Ceramide Synthesis Correlates with the Posttranscriptional Regulation of the Sterol-Regulatory Element-Binding Protein
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Department of Pathology (T.S.W.), Institute of Human Nutrition (T.S.W., R.A.J., T.S., R.J.D.), and Department of Pediatrics (R.J.D.), Columbia University, New York, NY.
ABSTRACT
Objective— Sterol-regulatory element-binding proteins (SREBPs) regulate transcription of genes of lipid metabolism. Ceramide decreases transcriptionally active SREBP levels independently of intracellular cholesterol levels. Mechanisms of the ceramide-mediated decrease of SREBP levels were investigated.
Methods and Results— Experiments were performed in Chinese hamster ovary cells. Inhibition of ceramide synthesis with myriocin, cycloserine, or fumonisin decreases levels of transcriptionally active SREBP and reduces SRE-mediated gene transcription. When ceramide synthesis is increased through exogenous sphingosine or inhibition of sphingosine kinase, SRE-mediated gene transcription is increased. The important role of ceramide synthesis in SRE-mediated gene transcription is confirmed in LY-B cells that do not synthesize ceramide de novo. LY-B cells fail to increase SRE-mediated gene transcription in sterol depletion.
Conclusions— Ceramide synthesis correlates with the generation of transcriptionally active SREBP and SRE-mediated gene transcription. Inhibition of ceramide synthesis decreases levels of transcriptionally active SREBP and SRE-mediated gene transcription. It is hypothesized that the process of ongoing ceramide synthesis contributes to the physiological processing of SREBP, perhaps affecting ER-to-Golgi trafficking. Taken together, modification of ceramide synthesis could be a novel target for drug development in the pharmacologic modification of SRE-dependent pathways.
Key Words: SREBP ? ceramide ? myriocin ? LY-Bsphingosine
Introduction
The sterol-regulatory element-binding proteins (SREBPs) are important transcription factors of genes of lipid metabolism. In sterol depletion, precursor SREBP is translocated from the endoplasmic reticulum by vesicular trafficking to the Golgi apparatus,1,2 where the transcriptionally active mature SREBP is generated. Mature SREBP binds to sterol-regulatory elements (SRE), cis-acting elements in the promoters of genes of cholesterol and fatty acid synthesis.3
Cholesterol and unsaturated fatty acids are known regulators of transcriptional and posttranscriptional processing of SREBP. We recently reported that unsaturated fatty acids-mediated decreases in SRE-mediated gene transcription are linked to cellular sphingolipid metabolism.4 Ceramide, a metabolite of sphingomyelin hydrolysis, also regulates mature SREBP levels. Regulation occurs even in the presence of inhibitors of intracellular cholesterol trafficking, suggesting a cholesterol-independent effect.4
Ceramide has multiple roles ranging from lipid second messenger to the induction of apoptosis, cell growth, and differentiation.5,6 Cellular ceramide levels are generated either by de novo synthesis from serine and palmitoyl-CoA or through a recycling pathway of sphingolipid hydrolysis. Ceramide also has a role in intracellular protein trafficking, can inhibit coated vesicle formation and exocytosis in Chinese hamster ovary (CHO) cells,7 and can modulate endocytosis in mammalian cells.8 In yeast, ongoing ceramide synthesis is critical in the vesicular ER-to-Golgi transport of GPI-anchored proteins.9–11
We investigated the effect of ceramide synthesis on SREBP levels and SRE-mediated gene transcription. Because increased ceramide levels decrease mature SREBP protein levels and SRE-mediated gene transcription,4 it was anticipated that inhibition of ceramide synthesis should increase mature SREBP levels and SRE-mediated gene transcription. Contrary to this hypothesis, inhibition of ceramide synthesis decreases SRE-mediated gene transcription. Increasing ceramide synthesis correlates with increased mature SREBP levels and SRE-mediated gene transcription. Consequently, the effect of ceramide on its own synthesis was investigated and shown to be inhibitory, providing an explanation that increased ceramide levels and decreased ceramide synthesis inhibit SRE-mediated gene transcription. The role of ceramide synthesis in SRE-mediated gene transcription is supported by experiments in CHO cells that lack ceramide de novo synthesis (LY-B cells) because of a mutation in a subunit of serine-palmitoyl transferase.12,13 LY-B cells fail to increase SRE-mediated gene transcription when they are cholesterol-depleted. Ceramide increases levels for precursor SREBP but decreases levels for mature SREBP, suggesting a block in the generation of mature SREBP. Our data provide evidence that ceramide synthesis is an important regulatory factor in the maturation cascade of SREBP.
Methods
3H-serine (555 Gbq-1.48 TBq; 0.1 mCi/mmol) and 3H-sphingosine (555 Gbq-1.11 TBq; 0.1 mCi/mL) were purchased from Perkin Elmer (Boston, Mass). CHO cells were obtained from American Type Culture Collection (Rockville, Md). LY-B cells were provided by Dr K. Hanada, National Institutes of Infectious Diseases, Tokyo, Japan.12 Ethanol, fatty acid free bovine serum (BSA), cholesterol, 25-hydroxycholesterol (25-OH cholesterol), and fumonisin B1 were obtained from Sigma, (St. Louis, Mo). D-MAPP (1S,2R)-D-erythro-2-(N-Myristoylamino)-1-phenyl-1-propanol, C6-ceramide (D-erythro hexanoylsphingosine), C8-ceramide (D-erythro N-octanoylsphingosine), C6-dihydroceramide (D-erythro N-hexanoyldihydrosphingosine), NB-DNJ, (N-Butyldeoxynojirimycin-HCl), PPMP (DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol HCl), and DMS (N,N-Dimethylsphingosine) were obtained from Biomol Research Laboratories (Plymouth Meeting, Pa). All cell culture reagents were obtained from Life Technologies (Grand Island, NY).
Plasmids
The pSyn-SRE luciferase reporter plasmid originates from the hamster HMG-CoA synthase promoter and contains three SRE elements (–326 to –225 bp) and has been described before.14,15 The pWLNeo plasmid was obtained from Stratagene (La Jolla, Calif).
Cell Culture and Stable Transfections
Cells were grown in F12-nutrient mixture medium containing 10% fetal bovine serum, 1% glutamine (v/v), 1% penicillin/streptomycin (v/v), and 10% fetal bovine serum (v/v) at 37°C in humidified CO2 (5%). The generation of stable transfectants has been previously described.4
Enzyme Assays
Luciferase activity was analyzed in aliquots of cell extracts using a luminometer (Berthold LB 9501; Wallac, Gaithersburg, Md). Cells were lysed with 0.1% Triton X-100, 50 mmol/L Hepes, 10 mmol/L MgSO4, pH 7.7. Luciferase activity in relative light units was divided by protein content (mg/mL) for each extract.
Protein Determination
Cellular protein was determined by the Biorad method. BSA was used as a standard.
Cell Survival
In all conditions, cell survival was assessed using the 3-(4,5-Dimethylthiazol-2yl)-2 to 5 diphenyltetrazolium bromide (MTT) assay16 and trypan blue exclusion. Cytotoxicity was assessed by measuring LDH release using a colorimetric assay (Roche, Indianapolis, Ind) in which addition of 1% Triton X was used as a positive control. A 3/7 caspase assay17 was used to assess induction of apoptosis and staurosporine was used as a positive control.
Ceramide de Novo Synthesis
Cells growing in the experimental conditions were incubated during the last 1.5 hours with 3H-serine (1 μL/mL) to allow incorporation into ceramide.18,19 Then, cells were washed twice with PBS, 0.2% BSA, twice with phosphate-buffered saline, lysed in 400 μL, 250 mmol/L Tris-Cl, scraped, and transferred to glass tubes. Then, 1 mL of ice-cold methanol, 2 mL of chloroform, and 0.5 mL of 0.1 N HCl was added, vortexed, and spun at 800g for 10 minutes. The organic phase was washed with 3x2 mL 0.001 N HCl. Lipids were dried under N2. Alkaline hydrolysis was performed by incubation in 2 mL of 0.1 N KOH in methanol at 37°C for 1 hour. Lipids were reextracted by adding 2 mL of chloroform and 1.2 mL of balanced salt solution (135 mmol/L NaCl, 4.5 mmol/L KCl, 1.5 mmol/L CaCl2, 0.5 mmol/L MgCl2, 5.6 mmol/L glucose, 10 mmol/L Hepes, pH 7.2)/EDTA 100 mmol/L (1.08 mL/0.12 mL). After vortexing and centrifugation at 800g for 5 minutes, the lower phase was dried under N2.20,21 The extracted lipids were then dissolved in 50 μL chloroform/methanol (1:1), spotted on TLC plates (Merck Silicagel 60; Darmstadt, Germany), and chromatographed with chloroform-methanol 0.22% aqueous CaCl2 (60:35:8 v/v).22 Ceramide and sphingomyelin (dissolved at 1 μg/μL) were run as standards. The TLC plate was cut at the corresponding lipid spots, mixed with scintillation fluid (Ultima Gold; Packard Instrument Company, CT), and analyzed in a scintillation counter (Perkin Elmer Wallac, Gaithersburg, Md). Results were expressed in dpm/mg protein as a percentage of total counts.
