Sphingosine-1-phosphate Lyase Is Involved in the Differentiation of F9 Embryonal Carcinoma Cells to Primitive Endoderm

Abstract

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.

Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation

“…Immunoblotting was performed as described previously (34,35) using anti-FLAG M2 (1.85 μg/mL) (Sigma), anti-calnexin 4F10 (1 μg/mL) (Medical & Biological Laboratories), or anti-GAPDH 6C5 (1 μg/mL; Ambion, Life Technologies) antibody as a primary antibody and an HRP-conjugated anti-mouse IgG F(ab') 2 fragment (1:7,500 dilution; GE Healthcare Life Sciences) as a secondary antibody. Labeling was detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).…”

Mouse Sphingosine Kinase Isoforms SPHK1a and SPHK1b Differ in Enzymatic Traits Including Stability, Localization, Modification, and Oligomerization

“…Immunoblotting-Immunoblotting was performed as described previously (30). Anti-SPHK1 (1/1000 dilution) (31), anti-FLAG M2 (1 g/ml; Stratagene), anti-Myc PL14 (1 g/ml; Medical & Biological Laboratories, Nagoya, Japan), anti-glyceraldehyde-3-phosphate dehydrogenase (1 g/ml; Ambion, Austin, TX), and anti-calnexin (H-10) antibodies (0.2 g/ml; Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies.…”

Changes in S1P1 and S1P2 expression during embryonal development and primitive endoderm differentiation of F9 cells

“…We recently reported that sphingosine kinase and S1P lyase, both of which are involved in S1P metabolism, were up-regulated during F9 PrE differentiation [19]. Moreover, S1P accumulation, resulting from the disruption of the S1P lyase gene or the overproduction of sphingosine kinase, resulted in accelerated of the PrE differentiation [19], suggesting that intracellular S1P is also involved in this process. Thus, it appears that extracellular and intracellular S1P cooperatively regulate PrE differentiation.…”

“…The pCE-puro plasmid, a mammalian expression vector, contains a puromycin-resistant gene used for selecting stable transformants [19]. The pCE-puro 3xFLAG-4 plasmid is a derivative of the pCE-puro plasmid and was designed to produce a C-terminal triple FLAG (3xFLAG)-tagged protein.…”

“…32 P-labeled probes were prepared from cDNA for each S1P receptor, which had been amplified using specific primers (for S1P 1 , Phalloidin staining. Phalloidin staining of F-actin was performed on F9 EC, PrE, and PE cells as described previously using a fluorescence microscopy AxioSkop 2 plus (Carl Zeiss, Oberkochen, Germany) [19]. and scrambled S1P 1 siRNA, 5'-AATTGATGATCGTTCCGGACT-3'.…”