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Assessment of Sphingosine-1-Phosphate Activity in Biological Samples by Receptor Internalization and Adherens Junction Formation

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Hideru Obinata
Hideru Obinata
Hideru Obinata
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Timothy Hla at Harvard Medical School/ Boston Children's Hospital
Timothy Hla
  • Harvard Medical School/ Boston Children's Hospital
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Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator involved in many biological actions, including vascular homeostasis and immune cell trafficking. S1P activity is mediated by specific G protein-coupled receptors, leading to multiple physiological responses including adherens junction formation in endothelial cells. Here, we describe bioassays for rapidly assessing S1P activity in biological fluids based on ligand-induced receptor internalization in transfected HEK293 cells and consequent adherens junction formation of vascular endothelial cells.
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Assessment of Sphingosine-1-Phosphate Activity in Biological
Samples by Receptor Internalization and Adherens Junction
Formation
Hideru Obinata and Timothy Hla
Abstract
Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator involved in many biological actions,
including vascular homeostasis and immune cell trafficking. S1P activity is mediated by specific
G protein-coupled receptors, leading to multiple physiological responses including adherens
junction formation in endothelial cells. Here, we describe bioassays for rapidly assessing S1P
activity in biological fluids based on ligand-induced receptor internalization in transfected
HEK293 cells and consequent adherens junction formation of vascular endothelial cells.
Keywords
Bioassay; Sphingosine-1-phosphate; Receptor; GFP; Internalization; Adherens junction;
Immunofluorescence staining
1. Introduction
Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator produced from sphingomyelin
by the sequential enzymatic actions of sphingomyelinase, ceramidase, and sphingosine
kinase (1, 2). S1P is enriched in blood and lymph in the submicromolar range, whereas S1P
in interstitial fluids of tissues is much lower, creating a steep S1P gradient (1). This vascular
S1P gradient is utilized to regulate trafficking of immune cells, such as lymphocytes,
hematopoietic progenitor cells, and dendritic cells (1, 3–6). S1P also plays important roles in
vessel maturation, angiogenesis, and vascular permeability both in the developmental stages
and in the adult (1, 7). S1P is also involved in cancer (8). Thus, it is critical to know when
and where S1P is produced for better understanding of its functions.
Several methods to measure S1P levels have been developed utilizing thin-layer
chromatography (9, 10), high-performance liquid chromatography (11–15), or liquid
chromatography-mass spectrometry (16, 17). Although these methods can provide
reasonable values of S1P concentration, they usually include specialized and time-
consuming procedures, such as radiolabeling, S1P extraction from crude samples, and
derivatization.
In this chapter, we describe bioassays to rapidly assess S1P activity in biological fluids
based on the functions of a specific receptor for S1P. Biological functions of S1P are
mediated by cell surface G protein-coupled receptors (18). Among five receptors identified
so far (S1P1-S1P5), the prototypical S1P1 receptor is well characterized. S1P1 is rapidly
internalized upon ligand stimulation via the endosomal pathway and gradually recycled back
to the plasma membrane in HEK293 cells (19). Activation of S1P1 evokes several
intracellular signaling cascades leading to proliferation, NO production, rearrangement of
actin cytoskeleton, and formation of adherens junctions in endothelial cells (1, 20). Based on
these observations, we describe here S1P1 internalization assay and visualization of
NIH Public Access
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Published in final edited form as:
Methods Mol Biol
. 2012 ; 874: 69–76. doi:10.1007/978-1-61779-800-9_6.
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adherens junction as tools for assessing S1P activity, utilizing the GFP fluorescence fused to
the C terminus of the receptor and the standard technique of immunofluorescence staining of
VE-cadherin, respectively. Although these bioassays give only rough estimates of S1P
activity, the specificity for S1P and the simplicity of the procedures provide good
opportunities as an initial assessment of S1P activity. These assays can be also applied for
the development of agonist/antagonist of S1P1 receptor.