Western Blot Analysis
Cells were incubated in control media (1% BSA) or with the respective conditions. After 8 hours, cells were scraped and pelleted at 1000g and resuspended in lysis buffer C (10 mmol/L Tris-Cl, 100 mmol/L NaCl, 1% SDS, pH 7.6) containing protease inhibitor CompleteTM (Roche Pharmaceuticals, Nutley, NJ). Two hours before harvesting, all cells had received 25 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN). An aliquot (30 μg of protein) was electrophoresed on a denaturing 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The monoclonal antibodies against SREBP-1 (BD Biosciences, San Jose, Calif) and actin (Sigma, St. Louis, Mo) and the peroxidase-labeled anti-mouse IgG (Amersham NIF 824) were used for Western blot analysis using the ECL method (Amersham, Arlington Heights, Ill).
Northern Bots
Cells were treated for 8 hours; 30 μg of total RNA were separated by 1.2% denaturing agarose/formaldehyde electrophoresis and transferred to Duralon UV-membranes (Stratagene, La Jolla, Calif). The cDNA probe for HMG-CoA synthase was obtained by RT-PCR using described primers15 and labeled by random priming (Stratagene Prime-It? Random priming labeling kit). The blot was hybridized in Quick-Hyb (Stratagene, La Jolla, Calif).
Data Analysis
Statistical significance was calculated by paired t tests. Results are given as mean±SD. All experiments were repeated on different days at least 3 times and each time in triplicate.
Results
We previously showed that increasing levels of cellular ceramide decreases mature SREBP and SRE-mediated gene transcription.4 We investigated potential mechanisms for this effect.
Increased Exogenous and Endogenous Ceramide Decreases Ceramide Synthesis
Cells were incubated for 8 hours in the presence of C6-ceramide or C8-ceramide (20 μmol/L), DH-C6-ceramide (20 μmol/L), D-MAPP (20 μmol/L), an inhibitor of alkaline ceramidase, or PPMP (20 μmol/L), an inhibitor of glucosylceramide synthesis.23 As a negative control, cells were incubated with NB-DNJ (40 μmol/L), an inhibitor of glucosylceramide synthesis that does not increase ceramide levels.24 For the last 1.5 hours of incubation, 3H-serine was added as a label to determine ceramide synthesis. All conditions, except incubation with NB-DNJ, significantly (P<0.05) decreased ceramide synthesis measured by incorporation of 3H-serine into ceramide (Figure 1).
Figure 1. Exogenous and endogenous ceramides, dihydroceramides, decrease incorporation of 3H serine into de novo synthesized ceramide. CHOs, stable transfectants for an SRE-reporter gene, were treated for 8 hours with control conditions (1% BSA) (negative control) and myriocin (1 μmol/L) (positive control), C6-Cer (20 μmol/L), C8-Cer (20 μmol/L), DHC6-Cer (20 μmol/L), D-MAPP (30 μmol/L), PPMP (20 μmol/L), or NB-DNJ (40 μmol/L). After 6.5 hours, 3H serine (1 μL/mL) was added to each condition to measure incorporation of label into de novo synthesized ceramide within 1.5 hours. All experimental conditions, except NB-DNJ, (which does not increase endogenous ceramide levels) significantly decrease 3H-serine incorporation into ceramide (P<0.05).
Ceramide Increases Levels of Precursor SREBP and Decreases Levels of Mature SREBP
Next, the effect of ceramide on SREBP levels was investigated by Western blot analysis. Incubation of CHO cells over 4 hours and 8 hours with C6-ceramide (20 μmol/L) increased cellular levels of precursor SREBP compared with controls at 4 hours and even more at 8 hours (Figure 2). At the same time, levels of mature SREBP decreased in the presence of C6 ceramide at 4 hours and at 8 hours compared with controls and compared with precursor SREBP of the same cell extract. To assure equal loading of the gel, the membrane was probed for actin.
Figure 2. C6-ceramide increases precursor SREBP levels. CHOs, stable transfectants for an SRE-reporter gene, were incubated for 4 and 8 hours with 1% BSA (control) or C6-ceramide (20 μmol/L). Whole-cell extracts (30 μg protein) were loaded on a 4% to 14% continuous gradient SDS-PAGE gel. P indicates the precursor (125 kd) form of SREBP-1. C6 ceramide increases the precursor form of SREBP-1 and decreases the mature form of SREBP-1. Probing for actin demonstrates equal loading of the samples.
Inhibition of Ceramide de Novo Synthesis Decreases SRE-Mediated Gene Transcription
The effect of decreased ceramide synthesis on SRE-mediated gene transcription was investigated next (Figure 3). CHO cells, stable transfectants for SRE-regulated reporter gene, were incubated for 8 hours with myriocin (1 μmol/L), a specific inhibitor of serine-palmitoyl transferase,25 cycloserine (500 mmol/L), another inhibitor of serine-palmitoyl transferase,26,27 or fumonisin B1 (10 μmol/L), an inhibitor of ceramide synthase.28 All 3 inhibitors significantly reduced SRE-mediated gene transcription by 8 hours (Figure 3). Cells were also incubated with PPMP (20 μmol/L), a glucosyltransferase inhibitor that increases intracellular ceramide levels and decreases ceramide synthesis as measured by incorporation of 3H-serine (Figure 1). As a negative control, cells were incubated with 40 μmol/L NB-DNJ, an inhibitor of glucosylceramide synthase that does not increase ceramide levels24 (Figure 3). The effects of myriocin were dose-dependent (Figure I, available online at http://atvb.ahajournals.org) and reversible within 8 hours and did not decrease the expression of a ?-gal control reporter gene (data not shown). All inhibitors shown did not affect cell survival at the concentrations used.
Figure 3. Inhibition of ceramide synthesis and SRE-mediated gene transcription. Decreased ceramide synthesis correlates with decreased levels of SRE-mediated gene transcription and mature SREBP. CHOs, stable transfectants for an SRE-reporter gene, were incubated for 8 hours in the presence of control conditions (1% BSA), cholesterol and 25-OH cholesterol (10 μg/mL and 1 μg/mL), myriocin (1 μmol/L), cycloserine (500 mmol/L), fumonisin B1 (20 μmol/L), PPMP (20 μmol/L), or NB-DNJ (negative control) (40 μmol/L); n=4 separate experiments. Compared with control, all conditions except NB-DNJ reduce luciferase expression (P<0.05) measured as relative light units (RLU).
Increased Ceramide Synthesis Increases SRE-Mediated Gene Transcription
Sphingosine can increase ceramide synthesis.29–32 Sphingosine levels can also be increased by DMS, an inhibitor of sphingosine-1-phosphate kinase.33,34 Cells were incubated with sphingosine (1.5 μmol/L) or DMS (1.5 to 5 μmol/L) for up to 6 hours. DMS dose-dependently increases SRE-mediated gene transcription (Figure 4) and levels of mature SREBP (Figure 4 inset). Sphingosine increases SRE-mediated gene transcription up to 2-fold (Figure 4). DMS dose-dependently increases incorporation of 3H-sphingosine label into ceramide by 70% within 5 hours (Figure II, available online at http://atvb.ahajournals.org).
