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Endocrinology Vol. 147, No. 11 5249-5258
Copyright © 2006 by The Endocrine Society

Sphingosine Regulates the Transcription of CYP17 by Binding to Steroidogenic Factor-1

Aarti N. Urs, Eric Dammer and Marion B. Sewer

School of Biology and the Parker H. Petit Institute for Bioengineering & Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0230

Address all correspondence and requests for reprints to: Marion B. Sewer, School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, Georgia 30332-0230. E-mail: marion.sewer{at}biology.gatech.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroidogenic factor (SF1, Ad4BP, NR5A1) is a nuclear receptor that is essential for steroid hormone biosynthesis and endocrine development. Recent crystallographic studies have found that phospholipids are ligands for SF1. In the present study, our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using tandem mass spectrometry, we show that in H295R adrenocortical cells, SF1 is bound to sphingosine (SPH) and lyso-sphingomyelin (lysoSM) under basal conditions and that cAMP stimulation decreases the amount of SPH and lysoSM bound to the receptor. Silencing both acid and neutral ceramidases using small interfering RNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. SPH antagonized the ability of cAMP and the coactivator steroid receptor coactivator-1 to increase CYP17 reporter gene activity. These studies demonstrate that SPH is a bonafide endogenous ligand for SF1 and a negative regulator of CYP17 gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BIOSYNTHESIS OF STEROID hormones requires a battery of oxidative enzymes located in both mitochondria and endoplasmic reticulum. The expression of these enzymes is transcriptionally regulated by a cAMP/cAMP-dependent protein kinase [protein kinase A (PKA)] pathway. In the adrenal cortex, this cAMP/PKA pathway is activated by ACTH. Increased cAMP/PKA signaling stimulates the binding of various transcription factors to the promoters of steroidogenic genes. One of the transcription factors required for the expression of steroidogenic genes is steroidogenic factor 1 (SF1) (1, 2). SF1 is a nuclear receptor that plays a key role not only in steroidogenesis, but also in endocrine development and sex differentiation (3, 4). Targeted disruption of SF1 in mice resulted in adrenal and gonadal agenesis, absence of the ventromedial hypothalamic nucleus, and impaired expression of pituitary gonadotropins (5, 6).

We have recently shown that ACTH and cAMP activate the metabolism of sphingolipids (7) and that one of the bioactive molecules produced during sphingolipid turnover, sphingosine-1-phosphate (S1P), induces CYP17 transcription by promoting the binding of the sterol regulatory element binding protein-1 to the CYP17 promoter (8). Although we found that the mechanism by which S1P activates CYP17 gene transcription is not dependent on SF1, these studies prompted us to examine the role of other sphingolipids in the regulation of CYP17 gene expression. We postulated that the ACTH- and cAMP-stimulated fluxes in the amounts of specific sphingolipids might affect CYP17 expression by modulating the binding of transcription factors other than sterol regulatory element binding protein-1 to the promoter. Furthermore, because SF1 regulates cAMP-dependent transcription of CYP17 (9, 10, 11), it is possible that cAMP-stimulated changes in cellular sphingolipid concentrations regulate the binding of SF1 to the CYP17 promoter.

SF1 is classified as an orphan nuclear receptor because a bonafide endogenous ligand has yet to be identified. Previous structural studies demonstrating that the ligand binding domain (LBD) of SF1 adopts an active conformation with helices 1 and 12 packed against the predicted {alpha}-helical bundle (12), led to the hypothesis that the receptor activates gene expression in a ligand-independent manner. However, recent x-ray crystallographic studies carried out using bacterially expressed SF1 have shown that phospholipids are present in the ligand binding pocket (13, 14, 15). Krylova et al. (13) demonstrated that phosphatidyl inositols interact with the LBD of SF1 and that ligand binding is required for maximal activity of the receptor. Li et al. found that the receptor has a large (~1600 Å) LBD that interacts with phospholipids that have fatty acid side chains between 12 and 18 carbons (14). Significantly, the large binding pocket led the authors to speculate that SF1 may readily exchange its ligands to respond to different cellular cues (14).

Although these structural studies have established the key role that ligand binding plays in regulating the ability of SF1 to activate gene expression, they were carried out using bacterially expressed receptor. Thus, the aim of the present study was to identify endogenous ligands bound to SF1 and to characterize the functional significance of these ligands in mediating cAMP-dependent transcription of CYP17 in the human adrenal cortex. Based on the studies described above demonstrating that both ACTH and cAMP alter the cellular content of various sphingolipid species (7, 8), we used tandem mass spectrometry to determine whether sphingolipids bind to SF1. We show that SPH and lysoSM bind to SF1 under basal conditions and that cAMP treatment decreases the amount of both sphingolipids bound to the receptor. We also show that SPH decreases SF1-dependent transcription of CYP17 reporter constructs by promoting the inhibitory effect of the corepressor SMRT (silencing mediator of retinoid and thyroid hormone receptor). Additionally, silencing ceramidase expression using RNA interference mimics cAMP-stimulated transcription of CYP17. In summary, these studies demonstrate that SPH is a bonafide endogenous ligand for SF1 and that cAMP stimulates transcription of CYP17 by promoting displacement of SPH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Dibutyryl cAMP (Bt2cAMP) was obtained from Sigma (St. Louis, MO). Phospholipids and sphingolipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL).

Cell culture
H295R adrenocortical cells (16, 17) were generously donated by Dr. William E. Rainey (Medical College of Georgia, Augusta, GA) and cultured in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% {nu}-Serum I (BD Biosciences, Palo Alto, CA), 0.5% ITS Plus (BD Biosciences), and antibiotics. Jeg3 human choriocarcinoma cells were obtained from Dr. Michael R. Waterman (Vanderbilt University School of Medicine, Nashville, TN) and cultured in DMEM containing 10% fetal bovine serum and antibiotics.