2. Materials
1. HEK293 cells.
2. Dulbecco's modified Eagle medium (DMEM).
3. Fetal bovine serum (FBS).
4. Human umbilical vein endothelial cells (HUVECs, passage 4–10).
5. Medium 199 (M199).
6. Phosphate-buffered saline (PBS).
7. Fibronectin solution: 50 μ g/ml in PBS.
8. Heparin.
9. Endothelial cell growth supplement (Biomedical Technologies, Inc.).
10. Expression vector for mammalian cells (see Note 1).
11. Lipofection reagent.
12. Charcoal (see Note 2).
13. Syringe filters, 0.45 and 0.2 μ m pore.
14. 35-mm glass-bottom dishes.
15. 4% Paraformaldehyde solution (see Note 3).
16. Permeabilization solution: 0.2% Triton X-100 in PBS.
17. Blocking solution: 2% bovine serum albumin and 0.1% Triton X-100 in PBS.
18. Anti-VE-cadherin antibody (see Note 4).
19. Fluorescent dye-conjugated secondary antibody (see Note 5).
20. Rhodamine phalloidin (see Note 6).
21. Nuclear staining dye (see Note 7).
22. Confocal microscope.
3. Methods
3.1. Cell Culture
HEK293 cells are cultured in DMEM supplemented with 10% FBS. HUVECs are cultured
on fibronectin-coated dishes in M199 supplemented with 10% FBS, 50 μ g/ml endothelial
cell growth supplement, and 5 U/ml heparin. Cells are maintained at 37°C in a humidified
5% CO2 incubator.
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3.2. Preparation of HEK293 Cells Stably Expressing S1P1-GFP Fusion Protein (293-S1P1-
GFP Cells)
Create an expression vector for S1P1-GFP fusion protein. C-terminal termination codon of
S1P1 should be deleted. Transfect HEK293 cells with the S1P1-GFP expression vector by
lipofection method according to the manufacturer's instructions. After selection by an
antibiotic of choice, isolate several individual clones by limiting dilution method. Further,
select the clones that show good surface localization of S1P1 by fluorescent microscopy.
3.3. Preparation of Charcoal-Stripped FBS
Because S1P content is high in serum, FBS should be treated with charcoal to remove S1P.
1. Take 2.5 g of activated charcoal into a 50-ml conical tube.
2. Wash the charcoal with distilled water three times.
3. Add 25 ml FBS to the tube.
4. Rotate overnight at 4°C.
5. Centrifuge at 1,200 ×
g
for 15 min.
6. Filter the supernatant with syringe filters twice, 0.45-μ m pore at first followed by
0.2-μ m pore.
7. Store the charcoal-stripped FBS at 4°C until use (see Note 8).
3.4. Receptor Internalization Assay
1. Coat 35-mm glass-bottom dishes with the fibronectin solution for at least 10 min at
room temperature (see Note 9).
2. Prepare the suspension of 293-S1P1-GFP cells at the density of 1.5 × 105/ml in
DMEM containing 2% charcoal-stripped FBS (see Note 10).
3. Aspirate the fibronectin solution from the dishes, and add 1 ml of the cell
suspension to each dish.
4. Incubate the dishes overnight in a CO2 incubator.
5. Replace the medium with plain DMEM for serum starvation (see Note 11).
6. Incubate for 2 h in a CO2 incubator.
7. Aspirate the medium, and add the solution of interest to the dishes (see Note 12).
8. Incubate for 1 h in a CO2 incubator (see Note 13).
9. Fix the cells with 1 ml/dish of 4% paraformaldehyde solution for 15 min at room
temperature (see Note 3).
10. Wash the cells twice with PBS (see Note 14).
11. Observe the cells with a confocal microscope (Fig. 1).
3.5. Adherens Junction Formation
3.5.1. Preparation and Stimulation of Cells
1. Coat 35-mm glass-bottom dishes with the fibronectin solution for at least 10 min at
room temperature.
2. Prepare the suspension of HUVEC at the density of 1 × 105/ml in M199 containing
1% charcoal-stripped FBS (see Note 15).
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3. Aspirate the fibronectin solution from the dishes, and add 1 ml of the cell
suspension to each dish.