Figure 4. Increased ceramide synthesis and SRE-mediated gene transcription. Increased ceramide synthesis correlates with increased SRE-mediated gene transcription. CHOs, stable transfectants for an SRE-reporter gene, were incubated for 6 hours in the presence of DMS (1.5 to 5 μmol/L) or with sphingosine (1.5 μmol/L). Inset, Western blot analysis. CHO cells were incubated for 6 hours with control medium (1% BSA) or DMS (5 μmol/L). Whole-cell extracts (30 μg protein) were loaded on a 4% to 14% continuous gradient SDS-PAGE gel. P and M indicate the precursor (125 kd) and mature (68 kd) forms of SREBP-1 in a representative experiment.
LY-B Cells That Do Not Synthesize Ceramide de Novo Fail to Increase SRE-Mediated Gene Transcription in Sterol Depletion
The role of ongoing ceramide synthesis on SRE-mediated gene transcription was investigated in LY-B cells.12 In LY-B cells, a mutation in the LCB1 subunit of serine-palmitoyl transferase results in a complete lack of enzyme activity and inability to de novo synthesize any sphingolipid species. SRE-mediated gene transcription was first suppressed by incubation for 16 hours in the presence of cholesterol (10 μg/mL) and 25-OH cholesterol (1 μg/mL). Then cells were switched for 6 hours to medium containing 1% BSA. Control cells increased SRE-mediated gene transcription but LY-B cells failed to do so (Figure 5). Control experiments were performed to demonstrate that LY-B cells are able to increase SRE-mediated gene transcription once a precursor for ceramide synthesis is supplied. This time medium was switched to 1% BSA containing 5 μmol/L DMS after previous incubation with cholesterol. Within 6 hours, cells significantly increased SRE-mediated gene transcription. This indicates that when the block in ceramide synthesis is bypassed by DMS, SRE-mediated gene transcription increases as expected, reflecting the physiological response to cellular sterol depletion.
Figure 5. LY-B cells fail to recover SRE-mediated gene transcription in cholesterol depletion but recover SRE-mediated gene transcription in the presence of DMS. LY-B and control CHO-cells, stably transfected with an SRE-reporter gene, were incubated for 16 hours with 1% BSA (control) or in the presence of cholesterol (10 μg/mL) and 25-OH cholesterol (1 μg/mL) to decrease SRE-mediated gene transcription. Then, medium was switched to 1% BSA or DMS (5 μmol/L) for 6 hours. Cholesterol decreases SRE-mediated gene transcription significantly stronger in LY-B cells (white bars) compared with control cells (black bars) (P<0.05). Incubation for 6 hours in the presence of 1% BSA fails to increase SRE-mediated gene transcription in LY-B cells compared with controls (P<0.05). Incubation with DMS (5 μmol/L) significantly (P<0.05) increases SRE-mediated gene transcription in LY-B and controls.
Inhibition of Ceramide de Novo Synthesis Decreases Levels of HMG-CoA Synthase mRNA
Northern blot analysis of HMG-CoA synthase was performed to confirm results obtained with SRE-reporter gene assays. HMG-CoA synthase is sensitively regulated by SREBP.35 We have previously shown that ceramide and D-MAPP decrease HMG-CoA synthase mRNA levels.4 Incubation with myriocin for 16 hours decreases HMG-CoA synthase mRNA levels to less than half (Figure 6).
Figure 6. Myriocin decreases levels of HMG-CoA synthase mRNA. CHO cells were incubated for 8 hours with control medium (1% BSA) or myriocin (1 μmol/L). Then 30 μg of total RNA were electrophoresed on a 1.2% agarose/formaldehyde gel, transferred to a nylon membrane, and hybridized with 32P-labeled probes for HMG-CoA synthase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lane 1 shows control (1% BSA); lane 2, myriocin (1 μmol/L). The level of HMG-CoA synthase mRNA relative to control values was calculated after quantitative phosphorimager analysis and correction of loading differences determined by the glyceraldehyde-3-phosphate dehydrogenase signal.
Discussion
In summary, the present study set out to investigate mechanisms how ceramide decreases transcriptionally active SREBP levels. Several lines of evidence demonstrate a prominent role for ongoing ceramide synthesis in the posttranscriptional regulation of SREBP and that ceramide reduces SREBP processing by decreasing de novo ceramide synthesis. Inhibition of ceramide synthesis results in accumulation of precursor SREBP and decreased mature SREBP protein leading to decreased SRE-dependent gene transcription.
We had previously shown that increasing cellular levels of ceramide decreases SRE-mediated gene transcription independent of cellular cholesterol levels, the classical feedback inhibitor of SRE-mediated gene transcription.4 Therefore, the initial hypothesis was that decreased ceramide synthesis should increase SRE-mediated gene transcription. On the contrary, inhibition of ceramide synthesis decreases SRE-mediated gene transcription (Figure 3) and levels of mature SREBP. Myriocin, a very specific inhibitor of serine-palmitoyl transferase, inhibits SRE-mediated gene transcription even more than cholesterol (Figure 3). Synthesis of ceramide occurs de novo through serine-palmitoyl transferase or through a recycling pathway via sphingosine.32,36 SRE-mediated gene transcription and mature SREBP levels are increased when ceramide synthesis is increased by sphingosine either added exogenously or increased endogenously through inhibition of sphingosine kinase (Figure 4). Taken together, the data suggest that increasing the flux into ceramide synthesis either de novo or through a recycling pathway modifies SREBP proteolysis and SRE-mediated gene transcription. The role of both pathways is demonstrated in LY-B cells. These cells, mutated in a subunit of serine-palmitoyl transferase and unable to synthesize sphingolipids de novo12,13 fail to increase SRE-mediated gene transcription in sterol depletion but recover this ability when sphingosine is present in the incubation medium (Figure 5). Our data do not implicate sphingosine 1-phosphate in SREBP proteolysis because inhibition of sphingosine kinase (DMS) increases SREBP proteolysis, and phosphatidylethanolamine, a metabolite of sphingosine 1-phosphate lyase, does not affect SRE-mediated gene transcription (data not shown).
To explain the finding that decreasing ceramide synthesis (this article) or increasing cellular ceramide levels4 both decrease SRE-mediated gene transcription, we questioned whether ceramide inhibits its own synthesis as shown for short-chain ceramides, dihydroceramides and dihydroceramide analogues.37,38 The data demonstrate that all ceramide analogues and inhibitors that increase intracellular cellular levels (ie, DMAPP, an inhibitor of ceramidase that increases cellular ceramide levels39 or PPMP, an inhibitor of glucosylceramide synthase) decrease ceramide de novo synthesis (Figure 1). Importantly, NB-DNJ, a glucosylceramide synthase inhibitor shown not to increase ceramide levels, equally does not affect ceramide de novo synthesis (Figure 1) or SRE-mediated gene transcription (Figure 3). These data support the role of ceramide in SRE-mediated gene regulation and furthermore suggests that glucosylceramide does not regulate SRE-mediated gene transcription. Therefore, inhibition of its own synthesis can be a mechanism of ceramide-mediated decrease of SRE-mediated gene expression.
Our data show that addition of exogenous ceramide increases levels of precursor SREBP and decreases levels of mature SREBP (Figure 2). This could suggest that the processing of precursor SREBP to mature SREBP is inhibited, potentially at multiple cellular sites. Ceramide synthesis has been shown to be obligatory in the ER to Golgi trafficking in yeast.10,11,40 Of relevance, increased levels of ceramides inhibit the formation of coated vesicles in CHO cells,7 glycoprotein traffic through the secretory pathway,41 and decrease endocytosis in mammalian cells.8 These observations also make it unlikely that the effect of ceramide on SREBP trafficking is unique.
We previously reported that ceramide decreases mature SREBP levels and SRE-mediated gene transcription even in the presence of inhibitors of intracellular cholesterol movement.4 Further evidence of a cholesterol-independent regulation of SREBP is found in Drosophila melanogaster, where SREBP levels are only regulated by palmitic acid and phosphatidylcholine but not by cholesterol or unsaturated fatty acids.42 Palmitic acid determines the rate of long-chain sphinganine synthesis,30 which can be further metabolized to ceramide. Hence, ceramide synthesis may also contribute to SREBP regulation in Drosophila. In mammalian cells, SREBP formation and cleavage occur by a number of metabolic pathways that can be modified by diet or by therapeutic agents. These "regulators" include cholesterol,35,43 fatty acids,15,44,45 and, as we show herein, modification of ceramide synthesis.
