Cyclic Adenosine 5'-Monophosphate-Dependent Sphingosine-1-Phosphate Biosynthesis Induces Human CYP17 Gene Transcription by Activat
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《内分泌学杂志》
School of Biology, Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332-0230
Abstract
In the human adrenal cortex, ACTH activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including CYP17) via a cAMP-dependent pathway. In the present study, we characterized the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) and the content of several sphingolipid species quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides, and sphingosine and stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (S1P) into the media. S1P increased CYP17 mRNA expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein (SREBP) 1. Chromatin immunoprecipitation assays revealed that Bt2cAMP and S1P increased acetylation of histone H3 and promoted binding of SREBP1 to the –520/–331 region of the CYP17 promoter. In summary, our studies demonstrate a role for sphingolipid metabolism and SREBP1 in ACTH-dependent CYP17 regulation and steroidogenesis.
Introduction
STEROID HORMONE biosynthesis in adrenal cortex involves the coordinate action of several cytochrome P450 enzymes, whose genes (CYP) are transcriptionally activated by the peptide hormone ACTH (1). In the zona fasciculata and zona reticularis, ACTH directs increased steroid hydroxylase gene transcription via the activation of adenylyl cyclase and subsequent increase in intracellular cAMP. This second messenger then activates protein kinase A (PKA), which induces gene transcription by phosphorylating transcription factors, coactivators, and/or other proteins in the ACTH signaling pathway.
We have previously shown that the ACTH/cAMP increases human CYP17 gene expression by promoting the binding of a protein complex containing steroidogenic factor-1 (SF-1), p54nrb, and polypyrimidine-tract binding protein-associated splicing factor (PSF) (2) to a 20-bp region between –57 and –37 of the CYP17 promoter. The affinity of this SF-1/p54nrb/PSF complex for region –57/–37 of the CYP17 promoter is induced by cAMP and is dependent on phosphatase activity (3, 4). Although ACTH/cAMP plays a central role in regulating CYP17 gene expression, other signaling cascades, and second-messenger systems have been shown to modulate CYP17 gene expression (5). Moreover, it is likely that ACTH/cAMP activates other signal transduction cascades that result in the induction of CYP17 transcription.
Sphingolipids are a diverse family of phospholipids and glycolipids that serve as structural components of the cell membrane and key mediators of numerous cellular processes (6, 7, 8, 9). Ceramide has been shown to act as a second messenger for events as diverse as differentiation, senescence, proliferation, cell cycle arrest, and apoptosis (7, 8, 9). Sphingosine-1-phosphate (S1P) also modulates a wide variety of physiological functions, including cell proliferation and survival (10, 11, 12, 13), chemotaxis (14), and protection against ceramide-mediated apoptosis (15).
Over the past few years, several studies have examined the role of sphingolipids on steroid hormone biosynthesis in both gonadal and adrenal cell lines (16, 17, 18, 19, 20, 21, 22, 23). Ceramide, bacterial sphingomyelinase [(SMase), which converts sphingomyelin (SM) to ceramide], and dihydroceramide all increase basal and human chorionic gonadotropin-stimulated progesterone synthesis in MA-10 murine Leydig cells (16). Exposure of glutaraldehyde-fixed MA-10 cells to recombinant SMase increases SM degradation, cholesterol movement from the cell surface into the mitochondria, and progesterone secretion (18). SMase also enhances cAMP-stimulated steroidogenesis, suggesting that activating sphingolipid turnover may promote steroid hormone biosynthesis by increasing cholesterol movement to the inner mitochondrial membrane (18). Similar stimulatory effects of ceramide on steroid hormone production have also been found in JEG-3 human choriocarcinoma cells (17). Finally, S1P was found to stimulate cortisol secretion in zona fasciculata bovine adrenal cells in a protein kinase C- and Ca2+-dependent manner (19).
Studies have also been published demonstrating an inhibitory role of sphingolipids on steroid hormone production (20, 21, 22, 23). Ceramide analogs have no effect on rat Leydig cell steroidogenesis, whereas SMase inhibits human chorionic gonadotropin-stimulated testosterone production (22). Budnick et al. (23) reported that TNF inhibits testosterone production by inducing ceramide accumulation, thereby resulting in decreased steroidogenic acute regulatory (StAR) protein expression levels. The StAR protein plays an essential role in steroidogenesis by enabling the transport of cholesterol to inner mitochondrial membrane in which P450 side chain cleavage enzyme (P450scc; encoded by CYP11A1) converts cholesterol to pregnenolone in the first enzymatic step of steroid hormone biosynthesis. In rat granulosa cells, both SMase and ceramide inhibit FSH-stimulated progesterone biosynthesis and the mRNA expression levels of CYP11A1 and 3-hydroxysteroid dehydrogenase (20).
Interestingly, sphingolipids have been shown to induce sterol regulatory element binding protein (SREBP) 1 cleavage, leading to increase in intracellular cholesterol (24). SREBP1a has been shown to activate transcription of the StAR protein (25). SREBPs are a family of transcription factors that regulate the genes that encode for more than 30 enzymes that are involved in cholesterol, triacylglyceride, phospholipid biosynthesis, fatty acid desaturation, and cholesterol uptake (26).
The aim of this study was to examine the relationship between ACTH/cAMP-dependent steroidogenesis and the sphingolipid metabolic pathways in the H295R human adrenocortical cell line. We used mass spectrometric analysis to determine the effects of ACTH and cAMP on the cellular content of several sphingolipid species. We also characterized the effect of S1P on CYP17 gene expression. We show that ACTH and cAMP rapidly alter the cellular sphingolipid profile by stimulating sphingolipid catabolism. ACTH and dibutyryl cAMP (Bt2cAMP) increased the activity of sphingosine kinase (SK). Moreover, the S1P produced in response to Bt2cAMP increased the transcription of CYP17 by stimulating binding of SREBP1 to the promoter. In summary, our studies establish a link between ACTH/cAMP-dependent steroidogenesis and sphingolipid metabolism in the human adrenal cortex and demonstrate that S1P can serve as a signaling mediator in ACTH/cAMP-stimulated CYP17 transcription by activating SREBP1.
Materials and Methods
Reagents
Bt2cAMP and N-acetyl-Leu-Leu-norleucine (ALLN) were obtained from Sigma (St. Louis, MO). Sphingolipids were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). N-Acetyl-D-erythro-sphingosine (C2-ceramide) was freshly prepared before each experiment by dissolving in ethanol. D-Erythro-sphingosine (SPH) was prepared by dissolving in ethanol, followed by dilution in fatty acid-free BSA (Calbiochem, La Jolla, CA). D-Erythro-S1P and dihydro-S1P (dhS1P) were prepared by dissolving in ethanol and dimethylamine, followed by evaporation and solubilization in 2 mM fatty acid-free BSA. ACTH was obtained from Calbiochem. Small interfering RNA (siRNA) oligonucleotides directed against SREBP1, SREBP2, SK1, and SK2 were obtained from Dharmacon (Lafayette, CO).
Cell culture
H295R adrenocortical cells (27, 28) were generously donated by Dr. William E. Rainey (Medical College of Georgia, Augusta, GA) and cultured in Dulbecco’s modified Eagle’s/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% -Serum I (BD Biosciences, Palo Alto, CA), 0.5% ITS Plus (BD Biosciences), antibiotics, and antimycotics.
Analysis of sphingolipid molecular species
For sphingolipid measurements, cells were treated for 30 min or 2 h with 50 nM ACTH or 1 mM Bt2cAMP and sphingolipids in the cells, and media were analyzed by liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS/MS) as described previously (29, 30). The internal standards for quantification of the sphingolipids were obtained from Avanti Polar Lipids.
SK assay
SK activity was assayed as described by Olivera et al. (31). Briefly, cells were plated onto 100-mm dishes and treated with 50 nM ACTH or 1 mM Bt2cAMP for 5–60 min. Alternatively, cells transfected with siRNA oligonucleotides directed against SK1 and SK2 were treated with 1 mM Bt2cAMP for 15 min. After the desired treatment period, the cells were washed with ice-cold PBS and then harvested into 200 μl of lysis buffer [20 mM Tris-HCl (pH 7.4), 20% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM MgCl2, 15 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM 4-deoxypyridoxine, and protease inhibitors (Calbiochem)]. The cytosolic fraction was incubated with 5 mM D-erythro-sphingosine and [-32P]ATP for 30 min at 37 C. Sphingosine used in the reactions was solubilized as a sphingosine-BSA complex as described above. Reactions were terminated by placing the tubes on ice and adding 20 μl of 1 M HCl and 800 μl of chloroform/methanol/HCl (100:200:1, vol/vol). The samples were vortexed for 10 min, followed by the addition of 200 μl chloroform and 200 μl of 2 M KCl, vortexing again for 10 min, and phase separation by centrifugation. An aliquot (50 μl) of the organic phase was spotted onto a Silica Gel 60 TLC plate and developed in 1-butanol/methanol/acetic acid/water (80:20:10:20, vol/vol). Unlabeled S1P was also spotted onto each plate and visualized by spraying with ninhydrin. The dried plates were exposed to a phosphor imager screen and the amount of radioactivity measured normalized to the total protein concentration of each sample.