4. Incubate the dishes overnight in a CO2 incubator.
5. Replace the medium with plain M199 (see Note 11).
6. Incubate for 2 h in a CO2 incubator.
7. Aspirate the medium, and add the solution of interest to the dishes (see Note 12).
8. Incubate for 1 h in a CO2 incubator (see Note 13).
9. Fix the cells with 1 ml/dish of 4% paraformaldehyde solution for 15 min at room
temperature (see Note 3).
10. Wash the cells twice with PBS (see Note 14).
3.5.2. Immunofluorescence Staining of VE-Cadherin—All procedures are carried
out at room temperature.
1. Aspirate the PBS, and add 1 ml/dish of the permeabilization solution. Incubate for
3 min.
2. Aspirate the permeabilization solution, and add 1 ml/dish of the blocking solution.
Incubate for 30 min.
3. Dilute anti-VE-cadherin antibody in the blocking solution (see Note 4).
4. Aspirate the blocking solution, and add 100 μ l/dish of the primary antibody
solution. Incubate for 1 h.
5. Wash with PBS for three times.
6. Dilute fluorescent dye-conjugated secondary antibody in the blocking solution (see
Note 5).
7. Add 100 μ l/dish of the secondary antibody solution. Incubate for 1 h.
8. Wash with PBS for three times.
When simultaneous visualization of cortical actin filaments and nuclei is
preferable, the following procedures can be carried out before proceeding to
microscopic observation.
9. Dilute rhodamine phalloidin in the blocking buffer (see Note 6).
10. Add 100 μ l/dish of the rhodamine phalloidin solution. Incubate for 20 min.
11. Wash with PBS for three times.
12. Dilute nuclear staining dye in PBS (see Note 7).
13. Add 100 μ l/dish of the nuclear staining solution. Incubate for 10 min.
14. Wash with PBS for three times.
15. Observe the cells with a confocal microscope (Fig. 2).
4. Notes
1. The vector should carry an antibiotic-resistance cassette that allows transfected
eukaryotic cells to be selected with an antibiotic of choice. We usually use a
pcDNA3.1 vector, and select transfected cells with 0.5 mg/ml G418.
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2. We use granular-activated charcoal (4–8 mesh), which is easier to remove than
powder-form charcoal.
3. Freshly prepare a 4% paraformaldehyde solution.
4. We use goat anti-VE-cadherin antibody (C-19) from Santa Cruz at the dilution
1:200.
5. We use Alexa488-conjugated donkey anti-goat IgG antibody from Invitrogen at the
dilution 1:1,000.
6. We use rhodamine phalloidin from Invitrogen at the dilution 1:500.
7. We use TO-PRO-3 dye from Invitrogen at the dilution 1:1,000.
8. Keep the charcoal-stripped FBS at −20°C for long-term storage.
9. Glass-bottom dishes can be coated by other types of adhesion molecules, such as
collagen, gelatin, and poly l-lysine.
10. HEK293 cells become extremely easy to come off the dish when they form sheet-
like structure. It is important to keep the cell density low and reduce the number of
medium change.
11. Do not aspirate the entire medium, but leave the medium in the glass-bottom region
to avoid cell damage and loss. Wash two to three times with plain DMEM to
remove FBS.
12. No need to cover the whole dish when the ligand solution is precious. To cover the
glass-bottom region, 100 μ l/dish is more than enough. When possible, titration
analysis of the samples is preferable.
13. Optimal time point should be determined.
14. The fixed cells can be stored in PBS at 4°C for several days before proceeding to
microscopic observation or immunofluorescent staining.
15. The cell density and serum-starvation conditions should be optimized so that cells
are close enough to each other to make adherens junctions but still do not complete
junction formations before stimulation.
Acknowledgments
We are grateful to Catherine H. Liu and Shobha Thangada for their efforts to establish these bioassays. This work is
supported by NIH grants HL-67330 and HL-89934.
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Fig. 1.
293-S1P1-GFP cells were stimulated with control solution (a) or 100 nM S1P for 1 h (b).
Scale bar, 10 μ m.
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Fig. 2.
HUVECs were stimulated with control solution (a) or 100 nM S1P for 1 h (b). VE-cadherin
(
green
), cortical actin filaments (
red
), and nuclei (
blue
) are visualized. Scale bar, 10 μ m.
