jte-013 and sphingosine-kinase

The recent identification of a cellular balance between ceramide and sphingosine 1-phosphate (S1P) as a critical regulator of cell growth and death has stimulated increasing research effort to clarify the role of ceramide and S1P in various diseases associated with dysregulated cell proliferation and apoptosis. S1P acts mainly, but not exclusively, by binding to and activating specific cell surface receptors, the so-called S1P receptors. These receptors belong to the class of G protein-coupled receptors that constitute five subtypes, denoted as S1P(1)-S1P(5), and represent attractive pharmacological targets to interfere with S1P action. Whereas classical receptor antagonists will directly block S1P action, S1P receptor agonists have also proven useful, as recently shown for the sphingolipid-like immunomodulatory substance FTY720. When phosphorylated by sphingosine kinase to yield FTY720 phosphate, it acutely acts as an agonist at S1P receptors, but upon prolonged presence, it displays antagonistic activity by specifically desensitizing the S1P(1) receptor subtype. This commentary will cover the most recent developments in the field of S1P receptor pharmacology and highlights the potential therapeutic benefit that can be expected from these novel drug targets in the future.

Topics: Animals; Fingolimod Hydrochloride; Humans; Lysophospholipids; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine; Sulfhydryl Compounds; Thiazolidines; Thiophenes

Lung alveolar epithelium is composed of alveolar type I (AT1) and type II (AT2) cells. AT1 cells mediate gas exchange, whereas AT2 cells act as progenitor cells to repair injured alveoli. Lung microvascular endothelial cells (LMVECs) play a crucial but still poorly understood role in regulating alveolar repair. Here, we studied the role of the LMVEC-derived bioactive lipid sphingosine-1-phosphate (S1P) in promoting alveolar repair using mice with endothelial-specific deletion of sphingosine kinase 1 (Sphk1), the key enzyme promoting S1P generation. These mutant lungs developed airspace-enlargement lesions and exhibited a reduced number of AT1 cells after Pseudomonas-aeruginosa-induced lung injury. We demonstrated that S1P released by LMVECs acted via its receptor, S1PR2, on AT2 cells and induced nuclear translocation of yes-associated protein (YAP), a regulator of AT2 to AT1 transition. Thus, angiocrine S1P released after injury acts via the S1PR2-YAP signaling axis on AT2 cells to promote AT2 to AT1 differentiation required for alveolar repair.

Topics: Adaptor Proteins, Signal Transducing; Alveolar Epithelial Cells; Animals; Cell Count; Cell Cycle Proteins; Down-Regulation; Female; Lung; Lysophospholipids; Male; Mice; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Pseudomonas aeruginosa; Pulmonary Surfactant-Associated Protein C; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Regeneration; Signal Transduction; Sphingosine; YAP-Signaling Proteins

The bioactive lipid sphingosine-1-phosphate (S1P) regulates smooth muscle (SM) contractility predominantly via three G protein-coupled receptors. The S1P1 receptor is associated with nitric oxide (NO)-mediated SM relaxation, while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho-kinase pathway. This study is to determine testosterone (T) modulating the expression and functional activity of S1P receptors in corpus cavernosum (CC). Adult male Sprague-Dawley rats were randomly divided into three groups: sham-operated controls, surgical castration and T supplemented group. Serum S1P levels were detected by high-performance liquid chromatography. The expression of S1P1-3 receptors and sphingosine kinases was detected by real-time RT-PCR. In vitro organ bath contractility and in vivo intracavernous pressure (ICP) measurement were also performed. T deprivation significantly decreased ICP rise. Meanwhile, surgical castration induced a significant increase in serum S1P level and the expression of S1P2-3 receptors by twofold (P < 0.05) but a decrease in the expression of S1P1 receptor. Castration also augmented exogenous phenylephrine (PE), S1P, S1P1,3 receptor agonist FTY720-P contractility and S1P2-specific antagonist JTE013 relaxation effect. T supplemented could restore the aforementioned changes. We provide novel data that castration increased serum S1P concentration and up-regulated the expression of S1P2-3 receptors in CC. Consistently, agonizing S1P receptors induced CCSM contraction and antagonizing mediated relaxation were augmented. This provides the first clear evidence that S1P system dysregulation may contribute to hypogonadism-related erectile dysfunction (ED), and S1P receptors may be expected as a potential target for treating ED.