Immunoprecipitation
For immunoprecipitation assays, H295R cells (150-mm dishes) were transfected with 45 µg of mutant or wild-type SF1-pCMV Tag1 per dish and treated with 1 mM Bt2cAMP for 4 h. Nuclear proteins were isolated using the NE-PER nuclear and cytoplasmic extraction reagent (Pierce, Rockford, IL) and incubated with anti-SF1 (Upstate, Lake Placid, NY) or anti-FLAG M2 antibody (Stratagene, La Jolla, CA) and protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C with rotation. The mixture was then centrifuged, the supernatant removed, and the precipitant subjected to a series of 5-min washes. Agarose beads were washed three times with a modified RIPA buffer [150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin] and ten times with PBS. The samples were then analyzed by mass spectrometry for detection of sphingolipids and by SDS-PAGE (10% gel) and Western blotting to confirm SF1 expression.

Analysis of sphingolipid and phospholipid molecular species
For sphingolipid measurements, immunoprecipitated SF1 was analyzed by liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS) as described previously (18, 19). Immunoprecipitated SF1 was pipetted into glass tubes and reconstituted in 500 µl methanol and 250 µl chloroform. Samples were mixed with internal standards (obtained from Avanti Polar Lipids), sonicated, and incubated overnight at 48 C. Samples were divided in half. Seventy-five microliters of KOH were added to one half of the sample, which was then centrifuged, dried, and reconstituted in free base reconstitution mixture [methanol:water:acetic acid (50:50:1, vol:vol:vol)]. The other half of the sample was neutralized (acetic acid, chloroform, and water) and centrifuged. The lower phase was dried and reconstituted in complex sphingolipid reconstitution mixture [methanol:chloroform (25:75, vol:vol). Samples were analyzed by LC-ESI-MS/MS using multiple reaction monitoring and sphingolipids quantified by calculating the area under the peaks.

RNA interference and real-time RT-PCR
Cells were subcultured into 12-well plates and 24 h later transfected with 150 nM of small interfering RNAs (siRNAs) directed against acid ceramidase (ASAH1), neutral ceramidase (ASAH2), alkaline ceramidase (ASAH3) (Dharmacon, Lafayatte, CO) using siIMPORTER (Upstate, Lake Placid, NY). Cells transfected with siRNA oligonucleotides directed against ASAH genes were incubated for 48 h then treated with 1 mM Bt2cAMP for 12 h. Expression of CYP17 was determined by real-time RT-PCR. For quantitative RT-PCR, total RNA was extracted using TRIzol (Invitrogen) and amplified using the iScript One-Step RT-PCR Kit (Bio-Rad, Hercules, CA) on an iCycler real-time thermocycler (Bio-Rad). TaqMan probes (Applied Biosystems, Foster City, CA) were used to detect CYP17. CYP17 expression is normalized to ß-actin (forward 5'-ACGGCTCCGGCATGTGCAAG-3' and reverse 5'-TGACGATGCCGTGCTGCATG-3') and calculated using the {delta}-{delta} cycle threshold ({Delta}{Delta}CT) method. Expression of ceramidases was determined by real-time RT-PCR using the following primers: ASAH1 (forward 5'-GCACAAGTTATGAAGAAGCCAAG-3' and reverse 5'-TCCAATGATTCCTTTCTGTCTCG-3'), ASAH2 (forward 5-GCGGCTGGCACTATTGATG-3' and reverse 5'-AGGATGGGCTTTGGTTTATGAC-3'), and ASAH3 (forward 5'-TCCGCCTGGTCTTCATCAC-3' and reverse 5'-GCTCCTTATTGCTGGTCTTCC-3').

Ceramidase activity assay
To confirm that siRNA oligonucleotides effectively suppressed ceramidase expression, we carried out ceramidase activity assays (20, 21). Briefly, cells were subcultured into 60-mm dishes and transfected with 150 nM siRNA oligonucleotides directed against ASAH1, ASAH2, or ASAH3. Forty-eight hours after transfection, cells were harvested into 500 µl of lysis buffer [10 mM Tris-Cl (pH 7.4), 0.2% Triton X-100, 1 mM 2-mercaptoethanol, 1 mM EDTA, 15 mM NaCl, 1x protease inhibitors]. Lysates were passed through a 25-gauge needle five times and a 20-µl aliquot removed for determination of protein concentration using the bicinchoninic acid assay (Pierce, Rockford, IL). To the remaining lysate, 1 µl 10 mM {omega}(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD)-ceramide was added and the samples vortexed and incubated on ice for 10 min to allow substrate to equilibrate among the membranes. One hundred-microliter aliquots were pipetted into borosilicate tubes containing buffers for assaying neutral [10 mM Tris-Cl (pH 7.4), 0.2% Triton X-100], acid (0.5 M acetate buffer, pH 4.5), or alkaline (10 mM HEPES, pH 9.0) ceramidase activity. The reactions were incubated at 37 C for 1 h in a water bath, then stopped by adding 10 µl of 10 mg/ml oleic acid (in chloroform:methanol, 2:1, vol/vol), 1 ml of chloroform:methanol (2:1, vol/vol) and 1 ml of Dole’s solution (Isopropanol:heptane:2 N H2SO4, 40:10:1, vol/vol). Samples were mixed and incubated 10 min, and then 400 µl heptane and 600 µl water were added and the samples vortexed 2 min. Samples were centrifuged for 10 min at 4000 rpm and the lower organic phase dried, resuspended in 15 µl chloroform:methanol (2:1, vol/vol) and spotted onto thin-layer chromatography plates along with NBD-ceramide and NBD-sphingosine standards. Plates were developed in chloroform:methanol:acetic acid (94:1:5, vol/vol) and reaction products visualized by fluorescent scanning on a Fluorescence/Phospho-Imager (Fuji Film, Japan).

