis. Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246–6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis. GlcCer 1The abbreviations used are: GlcCer, glucosyl ceramide; MDR1, multi-drug resistance protein 1 (P-glycoprotein); MRP1, multidrug resistance protein 1; CsA, cyclosporin A; GSL, glycosphingolipid; Cer, ceramide; LacCer, lactosyl ceramide (Galβ1–4Glc ceramide); GM3, monosialoganglioside (SAα2–3Galβ1–4Glc ceramide); Gb3, globotriaosyl ceramide (Galα1–4Galβ1–4Glc ceramide); Gb4, globotetraosyl ceramide (GalNacβ1–3Galα1–4Galβ1–4Glc ceramide); CTH, ceramide trihexoside; ER, endoplasmic reticulum; KOT, knock-out; VT1, verotoxin 1; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MDCK, Madin-Darby canine kidney cells. is the precursor of the majority of eukaryotic GSLs (1Stults C.L.M. Sweeley C.H. Macher B.A. Methods Enzymol. 1989; 179: 167-214Crossref PubMed Scopus (234) Google Scholar). The UDP galactose β1–4 glucosyl ceramide glycosyltransferase (LacCer synthase) within the Golgi lumen then provides the common precursor, LacCer for the synthesis of most complex GSL. Drug-resistant tumor cells have been shown to increase GlcCer synthesis (2Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 3Lucci A. Cho W. Han T. Giuliano A. Morton D. Cabot M. Anticancer Res. 1998; 18: 475-480PubMed Google Scholar), and this has been postulated as a mechanism for drug resistance by providing an alternative biosynthetic destination for ceramide, which might otherwise induce apoptosis (4Liu Y.-Y. Han T.-Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). GlcCer is synthesized from ER-derived ceramide, on the cytosolic cis-Golgi surface (5Coste H. Martel M.B. Got R. Biochim. Biophys. Acta. 1986; 858: 6-12Crossref PubMed Scopus (114) Google Scholar, 6Jeckel D. Karrenbauer A. Burger K.N.J. van Meer G. Wieland F. J. Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (259) Google Scholar, 7Warnock D. Lutz M. Blackburn W. Young W. Baenziger J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2708-2712Crossref PubMed Scopus (107) Google Scholar), and may be transported to the plasma membrane from this site (8van Helvoort A. Smith A. Sprong H. Fritzsche I. Schinkel A. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). However, the active sites of the glycosyltransferases responsible for further elongation of GSLs are located within the Golgi lumen (9Lannert H. Bunning C. Jeckel D. Wieland F. FEBS Lett. 1994; 342: 91-96Crossref PubMed Scopus (88) Google Scholar, 10Burger K. van der Bijl P. van Meer G. J. Cell Biol. 1996; 133: 15-28Crossref PubMed Scopus (90) Google Scholar). Thus, a mechanism for the translocation of GlcCer from the cytosolic surface of the Golgi to the lumen must exist. We have shown that transfection of cells with the mdr1 gene results in a marked elevation of GlcCer, LacCer, and Gb3 levels with a concomitant increase in cell sensitivity to the Gb3-binding verotoxin 1. Both effects were reversed in the presence of MDR1 inhibitors (11Lala P. Ito S. Lingwood C.A. J. Biol. Chem. 2000; 275: 6246-6251Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We proposed that MDR1 can function as a GlcCer translocase to flip GlcCer to the inner Golgi surface to enhance LacCer and subsequent Gb3 synthesis. MDR1 has been shown to function as a membrane translocase for chemically modified short chain fatty acid GlcCer (8van Helvoort A. Smith A. Sprong H. Fritzsche I. Schinkel A. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). However such derivatized, indicator GSLs have been considered possible drug substrates for MDR1, and the ability of MDR1 to translocate natural, long-chain GSLs has been questioned on the basis of lack of overt phenotype in MDR1 knockout mice (12Borst P. Zelcer N. van Helvoort A. Biochim. Biophys. Acta. 2000; 1486: 128-144Crossref PubMed Scopus (263) Google Scholar). In the present study, we show that fibroblasts from such mice are indeed defective in GSL synthesis, in the manner predicted from the in vitro MDR1 inhibition studies now reported. In this study, we address the question as to whether MDR1, which is widely expressed in normal tissues (13Lum B.L. Gosland M.P. Hematol. Oncol. Clin. North Am. 1995; 9: 319-336Abstract Full Text PDF PubMed Google Scholar), is involved in GSL synthesis in cultured cells in general. These studies fill a major gap in understanding the mechanism