RNA isolation and Northern blotting
Cells were cultured onto 12-well plates and treated with 1 mM Bt2cAMP, 0.1–10 μM SPH or 0.1–10 μM S1P for 1–12 h. Total RNA was prepared from treated cells by acid-phenol extraction (32). RNA was fractionated by agarose (1%) gel electrophoresis in the presence of 5% formaldehyde and transferred onto nylon transfer membrane filters (Millipore, Bedford, MA). A 1.2-kb cDNA fragment of CYP17 was used to detect CYP17 mRNA expression. cDNA fragments were radiolabeled with [a-32P]dCTP using a random primer labeling kit (Takara, Shiga, Japan). Blots were hybridized overnight at 42 C in 50% formamide, 5x sodium chloride/sodium phosphate/EDTA, 1% sodium dodecyl sulfate (SDS), 5x Denhardt’s, 50 μg/ml tRNA, and 32P-labeled cDNA fragments. The hybridized membranes were sequentially washed for 2 x 10 min in 2x saline sodium citrate, 0.2% SDS, and 2 x 5 min in 0.2x saline sodium citrate, 0.2% SDS at 42 C. The amount of probe bound to the filter was quantified using a fluorescence/phosphor imager (Fuji Film, Tokyo, Japan). Results were normalized to the content of glyceraldehyde-3-phosphate dehydrogenase mRNA.
RNA interference (RNAi) and real-time RT-PCR
For RNAi experiments, cells were subcultured onto 12-well plates and 24 h later media replaced by Optimem (Invitrogen). Cells were transfected with 150 nM of SREBP1, SREBP2, SK1, SK2 or nonspecific siRNA oligonucleotides using siIMPORTER (Upstate Biotechnology, Lake Placid, NY). Seventy-two hours (SREBP1 and SREBP2) or 48 h (SK1 and SK2) after transfection, cells were treated with 1 mM Bt2cAMP or 1 μM S1P for 6 h. Total RNA was extracted using TRIzol (Invitrogen) and amplified using the iScript one-step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, CA) and an iCycler real-time thermocycler (Bio-Rad). The following PCR primers were used: CYP17 (forward, 5'-CTCTTGCTGCTTCACCTA, and reverse, 5'-TCAAGGAGATGACATTGGTT); actin (forward, 5'-ACGGCTCCGGCATGTGCAAG-3', and reverse, 5'-TGACGATGCCGTGCTGCATG-3'), SK1 (forward, 5'-CTGGCAGCTTCCTTGAACCAT-3', and reverse, 5'-TGTGCAGAGACAGCAGGTTCA-3'); and SK2 (forward, 5'-CCAGTGTTGGAGAGCTGAAGGT-3', and reverse, 5'-GTCCATTCATCTGCTGGTCCTC-3'). CYP17 expression is normalized to actin and calculated using the - cycle threshold method. S1P receptor expression was determined using Taqman gene expression probes and the iScript one-step RT-PCR kit for probes (Bio-Rad).
Transient transfection and reporter gene analysis
Cells were subcultured onto 12-well plates and 24 h later transfected with 500 ng of reporter plasmids containing 1100, 700, 300, or 57 bp of the CYP17 promoter upstream of the start site (2) using GeneJuice (Novagen, Madison, WI). The CYP17 1100-, 700-, and 300-pGL3 constructs were generated by PCR using a plasmid containing a 1.8-kb fragment of the CYP17 promoter fused to the luciferase gene in the pGL3 vector [generously donated by Dr. Janette M. McAllister (Pennsylvania State University, Hershey, PA)]. The CYP17 57-pGL3 plasmid was constructed by ligating double stranded oligonucleotides corresponding to the region –57/–2 of the CYP17 5' flank upstream of the luciferase gene in the pGL3 vector (Promega, Madison, WI). Cells were cotransfected with 10 ng of the Renilla luciferase plasmid (pRL CMV, Promega) for normalization. Cells were then treated with 1 mM Bt2cAMP, 1 μM SPH, or 1 μM S1P for 6 h and harvested for dual luciferase assays (Promega).
Chromatin immunoprecipitation (ChIP)
For ChIP assays (33, 34), H295R cells (150-mm dishes) were stimulated with 50 nM ACTH, 1 mM Bt2cAMP, 1 μM C2-ceramide, 1 μM SPH, 1 μM S1P, or 500 nM trichostatin A (TSA) for 1 or 4 h. Cross-linking was performed by incubation in 1% formaldehyde (in PBS) for 10 min. The reaction was stopped by the addition of glycine (0.125 M final concentration), the cells washed, the nuclei harvested, and the lysates sonicated to obtain optimal DNA fragment lengths of 100-1000 bp. The purified chromatin solutions were immunoprecipitated using antiacetyl histone H3 (Upstate, Charlottesville, VA) or anti-SREBP1 (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A/G plus (Santa Cruz Biotechnology). The reaction was centrifuged at 4000 rpm for 5 min and 100 μl of the supernatant (input) retained. The antibody/protein/DNA bound beads were subjected to a series of 5-min washes: three times in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris, Cl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 150 nM aprotinin, 1 mM leupeptin, 1 mM E-64, 500 mM 4-(2-aminoethyl)benzenesulfonylfluoride], three times in RIPA buffer plus 500 mM NaCl, three times in washing buffer [10 mM Tris-Cl (pH 8), 0.25 M LiCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate, 10 mM sodium butyrate, 20 mM -glycerophosphate, and protease inhibitors], and three times in Tris/EDTA buffer. The cross-links were reversed and protein digested using proteinase K (100 μg/ml). DNA was purified by phenol-chloroform extraction and ethanol precipitation. Precipitated DNA was amplified by PCR using the following primer pairs: forward 5'-GGGGACCATTAACCCGACAGCCCTTATCGC-3' (–520/–491 CYP17), reverse 5'-GACAGATGACAGATTCAGGAGGGTCACAAG-3' (–331/–360 CYP17). PCRs were as follows: 1) 1 x 94 C, 5 min; 2) 35 x 95 C, 1 min, 55 C, 1 min, 72 C, 2 min; 3) 1 x 72 C, 10 min; 4) cool to 4 C. PCR products were then subjected to agarose (2%) gel electrophoresis.
SDS-PAGE and Western blot analysis
To confirm that RNAi of SK1, SK2, SREBP1, and SREBP2 was effective, cells were transfected with siRNA oligonucleotides for 48 h (SK1 and SK2) or 72 h (SREBP1 and SREBP2) and cell lysates harvested for SDS-PAGE and Western blotting. Twenty-five micrograms of each sample were run on 10% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes (PVDF, Pall Corp., Pensacola, FL) and probed with anti-SK1 (1:250), anti-SK2 (1:250), anti-SREBP1(K10) (1:500), or anti-SREBP2(H164) (1:500). Antibodies to SK1 and SK2 were obtained from Exalpha Biologicals Inc. (Watertown, MA), and antibodies to SREBP1 and SREBP2 were obtained from Santa Cruz Biotechnology. Protein expression was detected using an ECF Western blotting kit (Amersham Biosciences, Piscataway, NJ) and a fluor/Phosphor imager (Fuji Film).
To determine the effect of S1P on SREBP processing, cells were subcultured onto six-well plates and treated with 1 μM S1P for periods ranging from 1 to 4 h. Two hours before harvesting, 25 μg/ml ALLN were added to inhibit proteosomal degradation of mature SREBP. For the 1 h time point, cells were pretreated with ALLN before stimulation with S1P. Cells were washed twice in PBS, harvested into RIPA buffer, and lysed by passing 10 times through a 22-gauge needle. Lysates were centrifuged for 15 min at 4 C and the supernatant collected for analysis by SDS-PAGE. Aliquots of each sample (25 μg of protein) were run on 10% SDS-PAGE gels, transferred to PVDF membranes, and probed with anti-SREBP1. SREBP1 protein expression was detected and imaged as described above. For analysis of membrane and nuclear SREBP1, cells were plated onto 60-mm dishes and treated with 1 μM S1P or dhS1P. Cells were isolated and membrane and nuclear extracts prepared using NE-PER (Pierce, Rockford, IL) containing protease inhibitors. Twenty-five micrograms of nuclear or membrane extracts were run on 10% SDS-PAGE gels, transferred to PVDF membranes, probed with anti-SREBP1, and expression detected as described above.
Results
ACTH stimulates sphingolipid metabolism in H295R cells
Several studies have characterized the role of sphingolipids in steroid hormone biosynthesis and found both stimulatory and inhibitory roles for ceramide, SPH, and S1P on hormone secretion (16, 17, 18, 19, 20, 21, 22, 23). However, the effect of ACTH on cellular sphingolipid content is unknown. Thus, we treated H295R human adrenocortical cells for 2 h with 50 nM ACTH or 1 mM Bt2cAMP and quantified the amounts of several sphingolipids by LC-ESI-MS/MS. As shown in Fig. 1A, both ACTH and Bt2cAMP decreased cellular SM. Of the different SM molecules in H295R cells, both ACTH and Bt2cAMP decreased C16 and C18 SM (Fig. 1B).