Western blotting
Transfected cells (siRNA or SF-1 wild-type and mutant plasmids) were washed twice in PBS, harvested into 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] 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 and transferred to polyvinylidene difluoride membranes (PVDF, Pall Corp., Pensacola, FL). Blots were probed with anti-M2 FLAG (Stratagene) and expression detected using an ECF Western Blotting Kit (Amersham Biosciences, Piscataway, NJ). Protein expression was visualized by scanning blots on a Fluorescence/Phospho-Imager (Fuji Film, Japan).

Measurement of dehydroepiandrosterone (DHEA) secretion
Cells were cultured in 12-well plates and treated with 1 mM Bt2cAMP and/or 1 µM SPH for 8, 24, or 48 h. Media were collected and DHEA released into the media was determined in triplicate against standards made up in DMEM/F12 using a 96-well plate enzyme-linked immune assay (EIA; Diagnostic Systems Corp., Houston, TX). Results are expressed as nanomoles per milligram cellular protein. Cells were isolated for quantification of total cellular protein using the bicinchoninic acid protein assay (Pierce, Rockford, IL).

Bacterial expression of SF1
PCR was used to add six histidine residues to the LBD (amino acids 222–462) of SF1 and the PCR products cloned into the pET17b vector (Novagen, Madison, WI). The construct was transformed into BL21 cells and the cultures grown until the OD600nm equaled 0.4. Isopropyl-ß-D-thiogalactopyranoside (0.4 mM) was added and the cultures grown for 6 h at 28 C. His-tagged SF1 was purified using nickel affinity chromatography using a His-Bind purification kit (Novagen, Madison, WI) followed by a Q Sepharose column (GE Healthcare, Piscataway, NJ). Expression was verified by SDS-PAGE and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Scintillation proximity assay (SPA)
SPA (22, 23) experiments were carried out using purified and biotinylated SF1. SF1 (500 nM) was biotinylated using a EZ-Link Sulfo-NHS-LC-biotinylation kit (Pierce, Rockford, IL) for 2 h at 4 C, with rotation. The reaction was desalted to remove nonreacted biotinylation reagent as per the manufacturer’s instructions. Streptavidin-polyvinyl toluene SPA beads (Amersham Biosciences, Piscataway, NJ) were suspended in assay buffer (50 mM Tris-Cl, pH 8.0; 50 mM KCl; 1 mM EDTA; 1 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, 1 mM dithiothreitol, 0.1 mg/ml BSA) at 0.5 mg/ml. The biotinylated receptor was added to a final concentration of 100 nM and the mixture incubated for 4 h at 4 C with rotation. The reaction was centrifuged at 4000 rpm for 10 min and the uncoupled receptor (supernatant) was removed. The beads were resuspended in assay buffer, incubated for 15 min, and centrifuged again. Beads were resuspended in 1 ml assay buffer and used for subsequent reactions. To determine nonspecific binding, [3H]-SPH was added such that the final concentration of radioligand ranged from 1–500 nM.

For competition experiments, lipids were added at concentrations ranging from 0.1 nM to 100 µM to reactions containing 10 nM [3H]-SPH. Reactions were incubated for 2 h at room temperature followed by scintillation counting. All assays were performed at least three times in triplicate. Competition curves were generated by nonlinear regression analysis using GraphPad Prism 4.

Transient transfection and reporter gene analysis
To determine the effect of mutations in the LBD on SF1-dependent transcriptional activity, Jeg3 human choriocarcinoma cells were subcultured onto 12-well plates and 24 h later transfected with 250 ng of hCYP17 57-pGL3 reporter plasmid (9) and 50 ng of wild-type or mutants (H310A, G341K, Y436A, K440E) SF1-pCMV Tag1 (Invitrogen) using GeneJuice (Novagen, Madison, WI). wild-type bovine SF1 and human liver receptor homolog 1 (LRH1) were generously provided by Drs. Ken-ichiro Morohashi (National Institute for Basic Biology, Okazaki, Japan) and Matthew Redinbo (University of North Carolina, Chapel Hill, NC), respectively and cloned into the pCMV Tag1 vector. SF1 mutants were prepared using a QuikChange site-directed mutagenesis kit (Stratagene,La Jolla, CA) and were confirmed by sequencing. Cells were cotransfected with 10 ng of a Renilla luciferase plasmid under the control of the thymidine kinase (TK) promoter (TK-pRL, Promega, Madison, WI) for normalization. Twenty-four hours after transfection, the cells were treated with 1 mM Bt2cAMP and/or 1 µM SPH for 6 h and the transcriptional activity of the CYP17 reporter gene determined using a dual-luciferase assay (Promega, Madison, WI).

