tretinoin and sphingosine-1-phosphate

All-trans retinoic acid (ATRA) is a promising therapeutic agent, but exhibits low efficacy against human cancers. We investigated the effect of sphingosine-1-phosphate (S1P) on ATRA activity in human colon cancer HT-29 cells. S1P antagonized ATRA activity on HT-29 cell proliferation and retinoic acid receptor beta (RARβ) expression. S1P treatment or transient co-transfection with SphK2 expression vector antagonized ATRA-induced RARβ promoter activity. Proteasome inhibition prevented S1P-induced modulation of ATRA activity. Overall, S1P antagonized ATRA's inhibitory effects by down-regulating RARβ expression, likely via the proteasome-dependent pathway. Decreasing S1P production or inhibiting SphK2 activity could enhance the efficacy of retinoids in cancer treatments.

Topics: Cell Proliferation; Colonic Neoplasms; Down-Regulation; HT29 Cells; Humans; Leupeptins; Lysophospholipids; Proteasome Inhibitors; Receptors, Retinoic Acid; Sphingosine; Tretinoin

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1-/- mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.

Topics: Adenoviridae; Animals; Apolipoproteins E; Astrocytes; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Blotting, Western; Bucladesine; Cell Movement; Cells, Cultured; Culture Media, Conditioned; Genetic Vectors; Lipoproteins; Lipoproteins, HDL; Lysophospholipids; Mice; Mice, Inbred DBA; Mice, Knockout; Sphingosine; Stimulation, Chemical; Tretinoin

G protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) transduce signals to many functions of normal cells. Most human cancer cells upregulate S1P and LPA GPCRs, in patterns distinctive for each type of tumor. The findings that 1-alpha, 25-dihydroxy-vitamin D(3) (VD3) and all-trans retinoic acid (RA) differentially alter expression of the predominant S1P(3) (Edg-3) R and S1P(2) (Edg-5) R in human breast cancer cells (BCCs) permitted analyses of their individual activities, despite a lack of selective pharmacological probes. S1P-evoked increases in [Ca(2+)](i) in S1P(3) R-predominant BCCs were suppressed by concentrations of VD3 and RA which decreased expression of S1P(3) Rs, despite RA-induced increases in S1P(2) Rs. S1P-elicited chemokinetic migration of S1P(3) R-predominant BCCs across a type IV collagen-coated micropore filter also was inhibited by concentrations of VD3 and RA which decreased expression of S1P(3) Rs. The RA-induced increase in expression of S1P(2) Rs did not prevent suppression by RA of S1P-elicited chemokinesis, which appears to be mediated by S1P(3) Rs, but instead exposed S1P(2) R-mediated inhibition of epidermal growth factor-stimulated chemotaxis of BCCs. In contrast, expression of the predominant LPA(2) Rs, LPA-evoked increase in [Ca(2+)](i) and LPA-stimulated chemokinetic migration were suppressed concomitantly by RA but not VD3. Thus two structurally-homologous S1P Rs of BCCs differ in coupling to [Ca(2+)](i) signaling and have opposite effects on protein growth factor-stimulated chemotaxis.

Topics: Breast Neoplasms; Calcitriol; Calcium; Cell Movement; Cells, Cultured; Chemotaxis; Epidermal Growth Factor; Female; Gene Expression Regulation; Humans; Lysophospholipids; Male; Membrane Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Lysophospholipid; Signal Transduction; Sphingosine; Tretinoin

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

Recent studies have demonstrated that sphingosine 1-phosphate (SPP) plays a functional role as a signaling molecule in gene expression in several kinds of cells. The present study demonstrates selective expression by ceramide of retinoic acid receptor-alpha (RAR-alpha) and retinoic X receptor-alpha (RXR-alpha) in osteoblastic MC3T3-E1 cells and a functional role of SPP-mediated AP-1 in the signaling mechanism of ligand-dependent transcriptional activity of heterodimers of these receptors in the cells. C(2)- and C(6)-ceramides selectively stimulated the expression of RAR-alpha and RXR-alpha genes, but not that of beta and gamma isoform genes of RAR and RXR, in the cells. The C(2)-ceramide-induced stimulation was clearly inhibited by dl-threo-dihydrosphingosine, an inhibitor of sphingosine kinase. SPP also selectively stimulated the expression of both receptors and increased the specific binding of the nuclear proteins to direct repeat 5 (DR-5), a consensus sequence of RAR-RXR. In addition, SPP markedly stimulated transient chloramphenicol acetyltransferase (CAT) activity of retinoic acid-dependent transcriptional activity in the cells transfected with a DR-5-CAT reporter gene. The SPP stimulation was activation protein-1 (AP-1)-dependent, because the SPP stimulatory action toward these nuclear gene expressions and the transient CAT activity were inhibited by antisense c-fos and c-jun oligonucleotides. We observed that SPP actually stimulated AP-1 transcriptional activity in the cells. This study suggests an important role of SPP-mediated AP-1 in the selective expression of RAR-alpha and RXR-alpha in osteoblastic cells via the sphingosine pathway.

Topics: Animals; Ceramides; Dimerization; Gene Expression Regulation; Genes, fos; Genes, jun; Genes, Reporter; Lysophospholipids; Mice; Nuclear Proteins; Osteoblasts; Protein Isoforms; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Retinoid X Receptors; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingosine; Substrate Specificity; Transcription Factor AP-1; Transcription Factors; Tretinoin