Activation of sphingolipid turnover of complex sphingolipid molecules like SM is linked to the increased production of bioactive species such as ceramide, SPH, and S1P. Unexpectedly, however, we also observed ACTH/cAMP-stimulated reduction of cellular ceramides and SPH (Fig. 1C). Bt2cAMP treatment also decreased the amounts of multiple chain-length subspecies (Fig. 1D).
ACTH/cAMP activate SK activity
Because ACTH and Bt2cAMP evoked decreases in cellular ceramides and SPH, we hypothesized that ACTH and Bt2cAMP were activating SK. Increased SK activity would explain decreases in cellular SPH due to phosphorylation to form S1P. Thus, we carried out enzyme assays to determine whether ACTH/cAMP was activating sphingosine kinase activity. As shown in Fig. 2, exposure of H295R cells to ACTH and Bt2cAMP resulted in rapid increases in SK activity. Maximal increases in enzyme activity were observed at 15 min for both agents and returned to basal levels after 30 min. Incubation of H295R cells for 15 min with ACTH increased SK activity 3.2-fold, whereas Bt2cAMP resulted in a 4.8-fold increase in catalytic activity (Fig. 2).
ACTH/cAMP promote S1P secretion into the media
The increase in SK activity after exposure to ACTH or Bt2cAMP suggested that ACTH/cAMP may activate sphingolipid turnover and increase the cellular concentration of S1P. However, mass spectrometric analysis of cells treated for 30 min or 2 h with 50 nM ACTH or 1 mM Bt2cAMP revealed decreases in the cellular amounts of S1P (Fig. 3). Because some cell types secrete S1P, the amounts in the medium were also analyzed by tandem mass spectrometry. As shown in Fig. 3, S1P in the medium increased in a manner that mirrored the decreases in the cells; therefore, it appears that ACTH and cAMP are stimulating catabolism of SM, resulting in decreases in the cellular content of ceramide, SPH, and S1P and secretion of S1P into the media. Unexpectedly, no significant difference was observed in the amount of S1P in the media at the 30-min and 2-h time points. The cellular levels of most sphingolipids returned back to control levels after 6 h (data not shown).
S1P induces steroidogenic gene expression
Given that ACTH alters the sphingolipid profile in H295R cells (Fig. 1), we carried out studies to further explore the relationship between sphingolipid metabolism and a steroidogenic enzyme. CYP17 mRNA expression in Bt2cAMP-, S1P-, or SPH-treated cells was analyzed by Northern blotting (Fig. 4A) and real-time RT-PCR (Fig. 4, B and C). Densitometric analysis of Northern blots revealed that SPH and S1P increased CYP17 mRNA expression by 1.3- and 3.6-fold, respectively, whereas Bt2cAMP induced hCYP17 mRNA expression 5.2-fold (Fig. 4A). As shown in Fig. 4B, both S1P and Bt2cAMP increased CYP17 mRNA expression in a time-dependent manner; however, maximal stimulatory effects of S1P were observed at 6 h, whereas Bt2cAMP maximally increased CYP17 mRNA at the 12-h time point.
cAMP-dependent CYP17 mRNA expression requires SK1
Because SK activity was increased by Bt2cAMP treatment (Fig. 3) and S1P increased CYP17 expression (Fig. 4), we used RNAi to determine whether cAMP-stimulated CYP17 transcription required SK. H295R cells were transfected with SK1 and SK2 siRNAs and then treated with 1 mM Bt2cAMP for 12 h. Real-time RT-PCR revealed that silencing both SK1 and SK2 decreased the stimulatory effect of Bt2cAMP on CYP17 mRNA expression (Fig. 5A). We confirmed that RNAi suppressed SK1 and SK2 protein expression by Western blotting (Fig. 5B) and SK activity analysis (Fig. 5C).
Sphingolipids stimulate CYP17 transcriptional activity
To determine whether the stimulatory effects of sphingolipids on CYP17 mRNA (Fig. 4) were mediated at the level of transcription, we performed transient transfection assays using plasmids containing varying lengths of the CYP17 promoter fused to the Firefly luciferase gene. SPH and S1P increased the transcriptional activity of the 1100- and 700-pGL3 CYP17 reporter constructs, whereas having no significant effect on the CYP17 57- and 300-pGL3 plasmids (Fig. 6A). These findings suggest that the region of the CYP17 promoter required for sphingolipid-dependent gene transcription lies between –700 and –300 bp. In contrast to the effect of SPH and S1P, Bt2cAMP significantly stimulated the luciferase activity of all plasmids tested (Fig. 6A) as seen previously (2). In silico analysis (35) revealed a putative binding site for SREBPs (Fig. 6B). Thus, we determined the effect of mutating this site on the ability of S1P to stimulate CYP17 reporter gene activity. As shown in Fig. 6C, mutation of the putative SRE abolished the stimulatory effects of S1P.
Sphingolipids increase acetylation of histone H3 at the CYP17 promoter
Next we examined the effect of S1P on the acetylation state of histone H3 at the CYP17 promoter. Cells were treated with Bt2cAMP sphingolipids or the histone deacetylase inhibitor, TSA, for 4 h, followed by ChIP. PCR was performed using primers that amplified regions –142/+45 and –520/–360. Although we have previously shown that ACTH/cAMP-dependent gene transcription occurs on binding of a complex containing SF-1/p54nrb/PSF to region –57/–37 of the CYP17 promoter (2), studies described herein show that S1P activates CYP17 gene transcription by stimulating the binding of a trans-acting factor(s) to a more distal region of promoter (–436/–448).
As shown in Fig. 7A, acetylation of histone H3 at the –520/–360 region of the CYP17 gene is increased by Bt2cAMP, SPH, and S1P. TSA also increased the acetylation of histone H3 at the CYP17 promoter (Fig. 7A). These findings are in contrast to the results of PCR using primers designed to amplify the –142/+45 region of the CYP17 promoter, in which only Bt2cAMP and TSA significantly increased the acetylation of histone H3 (data not shown). Finally, we performed ChIP assays using antibodies to SREBP1. As shown in Fig. 7B, ACTH, Bt2cAMP, and S1P increased binding of SREBP1 to the –520/–60 region of the CYP17 gene.
S1P induces CYP17 transcription by activating SREBP1
Because sphingolipid-stimulated SREBP1a cleavage (24) mediates increased transcription of StAR (25), we investigated the role of SREBPs in S1P-evoked CYP17 gene expression. H295R cells were transfected with SREBP1 and SREBP2 siRNAs and treated with 1 mM S1P for 6 h followed by total RNA extraction and quantitative RT-PCR as described in Materials and Methods. As shown in Fig. 8A, SREBP1 siRNA abolished S1P-stimulated transcription of CYP17, whereas siRNAs targeted against SREBP2 decreased S1P-stimulated CYP17 mRNA expression by 31%. Small interfering oligonucleotides specifically decreased the protein expression SREBP1 and SREBP2 (Fig. 8B).
S1P stimulates SREBP1 cleavage and nuclear translocation
Nascent SREBPs contain two transmembrane domains that are integrally inserted into the endoplasmic reticulum. Processing of SREBP to generate the transcriptionally active N-terminal fragment involves the transport of the full-length protein to the Golgi apparatus in which it is cleaved sequentially by two membrane-associated proteases. The transcriptionally active fragment of SREBP is then translocated to the nucleus in which it binds to the promoters of SREBP target genes. To determine whether S1P stimulates the processing of SREBP1, we treated H295R cells with S1P for varying time points and measured the levels of mature and precursor forms by Western blotting. Because the mature form of SREBP is subjected to ubiquitination and subsequent proteasomal degradation (36), H295R cells were treated with the proteasome inhibitor ALLN as previously described by others (24, 37). As shown in Fig. 9A, S1P stimulates SREBP1 maturation in a time-dependent manner. To determine whether S1P activates SREBP cleavage by binding to a S1P receptor or acting intracellularly, we treated H295R cells with dhS1P. Like S1P, dhS1P binds to and activates S1P receptors (38, 39); however, because of its increased hydrophilic nature, dhS1P is unable to permeate the plasma membrane (40, 41). Both S1P and dhS1P stimulated an increase in the mature form of SREBP1 in the nucleus (Fig. 9B), suggesting that S1P acts extracellularly to induce SREBP1 maturation and induce CYP17 transcription.
Our observation that cAMP stimulates the secretion of S1P into the media (Fig. 3) coupled with the finding that dhS1P promotes SREBP1 translocation to the nucleus (Fig. 9B) suggests that S1P may act in a paracrine or autocrine manner to increase CYP17 transcription by binding to S1P receptors on surface of H295R cells. S1P regulates biological processes by serving as a ligand for the S1P family of G protein-coupled receptors (42, 43) and acting intracellularly (42). To date, five S1P receptors have been identified (44, 45, 46). These G protein-coupled receptors were initially called endothelial differentiation gene (EDG) receptors but have been renamed as S1P1 (EDG-1), S1P2 (EDG-5), S1P3 (EDG-3), S1P4 (EDG-6), and S1P5 (EDG-8) (44). As shown in Fig. 9C, all five receptors are expressed in H295R cells; however, S1P4 is not as highly expressed as the other four receptor subtypes. Finally, like Bt2cAMP and S1P, dhS1P is able to induce CYP17 mRNA expression (Fig. 9D), further supporting our hypothesis that S1P acts in a paracrine manner to stimulate SREBP1 binding to the CYP17 promoter by binding to a G protein-coupled receptor.