To examine the effect of sphingolipids and phospholipids on SF1-dependent CYP17 reporter gene activity, H295R human adrenocortical cells were transfected with 250 ng CYP17 57-pGL3, 50 ng SF1-pCMV Tag1, and 50 ng steroid receptor coactivator 1 (SRC1)-BK CMV (generously provided by Dr. Bert O’Malley, Baylor School of Medicine, Houston, TX), or 50 ng SMRT-pCMX (generously donated by Dr. Ronald Evans, The Salk Institute, La Jolla, CA), and 10 ng of the Renilla luciferase plasmid (for normalization). Twenty-four hours after transfection, cells were treated with 1 mM Bt2cAMP or SPH (0.1–10 µM) and harvested for dual-luciferase assays.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SF1 binds to sphingolipids in vivo
Mass spectrometric analysis was used to determine whether sphingolipids are ligands for SF1. The receptor was immunoprecipitated using an antibody to SF1 from control or cAMP-stimulated H295R cells and analyzed by LC-ESI-MS/MS. As shown in Fig. 1AGo, both SPH and lysoSM were bound to SF1 isolated from untreated cells. A pie chart shows the relative amounts of all sphingolipids bound to the receptor (Fig. 1CGo). There was 270 pmol of SPH bound to SF1 per milligram of total cellular protein and 325 pmol of lysoSM bound to SF1 per milligram of total cellular protein (Fig. 1AGo). However, when SF1 was immunoprecipitated from cells treated for 4 h with 1 mM Bt2cAMP, the amount of SPH and lysoSM decreased by 44% and 36%, respectively. These findings suggest that SPH and/or lysoSM bind to SF1 and maintain the receptor in an inactive conformation and that cAMP stimulation decreases sphingolipid binding, thereby activating the receptor. The relative amount of all sphingolipids quantified is shown in Fig. 1CGo. The amounts of other sphingolipids (ceramides, sphingomyelin, sphinganine, S1P, ceramide-1-phosphates) were present at less than 15 pmol/mg total protein.


Figure 1
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  		FIG. 1. SPH binds to SF1 in vivo. A, Quantification of LC-ESI-MS/MS analysis of SPH and lysoSM bound to SF1. H295R cells were transiently transfected with SF1-pCMVTag1 and treated for 4 h with 1 mM Bt2cAMP. The lysates were immunoprecipitated with anti-SF1 antibody and the immunoprecipitated receptor analyzed by LC-ESI-MS/MS. B, Structures of SPH and lysoSM. C, Relative amounts of all sphingolipid species bound to control or Bt2cAMP-treated H295R cells. Sa, Sphinganine; SaP, sphinganine-1-phosphate; lysodhSM, lysodihydrosphingomyelin; C1P, ceramide-1-phosphate; gluc-cer, glucosylceramides; SM, sphingomyelin. D, Competition binding assay. His-tagged SF1 was expressed in E. coli, purified, biotinylated and coupled to streptavidin-coated SPA beads as described in Materials and Methods. Receptor-bound SPA beads were incubated with 10 nM [3H]SPH and nonradiolabeled SPH (0.1 nM to 1 mM, filled squares) or lysoSM (0.1 nM to 1 mM, filled triangles). Data points represent the mean ± SD of four assays performed in triplicate.

 
SPH and lysoSM bind SF1 in vitro
To further characterize the interaction between SF1 and sphingolipids and to quantify relative binding affinities, we performed the SPA (22, 23, 24). His-tagged SF1 LBD was expressed in Escherichia coli, purified, biotinylated, and bound to streptavidin-coated polyvinyltoluene beads (see Materials and Methods). We determined the apparent equilibrium dissociation constant (Ki) for SPH and lysoSM in competition binding assays. The beads were incubated with 10 nM [3H]SPH in the presence of unlabeled SPH or lysoSM at concentrations ranging from 0.1 nM to 100 µM (Fig. 1DGo). [3H]SPH specifically bound to SF1 with a calculated Ki of approximately 325 nM, confirming LC-ESI-MS/MS studies and further demonstrating that [3H]SPH binds directly to SF1. LysoSM on the other hand, with an apparent Ki greater than 1 µM, was not as effective in competing with [3H]SPH for binding to SF1 (Fig. 1DGo). Based on the apparent Ki of 325 nM and the amount of SF1 used in each SPA reaction (100 nM), we estimate the binding stoichiometry to be 1:1.1, receptor to SPH.

Silencing ceramidase induces CYP17 transcription
Based on SPA competition assays demonstrating that SPH bound to SF1 with a higher affinity than lysoSM, we carried out studies to further characterize the role of SPH in SF1-dependent CYP17 transcription. SPH is produced when ceramidases cleave the amide bond of ceramide. To date, three types of ceramidases have been described and are classified as acid (ASAH1), neutral (ASAH2) and alkaline (ASAH3) according to the pH at which optimal activity is achieved (21, 25, 26, 27). Because cAMP decreases the amount of SPH bound to SF1 (Fig. 1AGo), we hypothesized that decreasing the cellular production of SPH would increase CYP17 gene expression. To test this hypothesis, H295R cells were transfected with siRNAs directed against ASAH1, ASAH2, and ASAH3 and the mRNA expression of CYP17 measured by real-time RT-PCR. As shown in Fig. 2AGo, silencing ASAH1 and ASAH2 increased CYP17 mRNA expression by 4.1- and 2.0-fold, respectively, further supporting a role for SPH as an inhibitory ligand for SF1. Real-time RT-PCR (Fig. 2BGo) and ceramidase activity assays (Fig. 2CGo) were carried out to confirm that RNA interference specifically suppressed the expression of each ceramidase gene.


Figure 2
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  		FIG. 2. Silencing ceramidase expression induces CYP17 mRNA. A, Cells were transfected with siRNA oligonucleotides targeted against ASAH1, ASAH2, or ASAH3 for 48 h. One group of untransfected cells was treated with 1 mM Bt2cAMP for 12 h. Total RNA was isolated and CYP17 mRNA expression was determined using real-time RT-PCR. Data graphed represent the mean ± SEM of three separate experiments, each performed in quadruplicate. *, P < 0.05 statistically different from untreated control. B, H295R cells were transfected with 150 nM ASAH1, ASAH2, or ASAH3 siRNA for 48 h and expression of all three ceramidases determined by real-time RT-PCR as described in Materials and Methods. Data graphed represent the mean ± SEM of three separate experiments, each performed in quadruplicate. *, P < 0.05 statistically different from untreated control. C, Ceramidase activity was determined by incubating lysates isolated from H295R cells transfected with siRNA oligonucleotides against ASAH1, ASAH2, or ASAH3 with NBD-ceramide in acid, neutral, or alkaline reaction buffer. Reactions were terminated and subjected to thin-layer chromatography. SPH formation was quantified by fluorescence scanning and densitometry and normalized to the protein content of each sample. Data graphed represent the mean ± SEM of two separate experiments, each performed in triplicate. *, P < 0.05 statistically different from untreated control.

