Differential Effects of Sphingomyelin Hydrolysis and Cholesterol Transport on Oxysterol-binding Protein Phosphorylation and Golgi Localization
1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês
10.1074/jbc.273.47.31621
ISSN1083-351X
AutoresNeale D. Ridgway, Thomas A. Lagace, Harold W. Cook, David M. Byers,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe deposition of de novo synthesized and lipoprotein-derived cholesterol at the plasma membrane and transport to the endoplasmic reticulum is dependent on sphingomyelin (SM) content. Here we show that hydrolysis of plasma membrane SM in Chinese hamster ovary cells by exogenous bacterial sphingomyelinase resulted in enhanced cholesterol esterification at the endoplasmic reticulum and rapid dephosphorylation of the oxysterol-binding protein (OSBP), a cytosolic/Golgi receptor for oxysterols such as 25-hydroxycholesterol. After sphingomyelinase treatment, restoration of OSBP phosphorylation closely paralleled resynthesis of SM and down-regulation of cholesterol ester synthesis. SM hydrolysis activated an okadaic acid-sensitive phosphatase that was not stimulated in Chinese hamster ovary cells by short chain ceramides. Agents that specifically blocked sphingomyelinase-mediated delivery of cholesterol to acyl-CoA:cholesterol acyltransferase (U18666A) or promoted cholesterol efflux to the medium (cyclodextrin) did not inhibit OSBP dephosphorylation. SM hydrolysis also promoted OSBP translocation from a vesicular compartment to the Golgi apparatus. Cyclodextrin and U18666A also caused OSBP translocation to the Golgi apparatus, suggesting that OSBP movement is coupled to changes in the cholesterol content of the plasma membrane or Golgi apparatus. These results identify OSBP as a potential target of SM turnover and cholesterol mobilization at the plasma membrane and/or Golgi apparatus. The deposition of de novo synthesized and lipoprotein-derived cholesterol at the plasma membrane and transport to the endoplasmic reticulum is dependent on sphingomyelin (SM) content. Here we show that hydrolysis of plasma membrane SM in Chinese hamster ovary cells by exogenous bacterial sphingomyelinase resulted in enhanced cholesterol esterification at the endoplasmic reticulum and rapid dephosphorylation of the oxysterol-binding protein (OSBP), a cytosolic/Golgi receptor for oxysterols such as 25-hydroxycholesterol. After sphingomyelinase treatment, restoration of OSBP phosphorylation closely paralleled resynthesis of SM and down-regulation of cholesterol ester synthesis. SM hydrolysis activated an okadaic acid-sensitive phosphatase that was not stimulated in Chinese hamster ovary cells by short chain ceramides. Agents that specifically blocked sphingomyelinase-mediated delivery of cholesterol to acyl-CoA:cholesterol acyltransferase (U18666A) or promoted cholesterol efflux to the medium (cyclodextrin) did not inhibit OSBP dephosphorylation. SM hydrolysis also promoted OSBP translocation from a vesicular compartment to the Golgi apparatus. Cyclodextrin and U18666A also caused OSBP translocation to the Golgi apparatus, suggesting that OSBP movement is coupled to changes in the cholesterol content of the plasma membrane or Golgi apparatus. These results identify OSBP as a potential target of SM turnover and cholesterol mobilization at the plasma membrane and/or Golgi apparatus. endoplasmic reticulum acyl-CoA:cholesterol acyltransferase brefeldin A cyclodextrin Chinese hamster ovary lipoprotein-deficient serum Dulbecco's modified Eagle's medium fetal calf serum oxysterol-binding protein phospholipase C phosphatidylcholine sphingomyelin sphingomyelinase low density lipoprotein polyacrylamide gel electrophoresis bovine serum albumin 12-O-tetradecanoylphorbol-13-acetate. Studies in cultured cell models have identified three organelles that figure prominently in cholesterol trafficking: the ER,1 the major site for cholesterol synthesis, regulation, and esterification; the plasma membrane, a prominent storage site for unesterified cholesterol; and lysosomes, where lipoprotein-derived cholesterol is liberated (reviewed in Ref. 1Liscum L. Underwood W.K. J. Biol. Chem. 1995; 270: 