sphingosine-kinase and Leiomyoma

Sphingosine 1-phosphate (S1P), a bioactive lipid generated by sphingosine kinases (SphK1/2), initiates different signalling pathways involved in physiological and pathological processes. We previously demonstrated that in rat myometrium at late (day 19) gestation, SphK1 increases the expression of COX2 via S1P generation and release. In rat uterine leiomyoma cells (ELT3), SphK1/S1P axis controls survival and proliferation. In the present study we demonstrate that PDBu activates SphK1 but not SphK2. SphK1 activation requires PKC and MAPK ERK1/2. S1P produced by PDBu is released in the medium. PDBu-induced S1P export is abolished by Ro-318220 and BIM (PKC inhibitors), by U0126 and PD98059 (MEK inhibitors), SKI-II (SphKI/2 inhibitor) and SphK1-siRNA, suggesting the involvement of PKC, ERK and SphK1 respectively. The release of S1P is insensitive to inhibitors of ATP Binding Cassette (ABC)A1 and ABCB1 transporters, but is abolished when ABCC1 transporters are inhibited by MK571 or down-regulated by ABCC1-siRNA. PDBu increases COX2 expression that is blocked by the inhibition of PKC, ERK1/2, SphK1, and when cells are treated with MK571 or transfected with ABCC1-siRNA. The induction of COX2 by the S1P release due to PDBu or by exogenous S1P involves S1P2 receptors coupled to Gi. In myometrium from rat at late gestation, the release of S1P is also strongly reduced when SphK and ABCC1 are inhibited. The data reveal that in rat leiomyoma cells and late pregnant rat myometrium, the release of S1P involves a similar signalling pathway and occurs through ABCC1.

Topics: Animals; Cell Line, Tumor; Cyclooxygenase 2; Enzyme Assays; Female; Gene Expression; Gene Knockdown Techniques; GTP-Binding Protein alpha Subunits, Gi-Go; Leiomyoma; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Multidrug Resistance-Associated Proteins; Myometrium; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Propionates; Protein Kinase C; Quinolines; Rats; Rats, Wistar; Receptors, Lysosphingolipid; RNA Interference; Sphingosine; Uterine Neoplasms

Sphingosine 1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed ( approximately 30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy.

Topics: Animals; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzymes; Female; Genes, Dominant; Humans; Immunologic Techniques; Leiomyoma; Lysophospholipids; Mutation; Myometrium; Myosin Light Chains; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Pregnancy, Animal; Progesterone; Progestins; Rats; Rats, Sprague-Dawley; Receptors, Lysophospholipid; RNA, Messenger; Signal Transduction; Sphingosine; Uterus

Uterine leiomyomas, or fibroids, are the most common tumors of the myometrium. The ELT3 cell line, derived from Eker rat leiomyoma, has been successfully used as a model for the study of leiomyomas. We have demonstrated previously the potent mitogenic properties of the peptidic hormone endothelin (ET)-1 in this cell line. Here we investigated the antiapoptotic effect of ET-1 in ELT3 cells. We found that 1) serum starvation of ELT3 cells induced an apoptotic process characterized by cytochrome c release from mitochondria, caspase-3/7 activation, nuclei condensation and DNA fragmentation; 2) ET-1 prevented the apoptotic process; and 3) this effect of ET-1 was fully reproduced by ETB agonists. In contrast, no antiapoptotic effect of ET-1 was observed in normal myometrial cells. A pharmacological approach showed that the effect of ET-1 on caspase-3/7 activation in ELT3 cells was not dependent on phosphatidylinositol 3-kinase, ERK1/2, or phospholipase D activities. However, inhibitors of sphingosine kinase-1 (SphK1), dimethylsphingosine and threo-dihydrosphingosine, reduced the effect of ET-1 by about 50%. Identical results were obtained when SphK1 expression was down-regulated in ELT3 cells transfected with SphK1 small interfering RNA. Furthermore, serum starvation induced a decrease in SphK1 activity that was prevented by ET-1 without affecting the level of SphK1 protein expression. Finally, sphingosine 1-phosphate, the product of SphK activity, was as efficient as ET-1 in inhibiting serum starvation-induced caspase-3/7 activation. Together, these results demonstrate that ET-1 possesses a potent antiapoptotic effect in ELT3 cells that involves sphingolipid metabolism through the activation of SphK1.

Topics: Animals; Apoptosis; Culture Media, Serum-Free; Cytoprotection; Endothelin-1; Female; Leiomyoma; Myometrium; Phosphotransferases (Alcohol Group Acceptor); Rats; Tumor Cells, Cultured; Uterine Neoplasms