Discussion
ACTH exerts its stimulatory actions on steroid hormone biosynthesis via two temporally distinct cAMP/PKA-dependent pathways. A rapid, acute response results in the transport of cholesterol into the inner mitochondrial membrane for conversion to pregnenolone by P450 11A1 (P450scc). During the acute response, an essential site of phosphorylation by PKA is cholesterol ester hydrolase, which on activation catalyzes the conversion of cholesterol esters to free cholesterol (47). Key regulators of the acute response, StAR protein (48, 49) and the peripheral benzodiazapene receptor (50, 51), facilitate cholesterol movement in the mitochondria. The chronic effect of ACTH is to increase the transcription of steroidogenic enzymes.
In this report, we provide evidence for a novel mechanism by which ACTH induces CYP17 transcription. We show herein that both ACTH and cAMP decrease the cellular amounts of several sphingolipid species, including SM and ceramides (Fig. 1). In addition, ACTH and cAMP rapidly and transiently activate SK catalytic activity (Fig. 2). Previous studies have demonstrated the relationship between SM and P450scc (52). P450scc catalyzes the first step in steroid hormone biosynthesis: conversion of cholesterol to pregnenolone. SM inhibits the ability of cholesterol to bind to P450scc (52). Furthermore, the interaction between cholesterol and SM is cooperative (52), indicating that interactions between cholesterol and lipids can play a role in steroidogenesis. In light of these previous findings, the studies presented herein suggest that in addition to increasing availability of cholesterol for steroidogenesis, ACTH activates sphingolipid metabolism to maintain optimal cholesterol to lipid ratios in the membranes of adrenocortical cells.
Because decreases in cellular levels of S1P were paralleled by increases in this bioactive molecule in the medium (Fig. 3) and S1P receptors are expressed in H295R cells, we speculated that S1P acts in a paracrine manner to increase steroidogenesis. Our findings that the S1P receptor agonist dhS1P mimicked the stimulatory effect of S1P on CYP17 gene expression suggest that S1P acts extracellularly by binding to one of the S1P receptors. Studies are underway to determine which one of the five S1P receptors mediates CYP17 transcription in response to S1P. The differences in signaling through these receptors are primarily a result of differential coupling to G proteins. S1P1 couples to Gi (53, 54), whereas S1P2 and S1P3 couple to Gi, Gq, and G13 (54). S1P4 has been shown to associate with Gi (39, 55) and G12/13 (56) and S1P5 couple to Gi/o and G12 (57). Interestingly, S1P2 activates adenylyl cyclase (58); however, because the receptor does not couple to Gs, the activation of adenylyl cyclase may occur through an indirect mechanism. It has also been shown that S1P induces MAPK phosphatase-1 (MKP-1) in mouse fibroblast CH10T1/2 cells (59). We have previously shown that induction of CYP17 gene expression in response to cAMP requires activation of MKP-1 (1). Thus, it is possible that cAMP-stimulated release of S1P into the media may activate CYP17 gene expression via MKP-1. Although our data demonstrating that S1P is secreted into the media (Fig. 3) and that dhS1P induces CYP17 transcription (Fig. 9C) suggest that S1P acts extracellularly, it is possible that S1P may also be acting intracellularly. The generation of fatty acids for prostaglandin synthesis by phospholipase A2 has been shown to have both extracellular and intracellular components (60, 61, 62, 63, 64).
SREBPs regulate genes involved in the sterol synthesis and uptake as well as fatty acid biosynthesis and desaturation (26, 65). When the levels of free cholesterol in the cell are low, SREBPs undergo proteolytic processing, resulting in the release of a mature transcription factor that translocates to the nucleus for increased expression of genes involved in cholesterol biosynthesis and fatty acid metabolism (26, 65). The activity of these transcription factors is controlled by transport from endoplasmic reticulum to Golgi by an escort protein called SREBP-cleavage activating protein (SCAP), which senses the absence of cholesterol (26). Recently sphingolipids have been shown to stimulate SREBP-1 cleavage by causing the cholesterol to be sequestered in endosomes or lysosomes, leading to a compartmentalization of cellular cholesterol (24). This redistribution of cholesterol is sensed by SCAP, which leads to the translocation of SREBPs to the Golgi for maturation (24). In agreement with these studies, we found that S1P promotes SREBP1 maturation (Fig. 9A) and nuclear expression (Fig. 9B). Moreover, transcription of StAR is increased by SREBP-1 (25). ChIP data presented in Fig. 7 showing increased acetylation of histone H3 and recruitment of SREBP1 to the –700/–300 region of the CYP17 promoter suggest that S1P stimulates chromatin remodeling, resulting in increased accessibility to this region of the promoter and SREBP binding. Of note, hyperacetylation of histone H3, but not histone H4, has been found to occur in chromatin at the promoters for the low-density lipoprotein receptor gene and hydroxymethyl glutaryl CoA reductase (66). Our findings agree with these findings and suggest that in addition to increasing the acetylation of histone H3 at the promoters of sterol-regulated genes, SREBP also promotes hyperacetylation of a steroidogenic gene. Additionally, recent studies by Hughes et al. (67), demonstrate that orthologs of SREBP and SCAP, sre1+, and scp1+, respectively, function as an oxygen sensor in fission yeast.
In these studies, microarray analysis carried out to identify genes activated by SREBP1 revealed that two genes required for the hydroxylation of sphingolipids were highly up-regulated (67). Previously it was proposed by Lawler et al. (68) that stimulation of human hepatocytes by TNF leads to the activation of neutral SMase, which in turn induces the maturation of SREBP1. This induction of SREBP1 cleavage occurred independent of cholesterol depletion. Furthermore, studies have suggested that whereas SRE-mediated gene transcription is decreased by the inhibition of ceramide synthesis, it is stimulated by increasing SPH levels (69).
Based on the findings presented herein, we propose a model (Fig. 10) for the role of S1P and SREBP1 in ACTH/cAMP-dependent CYP17 transcription and cortisol biosynthesis. Binding of ACTH to its receptor on cell surface activates adenylyl cyclase, which leads to the production of cAMP and activation of PKA. PKA promotes the turnover of SM, which leads increased sphingolipid turnover due to the activation of enzymes (such as SK) in the sphingolipid pathway, causing decreased levels of several sphingolipid species. The S1P released into extracellular space binds to an S1P receptor, thereby activating a signal transduction cascade that activates SREBP1 cleavage and maturation. Although we found that total cellular amounts of S1P decreased in cAMP-treated cells (Fig. 1), it is also possible that S1P concentrations are increased in distinct subcellular organelles, such as the endoplasmic reticulum or Golgi and that this organelle-specific increase may result in an intracellular pool of S1P that may contribute to the increase in CYP17 transcription. Cellular fractionation studies are ongoing to determine the effects of ACTH and cAMP on organellar concentrations of various sphingolipid moieties. Moreover, it is probable that ACTH/cAMP may affect de novo sphingolipid biosynthesis. Studies are underway to determine whether the decreases in cellular sphingolipid content are due to increased catabolism, decreased de novo synthesis, or both. In our model (Fig. 10), we propose that the S1P produced in response to cAMP promotes the migration of SREBP1 to the Golgi in which it is processed to the mature form. Mature SREBP1 then translocates to the nucleus, dimerizes, and binds to the SRE site on the CYP17 promoter causing the activation of transcription of CYP17 gene. In summary, our studies demonstrate that ACTH/cAMP rapidly activates sphingolipid catabolism and S1P production, which ultimately leads to induction of CYP17. These findings provide evidence for a role for sphingolipids in ACTH/cAMP-dependent cortisol biosynthesis and establish a novel mechanism by which S1P, acting as a paracrine or autocrine factor, can increase CYP17 gene transcription by activating SREBP1.
Acknowledgments
We thank Samuel Kelly, Elaine Wang, and Dr. Alfred H. Merrill, Jr. for mass spectrophotometric analysis of sphingolipid molecular species.
Footnotes
This work was supported by the National Science Foundation (MCB-0347682), the National Institutes of Health (GM073241), and the Georgia Cancer Coalition. Mass spectrometric analysis of cellular lipids was supported by the National Institutes of Health (PA-02-132 to A.H.M.).
The authors have nothing to declare.
First Published Online November 23, 2005
Abbreviations: ALLN, N-acetyl-Leu-Leu-norleucine; Bt2cAMP, dibutyryl cAMP; C2-ceramide, N-acetyl-D-erythro-sphingosine; ChIP, chromatin immunoprecipitation; dhS1P, dihydro-S1P; EDG, endothelial differentiation gene; LC-ESI-MS/MS, liquid chromatography, electrospray ionization, and tandem mass spectrometry; MKP-1, MAPK phosphatase-1; PKA, protein kinase A; P450scc, P450 side chain cleavage enzyme; PSF, protein-associated splicing factor; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; RNAi, RNA interference; SCAP, SREBP-cleavage activating protein; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; siRNA, small interfering RNA; SK, sphingosine kinase; SM, sphingomyelin; SMase, sphingomyelinase; S1P, sphingosine-1-phosphate; SPH, D-erythro-sphingosine; SRE, sterol regulatory element; SREBP, sterol regulatory element binding protein; StAR, steroidogenic acute regulatory; TSA, trichostatin A.