15443-15446Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Cholesterol made in the ER, as well as that released in the lysosome by lipoprotein catabolism, rapidly moves to the plasma membrane (1Liscum L. Underwood W.K. J. Biol. Chem. 1995; 270: 15443-15446Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 2Urbani L. Simoni R.D. J. Biol. Chem. 1990; 265: 1919-1923Abstract Full Text PDF PubMed Google Scholar, 3Brasaemle D.L. Attie A.D. J. Lipid Res. 1990; 31: 103-112Abstract Full Text PDF PubMed Google Scholar). Once the capacity of the plasma membrane to absorb cholesterol is exceeded, cholesterol is transported to the ER, where it is esterified, regulates 3-hydroxy-3-methylglutaryl-CoA reductase proteolysis, and inhibits proteolytic processing of sterol-regulatory element-binding proteins required for expression of sterol-regulated genes (4Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3002) Google Scholar). This distribution of cholesterol between sites of regulation, synthesis, and deposition provides for efficient control of cellular cholesterol levels. Sphingomyelin appears to play an important role in this process as demonstrated by the capacity of exogenous sphingomyelinase to degrade plasma membrane SM and stimulate cholesterol esterification (5Slotte J.P. Bierman E.L. Biochem. J. 1988; 250: 653-658Crossref PubMed Scopus (273) Google Scholar) and sterol-regulatory element-binding protein processing (6Scheek S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11179-11183Crossref PubMed Scopus (100) Google Scholar) in the ER. Cholesterol and SM are concentrated in the plasma membrane (7Lange Y.M. Swaisgood M.H. Ramos B.V. Steck T.L. J. Biol. Chem. 1989; 264: 3786-3793Abstract Full Text PDF PubMed Google Scholar) and are associated with caveolar membrane structures (8Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). Change in the stoichiometry of cholesterol and SM, either by SM depletion or cholesterol loading, is accompanied by alterations in cholesterol homeostasis. For example, varying the SM content of macrophages altered the stimulation of ACAT activity by acetyl-LDL (9Okwu A.K. Xu X.-X. Shiratori Y. Tabas I. J. Lipid Res. 1994; 35: 644-655Abstract Full Text PDF PubMed Google Scholar). The SM content of macrophages was also increased by acetyl-LDL cholesterol loading (10Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Abstract Full Text PDF PubMed Google Scholar) and by oxysterols in CHO cells (11Ridgway N.D. J. Lipid Res. 1995; 36: 1345-1358Abstract Full Text PDF PubMed Google Scholar). While there is evidence that SM and cholesterol levels are coordinately regulated and this is important in cholesterol homeostasis, precise mechanisms remain to be determined (9Okwu A.K. Xu X.-X. Shiratori Y. Tabas I. J. Lipid Res. 1994; 35: 644-655Abstract Full Text PDF PubMed Google Scholar, 10Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Abstract Full Text PDF PubMed Google Scholar, 11Ridgway N.D. J. Lipid Res. 1995; 36: 1345-1358Abstract Full Text PDF PubMed Google Scholar, 12Kudchodkar B.J. Albers J.J. Bierman E.L. Atherosclerosis. 1983; 46: 353-367Abstract Full Text PDF PubMed Scopus (19) Google Scholar, 13Chen H. Born E. Mathur S.N. Field F.J. J. Lipid Res. 1993; 34: 2159-2167Abstract Full Text PDF PubMed Google Scholar). The mechanism(s) for cholesterol transport from the plasma membrane in response to SM depletion or influx of cholesterol from the lysosomes is poorly understood. Delivery of β-very low density lipoprotein or LDL cholesterol from the lysosomes to ACAT has energy-dependent and -independent components (1Liscum L. Underwood W.K. J. Biol. Chem. 1995; 270: 15443-15446Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 14Skiba P.J. Zha X. Maxfield F.R. Schissel S.L. Tabas I. J. Biol. Chem. 1996; 271: 13392-13400Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and could involve a vesicle transport step (14Skiba P.J. Zha X. Maxfield F.R. Schissel S.L. Tabas I. J. Biol. Chem. 1996; 271: 13392-13400Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and the majority (>70%) transits through the plasma membrane (15Underwood K.W. Jacobs N.L. Howley A. Liscum