Acknowledgments
This work was supported by the National Institutes of Health Grants HL 40404. T. S. Worgall was supported in part by NIH training grant T32DK07715 and a grant-in aid awarded by the American Heart Association (0255656N). T. Seo was supported in part by NIH training grant HL07343–22. R. A. Juliano was supported in part by NIH training grant DK07715. The authors gratefully acknowledge Fannie Keyserman for her excellent technical assistance.
References
DeBose-Boyd RA, Brown MS, Li WP, Nohturfft A, Goldstein JL, Espenshade PJ. Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell. 1999; 99: 703–712.
Nohturfft A, Yabe D, Goldstein JL, Brown MS, Espenshade PJ. Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes. Cell. 2000; 102: 315–323.
Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997; 89: 331–340.
Worgall TS, Johnson RA, Seo T, Gierens H, Deckelbaum RJ. Unsaturated Fatty Acid-mediated Decreases in Sterol Regulatory Element-mediated Gene Transcription Are Linked to Cellular Sphingolipid Metabolism. J Biol Chem. 2002; 277: 3878–3885.
van Meer G, Holthuis JC. Sphingolipid transport in eukaryotic cells. Biochim Biophys Acta. 2000; 1486: 145–170.
Spiegel S, Merrill AH, Jr. Sphingolipid metabolism and cell growth regulation. FASEB J. 1996; 10: 1388–1397.
Abousalham A, Hobman TC, Dewald J, Garbutt M, Brindley DN. Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin-Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor. Biochem J. 2002; 361: 653–661.
Chen CS, Rosenwald AG, Pagano RE. Ceramide as a modulator of endocytosis. J Biol Chem. 1995; 270: 13291–13297.
Sutterlin C, Doering TL, Schimmoller F, Schroder S, Riezman H. Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast. J Cell Sci. 1997; 110: 2703–2714.
Zanolari B, Friant S, Funato K, Sutterlin C, Stevenson BJ, Riezman H. Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae. EMBO J. 2000; 19: 2824–2833.
Horvath A, Sutterlin C, Manning-Krieg U, Movva NR, Riezman H. Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast. EMBO J. 1994; 13: 3687–3695.
Hanada K, Hara T, Fukasawa M, Yamaji A, Umeda M, Nishijima M. Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase. J Biol Chem. 1998; 273: 33787–33794.
Fukasawa M, Nishijima M, Itabe H, Takano T, Hanada K. Reduction of Sphingomyelin Level without Accumulation of Ceramide in Chinese Hamster Ovary Cells Affects Detergent-resistant Membrane Domains and Enhances Cellular Cholesterol Efflux to Methyl-{beta}-cyclodextrin. J Biol Chem. 2000; 275: 34028–34034.
Dooley KA, Millinder S, Osborne TF. Sterol regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene through a direct interaction between sterol regulatory element binding protein and the trimeric CCAAT-binding factor/nuclear factor Y. J Biol Chem. 1998; 273: 1349–1356.
Worgall TS, Sturley SL, Seo T, Osborne TF, Deckelbaum RJ. Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein. J Biol Chem. 1998; 273: 25537–25540.
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987; 47: 936–942.
Carrasco RA, Stamm NB, Patel BK. One-step cellular caspase-3/7 assay. Biotechniques. 2003; 34: 1064–1067.
Gallaher WR, Weinstein DB, Blough HA. Rapid turnover of principal phospholipids in BHK-21 cells. Biochem Biophys Res Commun. 1973; 52: 1252–1256.
Gallaher WR, Blough HA. Synthesis and turnover of lipids in monolayer cultures of BHK-21 cells. Arch Biochem Biophys. 1975; 168: 104–114.
Slotte JP, Bierman EL. Movement of plasma-membrane sterols to the endoplasmic reticulum in cultured cells. Biochem J. 1987; 248: 237–242.
Santana P, Fanjul LF, Ruiz de Galarreta CM. Measurement of sphingomyelin and ceramide cellular levels after sphingomyelinase-mediated sphingomyelin hydrolysis. Methods Mol Biol. 1998; 105: 217–231.
van Echten-Deckert G, Klein A, Linke T, Heinemann T, Weisgerber J, Sandhoff K. Turnover of endogenous ceramide in cultured normal and Farber fibroblasts. J Lipid Res. 1997; 38: 2569–2579.
Abe A, Inokuchi J, Jimbo M, Shimeno H, Nagamatsu A, Shayman JA, Shukla GS, Radin NS. Improved inhibitors of glucosylceramide synthase. J Biochem (Tokyo). 1992; 111: 191–196.
Platt FM, Neises GR, Karlsson GB, Dwek RA, Butters TD. N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing. J Biol Chem. 1994; 269: 27108–27114.
Hanada K, Nishijima M, Fujita T, Kobayashi S. Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant. Biochem Pharmacol. 2000; 59: 1211–1216.
Sundaram KS, Lev M. Inhibition of sphingolipid synthesis by cycloserine in vitro and in vivo. J Neurochem. 1984; 42: 577–581.
Perez-Sala D, Cerdan S, Ballesteros P, Ayuso MS, Parrilla R. Pyruvate decarboxylating action of L-cycloserine. The significance of this in understanding its metabolic inhibitory action. J Biol Chem. 1986; 261: 13969–13972.
Wang E, Norred WP, Bacon CW, Riley RT, Merrill AH, Jr. Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J Biol Chem. 1991; 266: 14486–14490.
Merrill AH, Jr., Wang E. Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s). J Biol Chem. 1986; 261: 3764–3769.
Merrill AH, Jr., Wang E, Mullins RE. Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway. Biochemistry. 1988; 27: 340–345.
Kobayashi T, Shinnoh N, Goto I. Metabolism of free sphingoid bases in murine tissues and in cultured human fibroblasts. Eur J Biochem. 1989; 186: 493–499.
He X, Okino N, Dhami R, Dagan A, Gatt S, Schulze H, Sandhoff K, Schuchman EH. Purification and characterization of recombinant, human acid ceramidase. Catalytic reactions and interactions with acid sphingomyelinase. J Biol Chem. 2003; 278: 32978–32986.
Edsall LC, Van Brocklyn JR, Cuvillier O, Kleuser B, Spiegel S. N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide. Biochemistry. 1998; 37: 12892–12898.
Yatomi Y, Ruan F, Megidish T, Toyokuni T, Hakomori S, Igarashi Y. N,N-dimethylsphingosine inhibition of sphingosine kinase and sphingosine 1-phosphate activity in human platelets. Biochemistry. 1996; 35: 626–633.
Wang X, Sato R, Brown MS, Hua X, Goldstein JL. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 1994; 77: 53–62.
Smith WL, Merrill AH, Jr. Sphingolipid metabolism and signaling minireview series. J Biol Chem. 2002; 277: 25841–25842.
van Echten-Deckert G, Giannis A, Schwarz A, Futerman AH, Sandhoff K. 1-Methylthiodihydroceramide, a novel analog of dihydroceramide, stimulates sphinganine degradation resulting in decreased de novo sphingolipid biosynthesis. J Biol Chem. 1998; 273: 1184–1191.
Ridgway ND, Merriam DL. Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells. Biochim Biophys Acta. 1995; 1256: 57–70.
Bielawska A, Greenberg MS, Perry D, Jayadev S, Shayman JA, McKay C, Hannun YA. (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase. J Biol Chem. 1996; 271: 12646–12654.
Muniz M, Morsomme P, Riezman H. Protein sorting upon exit from the endoplasmic reticulum. Cell. 2001; 104: 313–320.
Rosenwald AG, Pagano RE. Inhibition of glycoprotein traffic through the secretory pathway by ceramide. J Biol Chem. 1993; 268: 4577–4579.
Seegmiller AC, Dobrosotskaya I, Goldstein JL, Ho YK, Brown MS, Rawson RB. The SREBP pathway in Drosophila: regulation by palmitate, not sterols. Dev Cell. 2002; 2: 229–238.
Edwards PA, Tabor D, Kast HR, Venkateswaran A. Regulation of gene expression by SREBP and SCAP. Biochim Biophys Acta. 2000; 1529: 103–113.
Ou J, Tu H, Shan B, Luk A, DeBose-Boyd RA, Bashmakov Y, Goldstein JL, Brown MS. Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR. Proc Natl Acad Sci U S A. 2001; 98: 6027–6032.