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Full-text available
  • May 1999
The endothelial-derived G-protein–coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1–green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP–EDG-1 interaction. SPP binds to EDG-1–GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1–GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a τ1/2 of ∼15 min. The EDG-1–GFP–containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1–GFP recycles back to the plasma membrane with a τ1/2 of ∼30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.
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Sphingosine 1-phosphate and cancer
Article
  • Jul 2010
There is substantial evidence that sphingosine 1-phosphate (S1P) is involved in cancer. S1P regulates processes such as inflammation, which can drive tumorigenesis; neovascularization, which provides cancer cells with nutrients and oxygen; and cell growth and survival. This occurs at multiple levels and involves S1P receptors, sphingosine kinases, S1P phosphatases and S1P lyase. This Review summarizes current research findings and examines the potential for new therapeutics designed to alter S1P signalling and function in cancer.
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Comprehensive Quantitative Analysis of Bioactive Sphingolipids by High-Performance Liquid Chromatography–Tandem Mass Spectrometry
Article
  • Aug 2009
There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1P) and this has necessitated the development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), ceramides (Cers), ceramide 1-phosphates (Cer-1P), glucosyl/galactosyl-ceramides (Glu-Cers), and sphingomyelins (SMs) is performed on a Thermo Fisher Scientific triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues, or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a LC/MS/MS system. Qualitative analysis (identification) of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target analyte, synthetic standards, and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration, using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit of subpicomole level. This methodology constitutes a "Lipidomic" approach to study the SPLs metabolism, defining a function of distinct subspecies of individual bioactive SPL classes.
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Quantitative analysis of sphingosine-1-phosphate by HPLC after napthalene-2,3-dicarboxaldehyde (NDA) derivatization
Article
  • May 2009
Sphingosine-1-phosphate (S1P) is an important sphingolipid signaling molecule that regulates numerous cellular processes. In this paper we report a new method to quantify the levels of S1P in biological samples that relies on derivatization with naphthalene-2,3-dicarboxaldehyde (NDA) and quantification by reverse-phase high performance liquid chromatography (HPLC). The limit of detection (LOD) using S1P standards was 20.9fmol (12.6nM), and the limit of quantification (LOQ) was 69.6fmol (41.7nM). The recovery of S1P standards was up to 97.5%. The mean relative standard deviations (RSD) for the intra- and inter-day assay were 4.1% and 7.7%, respectively. To validate this procedure, we quantified the S1P levels in plasma from human, horse, and mouse (mean values of 0.45, 0.25, and 1.23microM, respectively). We also used this technique to evaluate the S1P content in mouse tissues, as well as in rat neuronal cell cultures before and after sphingosine treatment. The results demonstrate that this new procedure can provide fast, sensitive, and reproducible S1P quantification, and offers several advantages over existing methods. The technique also may be used for determining the activity, as well as the inhibition, of sphingosine kinase. In the future it could be an important tool for investigators studying the role of S1P in signal transduction, cell growth and differentiation, and disease pathogenesis and treatment.
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Anti-atherogenic Actions of High-density Lipoprotein through Sphingosine 1-Phosphate Receptors and Scavenger Receptor Class B Type I
Article
  • Sep 2008
  • ENDOCR J
Plasma high-density lipoprotein (HDL) is a potent anti-atherogenic factor, a critical role of which is thought to be reverse cholesterol transport through the lipoprotein-associated apolipoprotein A-I (apoA-I). HDL also carries a potent bioactive lipid mediator, sphingosine 1-phophate (S1P), which exerts diverse physiological and pathophysiological actions in a variety of biological systems, including the cardiovascular system. In addition, HDL-associated apoA-I is known to stimulate intracellular signaling pathways unrelated to transporter activity. Mounting evidence indicates that multiple antiatherogenic or anti-inflammatory actions of HDL independent of cholesterol metabolism are mediated by the lipoprotein-associated S1P through S1P receptors and by apoA-I through scavenger receptor class B type I.