L. J. Biol. Chem. 1998; 273: 4266-4274Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 16Lange Y. Ye J. Chin J. J. Biol. Chem. 1997; 272: 17018-17022Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Stimulation of cholesterol esterification by SMase is energy-independent and involves vesiculation of the plasma membrane (14Skiba P.J. Zha X. Maxfield F.R. Schissel S.L. Tabas I. J. Biol. Chem. 1996; 271: 13392-13400Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Zha X. Pierini L.M. Leopold P.L. Skiba P.J. Tabas I. Maxfield F.R. J. Cell Biol. 1998; 140: 39-47Crossref PubMed Scopus (173) Google Scholar). SMase-stimulated cholesterol esterification is also insensitive to protease inhibitors that blocked β-very low density lipoprotein activation of ACAT (18Schissel S.L. Beatini N. Zha X. Maxfield F.R. Tabas I. Biochemistry. 1995; 34: 10463-10473Crossref PubMed Scopus (11) Google Scholar). These differences could simply reflect the lysosome-plasma membrane transport component for lipoprotein-derived cholesterol delivery to the ER. However, stimulation of cholesterol esterification by lipoproteins and SMase was inhibited by U18666A, suggesting a common component to both pathways (19Harmala A.-S. Porn I. Mattjus P. Slotte J.P. Biochim. Biophys. Acta. 1994; 1221: 317-325Crossref Scopus (44) Google Scholar, 20Underwood K.W. Andemaraim B. McWilliams G.L. Liscum L. J. Lipid Res. 1996; 37: 1556-1568Abstract Full Text PDF PubMed Google Scholar). Several proteins have been implicated in the regulation of cellular cholesterol trafficking. Caveolin, a scaffold protein found in caveolae, binds cholesterol and facilitates its movement between the plasma membrane and ER (8Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). The NPC-1 protein, which is defective in the Niemann-Pick C lysosomal cholesterol storage disorder, plays an undefined role in cholesterol egress from lysosomes (21Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Paven W.J. Krisman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T-Y. Liscum L. Strauss III, J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Markie D. O'Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Penchev P.G. Tagle D.A. Science. 1997; 277: 228-231Crossref PubMed Scopus (1216) Google Scholar). Sterol carrier protein 2 has also been implicated in cholesterol transport from the ER to plasma membrane (22Puglielli L. Rigotti A. Greco A.V. Santos M.J. Nervi F. J. Biol. Chem. 1995; 270: 18723-18726Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Still other unidentified proteins are involved in the delivery of LDL- and SMase-derived cholesterol to the ER (23Jacobs N.L. Andemariam B. Underwood K.W. Panchalingam K. Sternberg D. Kielian M. Liscum L. J. Lipid Res. 1997; 38: 1973-1987Abstract Full Text PDF PubMed Google Scholar). Another protein that could be involved in cholesterol trafficking and regulation is the oxysterol-binding protein (OSBP). OSBP was identified as a high affinity receptor for oxysterols such as 25-hydroxycholesterol (24Dawson P.A. Ridgway N.D. Slaughter C.A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 16798-16803Abstract Full Text PDF PubMed Google Scholar) that localized to the Golgi apparatus in the presence of its oxysterol ligand (25Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (239) Google Scholar). The involvement of OSBP in cholesterol regulation was suggested by the positive correlation between oxysterol affinity for OSBP and suppression of cholesterol synthesis (26Taylor F.R. Saucier S.E. Shown E.P. Parish E.J. Kandutsch A.A. J. Biol. Chem. 1984; 259: 12382-12387Abstract Full Text PDF PubMed Google Scholar). More recently, overexpression of OSBP in CHO cells was shown to have pleotropic effects on cholesterol synthesis, expression of sterol-regulated gene expression, and ACAT activity (27Lagace T.A. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1997; 326: 205-213Crossref PubMed Scopus (101) Google Scholar). Deletion of the OSBP pleckstrin homology domain resulted in loss of this phenotype and lack of association with the Golgi apparatus. OSBP is a phosphoprotein that can be identified on SDS-PAGE by a characteristic increase in apparent molecular mass of 2–3 kDa over the dephosphorylated form (28Ridgway N.D. Badiani K. Byers D.M. Cook H.W. Biochim. Biophys. Acta. 1997; 1390: 37-51Crossref Scopus (20) Google Scholar). While it is presently uncertain how OSBP affects cholesterol homeostasis, its position in the Golgi/vesicular compartment is suggestive of a role in cellular sterol or lipid trafficking. In this study, we investigated whether SM hydrolysis at the plasma membrane and perturbation of cholesterol trafficking affected OSBP. As expected, bacterial SMase digested 60–70% of cellular SM and promoted cholesterol esterification. This was accompanied by OSBP dephosphorylation, which was reversible upon SMase removal, and translocation to the Golgi apparatus. Furthermore, dephosphorylation of OSBP was directly linked to SM hydrolysis, but translocation to the Golgi apparatus was mediated by cholesterol depletion at the plasma membrane. Sphingomyelinase (Bacillus cereus), methyl-β-cyclodextrin, sphingosine, and fatty acid-free BSA were purchased from Sigma. Okadaic acid was from LC Laboratories. Phospholipase C (B. cereus) was purchased from Boehringer Mannheim. [32P]Phosphate, [1-3H]oleate, [G-3H]serine, and [methyl-3H]choline were from NEN Life Science Products. 25-Hydroxycholesterol was purchased from Steraloids (Wilton, NH). U18666A was kindly provided by Dr. M. E. Torkelson (Upjohn). Goat anti-rabbit and goat anti-mouse antibodies conjugated to horseradish peroxidase were from Bio-Rad. C2- and C6-ceramide were prepared by acylation of sphingosine and complexed with BSA prior to addition to cells (29Ridgway N.D. Merriam D.L. Biochim. Biophys. Acta. 1995; 1256: 57-70Crossref PubMed Scopus (50) Google Scholar). Enhanced chemiluminescence kits were from Amersham Pharmacia Biotech. Tissue culture reagents were from Life Technologies, Inc. Protein was determined with a micro-BCA kit according to the manufacturer's instructions (Pierce). CHO-K1 cells were grown at 37 °C in a humidified incubator in an atmosphere of 5% CO2. CHO-K1 cells overexpressing rabbit OSBP by 15–20-fold were prepared and cultured as described previously (27Lagace T.A. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1997; 326: 205-213Crossref PubMed Scopus (101) Google Scholar). Both control and overexpressing cells were seeded at 250,000 cells/60-mm dish in DMEM supplemented with 5% fetal bovine serum and 34 μg of proline/ml. Twenty-four hours prior to the start of experiments, cells received fresh DMEM containing either 5% FCS or 5% lipoprotein-deficient serum (LPDS). Stock solutions of reagents were prepared in the following manner. Cyclodextrin (250 mm) was dissolved in distilled water, U18666A (2 mg/ml) was dissolved in ethanol, okadaic acid (1 mm) was prepared in Me2SO, and SMase and PLC were diluted in phosphate-buffered saline to 10 milliunits/μl and 1 unit/μl, respectively. LPDS was prepared by ultracentrifugation at a density of 1.21 g/ml and dialyzed extensively against phosphate-buffered saline (30Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). Human LDL (1.018–1.063 g/ml) was isolated by ultracentrifugation (30Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). Rabbit OSBP overexpressed in CHO-K1 cells was detected by immunoblotting and immunoprecipitation using a monoclonal antibody 11H9 (25Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (239) Google Scholar, 27Lagace T.A. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1997; 326: 205-213Crossref PubMed Scopus (101) Google Scholar). Since 11H9 is specific for the rabbit OSBP, a polyclonal antibody was prepared that recognized the protein from CHO-K1 cells. Antibody 104 was raised in rabbits against a glutathioneS-transferase fusion protein expressing amino acids 201–309 of rabbit OSBP. The antibody was subsequently affinity-purified using the glutathione S-transferase-OSBP fusion protein coupled to Sepharose. At the completion of experiments, CHO cells were harvested in ice-cold PBS and collected by centrifugation (2000 × gfor 5 min). Cell pellets were solubilized in 10 mm sodium phosphate (pH 7.4), 150 mm NaCl, 2 mm EDTA, 2 mm EGTA, 10 mm NaF, 1 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 100 μm phenylmethanesulfonyl fluoride, 2 μg of aprotinin/ml, 2.5 μg of leupeptin/ml, and 0.3% (w/v) Triton X-100 (buffer B) on ice for 15 min followed by centrifugation for 15 min at 10,000 × g in a microcentrifuge. The supernatant, which contained all immunoreactive OSBP, was collected and analyzed by immunoblotting or immunoprecipitation. Triton X-100 extracts of CHO cells were resolved by SDS-PAGE on 6% gels and transferred to nitrocellulose, and OSBP was detected as described previously (27Lagace T.A. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1997; 326: 205-213Crossref PubMed Scopus (101) Google Scholar,28Ridgway N.D. Badiani K. Byers D.M. Cook H.W. Biochim. Biophys. Acta. 1997; 1390: 37-51Crossref Scopus (20) Google Scholar). Following metabolic labeling of cells with [32P]phosphate (refer to figure legends for specific details), OSBP was immunoprecipitated from Triton X-100 cell extracts by incubation with a 1:100 dilution of antibody 104 (whole serum) at 4 °C for 2 h in 200 μl of buffer B. A 50% slurry of protein A-Sepharose was added and incubated at 20 °C for an additional 30 min. Sepharose beads were collected by centrifugation; washed 8–10 times with 500 μl of PBS, 1% (w/v) Triton X-100; and separated by SDS-6% polyacrylamide gel electrophoresis. Immunofluorescence detection of endogenous OSBP in CHO-K1 cells was with affinity-purified antibody 104 and fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody. Cell manipulations, antibody treatments, and microscopy were essentially as described for detection of overexpressed OSBP (25Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (239) Google Scholar, 27Lagace T.A. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1997; 326: 205-213Crossref PubMed Scopus (101) Google Scholar). Cholesterol esterification in cultured cells was measured by the incorporation of [3H]oleate into cholesteryl ester (30Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). Cells were incubated in medium containing 100 μm [3H]oleate complexed to BSA at 37 °C. The reaction was terminated by extraction of cellular lipids with hexane/isopropyl alcohol (3:2, v/v), and radiolabeled cholesteryl ester and triglyceride were separated by thin layer chromatography and quantitated by liquid scintillation counting. To assess the extent of hydrolysis of plasma membrane SM and PtdCho by exogenous SMase and PLC, cells were incubated in DMEM with 5% FCS and 2 μCi of [methyl-3H]choline/ml for 24 h. Exogenous SMase or PLC was added directly to labeled cells, and at the indicated times medium was removed and cells were rinsed once with cold PBS and scraped in 2 ml of methanol/water (5:4, v/v). Lipids were extracted with 6 ml of chloroform/methanol (2:1, v/v) and 4 ml of 0.58% (w/v) NaCl and dried under nitrogen (11Ridgway N.D. J. Lipid Res. 1995; 36: 1345-1358Abstract Full Text PDF PubMed Google Scholar). [methyl- 3H]Choline-labeled SM and PtdCho were separated by thin layer chromatography in chloroform/methanol/water (65:25:4, v/v/v) and quantitated by liquid scintillation counting. [3H]Serine incorporation into SM and ceramide was quantitated by harvesting cells in methanol/water (5:4, v/v) and extracting lipids with chloroform/methanol (1:2, v/v) as described previously (11Ridgway N.D. J. Lipid Res. 1995; 36: 1345-1358Abstract Full Text PDF PubMed Google Scholar). SM and ceramide were resolved by thin layer chromatography in a solvent system of chloroform/methanol/water (65:25:4, v/v/v), identified by fluorography of the thin layer plate, and quantitated by liquid scintillation counting. A well characterized response to the depletion of SM in the plasma membrane is the internalization of cholesterol to the ER, where it is esterified by ACAT (6Scheek S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11179-11183Crossref PubMed Scopus (100) Google Scholar), down-regulates 3-hydroxy-3-methylglutaryl-CoA reductase activity (31Gupta A.K. Rudney H. J. Lipid Res. 1991; 32: 125-136Abstract Full Text PDF PubMed Google Scholar), and inhibits proteolysis of the precursor forms of sterol-regulatory element-binding proteins (7Lange Y.M. Swaisgood M.H. Ramos B.V. Steck T.L. J. Biol. Chem. 1989; 264: 3786-3793Abstract Full Text PDF PubMed Google Scholar). However, the sequence of events for delivery of cholesterol from the plasma membrane to the ER events following SM depletion is unknown. The localization of OSBP to a Golgi/vesicular compartment and its putative role in sterol and sphingomyelin regulation (11Ridgway N.D. J. Lipid Res. 1995; 36: 1345-1358Abstract Full Text PDF PubMed Google Scholar, 27Lagace T.A. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1997; 326: 205-213Crossref PubMed Scopus (101) Google Scholar) prompted an investigation of its role in SMase-mediated cholesterol mobilization. Initially, CHO-K1 cells overexpressing rabbit OSBP were treated with increasing amounts of bacterial SMase for 30 min, and SM hydrolysis, cholesterol esterification, and OSBP expression were examined (Fig. 1). As expected, cholesterol esterification was stimulated 3-fold in parallel with the hydrolysis of 50–70% of cellular SM by SMase concentrations > 5 milliunits/ml (Fig. 1 A). In untreated cells, OSBP migrated as a doublet of 97 and 101 kDa (Fig. 1 B); the higher molecular weight isoform had decreased mobility in SDS-PAGE as the result of extensive phosphorylation of serine residues (29Ridgway N.D. Merriam D.L. Biochim. Biophys. Acta. 1995; 1256: 57-70Crossref PubMed Scopus (50) Google Scholar). With increasing SMase, total OSBP expression did not change, but there was a pronounced shift to the lower molecular weight, dephosphorylated form (Fig. 1 B). When the phosphorylation status of OSBP was determined by [32P]phosphate incorporation and immunoprecipitation (Fig. 1 C), it was clear that SMase promoted rapid dephosphorylation of OSBP, which coincided with SM hydrolysis and stimulation of cholesterol esterification. Similar experiments were performed to determine the temporal relationship between SMase-mediated dephosphorylation of OSBP, SM hydrolysis, and cholesterol esterification (Fig. 2). SM hydrolysis was complete by 20 min, as was OSBP dephosphorylation, measured by immunoblotting (Fig. 2 B) and [32P]phosphate incorporation (Fig. 2 C). Cholesterol esterification was still increasing by 60 min following SMase addition. In the experiments shown in Figs. 1 and 2, SMase treatment inhibited [32P]phosphate incorporation into total cellular protein by <15% but blocked [32P]phosphate incorporation into OSBP by 70–90%. It could be argued that effects of SMase shown in Figs. 1 and 2 are the result of nonspecific changes in plasma membrane structure due to excessive loss of phospholipid. To rule out this possibility, we tested whether plasma membrane PtdCho hydrolysis by exogenous PLC in overexpressing CHO-K1 cells affected OSBP phosphorylation and cholesterol esterification. At the highest concentration of PLC tested, 60% of cellular PtdCho was hydrolyzed compared with only 15% of cellular SM (Fig. 3 A). However, PtdCho hydrolysis did not increase cholesterol esterification, nor did it stimulate OSBP dephosphorylation (as measured by immunoprecipitation of [32P]phosphate-labeled OSBP; Fig. 3 B). When SMase was removed from cells, restoration of SM to normal levels occurred within 2–6 h (32Slotte J.P. Harmala A.-S. Jansson C. Porn M.I. Biochim. Biophys. Acta. 1990; 1030: 251-257Crossref PubMed Scopus (56) Google Scholar), and cholesterol distribution was reestablished by 24–48 h (33Porn M.I. Slotte J.P. Biochem. J. 1990; 271: 121-126Crossref PubMed Scopus (38) Google Scholar). In wild type CHO-K1 cells, we examined whether SMase promoted dephosphorylation of endogenous hamster OSBP and whether rephosphorylation occurred concomitantly with resynthesis of SM and down-regulation of ACAT activity (Fig. 4). After a 30-min SMase treatment (25 milliunits/ml) and removal of enzyme, [3H]serine-labeled ceramide was gradually converted to SM over a 6-h period (Fig. 4 A). Resynthesis of SM was paralleled by a decline in cholesterol esterification that reached a minimum by 4 h (Fig. 4 B). Similar to the overexpressed rabbit protein, endogenous hamster OSBP was converted from the predominant high molecular weight phosphorylated form to the