Hannah VC, Ou J, Luong A, Goldstein JL, Brown MS. Unsaturated Fatty Acids Down-Regulate SREBP Isoforms 1a and 1c by Two Mechanisms in HEK-293 Cells. J Biol Chem. 2000; 276: 4365–4372.(Tilla S. Worgall; Rebecca)
ABSTRACT
Objective— Sterol-regulatory element-binding proteins (SREBPs) regulate transcription of genes of lipid metabolism. Ceramide decreases transcriptionally active SREBP levels independently of intracellular cholesterol levels. Mechanisms of the ceramide-mediated decrease of SREBP levels were investigated.
Methods and Results— Experiments were performed in Chinese hamster ovary cells. Inhibition of ceramide synthesis with myriocin, cycloserine, or fumonisin decreases levels of transcriptionally active SREBP and reduces SRE-mediated gene transcription. When ceramide synthesis is increased through exogenous sphingosine or inhibition of sphingosine kinase, SRE-mediated gene transcription is increased. The important role of ceramide synthesis in SRE-mediated gene transcription is confirmed in LY-B cells that do not synthesize ceramide de novo. LY-B cells fail to increase SRE-mediated gene transcription in sterol depletion.
Conclusions— Ceramide synthesis correlates with the generation of transcriptionally active SREBP and SRE-mediated gene transcription. Inhibition of ceramide synthesis decreases levels of transcriptionally active SREBP and SRE-mediated gene transcription. It is hypothesized that the process of ongoing ceramide synthesis contributes to the physiological processing of SREBP, perhaps affecting ER-to-Golgi trafficking. Taken together, modification of ceramide synthesis could be a novel target for drug development in the pharmacologic modification of SRE-dependent pathways.
Key Words: SREBP ? ceramide ? myriocin ? LY-Bsphingosine
Introduction
The sterol-regulatory element-binding proteins (SREBPs) are important transcription factors of genes of lipid metabolism. In sterol depletion, precursor SREBP is translocated from the endoplasmic reticulum by vesicular trafficking to the Golgi apparatus,1,2 where the transcriptionally active mature SREBP is generated. Mature SREBP binds to sterol-regulatory elements (SRE), cis-acting elements in the promoters of genes of cholesterol and fatty acid synthesis.3
Cholesterol and unsaturated fatty acids are known regulators of transcriptional and posttranscriptional processing of SREBP. We recently reported that unsaturated fatty acids-mediated decreases in SRE-mediated gene transcription are linked to cellular sphingolipid metabolism.4 Ceramide, a metabolite of sphingomyelin hydrolysis, also regulates mature SREBP levels. Regulation occurs even in the presence of inhibitors of intracellular cholesterol trafficking, suggesting a cholesterol-independent effect.4
Ceramide has multiple roles ranging from lipid second messenger to the induction of apoptosis, cell growth, and differentiation.5,6 Cellular ceramide levels are generated either by de novo synthesis from serine and palmitoyl-CoA or through a recycling pathway of sphingolipid hydrolysis. Ceramide also has a role in intracellular protein trafficking, can inhibit coated vesicle formation and exocytosis in Chinese hamster ovary (CHO) cells,7 and can modulate endocytosis in mammalian cells.8 In yeast, ongoing ceramide synthesis is critical in the vesicular ER-to-Golgi transport of GPI-anchored proteins.9–11
We investigated the effect of ceramide synthesis on SREBP levels and SRE-mediated gene transcription. Because increased ceramide levels decrease mature SREBP protein levels and SRE-mediated gene transcription,4 it was anticipated that inhibition of ceramide synthesis should increase mature SREBP levels and SRE-mediated gene transcription. Contrary to this hypothesis, inhibition of ceramide synthesis decreases SRE-mediated gene transcription. Increasing ceramide synthesis correlates with increased mature SREBP levels and SRE-mediated gene transcription. Consequently, the effect of ceramide on its own synthesis was investigated and shown to be inhibitory, providing an explanation that increased ceramide levels and decreased ceramide synthesis inhibit SRE-mediated gene transcription. The role of ceramide synthesis in SRE-mediated gene transcription is supported by experiments in CHO cells that lack ceramide de novo synthesis (LY-B cells) because of a mutation in a subunit of serine-palmitoyl transferase.12,13 LY-B cells fail to increase SRE-mediated gene transcription when they are cholesterol-depleted. Ceramide increases levels for precursor SREBP but decreases levels for mature SREBP, suggesting a block in the generation of mature SREBP. Our data provide evidence that ceramide synthesis is an important regulatory factor in the maturation cascade of SREBP.
Methods
3H-serine (555 Gbq-1.48 TBq; 0.1 mCi/mmol) and 3H-sphingosine (555 Gbq-1.11 TBq; 0.1 mCi/mL) were purchased from Perkin Elmer (Boston, Mass). CHO cells were obtained from American Type Culture Collection (Rockville, Md). LY-B cells were provided by Dr K. Hanada, National Institutes of Infectious Diseases, Tokyo, Japan.12 Ethanol, fatty acid free bovine serum (BSA), cholesterol, 25-hydroxycholesterol (25-OH cholesterol), and fumonisin B1 were obtained from Sigma, (St. Louis, Mo). D-MAPP (1S,2R)-D-erythro-2-(N-Myristoylamino)-1-phenyl-1-propanol, C6-ceramide (D-erythro hexanoylsphingosine), C8-ceramide (D-erythro N-octanoylsphingosine), C6-dihydroceramide (D-erythro N-hexanoyldihydrosphingosine), NB-DNJ, (N-Butyldeoxynojirimycin-HCl), PPMP (DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol HCl), and DMS (N,N-Dimethylsphingosine) were obtained from Biomol Research Laboratories (Plymouth Meeting, Pa). All cell culture reagents were obtained from Life Technologies (Grand Island, NY).
Plasmids
The pSyn-SRE luciferase reporter plasmid originates from the hamster HMG-CoA synthase promoter and contains three SRE elements (–326 to –225 bp) and has been described before.14,15 The pWLNeo plasmid was obtained from Stratagene (La Jolla, Calif).
Cell Culture and Stable Transfections
Cells were grown in F12-nutrient mixture medium containing 10% fetal bovine serum, 1% glutamine (v/v), 1% penicillin/streptomycin (v/v), and 10% fetal bovine serum (v/v) at 37°C in humidified CO2 (5%). The generation of stable transfectants has been previously described.4
Enzyme Assays
Luciferase activity was analyzed in aliquots of cell extracts using a luminometer (Berthold LB 9501; Wallac, Gaithersburg, Md). Cells were lysed with 0.1% Triton X-100, 50 mmol/L Hepes, 10 mmol/L MgSO4, pH 7.7. Luciferase activity in relative light units was divided by protein content (mg/mL) for each extract.
Protein Determination
Cellular protein was determined by the Biorad method. BSA was used as a standard.
Cell Survival
In all conditions, cell survival was assessed using the 3-(4,5-Dimethylthiazol-2yl)-2 to 5 diphenyltetrazolium bromide (MTT) assay16 and trypan blue exclusion. Cytotoxicity was assessed by measuring LDH release using a colorimetric assay (Roche, Indianapolis, Ind) in which addition of 1% Triton X was used as a positive control. A 3/7 caspase assay17 was used to assess induction of apoptosis and staurosporine was used as a positive control.
Ceramide de Novo Synthesis
Cells growing in the experimental conditions were incubated during the last 1.5 hours with 3H-serine (1 μL/mL) to allow incorporation into ceramide.18,19 Then, cells were washed twice with PBS, 0.2% BSA, twice with phosphate-buffered saline, lysed in 400 μL, 250 mmol/L Tris-Cl, scraped, and transferred to glass tubes. Then, 1 mL of ice-cold methanol, 2 mL of chloroform, and 0.5 mL of 0.1 N HCl was added, vortexed, and spun at 800g for 10 minutes. The organic phase was washed with 3x2 mL 0.001 N HCl. Lipids were dried under N2. Alkaline hydrolysis was performed by incubation in 2 mL of 0.1 N KOH in methanol at 37°C for 1 hour. Lipids were reextracted by adding 2 mL of chloroform and 1.2 mL of balanced salt solution (135 mmol/L NaCl, 4.5 mmol/L KCl, 1.5 mmol/L CaCl2, 0.5 mmol/L MgCl2, 5.6 mmol/L glucose, 10 mmol/L Hepes, pH 7.2)/EDTA 100 mmol/L (1.08 mL/0.12 mL). After vortexing and centrifugation at 800g for 5 minutes, the lower phase was dried under N2.20,21 The extracted lipids were then dissolved in 50 μL chloroform/methanol (1:1), spotted on TLC plates (Merck Silicagel 60; Darmstadt, Germany), and chromatographed with chloroform-methanol 0.22% aqueous CaCl2 (60:35:8 v/v).22 Ceramide and sphingomyelin (dissolved at 1 μg/μL) were run as standards. The TLC plate was cut at the corresponding lipid spots, mixed with scintillation fluid (Ultima Gold; Packard Instrument Company, CT), and analyzed in a scintillation counter (Perkin Elmer Wallac, Gaithersburg, Md). Results were expressed in dpm/mg protein as a percentage of total counts.