of GSL biosynthesis and demonstrate a new, unsuspected distinction between acidic and neutral GSL synthesis. MDCK cells transfected with the human MDR1 cDNA were a gift from Dr. M. Gottesman (National Institutes of Health, Bethesda, MD) (14Pastan I. Gottesman M. Ueda K. Lovelace E. Rutherford A. Willingham M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4486-4490Crossref PubMed Scopus (476) Google Scholar). The human astrocytoma cell line SF-539, and IOMM Lee and CH157 MN meningioma cell lines were kindly provided by Dr. J. Rutka (Hospital for Sick of the cell line G. M. Res. 1989; Google cells were in with or and and 1 HeLa cells, cells, and meningioma IOMM Lee and CH157 MN cell lines were in modified of for HeLa and and with 1 of for meningioma cell with and cells were in with cell line Y. M. H. J. 1999; PubMed Scopus Google was in with and cells and are fibroblasts from a knock-out mouse J. van C.A. van der M. Schinkel Borst P. J. Clin. 2000; PubMed Scopus Google Scholar, Res. Google and were a gift from Dr. J. of were which the cells were for to in the present MDR1 inhibition, cells were in the or for or or for which studies shown to have on cell was as previously A. Lingwood C.A. Methods Google Scholar). cells in were cultured in and in with of the cells were with and a as previously W. E. U. 1989; PubMed Scopus Google Scholar). and cell were by MDR1 inhibition or was added to cells or to and the GSLs were from cells. cells were and cells were by for The cell was with of as A. G. Lingwood C.A. Biophys. PubMed Scopus Google Scholar). The was and the was to a in The was with and the with A. Lingwood C. Methods Google Scholar). were from and by and in G. S. PubMed Scopus Google Scholar). GSL on cell were by and the were and with in with were with for 1 the were with J. M. Lingwood C.A. H. M. M. J. Clin. PubMed Google by was with A. Lingwood C. Methods Google Scholar). cells were PBS, and for The was in and and a The was for and the was further for The was for and the was in of the a cell was by GSLs in were with of the of was in and for with of was then added to a and for a further with liposomal GlcCer in of was added the of cells were with for were with and by were and on as GSLs were and were with G. S. PubMed Scopus Google Scholar). were by and by and type and were in in the presence of or 1 for in modified fetal was and with of and was to for was then to an and of of and of acid were were then and the was The was and by and by and to type and fibroblasts were with of a of S. R. S. A. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In this the was in of in for with of of of and Both were and were on a The was then by as of were with and in 1 for 1 with for PubMed Scopus Google and were on protein by a R. H. S. Res. 1994; of Cell IOMM and CH157 MN cells were on cells were with for 1 by for 1 with PBS, the were in in for with and and a were the same cells were and in in for were then by for in in and with for The were then on and in were with a a were to in a of and the were in and and were then with a Golgi A. A. J. PubMed Scopus Google for 1 a in were in a complex for 1 was on the same was used as the and complex as the were then with by and in a of the of and and the of an or acidic were then in a and were a were for was on a of from a of an to cell and was as and within of and in for of the cell and with and ketoconazole, were by and for as as the were by A of was considered MDR1 and cell HeLa Lee CH157 MN and cell lines were for neutral GSL and Gb3 and and or 1 and cell were used the which on cell of MDR1 drug cell lines HeLa The GSL from or cells was by and Gb3 by In HeLa the Gb3 LacCer and was in cells, with the In HeLa cells, sensitivity and their Gb3 was largely unaffected by MDR1 The Gb3 and sensitivity of and cells was CsA, as to consistent with that the sensitivity of cell lines was by the that Gb3 synthesis in and cells was inhibited by CsA, a on sensitivity was cells are and Gb3 I. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The neutral GSLs of mouse cells were by human cells do not express Gb3 A. M. Lingwood C.A. PubMed Scopus Google and the neutral GSLs of the cell line were with 1 and of or on cell line sensitivity The of MDR1 inhibition on the sensitivity of cell to was The required to of cells from the was for cells of cells with or as in 1. the on cell sensitivity to was to to drug in not not in not not in a of the of MDR1 inhibition on and GSL cellular GSL A, and neutral GSL and ganglioside A and to cells, and to cells, and and to cells. A, and GlcCer, LacCer, Gb3, and and and GSLs from and GSLs from or and cells. GSLs were by and for were results were in labeling cells were with for and the neutral and acidic GSLs were and by and by A, neutral 1 and and cells. cells, and The in the neutral GSL with and is with the for was of on synthesis (LacCer and in a by was inhibited by of GlcCer LacCer but inhibition was of as to is the precursor of and neutral We the of or on the synthesis of Gb3 and GM3, the in and cells. The of on cell GlcCer, the neutral GSL and ganglioside was In cells cultured with and cells cultured with CsA, Gb3 levels were inhibited by the of GM3, the ganglioside was In cells, GlcCer was by GM3, and ganglioside were of the neutral and acidic GSL of cells cultured with or CsA, Gb3 synthesis was the synthesis of and the complex were not inhibited by was in the neutral GSL and labeling was not in cells. of MDR1 which have been in with J. van C.A. van der M. Schinkel Borst P. J. Clin. 2000; PubMed Scopus Google Scholar, Res. Google Scholar). of the labeling of GSLs of fibroblasts from knockout mice and wild type fibroblasts and showed the same we with and of cell and labeling of GlcCer and LacCer in the cells was and as with wild type of an was but In contrast, labeling of ganglioside was not labeling was in the labeling was and labeling with was labeling from or was in of labeling of GSL of MDR1 knockout fibroblasts with or The from cells and by were by and the synthesis of neutral GSLs GlcCer and LacCer was ganglioside synthesis was not inhibited in the cells, as to the wild type synthesis was unaffected for synthesis and a marked in labeling from labeling was in of to for wild type ceramide not further ceramide not further in in of to for wild type CTH, ceramide not further in in a a of 1 S. R. S. A. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar), on GSL labeling with in wild type or knockout fibroblasts of on GSL labeling in wild type and knockout fibroblasts that the lack of in cells role in the neutral GSL synthesis in cells, the of cell with an on GSL labeling with was in wild type and cells in in were from cells, by and by was to have on neutral as in as in as in in a Cell of HeLa cell GSL biosynthesis and sensitivity were unaffected by MDR1 inhibitors, we whether MDR1 was expressed by cells. a MDR1 was in the cell but was in the HeLa cell HeLa cells showed surface for MDR1 as with a of cell lines in which MDR1 inhibitors neutral GSL synthesis SF-539, and CH157 MN which as has shown a lack of MDR1 in HeLa cells G. A. J. C. K. J. C. M. P. D. Biol. Cell. PubMed Scopus Google Scholar). In cells surface MDR1 is consistent with a M. J. D. R. 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PubMed Scopus Google and apoptosis GlcCer Golgi translocation has been implicated to this Google Scholar). In to drug MDR1 has been shown to be a (8van Helvoort A. Smith A. Sprong H. Fritzsche I. Schinkel A. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). MDR1 can R. I. van Meer G. J. PubMed Scopus Google Scholar), but a role for MDR1 in has (12Borst P. Zelcer N. van Helvoort A. Biochim. Biophys. Acta. 2000; 1486: 128-144Crossref PubMed Scopus (263) Google Scholar). mice are and GSL or phenotype has been reported. The and are A. J. R. R. Schinkel A. Borst P. 1998; PubMed Scopus Google Scholar), and has activity for GSLs (8van Helvoort A. Smith A. Sprong H. Fritzsche I. Schinkel A. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). MDR1 function as in and A A. C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, G. T. H. S. van J. 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PubMed Scopus Google is a function of such as we for HeLa cells in the present The labeling of in cells from an ceramide subsequent to inhibition of neutral GSL synthesis. The labeling of the neutral was in the cells. The may to the role for MDR1 proposed in D. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. D. J. Res. Full Text Full Text PDF PubMed Google Scholar, Full Text Full Text PDF PubMed Scopus Google and A. A.E. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In (11Lala P. Ito S. Lingwood C.A. J. Biol. 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