lower molecular weight dephosphorylated form by exogenous SMase (Fig. 4 C). The proportion of phosphorylated OSBP slowly increased following removal of SMase and by 6 h was similar to the pretreatment distribution. OSBP is phosphorylated on at least five distinct sites on serine residues, and approximately 70% of the phosphates at these sites turnover within 20–30 min (28Ridgway N.D. Badiani K. Byers D.M. Cook H.W. Biochim. Biophys. Acta. 1997; 1390: 37-51Crossref Scopus (20) Google Scholar). Given this rapid phosphorylation cycle for OSBP, SMase hydrolysis could either be inhibiting an OSBP kinase or stimulating dephosphorylation via an okadaic acid-sensitive phosphatase (28Ridgway N.D. Badiani K. Byers D.M. Cook H.W. Biochim. Biophys. Acta. 1997; 1390: 37-51Crossref Scopus (20) Google Scholar), thus resulting in net dephosphorylation. Given the previous reports of protein phosphatase 2A activation by ceramide in vitro (34Dobrowsky R.T. Kamibayashi C. Mumby M.C. Hannun Y.A. J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar, 35Dobrowsky R.T. Hannun Y.A. J. Biol. Chem. 1992; 267: 5048-5051Abstract Full Text PDF PubMed Google Scholar, 36Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1495) Google Scholar), we chose to test whether the okadaic acid-sensitive OSBP phosphatase was stimulated by SMase treatment (Fig. 5). In these experiments, OSBP from overexpressing cells was measured by immunoblotting after SMase treatment and either pre- or post-treatment with okadaic acid. As shown in previous experiments, as little as 10 milliunits/ml SMase promoted dephosphorylation of OSBP, as determined by a shift to the low molecular weight isoform. When cells were pretreated with okadaic acid (500 nm), dephosphorylation was blocked, and OSBP was predominately in the high molecular weight isoform. More importantly, after OSBP was first dephosphorylated with SMase for 60 min, okadaic acid (500 nm) treatment for 30 min was effective in reversing dephosphorylation. We tested if the okadaic acid-sensitive phosphatase could be stimulated to dephosphorylate OSBP in response to short chain analogues of ceramide in CHO cells (Fig. 6). In these experiments, SMase (50 milliunits/ml for 60 min) inhibited [32P]phosphate incorporation into OSBP from overexpressing cells by 80%. However, this effect could not be recapitulated with C2- and C6-ceramides, even at concentrations up to 25 μm for 1 h. Phosphorylation of endogenous OSBP in CHO-K1 cells was also unaffected by treatment with these short chain ceramides under similar conditions (results not shown). We previously reported that OSBP overexpressed in CHO-K1 cells translocated to the Golgi apparatus in response to oxysterols but not LDL or other agents that alter cholesterol homeostasis (25Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (239) Google Scholar). However, this was not confirmed for the endogenous protein due to the lack of a suitable antibody. We developed an affinity-purified antibody (antibody 104) that is capable of detecting endogenous OSBP in CHO-K1 cells and used this antibody to assess changes in localization of OSBP in response to SMase treatment by indirect immunofluorescence (Fig. 7). These experiments were performed on CHO-K1 cells cultured in DMEM with 5% LPDS and supplemented with human LDL. In untreated cells (NO ADDITION) the distribution of OSBP was diffuse and appeared localized to small vesicles, which in some instances were clustered around the nucleus. Following SMase treatment for 45 min, OSBP was associated with a structure that appeared at one pole of the nucleus. BFA disrupted this staining pattern, confirming it as the Golgi apparatus (Ref. 25Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (239) Google Scholar; results not shown). Interestingly, when cells were treated with PLC, which did not promote ACAT activation or hydrolyze SM, OSBP strongly localized to the Golgi apparatus. Similarly, the protein kinase C activator TPA (100 nm) caused OSBP localization to the Golgi complex. SM turnover at the plasma membrane results in the generation of ceramide and possibly other bioactive metabolites such as sphingo
Referência(s)