Western Blot Analysis
Cells were incubated in control media (1% BSA) or with the respective conditions. After 8 hours, cells were scraped and pelleted at 1000g and resuspended in lysis buffer C (10 mmol/L Tris-Cl, 100 mmol/L NaCl, 1% SDS, pH 7.6) containing protease inhibitor CompleteTM (Roche Pharmaceuticals, Nutley, NJ). Two hours before harvesting, all cells had received 25 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN). An aliquot (30 μg of protein) was electrophoresed on a denaturing 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The monoclonal antibodies against SREBP-1 (BD Biosciences, San Jose, Calif) and actin (Sigma, St. Louis, Mo) and the peroxidase-labeled anti-mouse IgG (Amersham NIF 824) were used for Western blot analysis using the ECL method (Amersham, Arlington Heights, Ill).
Northern Bots
Cells were treated for 8 hours; 30 μg of total RNA were separated by 1.2% denaturing agarose/formaldehyde electrophoresis and transferred to Duralon UV-membranes (Stratagene, La Jolla, Calif). The cDNA probe for HMG-CoA synthase was obtained by RT-PCR using described primers15 and labeled by random priming (Stratagene Prime-It? Random priming labeling kit). The blot was hybridized in Quick-Hyb (Stratagene, La Jolla, Calif).
Data Analysis
Statistical significance was calculated by paired t tests. Results are given as mean±SD. All experiments were repeated on different days at least 3 times and each time in triplicate.
Results
We previously showed that increasing levels of cellular ceramide decreases mature SREBP and SRE-mediated gene transcription.4 We investigated potential mechanisms for this effect.
Increased Exogenous and Endogenous Ceramide Decreases Ceramide Synthesis
Cells were incubated for 8 hours in the presence of C6-ceramide or C8-ceramide (20 μmol/L), DH-C6-ceramide (20 μmol/L), D-MAPP (20 μmol/L), an inhibitor of alkaline ceramidase, or PPMP (20 μmol/L), an inhibitor of glucosylceramide synthesis.23 As a negative control, cells were incubated with NB-DNJ (40 μmol/L), an inhibitor of glucosylceramide synthesis that does not increase ceramide levels.24 For the last 1.5 hours of incubation, 3H-serine was added as a label to determine ceramide synthesis. All conditions, except incubation with NB-DNJ, significantly (P<0.05) decreased ceramide synthesis measured by incorporation of 3H-serine into ceramide (Figure 1).
Figure 1. Exogenous and endogenous ceramides, dihydroceramides, decrease incorporation of 3H serine into de novo synthesized ceramide. CHOs, stable transfectants for an SRE-reporter gene, were treated for 8 hours with control conditions (1% BSA) (negative control) and myriocin (1 μmol/L) (positive control), C6-Cer (20 μmol/L), C8-Cer (20 μmol/L), DHC6-Cer (20 μmol/L), D-MAPP (30 μmol/L), PPMP (20 μmol/L), or NB-DNJ (40 μmol/L). After 6.5 hours, 3H serine (1 μL/mL) was added to each condition to measure incorporation of label into de novo synthesized ceramide within 1.5 hours. All experimental conditions, except NB-DNJ, (which does not increase endogenous ceramide levels) significantly decrease 3H-serine incorporation into ceramide (P<0.05).
Ceramide Increases Levels of Precursor SREBP and Decreases Levels of Mature SREBP
Next, the effect of ceramide on SREBP levels was investigated by Western blot analysis. Incubation of CHO cells over 4 hours and 8 hours with C6-ceramide (20 μmol/L) increased cellular levels of precursor SREBP compared with controls at 4 hours and even more at 8 hours (Figure 2). At the same time, levels of mature SREBP decreased in the presence of C6 ceramide at 4 hours and at 8 hours compared with controls and compared with precursor SREBP of the same cell extract. To assure equal loading of the gel, the membrane was probed for actin.
Figure 2. C6-ceramide increases precursor SREBP levels. CHOs, stable transfectants for an SRE-reporter gene, were incubated for 4 and 8 hours with 1% BSA (control) or C6-ceramide (20 μmol/L). Whole-cell extracts (30 μg protein) were loaded on a 4% to 14% continuous gradient SDS-PAGE gel. P indicates the precursor (125 kd) form of SREBP-1. C6 ceramide increases the precursor form of SREBP-1 and decreases the mature form of SREBP-1. Probing for actin demonstrates equal loading of the samples.
Inhibition of Ceramide de Novo Synthesis Decreases SRE-Mediated Gene Transcription
The effect of decreased ceramide synthesis on SRE-mediated gene transcription was investigated next (Figure 3). CHO cells, stable transfectants for SRE-regulated reporter gene, were incubated for 8 hours with myriocin (1 μmol/L), a specific inhibitor of serine-palmitoyl transferase,25 cycloserine (500 mmol/L), another inhibitor of serine-palmitoyl transferase,26,27 or fumonisin B1 (10 μmol/L), an inhibitor of ceramide synthase.28 All 3 inhibitors significantly reduced SRE-mediated gene transcription by 8 hours (Figure 3). Cells were also incubated with PPMP (20 μmol/L), a glucosyltransferase inhibitor that increases intracellular ceramide levels and decreases ceramide synthesis as measured by incorporation of 3H-serine (Figure 1). As a negative control, cells were incubated with 40 μmol/L NB-DNJ, an inhibitor of glucosylceramide synthase that does not increase ceramide levels24 (Figure 3). The effects of myriocin were dose-dependent (Figure I, available online at http://atvb.ahajournals.org) and reversible within 8 hours and did not decrease the expression of a ?-gal control reporter gene (data not shown). All inhibitors shown did not affect cell survival at the concentrations used.
Figure 3. Inhibition of ceramide synthesis and SRE-mediated gene transcription. Decreased ceramide synthesis correlates with decreased levels of SRE-mediated gene transcription and mature SREBP. CHOs, stable transfectants for an SRE-reporter gene, were incubated for 8 hours in the presence of control conditions (1% BSA), cholesterol and 25-OH cholesterol (10 μg/mL and 1 μg/mL), myriocin (1 μmol/L), cycloserine (500 mmol/L), fumonisin B1 (20 μmol/L), PPMP (20 μmol/L), or NB-DNJ (negative control) (40 μmol/L); n=4 separate experiments. Compared with control, all conditions except NB-DNJ reduce luciferase expression (P<0.05) measured as relative light units (RLU).
Increased Ceramide Synthesis Increases SRE-Mediated Gene Transcription
Sphingosine can increase ceramide synthesis.29–32 Sphingosine levels can also be increased by DMS, an inhibitor of sphingosine-1-phosphate kinase.33,34 Cells were incubated with sphingosine (1.5 μmol/L) or DMS (1.5 to 5 μmol/L) for up to 6 hours. DMS dose-dependently increases SRE-mediated gene transcription (Figure 4) and levels of mature SREBP (Figure 4 inset). Sphingosine increases SRE-mediated gene transcription up to 2-fold (Figure 4). DMS dose-dependently increases incorporation of 3H-sphingosine label into ceramide by 70% within 5 hours (Figure II, available online at http://atvb.ahajournals.org).
Figure 4. Increased ceramide synthesis and SRE-mediated gene transcription. Increased ceramide synthesis correlates with increased SRE-mediated gene transcription. CHOs, stable transfectants for an SRE-reporter gene, were incubated for 6 hours in the presence of DMS (1.5 to 5 μmol/L) or with sphingosine (1.5 μmol/L). Inset, Western blot analysis. CHO cells were incubated for 6 hours with control medium (1% BSA) or DMS (5 μmol/L). Whole-cell extracts (30 μg protein) were loaded on a 4% to 14% continuous gradient SDS-PAGE gel. P and M indicate the precursor (125 kd) and mature (68 kd) forms of SREBP-1 in a representative experiment.