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Sphingosine-1-Phosphate Regulation of Mammalian Development
Article
  • Oct 2008
  • Biochim Biophys Acta
Sphingosine-1-phosphate (S1P) was first identified as a lysophospholipid metabolite whose formation is required for the irreversible degradation of sphingolipids. Years later, it was discovered that S1P is a bioactive lipid that provokes varied cell responses by acting through cell-surface receptors to drive cell signaling. More recent findings in model organisms have now established that S1P metabolism and signaling are integrated into many physiological systems. We describe here the surprising breadth of function of S1P in mammalian development and the underlying biologic processes that S1P regulates.
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The vascular S1P gradient-Cellular sources and biological significance
Article
  • Oct 2008
  • Biochim Biophys Acta
Sphingosine 1-phosphate (S1P), a product of sphingomyelin metabolism, is enriched in the circulatory system whereas it is estimated to be much lower in interstitial fluids of tissues. This concentration gradient, termed the vascular S1P gradient appears to form as a result of substrate availability and the action of metabolic enzymes. S1P levels in blood and lymph are estimated to be in the muM range. In the immune system, the S1P gradient is needed as a spatial cue for lymphocyte and hematopoietic cell trafficking. During inflammatory reactions in which enhanced vascular permeability occurs, a burst of S1P becomes available to its receptors in the extravascular compartment, which likely contributes to the tissue reactions. Thus, the presence of the vascular S1P gradient is thought to contribute to physiological and pathological conditions. From an evolutionary perspective, S1P receptors may have co-evolved with the advent of a closed vascular system and the trafficking paradigms for hematopoietic cells to navigate in and out of the vascular system.
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Yatomi Y, Ruan F, Ohta J, Welch RJ, Hakomori S & Igarashi Y.Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride. Anal Biochem 230: 315−320
Article
  • Oct 1995
We describe here in detail the development of a method to quantitatively measure sphingosine 1-phosphate (Sph-1-P), a bioactive sphingolipid. Sph-1-P was first extracted from cells into the upper aqueous phase under alkaline conditions by Folch's phase separation and then reextracted into the lower chloroform phase under acidic conditions. This phosphorylated sphingoid base extracted was quantitatively converted to N-[3H]-acetylated Sph-1-P, that is [3H]C2-ceramide 1-phosphate (C2-Cer-1-P), by N-acylation with [3H]acetic anhydride. The [3H]C2-Cer-1-P formed with the acylation was resolved by thin-layer chromatography, detected with autoradiography, and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. This assay allows quantification of Sph-1-P over a range from at least 100 pmol (often 30 pmol) to 10 nmol (the highest level tested). The utility and validity of our assay were demonstrated using human platelets. The amount of Sph-1-P in platelet extracts was proportional to the cell number and calculated as 141 +/- 4 pmol/10(8) cells (mean +/- SD, n = 3), which was about four times higher than that of sphingosine. The potent agonist thrombin did not affect the total Sph-1-P amounts in platelet suspensions but induced the release of Sph-1-P stored in the cells into the medium.
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Simultaneous Quantitative Determination Method for Sphingolipid Metabolites by Liquid Chromatography/Ionspray Ionization Tandem Mass Spectrometry
Article
  • Feb 1997
Sphingolipid metabolites ceramide, sphingomyelin, sphingosine, psycosine, sphingosylphosphorylcholine, and dimethylsphingosine were separated and simulataneously quantitated by liquid chromatography/ionspray ionization tandem mass spectrometry (LC/MS/ MS). The use of glassware throughout minimized losses due to adsorption and the pretreatment of this method consisted of simple liquid-liquid extraction procedure with a mixture of chloroform and methanol. After separation on a short C18 silica column eluted in a gradient mode, the metabolites were detected by MS/ MS. This assay allows simultaneously quantification of these metabolites over a range of at least 0.1 to 100 ng/ 10(6) cells. The LC/MS/MS analyses took 10 to 15 min per sample and we could examine up to 50 samples per day. We also detected endogenous sphingosine 1-phosphate in HL-60 cells. The utility of the method was demonstrated by examining changes in metabolites levels in HL-60 cells after treatment with sphingomyelinase. It was found that sphingomyelinase from Bacillus cereus may have selectivity for acyl chain length.
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