Accepted for publication November 14, 2005.
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Abstract
In the human adrenal cortex, ACTH activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including CYP17) via a cAMP-dependent pathway. In the present study, we characterized the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) and the content of several sphingolipid species quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides, and sphingosine and stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (S1P) into the media. S1P increased CYP17 mRNA expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein (SREBP) 1. Chromatin immunoprecipitation assays revealed that Bt2cAMP and S1P increased acetylation of histone H3 and promoted binding of SREBP1 to the –520/–331 region of the CYP17 promoter. In summary, our studies demonstrate a role for sphingolipid metabolism and SREBP1 in ACTH-dependent CYP17 regulation and steroidogenesis.
Introduction
STEROID HORMONE biosynthesis in adrenal cortex involves the coordinate action of several cytochrome P450 enzymes, whose genes (CYP) are transcriptionally activated by the peptide hormone ACTH (1). In the zona fasciculata and zona reticularis, ACTH directs increased steroid hydroxylase gene transcription via the activation of adenylyl cyclase and subsequent increase in intracellular cAMP. This second messenger then activates protein kinase A (PKA), which induces gene transcription by phosphorylating transcription factors, coactivators, and/or other proteins in the ACTH signaling pathway.
We have previously shown that the ACTH/cAMP increases human CYP17 gene expression by promoting the binding of a protein complex containing steroidogenic factor-1 (SF-1), p54nrb, and polypyrimidine-tract binding protein-associated splicing factor (PSF) (2) to a 20-bp region between –57 and –37 of the CYP17 promoter. The affinity of this SF-1/p54nrb/PSF complex for region –57/–37 of the CYP17 promoter is induced by cAMP and is dependent on phosphatase activity (3, 4). Although ACTH/cAMP plays a central role in regulating CYP17 gene expression, other signaling cascades, and second-messenger systems have been shown to modulate CYP17 gene expression (5). Moreover, it is likely that ACTH/cAMP activates other signal transduction cascades that result in the induction of CYP17 transcription.
Sphingolipids are a diverse family of phospholipids and glycolipids that serve as structural components of the cell membrane and key mediators of numerous cellular processes (6, 7, 8, 9). Ceramide has been shown to act as a second messenger for events as diverse as differentiation, senescence, proliferation, cell cycle arrest, and apoptosis (7, 8, 9). Sphingosine-1-phosphate (S1P) also modulates a wide variety of physiological functions, including cell proliferation and survival (10, 11, 12, 13), chemotaxis (14), and protection against ceramide-mediated apoptosis (15).
Over the past few years, several studies have examined the role of sphingolipids on steroid hormone biosynthesis in both gonadal and adrenal cell lines (16, 17, 18, 19, 20, 21, 22, 23). Ceramide, bacterial sphingomyelinase [(SMase), which converts sphingomyelin (SM) to ceramide], and dihydroceramide all increase basal and human chorionic gonadotropin-stimulated progesterone synthesis in MA-10 murine Leydig cells (16). Exposure of glutaraldehyde-fixed MA-10 cells to recombinant SMase increases SM degradation, cholesterol movement from the cell surface into the mitochondria, and progesterone secretion (18). SMase also enhances cAMP-stimulated steroidogenesis, suggesting that activating sphingolipid turnover may promote steroid hormone biosynthesis by increasing cholesterol movement to the inner mitochondrial membrane (18). Similar stimulatory effects of ceramide on steroid hormone production have also been found in JEG-3 human choriocarcinoma cells (17). Finally, S1P was found to stimulate cortisol secretion in zona fasciculata bovine adrenal cells in a protein kinase C- and Ca2+-dependent manner (19).
Studies have also been published demonstrating an inhibitory role of sphingolipids on steroid hormone production (20, 21, 22, 23). Ceramide analogs have no effect on rat Leydig cell steroidogenesis, whereas SMase inhibits human chorionic gonadotropin-stimulated testosterone production (22). Budnick et al. (23) reported that TNF inhibits testosterone production by inducing ceramide accumulation, thereby resulting in decreased steroidogenic acute regulatory (StAR) protein expression levels. The StAR protein plays an essential role in steroidogenesis by enabling the transport of cholesterol to inner mitochondrial membrane in which P450 side chain cleavage enzyme (P450scc; encoded by CYP11A1) converts cholesterol to pregnenolone in the first enzymatic step of steroid hormone biosynthesis. In rat granulosa cells, both SMase and ceramide inhibit FSH-stimulated progesterone biosynthesis and the mRNA expression levels of CYP11A1 and 3-hydroxysteroid dehydrogenase (20).
Interestingly, sphingolipids have been shown to induce sterol regulatory element binding protein (SREBP) 1 cleavage, leading to increase in intracellular cholesterol (24). SREBP1a has been shown to activate transcription of the StAR protein (25). SREBPs are a family of transcription factors that regulate the genes that encode for more than 30 enzymes that are involved in cholesterol, triacylglyceride, phospholipid biosynthesis, fatty acid desaturation, and cholesterol uptake (26).
The aim of this study was to examine the relationship between ACTH/cAMP-dependent steroidogenesis and the sphingolipid metabolic pathways in the H295R human adrenocortical cell line. We used mass spectrometric analysis to determine the effects of ACTH and cAMP on the cellular content of several sphingolipid species. We also characterized the effect of S1P on CYP17 gene expression. We show that ACTH and cAMP rapidly alter the cellular sphingolipid profile by stimulating sphingolipid catabolism. ACTH and dibutyryl cAMP (Bt2cAMP) increased the activity of sphingosine kinase (SK). Moreover, the S1P produced in response to Bt2cAMP increased the transcription of CYP17 by stimulating binding of SREBP1 to the promoter. In summary, our studies establish a link between ACTH/cAMP-dependent steroidogenesis and sphingolipid metabolism in the human adrenal cortex and demonstrate that S1P can serve as a signaling mediator in ACTH/cAMP-stimulated CYP17 transcription by activating SREBP1.
Materials and Methods
Reagents
Bt2cAMP and N-acetyl-Leu-Leu-norleucine (ALLN) were obtained from Sigma (St. Louis, MO). Sphingolipids were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). N-Acetyl-D-erythro-sphingosine (C2-ceramide) was freshly prepared before each experiment by dissolving in ethanol. D-Erythro-sphingosine (SPH) was prepared by dissolving in ethanol, followed by dilution in fatty acid-free BSA (Calbiochem, La Jolla, CA). D-Erythro-S1P and dihydro-S1P (dhS1P) were prepared by dissolving in ethanol and dimethylamine, followed by evaporation and solubilization in 2 mM fatty acid-free BSA. ACTH was obtained from Calbiochem. Small interfering RNA (siRNA) oligonucleotides directed against SREBP1, SREBP2, SK1, and SK2 were obtained from Dharmacon (Lafayette, CO).
Cell culture
H295R adrenocortical cells (27, 28) were generously donated by Dr. William E. Rainey (Medical College of Georgia, Augusta, GA) and cultured in Dulbecco’s modified Eagle’s/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% -Serum I (BD Biosciences, Palo Alto, CA), 0.5% ITS Plus (BD Biosciences), antibiotics, and antimycotics.
Analysis of sphingolipid molecular species
For sphingolipid measurements, cells were treated for 30 min or 2 h with 50 nM ACTH or 1 mM Bt2cAMP and sphingolipids in the cells, and media were analyzed by liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS/MS) as described previously (29, 30). The internal standards for quantification of the sphingolipids were obtained from Avanti Polar Lipids.
SK assay
SK activity was assayed as described by Olivera et al. (31). Briefly, cells were plated onto 100-mm dishes and treated with 50 nM ACTH or 1 mM Bt2cAMP for 5–60 min. Alternatively, cells transfected with siRNA oligonucleotides directed against SK1 and SK2 were treated with 1 mM Bt2cAMP for 15 min. After the desired treatment period, the cells were washed with ice-cold PBS and then harvested into 200 μl of lysis buffer [20 mM Tris-HCl (pH 7.4), 20% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM MgCl2, 15 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM 4-deoxypyridoxine, and protease inhibitors (Calbiochem)]. The cytosolic fraction was incubated with 5 mM D-erythro-sphingosine and [-32P]ATP for 30 min at 37 C. Sphingosine used in the reactions was solubilized as a sphingosine-BSA complex as described above. Reactions were terminated by placing the tubes on ice and adding 20 μl of 1 M HCl and 800 μl of chloroform/methanol/HCl (100:200:1, vol/vol). The samples were vortexed for 10 min, followed by the addition of 200 μl chloroform and 200 μl of 2 M KCl, vortexing again for 10 min, and phase separation by centrifugation. An aliquot (50 μl) of the organic phase was spotted onto a Silica Gel 60 TLC plate and developed in 1-butanol/methanol/acetic acid/water (80:20:10:20, vol/vol). Unlabeled S1P was also spotted onto each plate and visualized by spraying with ninhydrin. The dried plates were exposed to a phosphor imager screen and the amount of radioactivity measured normalized to the total protein concentration of each sample.