LY-B Cells That Do Not Synthesize Ceramide de Novo Fail to Increase SRE-Mediated Gene Transcription in Sterol Depletion
The role of ongoing ceramide synthesis on SRE-mediated gene transcription was investigated in LY-B cells.12 In LY-B cells, a mutation in the LCB1 subunit of serine-palmitoyl transferase results in a complete lack of enzyme activity and inability to de novo synthesize any sphingolipid species. SRE-mediated gene transcription was first suppressed by incubation for 16 hours in the presence of cholesterol (10 μg/mL) and 25-OH cholesterol (1 μg/mL). Then cells were switched for 6 hours to medium containing 1% BSA. Control cells increased SRE-mediated gene transcription but LY-B cells failed to do so (Figure 5). Control experiments were performed to demonstrate that LY-B cells are able to increase SRE-mediated gene transcription once a precursor for ceramide synthesis is supplied. This time medium was switched to 1% BSA containing 5 μmol/L DMS after previous incubation with cholesterol. Within 6 hours, cells significantly increased SRE-mediated gene transcription. This indicates that when the block in ceramide synthesis is bypassed by DMS, SRE-mediated gene transcription increases as expected, reflecting the physiological response to cellular sterol depletion.
Figure 5. LY-B cells fail to recover SRE-mediated gene transcription in cholesterol depletion but recover SRE-mediated gene transcription in the presence of DMS. LY-B and control CHO-cells, stably transfected with an SRE-reporter gene, were incubated for 16 hours with 1% BSA (control) or in the presence of cholesterol (10 μg/mL) and 25-OH cholesterol (1 μg/mL) to decrease SRE-mediated gene transcription. Then, medium was switched to 1% BSA or DMS (5 μmol/L) for 6 hours. Cholesterol decreases SRE-mediated gene transcription significantly stronger in LY-B cells (white bars) compared with control cells (black bars) (P<0.05). Incubation for 6 hours in the presence of 1% BSA fails to increase SRE-mediated gene transcription in LY-B cells compared with controls (P<0.05). Incubation with DMS (5 μmol/L) significantly (P<0.05) increases SRE-mediated gene transcription in LY-B and controls.
Inhibition of Ceramide de Novo Synthesis Decreases Levels of HMG-CoA Synthase mRNA
Northern blot analysis of HMG-CoA synthase was performed to confirm results obtained with SRE-reporter gene assays. HMG-CoA synthase is sensitively regulated by SREBP.35 We have previously shown that ceramide and D-MAPP decrease HMG-CoA synthase mRNA levels.4 Incubation with myriocin for 16 hours decreases HMG-CoA synthase mRNA levels to less than half (Figure 6).
Figure 6. Myriocin decreases levels of HMG-CoA synthase mRNA. CHO cells were incubated for 8 hours with control medium (1% BSA) or myriocin (1 μmol/L). Then 30 μg of total RNA were electrophoresed on a 1.2% agarose/formaldehyde gel, transferred to a nylon membrane, and hybridized with 32P-labeled probes for HMG-CoA synthase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lane 1 shows control (1% BSA); lane 2, myriocin (1 μmol/L). The level of HMG-CoA synthase mRNA relative to control values was calculated after quantitative phosphorimager analysis and correction of loading differences determined by the glyceraldehyde-3-phosphate dehydrogenase signal.
Discussion
In summary, the present study set out to investigate mechanisms how ceramide decreases transcriptionally active SREBP levels. Several lines of evidence demonstrate a prominent role for ongoing ceramide synthesis in the posttranscriptional regulation of SREBP and that ceramide reduces SREBP processing by decreasing de novo ceramide synthesis. Inhibition of ceramide synthesis results in accumulation of precursor SREBP and decreased mature SREBP protein leading to decreased SRE-dependent gene transcription.
We had previously shown that increasing cellular levels of ceramide decreases SRE-mediated gene transcription independent of cellular cholesterol levels, the classical feedback inhibitor of SRE-mediated gene transcription.4 Therefore, the initial hypothesis was that decreased ceramide synthesis should increase SRE-mediated gene transcription. On the contrary, inhibition of ceramide synthesis decreases SRE-mediated gene transcription (Figure 3) and levels of mature SREBP. Myriocin, a very specific inhibitor of serine-palmitoyl transferase, inhibits SRE-mediated gene transcription even more than cholesterol (Figure 3). Synthesis of ceramide occurs de novo through serine-palmitoyl transferase or through a recycling pathway via sphingosine.32,36 SRE-mediated gene transcription and mature SREBP levels are increased when ceramide synthesis is increased by sphingosine either added exogenously or increased endogenously through inhibition of sphingosine kinase (Figure 4). Taken together, the data suggest that increasing the flux into ceramide synthesis either de novo or through a recycling pathway modifies SREBP proteolysis and SRE-mediated gene transcription. The role of both pathways is demonstrated in LY-B cells. These cells, mutated in a subunit of serine-palmitoyl transferase and unable to synthesize sphingolipids de novo12,13 fail to increase SRE-mediated gene transcription in sterol depletion but recover this ability when sphingosine is present in the incubation medium (Figure 5). Our data do not implicate sphingosine 1-phosphate in SREBP proteolysis because inhibition of sphingosine kinase (DMS) increases SREBP proteolysis, and phosphatidylethanolamine, a metabolite of sphingosine 1-phosphate lyase, does not affect SRE-mediated gene transcription (data not shown).
To explain the finding that decreasing ceramide synthesis (this article) or increasing cellular ceramide levels4 both decrease SRE-mediated gene transcription, we questioned whether ceramide inhibits its own synthesis as shown for short-chain ceramides, dihydroceramides and dihydroceramide analogues.37,38 The data demonstrate that all ceramide analogues and inhibitors that increase intracellular cellular levels (ie, DMAPP, an inhibitor of ceramidase that increases cellular ceramide levels39 or PPMP, an inhibitor of glucosylceramide synthase) decrease ceramide de novo synthesis (Figure 1). Importantly, NB-DNJ, a glucosylceramide synthase inhibitor shown not to increase ceramide levels, equally does not affect ceramide de novo synthesis (Figure 1) or SRE-mediated gene transcription (Figure 3). These data support the role of ceramide in SRE-mediated gene regulation and furthermore suggests that glucosylceramide does not regulate SRE-mediated gene transcription. Therefore, inhibition of its own synthesis can be a mechanism of ceramide-mediated decrease of SRE-mediated gene expression.
Our data show that addition of exogenous ceramide increases levels of precursor SREBP and decreases levels of mature SREBP (Figure 2). This could suggest that the processing of precursor SREBP to mature SREBP is inhibited, potentially at multiple cellular sites. Ceramide synthesis has been shown to be obligatory in the ER to Golgi trafficking in yeast.10,11,40 Of relevance, increased levels of ceramides inhibit the formation of coated vesicles in CHO cells,7 glycoprotein traffic through the secretory pathway,41 and decrease endocytosis in mammalian cells.8 These observations also make it unlikely that the effect of ceramide on SREBP trafficking is unique.
We previously reported that ceramide decreases mature SREBP levels and SRE-mediated gene transcription even in the presence of inhibitors of intracellular cholesterol movement.4 Further evidence of a cholesterol-independent regulation of SREBP is found in Drosophila melanogaster, where SREBP levels are only regulated by palmitic acid and phosphatidylcholine but not by cholesterol or unsaturated fatty acids.42 Palmitic acid determines the rate of long-chain sphinganine synthesis,30 which can be further metabolized to ceramide. Hence, ceramide synthesis may also contribute to SREBP regulation in Drosophila. In mammalian cells, SREBP formation and cleavage occur by a number of metabolic pathways that can be modified by diet or by therapeutic agents. These "regulators" include cholesterol,35,43 fatty acids,15,44,45 and, as we show herein, modification of ceramide synthesis.
Acknowledgments
This work was supported by the National Institutes of Health Grants HL 40404. T. S. Worgall was supported in part by NIH training grant T32DK07715 and a grant-in aid awarded by the American Heart Association (0255656N). T. Seo was supported in part by NIH training grant HL07343–22. R. A. Juliano was supported in part by NIH training grant DK07715. The authors gratefully acknowledge Fannie Keyserman for her excellent technical assistance.