Topics: Animals; Gene Expression Regulation; Lysophospholipids; Male; Muscle Contraction; Muscle, Smooth; Orchiectomy; Organ Culture Techniques; Organ Size; Organophosphates; Penile Erection; Penis; Phenylephrine; Phosphotransferases (Alcohol Group Acceptor); Prostate; Protein Isoforms; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Testis; Testosterone

Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.

Topics: Cell Movement; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; HeLa Cells; Humans; Immunoblotting; Lysophospholipids; Microscopy, Confocal; Mutation; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; RNA Interference; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

The sphingosine kinase (SPK)/sphingosine-1-phosphate (S1P) pathway recently has been associated with a variety of inflammatory-based diseases. The majority of these studies have been performed in vitro. Here, we have addressed the relevance of the SPK/S1P pathway in the acute inflammatory response in vivo by using different well known preclinical animal models. The study has been performed by operating a pharmacological modulation using 1) L-cycloserine and DL-threo-dihydrosphingosine (DTD), S1P synthesis inhibitors or 2) 2-undecyl-thiazolidine-4-carboxylic acid (BML-241) and N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide (JTE-013), specific S1P(2) and S1P(3) receptor antagonists. After local injection of carrageenan in mouse paw S1P release significantly increases locally and decreases during the resolution phase. Expression of SPKs and S1P(2) and S1P(3) receptors is increased in inflamed tissues. Administration of L-cycloserine or DTD caused a significant anti-inflammatory effect. By using different animal models we have also demonstrated that the SPK/S1P pathway contributes to changes in vascular permeability and promotes cell recruitment. The S1P effect on cell recruitment results is receptor-mediated because both JTE-013 and BML-241 inhibited zymosan-induced cell chemotaxis without effect on vascular leakage. Conversely, changes in vascular permeability involve mainly SPK activity, because compound 48/80-induced vascular leakage was significantly inhibited by DTD. In conclusion, the SPK/S1P pathway is involved in acute inflammation and could represent a valuable therapeutic target for developing a new class of anti-inflammatory drugs.

Topics: Animals; Capillary Permeability; Chemotaxis, Leukocyte; Cycloserine; Edema; Inflammation; Lysophospholipids; Male; Mice; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiazolidines

Lung parenchymal strips isolated from ovalbumin-sensitized rats manifest a mast cell-dependent, biphasic contraction when challenged with allergen. The first phase is mediated by the release of preformed 5-HT while the second phase is dependent on de novo synthesis of leukotrienes. Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite which is readily generated in mast cells and has been demonstrated to be an important regulator of allergen-induced mast cell activation. We have used the parenchymal strip to explore the role of sphingosine 1-phosphate and the S1P(2) receptor in the two components of the acute response to allergen. Lung parenchymal strips were prepared from Brown Norway rats actively sensitized to ovalbumin. The strips were set up in organ baths and contractile responses measured isometrically. The inhibitors of sphingosine kinase, D-erythro-NN-dimethylsphingosine (dimethylsphingosine) and 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) inhibited concentration-dependently both phases of the contractile response induced by 0.1 microg ml(-1) ovalbumin. The effects were seen at concentrations similar to those which inhibit the purified enzyme and were selective in that neither the contractile response to adenosine nor that to 5-hydroxytryptamine was affected. JTE-013 (a selective S1P(2) receptor antagonist) also blocked the response to ovalbumin (0.1 microg ml(-1)). However, the concentrations of JTE-013 required (microM) were substantially higher than its affinity for the S1P(2) receptors (nM). However, when tested against a lower concentration of ovalbumin (0.03 microg ml(-1)), JTE-013 inhibited the response with nM potency. These data demonstrate the importance of S1P and the S1P(2) receptor as regulators of allergen-induced activation of mast cells in their natural environment in the rat lung.

Topics: Acetates; Adenosine; Allergens; Animals; Bronchoconstriction; Cyclopropanes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Injections, Subcutaneous; Leukotriene Antagonists; Lung; Lysophospholipids; Male; Mast Cells; Methysergide; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Quinolines; Rats; Rats, Inbred BN; Receptors, Lysosphingolipid; Serotonin; Serotonin Antagonists; Signal Transduction; Sphingosine; Sulfides; Thiazoles