 
SPH antagonizes cAMP-stimulated CYP17 reporter gene activity
Based on LC-ESI-MS/MS data demonstrating that cAMP treatment decreased the amount of SPH bound to SF1, we hypothesized that SPH may act as an antagonist and inhibit the ability of cAMP to activate SF1-dependent CYP17 reporter gene activity. As shown in Fig. 3AGo, increasing concentrations (0.1–10 µM) of SPH attenuated the stimulatory effect of Bt2cAMP on CYP17 transcriptional activity, whereas SPH had no effect on the ability of LRH-1 to increase CYP17 reporter gene activity. In additional experiments carried out to determine the specificity of SPH on reporter gene activity, cells were transfected with a plasmid containing the human CYP1B1 promoter (regulated by the aryl hydrocarbon receptor) fused to the luciferase gene and treated with 2,3,7,8-tetrachlorodibenxodioxin (TCDD; ligand for the aryl hydrocarbon receptor). SPH had no effect on TCDD-stimulated CYP1B1 reporter gene activity (data not shown). LysoSM had no significant effect on the ability of Bt2cAMP to stimulate SF1-dependent CYP17 reporter gene activity (data not shown).


Figure 3
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  		FIG. 3. SPH antagonizes cAMP-stimulated CYP17 transcription, expression, and P450c17 activity. A, Jeg3 cells were transfected with CYP17 57-pGL3, SF1-pCMVTag1, and LRH1-pCMVTag1 using Gene Juice. Twenty-four hours after transfection, the cells were treated for 6 h with 1 mM Bt2cAMP in the presence and absence of SPH (0.1-, 1-, or 10 µM). Data graphed are normalized to Renilla activity (TKpRL) and is expressed as fold increase of CYP17 57-pGL3 Firefly luciferase activity over TKpRL Renilla luciferase activity. Data graphed represent the mean ± SEM of three experiments, each performed in triplicate. *, P < 0.05 statistically different from untreated control. B, H295R cells were treated with 1 mM Bt2cAMP and/or 1 µM SPH for 12 h and total RNA was isolated for analysis of CYP17 and actin mRNA expression by real-time RT-PCR. Data graphed are expressed as percent of control group mean of three experiments, each performed in triplicate. *, P < 0.05 statistically different from untreated control. C, H295R cells were cultured into 12-well plates and treated with 1 mM Bt2cAMP in the presence and absence of 1 µM SPH for 8, 24, or 48 h and media collected for quantification of DHEA production by EIA. DHEA secreted was normalized to the total cellular protein content.

 
We also carried out studies to determine the effect of SPH on endogenous CYP17 mRNA expression. H295R cells were treated with Bt2cAMP in the presence and absence of 1 µM SPH for 12 h and CYP17 mRNA quantified by real-time RT-PCR. As shown in Fig. 3BGo, Bt2cAMP increased CYP17 mRNA expression 480% compared with the untreated control group. SPH attenuated the stimulatory effect of Bt2cAMP on CYP17 mRNA expression and decreased basal expression by 25%. To determine the effect of SPH on P450c17 enzymatic activity, cells were treated with Bt2cAMP and SPH for 8, 24, or 48 h and media collected for analysis of DHEA content by EIA. Bt2cAMP increased DHEA secretion at all time points tested (Fig. 3CGo). Coincubation with SPH completely inhibited Bt2cAMP-stimulated DHEA biosynthesis at the 8-h time point and decreased DHEA secretion at later time points.

SPH antagonizes coactivator recruitment
The ability of nuclear receptors to activate gene transcription is dependent on the recruitment of coactivator proteins such as SRC-1 and the dissociation of corepressors such as SMRT. To determine the effect of SPH on the coregulator recruitment to the CYP17 promoter, Jeg3 cells were transfected with the CYP17 reporter plasmid and SRC1-pBK CMV or the corepressor SMRT. As shown in Fig. 4Go, cotransfection of SF1 increased CYP17–57pGL3 luciferase activity. Overexpression of SRC-1 further stimulated the ability of SF1 to transactivate the CYP17 reporter gene, whereas SMRT decreased reporter gene activity. Exposure of cells to 1 µM SPH attenuated the ability of SF1 to increase CYP17–57pGL3 transcriptional activity (Fig. 4Go). Additionally, SPH antagonized the stimulatory effect of Bt2cAMP on CYP17 reporter gene expression.


Figure 4
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  		FIG. 4. Effect of SPH on coregulator-mediated CYP17 reporter gene activity. Jeg3 cells were transfected with CYP17 57-pGL3, SF1-pCMV Tag1, SRC1-pBK CMV, SMRT-pCMX and pRL-TK. Twenty-four hours after transfection, cells were treated with 1 mM Bt2cAMP and/or 1 µM SPH and harvested for dual-luciferase assays. Data are normalized to the luciferase activity of the Renilla gene (TKpRL) and are expressed as the fold increase of CYP17 57-pGL3 activity over TKpRL activity. Data graphed represent the mean ± SEM of two separate experiments, each performed in triplicate.