RNA isolation and Northern blotting
Cells were cultured onto 12-well plates and treated with 1 mM Bt2cAMP, 0.1–10 μM SPH or 0.1–10 μM S1P for 1–12 h. Total RNA was prepared from treated cells by acid-phenol extraction (32). RNA was fractionated by agarose (1%) gel electrophoresis in the presence of 5% formaldehyde and transferred onto nylon transfer membrane filters (Millipore, Bedford, MA). A 1.2-kb cDNA fragment of CYP17 was used to detect CYP17 mRNA expression. cDNA fragments were radiolabeled with [a-32P]dCTP using a random primer labeling kit (Takara, Shiga, Japan). Blots were hybridized overnight at 42 C in 50% formamide, 5x sodium chloride/sodium phosphate/EDTA, 1% sodium dodecyl sulfate (SDS), 5x Denhardt’s, 50 μg/ml tRNA, and 32P-labeled cDNA fragments. The hybridized membranes were sequentially washed for 2 x 10 min in 2x saline sodium citrate, 0.2% SDS, and 2 x 5 min in 0.2x saline sodium citrate, 0.2% SDS at 42 C. The amount of probe bound to the filter was quantified using a fluorescence/phosphor imager (Fuji Film, Tokyo, Japan). Results were normalized to the content of glyceraldehyde-3-phosphate dehydrogenase mRNA.
RNA interference (RNAi) and real-time RT-PCR
For RNAi experiments, cells were subcultured onto 12-well plates and 24 h later media replaced by Optimem (Invitrogen). Cells were transfected with 150 nM of SREBP1, SREBP2, SK1, SK2 or nonspecific siRNA oligonucleotides using siIMPORTER (Upstate Biotechnology, Lake Placid, NY). Seventy-two hours (SREBP1 and SREBP2) or 48 h (SK1 and SK2) after transfection, cells were treated with 1 mM Bt2cAMP or 1 μM S1P for 6 h. Total RNA was extracted using TRIzol (Invitrogen) and amplified using the iScript one-step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, CA) and an iCycler real-time thermocycler (Bio-Rad). The following PCR primers were used: CYP17 (forward, 5'-CTCTTGCTGCTTCACCTA, and reverse, 5'-TCAAGGAGATGACATTGGTT); actin (forward, 5'-ACGGCTCCGGCATGTGCAAG-3', and reverse, 5'-TGACGATGCCGTGCTGCATG-3'), SK1 (forward, 5'-CTGGCAGCTTCCTTGAACCAT-3', and reverse, 5'-TGTGCAGAGACAGCAGGTTCA-3'); and SK2 (forward, 5'-CCAGTGTTGGAGAGCTGAAGGT-3', and reverse, 5'-GTCCATTCATCTGCTGGTCCTC-3'). CYP17 expression is normalized to actin and calculated using the - cycle threshold method. S1P receptor expression was determined using Taqman gene expression probes and the iScript one-step RT-PCR kit for probes (Bio-Rad).
Transient transfection and reporter gene analysis
Cells were subcultured onto 12-well plates and 24 h later transfected with 500 ng of reporter plasmids containing 1100, 700, 300, or 57 bp of the CYP17 promoter upstream of the start site (2) using GeneJuice (Novagen, Madison, WI). The CYP17 1100-, 700-, and 300-pGL3 constructs were generated by PCR using a plasmid containing a 1.8-kb fragment of the CYP17 promoter fused to the luciferase gene in the pGL3 vector [generously donated by Dr. Janette M. McAllister (Pennsylvania State University, Hershey, PA)]. The CYP17 57-pGL3 plasmid was constructed by ligating double stranded oligonucleotides corresponding to the region –57/–2 of the CYP17 5' flank upstream of the luciferase gene in the pGL3 vector (Promega, Madison, WI). Cells were cotransfected with 10 ng of the Renilla luciferase plasmid (pRL CMV, Promega) for normalization. Cells were then treated with 1 mM Bt2cAMP, 1 μM SPH, or 1 μM S1P for 6 h and harvested for dual luciferase assays (Promega).
Chromatin immunoprecipitation (ChIP)
For ChIP assays (33, 34), H295R cells (150-mm dishes) were stimulated with 50 nM ACTH, 1 mM Bt2cAMP, 1 μM C2-ceramide, 1 μM SPH, 1 μM S1P, or 500 nM trichostatin A (TSA) for 1 or 4 h. Cross-linking was performed by incubation in 1% formaldehyde (in PBS) for 10 min. The reaction was stopped by the addition of glycine (0.125 M final concentration), the cells washed, the nuclei harvested, and the lysates sonicated to obtain optimal DNA fragment lengths of 100-1000 bp. The purified chromatin solutions were immunoprecipitated using antiacetyl histone H3 (Upstate, Charlottesville, VA) or anti-SREBP1 (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A/G plus (Santa Cruz Biotechnology). The reaction was centrifuged at 4000 rpm for 5 min and 100 μl of the supernatant (input) retained. The antibody/protein/DNA bound beads were subjected to a series of 5-min washes: three times in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris, Cl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 150 nM aprotinin, 1 mM leupeptin, 1 mM E-64, 500 mM 4-(2-aminoethyl)benzenesulfonylfluoride], three times in RIPA buffer plus 500 mM NaCl, three times in washing buffer [10 mM Tris-Cl (pH 8), 0.25 M LiCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate, 10 mM sodium butyrate, 20 mM -glycerophosphate, and protease inhibitors], and three times in Tris/EDTA buffer. The cross-links were reversed and protein digested using proteinase K (100 μg/ml). DNA was purified by phenol-chloroform extraction and ethanol precipitation. Precipitated DNA was amplified by PCR using the following primer pairs: forward 5'-GGGGACCATTAACCCGACAGCCCTTATCGC-3' (–520/–491 CYP17), reverse 5'-GACAGATGACAGATTCAGGAGGGTCACAAG-3' (–331/–360 CYP17). PCRs were as follows: 1) 1 x 94 C, 5 min; 2) 35 x 95 C, 1 min, 55 C, 1 min, 72 C, 2 min; 3) 1 x 72 C, 10 min; 4) cool to 4 C. PCR products were then subjected to agarose (2%) gel electrophoresis.
SDS-PAGE and Western blot analysis
To confirm that RNAi of SK1, SK2, SREBP1, and SREBP2 was effective, cells were transfected with siRNA oligonucleotides for 48 h (SK1 and SK2) or 72 h (SREBP1 and SREBP2) and cell lysates harvested for SDS-PAGE and Western blotting. Twenty-five micrograms of each sample were run on 10% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes (PVDF, Pall Corp., Pensacola, FL) and probed with anti-SK1 (1:250), anti-SK2 (1:250), anti-SREBP1(K10) (1:500), or anti-SREBP2(H164) (1:500). Antibodies to SK1 and SK2 were obtained from Exalpha Biologicals Inc. (Watertown, MA), and antibodies to SREBP1 and SREBP2 were obtained from Santa Cruz Biotechnology. Protein expression was detected using an ECF Western blotting kit (Amersham Biosciences, Piscataway, NJ) and a fluor/Phosphor imager (Fuji Film).
To determine the effect of S1P on SREBP processing, cells were subcultured onto six-well plates and treated with 1 μM S1P for periods ranging from 1 to 4 h. Two hours before harvesting, 25 μg/ml ALLN were added to inhibit proteosomal degradation of mature SREBP. For the 1 h time point, cells were pretreated with ALLN before stimulation with S1P. Cells were washed twice in PBS, harvested into RIPA buffer, and lysed by passing 10 times through a 22-gauge needle. Lysates were centrifuged for 15 min at 4 C and the supernatant collected for analysis by SDS-PAGE. Aliquots of each sample (25 μg of protein) were run on 10% SDS-PAGE gels, transferred to PVDF membranes, and probed with anti-SREBP1. SREBP1 protein expression was detected and imaged as described above. For analysis of membrane and nuclear SREBP1, cells were plated onto 60-mm dishes and treated with 1 μM S1P or dhS1P. Cells were isolated and membrane and nuclear extracts prepared using NE-PER (Pierce, Rockford, IL) containing protease inhibitors. Twenty-five micrograms of nuclear or membrane extracts were run on 10% SDS-PAGE gels, transferred to PVDF membranes, probed with anti-SREBP1, and expression detected as described above.
Results
ACTH stimulates sphingolipid metabolism in H295R cells
Several studies have characterized the role of sphingolipids in steroid hormone biosynthesis and found both stimulatory and inhibitory roles for ceramide, SPH, and S1P on hormone secretion (16, 17, 18, 19, 20, 21, 22, 23). However, the effect of ACTH on cellular sphingolipid content is unknown. Thus, we treated H295R human adrenocortical cells for 2 h with 50 nM ACTH or 1 mM Bt2cAMP and quantified the amounts of several sphingolipids by LC-ESI-MS/MS. As shown in Fig. 1A, both ACTH and Bt2cAMP decreased cellular SM. Of the different SM molecules in H295R cells, both ACTH and Bt2cAMP decreased C16 and C18 SM (Fig. 1B).
Activation of sphingolipid turnover of complex sphingolipid molecules like SM is linked to the increased production of bioactive species such as ceramide, SPH, and S1P. Unexpectedly, however, we also observed ACTH/cAMP-stimulated reduction of cellular ceramides and SPH (Fig. 1C). Bt2cAMP treatment also decreased the amounts of multiple chain-length subspecies (Fig. 1D).
