Sphingolipids, characterized with the presence of sphingoid bases (sphingosine or dihydrosphingosine), are important components of eukaryotic cells, many of which function as bioactive signaling molecules. Sphingosine-1-phosphate (S1P) is derived from sphingosine, and it is a ligand for a family of five G-protein-coupled receptors (S1P1-S1P5). It has been emerging as a vital lipid mediator that enhances cell growth and survival. By contrast, its precursors –Cer and sphingosine– are believed to inhibit proliferation and/or promote apoptosis. As these metabolites are interconvertible, it has been proposed that it is not their absolute amounts, but rather their relative levels, that determine cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. This section focuses on the regulation of cell fate by sphingolipids including S1P and Cer, and suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate is illustrated using a cell culture model. Moreover, the experimental handling of lipophilic sphingolipids and detection of cell death are also documented. |
Category | Biosynthesis & Metabolism |
Protocol Name | Cell death/survival signal by ceramide and sphingosin-1-phosphate |
Authors
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Kitatani, Kazuyuki
Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University
Taniguchi, Makoto
Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University
Okazaki, Toshiro
*
Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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N-acetyl-D-sphingosine (C2-Cer) (Matreya #1901, Matreya LLC, Pleasant Gap, PA) |
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N-Hexanoyl-D-erythro-sphingosine (C6-Cer) (Matreya #1900, Matreya LLC) |
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D-erythro-S1P (Matreya #1803, Matreya LLC) |
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Ethanol (Wako Pure Chemical Industries Ltd., Osaka, Japan) |
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RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS) |
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4', 6-Diamidino-2-phenylindole (DAPI) (Roche, Basel, Switzerland) |
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Instruments
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Fluorescent microscopy Leica DMRB (Leica Microsystems GmbH, Watzlar, Germany) |
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Methods |
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Making stock solution of C2-Cer or C6-Cer, and their preservation
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Dissolve C2-Cer (10 mg) and C6-Cer (10 mg) in 1.46 mL and 1.25 mL of 100% ethanol and mixed, respectively. |
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Dispense as each 200 μL of these solutions to dark glass vial tube and dry under N2 gas. |
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Reconstitute Cer with 200 μL ethanol (final concentration: 20 mM). |
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Making stock solution of S1P and their preservation
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Dissolve S1P (5 mg) in 1.32 mL of phosphate-buffered saline (PBS) containing 0.4% fatty acid free BSA (PBS/BSA) (final concentration: 10 mM). |
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Dispense and store tubes at −80°C. |
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Detection of cell death by DAPI staining after treatment with C2-Cer and S1P
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HL-60 cells were seeded at a density of 3 × 105 cells/mL in 35 mm non-treated dishes in RPMI 1640 containing 2% FCS. |
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After 30 min, S1P (Final concentration: 0–20 mM), C2-Cer (Final concentration: 10 μM), or combination of both was added with various concentrations. |
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After 24 h, cells were harvested, washed with PBS, and re-suspended in PBS. |
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The cells were attached on the slide glass by cytospin at 200 × g for 5 min. |
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The cells were fixed with PBS containing 1% paraformaldehyde for 15 min at 4°C. |
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After washing twice with PBS, cells were treated with DAPI (Final concentration: 2 μg/mL) in PBS for 20 min at room temperature. |
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Cells were washed three times with PBS, rinsed with distilled water, and amounted with cover glass. |
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Condensation of DNA was detected by fluorescent microscopy using a UV-filter (excitation at 365 nm, emission at 480 nm). |
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At least 200 cells for determination were counted under a fluorescent microscope, and the cells with nuclear condensation and fragmentation were expressed as dead cells. |
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Copyrights |
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Date of registration:2014-11-12 09:52:23 |
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- Iwai, K., Kondo, T., Watanabe, M., Yabu, T., Kitano, T., Taguchi, Y., Umehara, H., Takahashi, A., Uchiyama, T., and Okazaki, T. (2003) Ceramide increases oxidative damage due to inhibition of catalase by caspase-3-dependent proteolysis in HL-60 cell apoptosis. J Biol Chem. 278, 9813–9822 [PMID : 12511568]
- Spiegel, S., and Milstin, S. (2003) Sphingosine-1-phosphate: an enigmatic signaling lipid. Nature Rev Mol Cell Biol. 4, 397–407 [PMID : 12728273]
- Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P.G., Coso, O.A., Gutkind JS., and Spiegel S. (1996) Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature. 381, 800–803 [PMID : 8657285]
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Okazaki, Toshiro,
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Taniguchi, Makoto,
Okazaki, Toshiro,
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Cell death/survival signal by ceramide and sphingosin-1-phosphate.
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