 
Mutations in the LBD alter SPH binding in vivo
Based on recent structural studies identifying residues that contact the ligand (13), we generated constructs containing mutations in four amino acid residues (H310A, G341K, K440E, and Y436A). Residues G341, Y436, and K440 are at the entry of the ligand binding pocket and amino acid H310 lies on the inner surface of the pocket. Both wild-type and mutant FLAG-tagged constructs were transfected into H295R cells and immunoprecipitated for analysis by LC-ESI-MS/MS. The H310A mutant decreased binding of SPH to SF1 in control cells by 30%, while decreasing the binding of SPH to SF1 isolated from cAMP-treated cells by only 14% (Fig. 5AGo). Similar findings were observed with the G341K mutation. Bt2cAMP had no significant effect on the amount of SPH bound to the H310A and G341K mutants, suggesting that mutation of these two amino acid residues prevents the exchange of SPH for an activating ligand. The K440E and Y436A mutants exhibited approximately a 75% and 50% decrease in binding to SPH in control cells (Fig. 5AGo). Collectively, these observations suggest that K440 and Y436 are important for binding SPH and that H310 and G341 may play a role in the cAMP-stimulated exchange of SPH for a lipid whose binding is required for activation of the receptor. All mutants were expressed at similar levels (Fig. 5BGo).


Figure 5
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  		FIG. 5. Effect of mutations in the LBD of SF1 on SPH binding in vivo. Cells were transiently transfected with wild-type or mutant SF1 (in pCMV Tag1 vector) and treated for 4 h with 1 mM Bt2cAMP. A, The lysates were immunoprecipitated with anti-FLAG antibody and sphingolipid binding analyzed by LC-ESI-MS/MS. SPH amounts are expressed in picomoles per milligram of total protein and determined by comparison to a standard. Data graphed represent the mean ± SEM of two separate experiments, each performed in duplicate. B, Expression of wild-type and mutant receptor in transfected H295R cells. A fraction (10 µl of a 50% slurry) of the immunoprecipitated receptor was subjected to SDS-PAGE and Coomassie staining.

 
Effect of mutations in the LBD on CYP17-reporter gene activity
We next characterized the effect of mutations in the LBD of SF1 on CYP17 reporter gene expression. Jeg3 cells (which lack endogenous SF1) were transiently transfected with plasmids containing wild-type or mutant SF1 and the CYP17 pGL3 reporter plasmid. Cells were treated for 6 h with 1 mM Bt2cAMP and/or 1 µM SPH. As shown in Fig. 6Go, transfection of wild-type SF1 resulted in a 3.2-fold increase in luciferase activity, which was further stimulated by Bt2cAMP. Cotreatment with SPH inhibited the ability of Bt2cAMP-stimulated luciferase activity. Mutations to H310 and G341 attenuated both basal and Bt2cAMP-stimulated SF1-dependent transcriptional activity. Interestingly, mutations at K440 and Y436 increased the ability of SF1 to trans-activate the CYP17 reporter gene (Fig. 6Go), consistent with a decrease in the ability of these mutants to bind SPH (Fig. 5AGo). SPH inhibited Bt2cAMP-stimulated SF1 K440E-dependent transcriptional activity, while having no effect on SF1 Y436A-mediated reporter gene activity. These findings further suggest that SPH acts as an inhibitory ligand for SF1. Similar results were observed in H295R cells (data not shown).


Figure 6
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  		FIG. 6. LBD mutants of SF1 show reduced transcriptional activity. Jeg3 cells were transiently transfected with 250 ng CYP17 57-pGL3, and 100 ng wild-type or mutant SF1 (in pCMV Tag1 vector) and treated with 1 mM Bt2cAMP and/or 1 µM SPH for 6 h. Data graphed are normalized to Renilla activity (TKpRL) and are expressed as fold increase of CYP17 57-pGL3 Firefly luciferase activity over TKpRL Renilla luciferase activity. Data graphed represent the mean ± SEM of three experiments, each performed in triplicate.

 
Both sphingolipids and phospholipids compete with SPH for binding to SF1
Because recent studies have shown that phospholipids are ligands for SF1 and that ligand binding is required for maximal activity (13, 14), we determined the ability of several phospholipids and sphingolipids to displace [3H]SPH using the SPA. As shown in Fig. 7Go, most of the lipids tested displace [3H]SPH from the receptor. Phosphatidylinositols, which were bound to bacterially expressed SF1 used for crystallographic studies (13), showed varying abilities to decrease [3H]SPH binding to the receptor (Fig. 7Go). Phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], and phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] inhibited [3H]SPH binding by 40%, 48%, and 57%, respectively, whereas phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] only decreased [3H]SPH binding by 11%. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were also found in the LBD of SF1 in structural studies (14). PC (16:0–18:1) displaced 78% of [3H]SPH, whereas PE (16:0–18:1) decreased [3H]SPH binding to SF1 by 57%. Phosphatidic acid [PA; 16:0–18:1] inhibited 82% of [3H]SPH binding to SF1, whereas S1P displaced 93% of labeled SPH (Fig. 7Go).