ACTH/cAMP activate SK activity
Because ACTH and Bt2cAMP evoked decreases in cellular ceramides and SPH, we hypothesized that ACTH and Bt2cAMP were activating SK. Increased SK activity would explain decreases in cellular SPH due to phosphorylation to form S1P. Thus, we carried out enzyme assays to determine whether ACTH/cAMP was activating sphingosine kinase activity. As shown in Fig. 2, exposure of H295R cells to ACTH and Bt2cAMP resulted in rapid increases in SK activity. Maximal increases in enzyme activity were observed at 15 min for both agents and returned to basal levels after 30 min. Incubation of H295R cells for 15 min with ACTH increased SK activity 3.2-fold, whereas Bt2cAMP resulted in a 4.8-fold increase in catalytic activity (Fig. 2).
ACTH/cAMP promote S1P secretion into the media
The increase in SK activity after exposure to ACTH or Bt2cAMP suggested that ACTH/cAMP may activate sphingolipid turnover and increase the cellular concentration of S1P. However, mass spectrometric analysis of cells treated for 30 min or 2 h with 50 nM ACTH or 1 mM Bt2cAMP revealed decreases in the cellular amounts of S1P (Fig. 3). Because some cell types secrete S1P, the amounts in the medium were also analyzed by tandem mass spectrometry. As shown in Fig. 3, S1P in the medium increased in a manner that mirrored the decreases in the cells; therefore, it appears that ACTH and cAMP are stimulating catabolism of SM, resulting in decreases in the cellular content of ceramide, SPH, and S1P and secretion of S1P into the media. Unexpectedly, no significant difference was observed in the amount of S1P in the media at the 30-min and 2-h time points. The cellular levels of most sphingolipids returned back to control levels after 6 h (data not shown).
S1P induces steroidogenic gene expression
Given that ACTH alters the sphingolipid profile in H295R cells (Fig. 1), we carried out studies to further explore the relationship between sphingolipid metabolism and a steroidogenic enzyme. CYP17 mRNA expression in Bt2cAMP-, S1P-, or SPH-treated cells was analyzed by Northern blotting (Fig. 4A) and real-time RT-PCR (Fig. 4, B and C). Densitometric analysis of Northern blots revealed that SPH and S1P increased CYP17 mRNA expression by 1.3- and 3.6-fold, respectively, whereas Bt2cAMP induced hCYP17 mRNA expression 5.2-fold (Fig. 4A). As shown in Fig. 4B, both S1P and Bt2cAMP increased CYP17 mRNA expression in a time-dependent manner; however, maximal stimulatory effects of S1P were observed at 6 h, whereas Bt2cAMP maximally increased CYP17 mRNA at the 12-h time point.
cAMP-dependent CYP17 mRNA expression requires SK1
Because SK activity was increased by Bt2cAMP treatment (Fig. 3) and S1P increased CYP17 expression (Fig. 4), we used RNAi to determine whether cAMP-stimulated CYP17 transcription required SK. H295R cells were transfected with SK1 and SK2 siRNAs and then treated with 1 mM Bt2cAMP for 12 h. Real-time RT-PCR revealed that silencing both SK1 and SK2 decreased the stimulatory effect of Bt2cAMP on CYP17 mRNA expression (Fig. 5A). We confirmed that RNAi suppressed SK1 and SK2 protein expression by Western blotting (Fig. 5B) and SK activity analysis (Fig. 5C).
Sphingolipids stimulate CYP17 transcriptional activity
To determine whether the stimulatory effects of sphingolipids on CYP17 mRNA (Fig. 4) were mediated at the level of transcription, we performed transient transfection assays using plasmids containing varying lengths of the CYP17 promoter fused to the Firefly luciferase gene. SPH and S1P increased the transcriptional activity of the 1100- and 700-pGL3 CYP17 reporter constructs, whereas having no significant effect on the CYP17 57- and 300-pGL3 plasmids (Fig. 6A). These findings suggest that the region of the CYP17 promoter required for sphingolipid-dependent gene transcription lies between –700 and –300 bp. In contrast to the effect of SPH and S1P, Bt2cAMP significantly stimulated the luciferase activity of all plasmids tested (Fig. 6A) as seen previously (2). In silico analysis (35) revealed a putative binding site for SREBPs (Fig. 6B). Thus, we determined the effect of mutating this site on the ability of S1P to stimulate CYP17 reporter gene activity. As shown in Fig. 6C, mutation of the putative SRE abolished the stimulatory effects of S1P.
Sphingolipids increase acetylation of histone H3 at the CYP17 promoter
Next we examined the effect of S1P on the acetylation state of histone H3 at the CYP17 promoter. Cells were treated with Bt2cAMP sphingolipids or the histone deacetylase inhibitor, TSA, for 4 h, followed by ChIP. PCR was performed using primers that amplified regions –142/+45 and –520/–360. Although we have previously shown that ACTH/cAMP-dependent gene transcription occurs on binding of a complex containing SF-1/p54nrb/PSF to region –57/–37 of the CYP17 promoter (2), studies described herein show that S1P activates CYP17 gene transcription by stimulating the binding of a trans-acting factor(s) to a more distal region of promoter (–436/–448).
As shown in Fig. 7A, acetylation of histone H3 at the –520/–360 region of the CYP17 gene is increased by Bt2cAMP, SPH, and S1P. TSA also increased the acetylation of histone H3 at the CYP17 promoter (Fig. 7A). These findings are in contrast to the results of PCR using primers designed to amplify the –142/+45 region of the CYP17 promoter, in which only Bt2cAMP and TSA significantly increased the acetylation of histone H3 (data not shown). Finally, we performed ChIP assays using antibodies to SREBP1. As shown in Fig. 7B, ACTH, Bt2cAMP, and S1P increased binding of SREBP1 to the –520/–60 region of the CYP17 gene.
S1P induces CYP17 transcription by activating SREBP1
Because sphingolipid-stimulated SREBP1a cleavage (24) mediates increased transcription of StAR (25), we investigated the role of SREBPs in S1P-evoked CYP17 gene expression. H295R cells were transfected with SREBP1 and SREBP2 siRNAs and treated with 1 mM S1P for 6 h followed by total RNA extraction and quantitative RT-PCR as described in Materials and Methods. As shown in Fig. 8A, SREBP1 siRNA abolished S1P-stimulated transcription of CYP17, whereas siRNAs targeted against SREBP2 decreased S1P-stimulated CYP17 mRNA expression by 31%. Small interfering oligonucleotides specifically decreased the protein expression SREBP1 and SREBP2 (Fig. 8B).
S1P stimulates SREBP1 cleavage and nuclear translocation
Nascent SREBPs contain two transmembrane domains that are integrally inserted into the endoplasmic reticulum. Processing of SREBP to generate the transcriptionally active N-terminal fragment involves the transport of the full-length protein to the Golgi apparatus in which it is cleaved sequentially by two membrane-associated proteases. The transcriptionally active fragment of SREBP is then translocated to the nucleus in which it binds to the promoters of SREBP target genes. To determine whether S1P stimulates the processing of SREBP1, we treated H295R cells with S1P for varying time points and measured the levels of mature and precursor forms by Western blotting. Because the mature form of SREBP is subjected to ubiquitination and subsequent proteasomal degradation (36), H295R cells were treated with the proteasome inhibitor ALLN as previously described by others (24, 37). As shown in Fig. 9A, S1P stimulates SREBP1 maturation in a time-dependent manner. To determine whether S1P activates SREBP cleavage by binding to a S1P receptor or acting intracellularly, we treated H295R cells with dhS1P. Like S1P, dhS1P binds to and activates S1P receptors (38, 39); however, because of its increased hydrophilic nature, dhS1P is unable to permeate the plasma membrane (40, 41). Both S1P and dhS1P stimulated an increase in the mature form of SREBP1 in the nucleus (Fig. 9B), suggesting that S1P acts extracellularly to induce SREBP1 maturation and induce CYP17 transcription.
Our observation that cAMP stimulates the secretion of S1P into the media (Fig. 3) coupled with the finding that dhS1P promotes SREBP1 translocation to the nucleus (Fig. 9B) suggests that S1P may act in a paracrine or autocrine manner to increase CYP17 transcription by binding to S1P receptors on surface of H295R cells. S1P regulates biological processes by serving as a ligand for the S1P family of G protein-coupled receptors (42, 43) and acting intracellularly (42). To date, five S1P receptors have been identified (44, 45, 46). These G protein-coupled receptors were initially called endothelial differentiation gene (EDG) receptors but have been renamed as S1P1 (EDG-1), S1P2 (EDG-5), S1P3 (EDG-3), S1P4 (EDG-6), and S1P5 (EDG-8) (44). As shown in Fig. 9C, all five receptors are expressed in H295R cells; however, S1P4 is not as highly expressed as the other four receptor subtypes. Finally, like Bt2cAMP and S1P, dhS1P is able to induce CYP17 mRNA expression (Fig. 9D), further supporting our hypothesis that S1P acts in a paracrine manner to stimulate SREBP1 binding to the CYP17 promoter by binding to a G protein-coupled receptor.
