Products by the sphingosine kinase/sphingosine 1‐phosphate (S1P) lyase pathway but not S1P stimulate mitogenesis

Abstract

Sphingosine 1-phosphate (S1P) functions as a ligand for the S1P/EDG family receptors. For years, intracellular signaling roles for S1P have also been suggested, especially in cell proliferation. Now, we have generated several mouse F9 embryonic carcinoma cell lines varying in expression of the S1P-degrading enzyme, S1P lyase (SPL) and/or sphingosine kinase (SPHK1). All these cell lines accumulated S1P compared to the wild-type F9 cells, but the amounts varied. We investigated the ability of these cells to proliferate under low serum conditions, as measured by a thymidine uptake assay. Although F9 cells over-expressing SPHK1 did exhibit enhanced DNA synthesis, other S1P-accumulating cells ( SPL -null cells and SPL -null cells over-expressing SPHK1) did not. The overproduction of both SPL and SPHK1 resulted in the most striking mitogenic effect. Moreover, nM concentrations of sphingosine (or dihydrosphingosine) stimulated DNA synthesis in an SPL -dependent manner. These results indicate that products by the SPL pathway, not S1P itself, function in mitogenesis.

Sphingosine-1–Phosphate, FTY720, and Sphingosine-1–Phosphate Receptors in the Pathobiology of Acute Lung Injury

“…In murine F9 embryonic carcinoma and Henrietta Lacks (HeLa) cells, the overexpression of SphK1 enhanced DNA synthesis. However, in Sgpl1 2/2 null cells or Sgpl1 2/2 null cells overexpressing SphK1, no effect on mitogenesis was evident, suggesting that the products of the S1PL pathway, and not S1P itself, stimulated mitogenesis (21,60). The addition of trans-2-hexadecenal to human embryonic kidney293 (HEK293), National Institutes of Health 3-day transfer, inoculum 3 3 10,000 (NIH 3T3), and HeLa cells induced a cytoskeletal reorganization and apoptosis that was dependent on c-Jun N-terminal kinase (JNK) activation and c-Jun phosphorylation (61).…”

“…Both FTY720 (left panel) and FTY720-P (right panel) potently increase endothelial cell (EC) barrier function in vitro when concentrations of less than 10 mM are used (higher concentrations or prolonged stimulation over hours to days may disrupt barrier function), but multiple aspects differ in the signaling pathways involved. FTY720-P rapidly induces a series of events similar to the actions of S1P to enhance barrier function, including S1P 1 ligation, Gi-coupled signaling, lipid raft membrane platforms, increased intracellular Ca 21 , Rac1 activation, and dynamic actin changes, producing increased cortical actin linked to adherens junction and focal adhesion complex formation and stabilization. However, FTY720-P also induces the ubiquitination and subsequent proteosomal degradation of barrier-promoting S1P 1 , eventually leading to increased permeability after prolonged exposure.…”

“…Alternatively, S1P is irreversibly cleaved by S1P lyase (S1PL), a pyridoxal phosphate-dependent enzyme, to ethanolamine phosphate and trans-2-hexadecenal. Subsequently, trans-2-hexadecenal is oxidized by fatty aldehyde dehydrogenase to trans-2-hexadecenoic acid, which is recycled into glycerolipid or sphingolipid metabolic pathways, whereas ethanolamine phosphate is used for the biosynthesis of ethanolamine phospholipids ( Figure 1) (19)(20)(21). Moreover, in response to TNF-a and other agonists, SM is hydrolyzed to ceramides of variable N-acyl chain lengths by one of the three (acid, neutral, or alkaline) sphingomyelinases (SMases) (Figure 2) (22).…”

Sphingosine-1-Phosphate Metabolism and Intestinal Tumorigenesis: Lipid Signaling Strikes Again

“…59 Many of the effects of SPL appear to be due to its catabolism of S1P, but other effects are S1P-independent and instead rely on the generation of EP and/or long chain aldehydes that can be incorporated into phospholipids. 51,60 …”

Mouse aldehyde dehydrogenase ALDH3B2 is localized to lipid droplets via two C-terminal tryptophan residues and lipid modification

“…The in vitro ALDH assay was performed by incubating 10 ng of the affinity-purified protein with 500 μM NAD + and 100 μM hexadecanal [23], octanal (Wako Pure Chemical Industries, Osaka, Japan), or acetaldehyde (Wako Pure Chemical Industries) in buffer B (50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100) for 15 min at 37 °C. The reaction was monitored by measuring the fluorescence of the NADH product (excitation at 356 nm and emission at 460 nm) using a monochromator Infinite M200 (Tecan, Männedorf, Switzerland).…”