References
DeBose-Boyd RA, Brown MS, Li WP, Nohturfft A, Goldstein JL, Espenshade PJ. Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell. 1999; 99: 703–712.
Nohturfft A, Yabe D, Goldstein JL, Brown MS, Espenshade PJ. Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes. Cell. 2000; 102: 315–323.
Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997; 89: 331–340.
Worgall TS, Johnson RA, Seo T, Gierens H, Deckelbaum RJ. Unsaturated Fatty Acid-mediated Decreases in Sterol Regulatory Element-mediated Gene Transcription Are Linked to Cellular Sphingolipid Metabolism. J Biol Chem. 2002; 277: 3878–3885.
van Meer G, Holthuis JC. Sphingolipid transport in eukaryotic cells. Biochim Biophys Acta. 2000; 1486: 145–170.
Spiegel S, Merrill AH, Jr. Sphingolipid metabolism and cell growth regulation. FASEB J. 1996; 10: 1388–1397.
Abousalham A, Hobman TC, Dewald J, Garbutt M, Brindley DN. Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin-Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor. Biochem J. 2002; 361: 653–661.
Chen CS, Rosenwald AG, Pagano RE. Ceramide as a modulator of endocytosis. J Biol Chem. 1995; 270: 13291–13297.
Sutterlin C, Doering TL, Schimmoller F, Schroder S, Riezman H. Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast. J Cell Sci. 1997; 110: 2703–2714.
Zanolari B, Friant S, Funato K, Sutterlin C, Stevenson BJ, Riezman H. Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae. EMBO J. 2000; 19: 2824–2833.
Horvath A, Sutterlin C, Manning-Krieg U, Movva NR, Riezman H. Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast. EMBO J. 1994; 13: 3687–3695.
Hanada K, Hara T, Fukasawa M, Yamaji A, Umeda M, Nishijima M. Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase. J Biol Chem. 1998; 273: 33787–33794.
Fukasawa M, Nishijima M, Itabe H, Takano T, Hanada K. Reduction of Sphingomyelin Level without Accumulation of Ceramide in Chinese Hamster Ovary Cells Affects Detergent-resistant Membrane Domains and Enhances Cellular Cholesterol Efflux to Methyl-{beta}-cyclodextrin. J Biol Chem. 2000; 275: 34028–34034.
Dooley KA, Millinder S, Osborne TF. Sterol regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene through a direct interaction between sterol regulatory element binding protein and the trimeric CCAAT-binding factor/nuclear factor Y. J Biol Chem. 1998; 273: 1349–1356.
Worgall TS, Sturley SL, Seo T, Osborne TF, Deckelbaum RJ. Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein. J Biol Chem. 1998; 273: 25537–25540.
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987; 47: 936–942.
Carrasco RA, Stamm NB, Patel BK. One-step cellular caspase-3/7 assay. Biotechniques. 2003; 34: 1064–1067.
Gallaher WR, Weinstein DB, Blough HA. Rapid turnover of principal phospholipids in BHK-21 cells. Biochem Biophys Res Commun. 1973; 52: 1252–1256.
Gallaher WR, Blough HA. Synthesis and turnover of lipids in monolayer cultures of BHK-21 cells. Arch Biochem Biophys. 1975; 168: 104–114.
Slotte JP, Bierman EL. Movement of plasma-membrane sterols to the endoplasmic reticulum in cultured cells. Biochem J. 1987; 248: 237–242.
Santana P, Fanjul LF, Ruiz de Galarreta CM. Measurement of sphingomyelin and ceramide cellular levels after sphingomyelinase-mediated sphingomyelin hydrolysis. Methods Mol Biol. 1998; 105: 217–231.
van Echten-Deckert G, Klein A, Linke T, Heinemann T, Weisgerber J, Sandhoff K. Turnover of endogenous ceramide in cultured normal and Farber fibroblasts. J Lipid Res. 1997; 38: 2569–2579.
Abe A, Inokuchi J, Jimbo M, Shimeno H, Nagamatsu A, Shayman JA, Shukla GS, Radin NS. Improved inhibitors of glucosylceramide synthase. J Biochem (Tokyo). 1992; 111: 191–196.
Platt FM, Neises GR, Karlsson GB, Dwek RA, Butters TD. N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing. J Biol Chem. 1994; 269: 27108–27114.
Hanada K, Nishijima M, Fujita T, Kobayashi S. Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant. Biochem Pharmacol. 2000; 59: 1211–1216.
Sundaram KS, Lev M. Inhibition of sphingolipid synthesis by cycloserine in vitro and in vivo. J Neurochem. 1984; 42: 577–581.
Perez-Sala D, Cerdan S, Ballesteros P, Ayuso MS, Parrilla R. Pyruvate decarboxylating action of L-cycloserine. The significance of this in understanding its metabolic inhibitory action. J Biol Chem. 1986; 261: 13969–13972.
Wang E, Norred WP, Bacon CW, Riley RT, Merrill AH, Jr. Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J Biol Chem. 1991; 266: 14486–14490.
Merrill AH, Jr., Wang E. Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s). J Biol Chem. 1986; 261: 3764–3769.
Merrill AH, Jr., Wang E, Mullins RE. Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway. Biochemistry. 1988; 27: 340–345.
Kobayashi T, Shinnoh N, Goto I. Metabolism of free sphingoid bases in murine tissues and in cultured human fibroblasts. Eur J Biochem. 1989; 186: 493–499.
He X, Okino N, Dhami R, Dagan A, Gatt S, Schulze H, Sandhoff K, Schuchman EH. Purification and characterization of recombinant, human acid ceramidase. Catalytic reactions and interactions with acid sphingomyelinase. J Biol Chem. 2003; 278: 32978–32986.
Edsall LC, Van Brocklyn JR, Cuvillier O, Kleuser B, Spiegel S. N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide. Biochemistry. 1998; 37: 12892–12898.
Yatomi Y, Ruan F, Megidish T, Toyokuni T, Hakomori S, Igarashi Y. N,N-dimethylsphingosine inhibition of sphingosine kinase and sphingosine 1-phosphate activity in human platelets. Biochemistry. 1996; 35: 626–633.
Wang X, Sato R, Brown MS, Hua X, Goldstein JL. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 1994; 77: 53–62.
Smith WL, Merrill AH, Jr. Sphingolipid metabolism and signaling minireview series. J Biol Chem. 2002; 277: 25841–25842.
van Echten-Deckert G, Giannis A, Schwarz A, Futerman AH, Sandhoff K. 1-Methylthiodihydroceramide, a novel analog of dihydroceramide, stimulates sphinganine degradation resulting in decreased de novo sphingolipid biosynthesis. J Biol Chem. 1998; 273: 1184–1191.
Ridgway ND, Merriam DL. Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells. Biochim Biophys Acta. 1995; 1256: 57–70.
Bielawska A, Greenberg MS, Perry D, Jayadev S, Shayman JA, McKay C, Hannun YA. (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase. J Biol Chem. 1996; 271: 12646–12654.
Muniz M, Morsomme P, Riezman H. Protein sorting upon exit from the endoplasmic reticulum. Cell. 2001; 104: 313–320.
Rosenwald AG, Pagano RE. Inhibition of glycoprotein traffic through the secretory pathway by ceramide. J Biol Chem. 1993; 268: 4577–4579.
Seegmiller AC, Dobrosotskaya I, Goldstein JL, Ho YK, Brown MS, Rawson RB. The SREBP pathway in Drosophila: regulation by palmitate, not sterols. Dev Cell. 2002; 2: 229–238.
Edwards PA, Tabor D, Kast HR, Venkateswaran A. Regulation of gene expression by SREBP and SCAP. Biochim Biophys Acta. 2000; 1529: 103–113.
Ou J, Tu H, Shan B, Luk A, DeBose-Boyd RA, Bashmakov Y, Goldstein JL, Brown MS. Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR. Proc Natl Acad Sci U S A. 2001; 98: 6027–6032.
Hannah VC, Ou J, Luong A, Goldstein JL, Brown MS. Unsaturated Fatty Acids Down-Regulate SREBP Isoforms 1a and 1c by Two Mechanisms in HEK-293 Cells. J Biol Chem. 2000; 276: 4365–4372.(Tilla S. Worgall; Rebecca)