Discussion
ACTH exerts its stimulatory actions on steroid hormone biosynthesis via two temporally distinct cAMP/PKA-dependent pathways. A rapid, acute response results in the transport of cholesterol into the inner mitochondrial membrane for conversion to pregnenolone by P450 11A1 (P450scc). During the acute response, an essential site of phosphorylation by PKA is cholesterol ester hydrolase, which on activation catalyzes the conversion of cholesterol esters to free cholesterol (47). Key regulators of the acute response, StAR protein (48, 49) and the peripheral benzodiazapene receptor (50, 51), facilitate cholesterol movement in the mitochondria. The chronic effect of ACTH is to increase the transcription of steroidogenic enzymes.
In this report, we provide evidence for a novel mechanism by which ACTH induces CYP17 transcription. We show herein that both ACTH and cAMP decrease the cellular amounts of several sphingolipid species, including SM and ceramides (Fig. 1). In addition, ACTH and cAMP rapidly and transiently activate SK catalytic activity (Fig. 2). Previous studies have demonstrated the relationship between SM and P450scc (52). P450scc catalyzes the first step in steroid hormone biosynthesis: conversion of cholesterol to pregnenolone. SM inhibits the ability of cholesterol to bind to P450scc (52). Furthermore, the interaction between cholesterol and SM is cooperative (52), indicating that interactions between cholesterol and lipids can play a role in steroidogenesis. In light of these previous findings, the studies presented herein suggest that in addition to increasing availability of cholesterol for steroidogenesis, ACTH activates sphingolipid metabolism to maintain optimal cholesterol to lipid ratios in the membranes of adrenocortical cells.
Because decreases in cellular levels of S1P were paralleled by increases in this bioactive molecule in the medium (Fig. 3) and S1P receptors are expressed in H295R cells, we speculated that S1P acts in a paracrine manner to increase steroidogenesis. Our findings that the S1P receptor agonist dhS1P mimicked the stimulatory effect of S1P on CYP17 gene expression suggest that S1P acts extracellularly by binding to one of the S1P receptors. Studies are underway to determine which one of the five S1P receptors mediates CYP17 transcription in response to S1P. The differences in signaling through these receptors are primarily a result of differential coupling to G proteins. S1P1 couples to Gi (53, 54), whereas S1P2 and S1P3 couple to Gi, Gq, and G13 (54). S1P4 has been shown to associate with Gi (39, 55) and G12/13 (56) and S1P5 couple to Gi/o and G12 (57). Interestingly, S1P2 activates adenylyl cyclase (58); however, because the receptor does not couple to Gs, the activation of adenylyl cyclase may occur through an indirect mechanism. It has also been shown that S1P induces MAPK phosphatase-1 (MKP-1) in mouse fibroblast CH10T1/2 cells (59). We have previously shown that induction of CYP17 gene expression in response to cAMP requires activation of MKP-1 (1). Thus, it is possible that cAMP-stimulated release of S1P into the media may activate CYP17 gene expression via MKP-1. Although our data demonstrating that S1P is secreted into the media (Fig. 3) and that dhS1P induces CYP17 transcription (Fig. 9C) suggest that S1P acts extracellularly, it is possible that S1P may also be acting intracellularly. The generation of fatty acids for prostaglandin synthesis by phospholipase A2 has been shown to have both extracellular and intracellular components (60, 61, 62, 63, 64).
SREBPs regulate genes involved in the sterol synthesis and uptake as well as fatty acid biosynthesis and desaturation (26, 65). When the levels of free cholesterol in the cell are low, SREBPs undergo proteolytic processing, resulting in the release of a mature transcription factor that translocates to the nucleus for increased expression of genes involved in cholesterol biosynthesis and fatty acid metabolism (26, 65). The activity of these transcription factors is controlled by transport from endoplasmic reticulum to Golgi by an escort protein called SREBP-cleavage activating protein (SCAP), which senses the absence of cholesterol (26). Recently sphingolipids have been shown to stimulate SREBP-1 cleavage by causing the cholesterol to be sequestered in endosomes or lysosomes, leading to a compartmentalization of cellular cholesterol (24). This redistribution of cholesterol is sensed by SCAP, which leads to the translocation of SREBPs to the Golgi for maturation (24). In agreement with these studies, we found that S1P promotes SREBP1 maturation (Fig. 9A) and nuclear expression (Fig. 9B). Moreover, transcription of StAR is increased by SREBP-1 (25). ChIP data presented in Fig. 7 showing increased acetylation of histone H3 and recruitment of SREBP1 to the –700/–300 region of the CYP17 promoter suggest that S1P stimulates chromatin remodeling, resulting in increased accessibility to this region of the promoter and SREBP binding. Of note, hyperacetylation of histone H3, but not histone H4, has been found to occur in chromatin at the promoters for the low-density lipoprotein receptor gene and hydroxymethyl glutaryl CoA reductase (66). Our findings agree with these findings and suggest that in addition to increasing the acetylation of histone H3 at the promoters of sterol-regulated genes, SREBP also promotes hyperacetylation of a steroidogenic gene. Additionally, recent studies by Hughes et al. (67), demonstrate that orthologs of SREBP and SCAP, sre1+, and scp1+, respectively, function as an oxygen sensor in fission yeast.
In these studies, microarray analysis carried out to identify genes activated by SREBP1 revealed that two genes required for the hydroxylation of sphingolipids were highly up-regulated (67). Previously it was proposed by Lawler et al. (68) that stimulation of human hepatocytes by TNF leads to the activation of neutral SMase, which in turn induces the maturation of SREBP1. This induction of SREBP1 cleavage occurred independent of cholesterol depletion. Furthermore, studies have suggested that whereas SRE-mediated gene transcription is decreased by the inhibition of ceramide synthesis, it is stimulated by increasing SPH levels (69).
Based on the findings presented herein, we propose a model (Fig. 10) for the role of S1P and SREBP1 in ACTH/cAMP-dependent CYP17 transcription and cortisol biosynthesis. Binding of ACTH to its receptor on cell surface activates adenylyl cyclase, which leads to the production of cAMP and activation of PKA. PKA promotes the turnover of SM, which leads increased sphingolipid turnover due to the activation of enzymes (such as SK) in the sphingolipid pathway, causing decreased levels of several sphingolipid species. The S1P released into extracellular space binds to an S1P receptor, thereby activating a signal transduction cascade that activates SREBP1 cleavage and maturation. Although we found that total cellular amounts of S1P decreased in cAMP-treated cells (Fig. 1), it is also possible that S1P concentrations are increased in distinct subcellular organelles, such as the endoplasmic reticulum or Golgi and that this organelle-specific increase may result in an intracellular pool of S1P that may contribute to the increase in CYP17 transcription. Cellular fractionation studies are ongoing to determine the effects of ACTH and cAMP on organellar concentrations of various sphingolipid moieties. Moreover, it is probable that ACTH/cAMP may affect de novo sphingolipid biosynthesis. Studies are underway to determine whether the decreases in cellular sphingolipid content are due to increased catabolism, decreased de novo synthesis, or both. In our model (Fig. 10), we propose that the S1P produced in response to cAMP promotes the migration of SREBP1 to the Golgi in which it is processed to the mature form. Mature SREBP1 then translocates to the nucleus, dimerizes, and binds to the SRE site on the CYP17 promoter causing the activation of transcription of CYP17 gene. In summary, our studies demonstrate that ACTH/cAMP rapidly activates sphingolipid catabolism and S1P production, which ultimately leads to induction of CYP17. These findings provide evidence for a role for sphingolipids in ACTH/cAMP-dependent cortisol biosynthesis and establish a novel mechanism by which S1P, acting as a paracrine or autocrine factor, can increase CYP17 gene transcription by activating SREBP1.
Acknowledgments
We thank Samuel Kelly, Elaine Wang, and Dr. Alfred H. Merrill, Jr. for mass spectrophotometric analysis of sphingolipid molecular species.
Footnotes
This work was supported by the National Science Foundation (MCB-0347682), the National Institutes of Health (GM073241), and the Georgia Cancer Coalition. Mass spectrometric analysis of cellular lipids was supported by the National Institutes of Health (PA-02-132 to A.H.M.).
The authors have nothing to declare.
First Published Online November 23, 2005
Abbreviations: ALLN, N-acetyl-Leu-Leu-norleucine; Bt2cAMP, dibutyryl cAMP; C2-ceramide, N-acetyl-D-erythro-sphingosine; ChIP, chromatin immunoprecipitation; dhS1P, dihydro-S1P; EDG, endothelial differentiation gene; LC-ESI-MS/MS, liquid chromatography, electrospray ionization, and tandem mass spectrometry; MKP-1, MAPK phosphatase-1; PKA, protein kinase A; P450scc, P450 side chain cleavage enzyme; PSF, protein-associated splicing factor; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; RNAi, RNA interference; SCAP, SREBP-cleavage activating protein; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; siRNA, small interfering RNA; SK, sphingosine kinase; SM, sphingomyelin; SMase, sphingomyelinase; S1P, sphingosine-1-phosphate; SPH, D-erythro-sphingosine; SRE, sterol regulatory element; SREBP, sterol regulatory element binding protein; StAR, steroidogenic acute regulatory; TSA, trichostatin A.
Accepted for publication November 14, 2005.
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