Figure 7
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  		FIG. 7. Phospholipids and sphingolipids compete with [3H]SPH for binding to SF1. The LBD of SF1 was His-tagged, expressed in E. coli, biotinylated, and coupled to streptavidin-coated SPA beads as described in Materials and Methods. Competition binding assays were performed with 10 nM [3H]SPH and 10 µM of each lipid. The following lipids were assayed: PA, PC, PE, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P2], lysoSM, diacylglycerol pyrophosphate (DAGPP), lysophosphatidic acid (lysoPA), N-acetoyl ceramide-1-phosphate (act. C1P), N-octanoyl ceramide-1-phosphate (oct. C1P), S1P, and SPH. Data represent the mean of three assays performed in triplicate ± SD and are plotted as percent inhibition of [3H]SPH binding where competition with unlabeled SPH is defined as 100%.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nuclear hormone receptors constitute a superfamily of transcription regulators that are involved in widely diverse physiological functions including control of embryonic development, cell differentiation, and homeostasis (28, 29, 30, 31). SF1 is a member of the orphan nuclear receptor family because it was cloned before the identification of a ligand. SF1 is required for development of endocrine glands and sexual development and is also essential for steroid hormone biosynthesis and for the expression of steroidogenic genes (3, 4). In the adrenal gland, steroidogenic gene transcription occurs in response to ACTH, a peptide hormone that binds to G protein-coupled receptors on the surface of adrenocortical cells and activates a cAMP-dependent signal transduction cascade (2). Increased intracellular cAMP results in the activation of PKA, and ultimately, a coordinate increase in the transcription of steroidogenic genes. The direct target of PKA has yet to be determined; however, several transcription factors, including SF1, are required for conveying cAMP-dependent transcription (2).

Recently, crystallographic studies of bacterially expressed SF1 have found that phospholipids are ligands for SF1 (13, 14, 15). However, based on our studies establishing a role for sphingolipids in cAMP-dependent CYP17 gene expression (7, 8) and our findings that ACTH and cAMP promote sphingolipid metabolism (7, 8), we postulated that sphingolipids regulate CYP17 by modulating the ability of SF1 to activate gene expression. Thus, we used LC-ESI-MS/MS to determine whether sphingolipids bind to SF1 and characterized the effect of cAMP on the ability of sphingolipids to regulate SF1 function.

Our findings herein demonstrate that SF1 is a lipid binding protein. Specifically, we have shown that both SPH and lysoSM are bound to SF1 immunoprecipitated from H295R cells (Fig. 1AGo). Moreover, the amount of both sphingolipids bound to SF1 is regulated by cAMP because SF1 purified from cells treated with Bt2cAMP contained decreased amounts of SPH and lysoSM. Competition binding assays, however, demonstrated that lysoSM bound to SF1 with a lower affinity than SPH (Fig. 1DGo). Additionally, lysoSM had no effect on the ability of Bt2cAMP to stimulate CYP17 reporter gene activity (data not shown). Thus, these findings suggest that SPH is an inhibitory ligand for SF1 and that activation of the receptor takes place by the exchange of SPH for an activating ligand. This hypothesis is supported by RNA interference data demonstrating that silencing the expression of acid and neutral ceramidases increases CYP17 mRNA expression (Fig. 2Go). SPH is not synthesized de novo (32, 33). Thus, the sole cellular source of SPH is through the hydrolysis of ceramide, a reaction catalyzed by ceramidases (25, 26, 27). Acid ceramidase has a pH optimum of approximately 4.5; therefore, the hydrolytic activity of this enzyme is thought to occur within lysosomes and/or late endosomes, whereas neutral and alkaline ceramidases are active at pH values ranging from 6.5 to 9.0 (20). Studies using cloned neutral and alkaline ceramidases have shown that these enzymes can catalyze both the forward and reverse reactions (ceramide degradation and synthesis) in vitro (34, 35, 36). The neutral ceramidase is membrane bound; however, it has yet to be determined whether this enzyme is associated with the nuclear membrane. Of note, studies have shown that PA and phosphatidylserine stimulate the hydrolytic activity of neutral ceramidase (37).

Although lysoSM did not have any antagonistic effects on SF1-dependent CYP17 transcription, lysoSM regulates diverse biological responses including cytoskeletal rearrangement (38), cell proliferation (39, 40, 41, 42, 43), growth inhibition (44, 45, 46), cell migration (47), and wound healing (48). LysoSM belongs to a class of lipid signaling molecules, which includes lysophosphatidic acid and S1P (49, 50). These bioactive lipids exert their effects by either binding to G protein-coupled receptors or by acting as intracellular second messengers (49, 50). LysoSM is a high-affinity ligand for the ovarian cancer G protein-coupled receptor-1 (OGR1) (51). Upon binding to OGR1, lysoSM activates MAPKs, increases intracellular calcium, and inhibits the proliferation of MCF7 and HEK293 cells (51). Interestingly, OGR1 has been also characterized as a proton-sensing receptor that stimulates inositol phosphate production (52). The receptor was found to be inactive at pH 7.8 and activated at pH 6.8, leading the researchers to propose that OGR1 is a regulator of pH homeostasis (52). In the presence of lysoSM, however, the proton-stimulated inositol phosphate production is attenuated (53). When acting as an intracellular second messenger, lysoSM potently stimulates smooth muscle cell contraction (54). Although lysoSM had no effect on the ability of SF1 to promote CYP17 expression, the identification of this bioactive lipid bound to the receptor suggests that the interaction of lysoSM and SF1 may have functional significance. Clearly more studies are necessary to determine the role of lysoSM in SF1-dependent steroid hormone biosynthesis in the human adrenal cortex.

