tretinoin and sphingosine-kinase

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.

Topics: Aldehyde-Lyases; Animals; Cell Differentiation; Cell Membrane; Cyclic AMP; DNA, Complementary; Endoderm; Exons; Immunoblotting; Kinetics; Lysophospholipids; Mice; Mice, Knockout; Models, Biological; Models, Genetic; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Tretinoin; Tumor Cells, Cultured; Up-Regulation

In hepatoma Huh-7 cells, inhibition of sphingosine kinase (SphK) activity by N,N-dimethylsphingosine (DMS) resulted in up-regulated production of liver-specific serum proteins including albumin and alpha-fetoprotein (AFP). The changes in these protein levels coincided well with those of two liver-enriched transcription factors, hepatocyte nuclear factor (HNF)-1 and -4, which regulate a number of liver-specific genes at the transcriptional level. Moreover, DMS induced the expression of retinoic acid receptor-alpha and retinoid X receptor-alpha. In DMS-treated cells, 9-cis retinoic acid (RA) further enhanced HNF-4alpha and albumin expression but it inhibited AFP accumulation. These results suggest that activation of SphK disengages cells from their liver-specific phenotype, and that 9-cis RA further induces differentiation of hepatoma cells when SphK activity is inhibited.

Topics: Alitretinoin; alpha-Fetoproteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Carcinoma, Hepatocellular; Cell Differentiation; DNA-Binding Proteins; Enzyme Inhibitors; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 1-alpha; Hepatocyte Nuclear Factor 1-beta; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver; Lysophospholipids; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Serum Albumin; Sphingosine; Transcription Factors; Tretinoin; Tumor Cells, Cultured