Our reporter gene studies in Jeg3 cells, which lack endogenous SF1 (Fig. 6Go) as well as in H295R human adrenocortical cells (data not shown) with SF1-containing mutations in the ligand binding pocket reveal that H310, G341, Y436, and K440 are critical for basal, and cAMP-stimulated transactivation potential. These four residues were among the 33 that made contact with phospholipid ligands identified in crystallographic studies by Krylova et al. (13). Residues G341, Y436, and K440 are at the entrance to the binding pocket and form hydrogen bonds with the phosphate head group at the entry of the binding pocket (13), whereas H310 lies within the pocket and interacts with the fatty acid tails of the phospholipids (14). Because SPH lacks the negatively charged phosphate head group found in the phospholipids isolated in crystallographic studies, residues G341, Y436, and K440 do not coordinate SPH by interacting with the amine group at carbon; however, an interaction between the terminal hydroxyl group on SPH and these three residues may be important for stabilizing the ligand-bound receptor. Although mutation of all three residues (G341, Y436, and K440) at the entry decreases the amount of SPH bound to the receptor, the K440E and Y436A mutations have the most profound effect on the interaction between SF1 and SPH (Fig. 5Go). Interestingly, these two mutations increase the ability of the receptor to activate CYP17 reporter gene activity (Fig. 6Go). However, SPH inhibited Bt2cAMP-stimulated K440E luciferase activity while having no effect on Y436A-dependent transcriptional activity. Modeling studies are warranted to determine the precise mechanism by which SPH makes contact with the ligand binding pocket of SF1.

As mentioned above, H310 lies within the binding pocket. Based on the amount of SPH bound to the H310A mutant immunoprecipitated from control and Bt2cAMP-treated cells (Fig. 5Go), we postulate that mutation of this residue neutralizes the positive charge near the fatty acid tail of SPH, which increases the stability of SPH in the binding pocket. In the crystal structure, H310 is covered with four water molecules, which neutralize the charge and are thought to stabilize the protein (14). We speculate that mutating H310 to alanine mimics the neutralizing effect of the water molecules found in the crystal structure. Of note, the mouse LRH-1, which is thought not to bind to ligands, has an acidic glutamate residue in place of the equivalent glycine at position 341 in SF1. Crystallographic analysis of mouse LRH-1 reveals that this negatively charged amino acid forms a strong ionic interaction with lysine-539, thereby closing the binding pocket and allowing mouse LRH-1 to be stabilized in an active conformation without ligand binding (13), whereas human SF1 lacks this structural bridge within the ligand binding pocket.

As previously discussed, both recently published crystal structures have revealed that SF1 contains a large ligand binding pocket that is filled by a phospholipid ligand (13, 14). Li et al. (14) show that the interaction of SF1 with coactivators is reduced both by the absence of phospholipids and by phospholipids with longer fatty acids, that are predicted to project out of the pocket and interfere with the folding of the activation function-2 helix into the active conformation. Although both crystallographic studies identified different phospholipids in the SF1 ligand binding pocket, neither study showed the presence of SPH or lysoSM because bacteria do not synthesize sphingolipids. Thus, our findings provide insight into an endogenous ligand for SF1 in a steroidogenic mammalian cell.

Significantly, our studies demonstrate that LC-ESI-MS/MS can be a useful tool for characterizing lipid-protein binding in vivo. Additionally, our findings suggest that ACTH may activate a cAMP-dependent signaling cascade leading to the exchange of SPH for a stimulatory ligand. We postulate that in the absence of ACTH/cAMP activation, the binding of SPH to SF1 stabilizes the receptor in an inactive conformation and prevents CYP17 transcription by impairing the ability of coactivators such as SRC-1 to promote transcription (Fig. 4Go). SPA data showing that several sphingolipids and phospholipids can compete with SPH for binding to the receptor (Fig. 7Go) suggest that SF1 may respond to different lipids in a context-specific manner to direct the expression of genes involved in development and steroidogenesis.

Our findings showing cAMP-stimulated decreased SPH binding to SF1 (Fig. 1AGo) are in agreement with the prediction of Li et al. (14) that SF1 readily exchanges its ligands. It is plausible that cAMP alters lipid amounts in distinct organelles (such as nucleus) and that these changes are essential for steroidogenic gene transcription. Studies are underway to determine the effect of cAMP on the subcellular populations of phospholipids and sphingolipids and to identify the endogenous activating ligand for SF1. In summary, our findings demonstrate that SPH is an endogenous ligand for the nuclear receptor SF1. To our knowledge, this is the first study to demonstrate that SPH regulates a transcription factor by directly binding to the protein. SPH binding to SF1 antagonizes the ability of cAMP to activate CYP17 gene transcription by promoting corepressor binding. We propose that ACTH/cAMP activates CYP17 in the human adrenal cortex by activating a signaling pathway leading to the dissociation of SPH from SF1 and the binding of a yet-to-be-identified activating ligand.


   Acknowledgments
 
We are grateful for mass spectrometric analysis of sphingolipid species carried out by Elaine Wang, Samuel Kelly, and Alfred H. Merrill, Jr.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grant PA02132, NIH Grant GM073241, National Science Foundation CAREER MCB0347682, and a Georgia Cancer Coalition Distinguished Scientist Award (to M.B.S.).

Disclosure summary: A.U., E.D., and M.B.S. have nothing to declare.

First Published Online August 3, 2006

Abbreviations: ASAH1, Acid ceramidase; ASAH2, neutral ceramidase; ASAH3, alkaline ceramidase; Bt2cAMP, dibutyryl cAMP; DHEA, dehydroepiandrosterone; EIA, enzyme-linked immune assay; LBD, ligand binding domain; LC-ESI-MS/MS, liquid chromatography, electrospray ionization, tandem mass spectrometry; LRH1, liver receptor homolog 1; lysoSM, lyso-sphingomyelin; NBD, {omega}(7-nitro-2-1,3-benzoxadiazol-4-yl); PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PKA, protein kinase A; SF 1, steroidogenic factor 1; siRNA, small interfering RNA; SMRT, silencing mediator of retinoid and thyroid hormone receptor; S1P, sphingosine-1-phosphate; SPA, scintillation proximity assay; SPH, sphingosine; SRC-1, steroid receptor coactivator-1; TK, thymidine kinase.

Received March 20, 2006.

Accepted for publication July 26, 2006.


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