In our effort to develop selective sphingosine kinase-2 (SphK2) inhibitors as pharmacological tools, a thiazolidine-2,4-dione analogue, 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145), was synthesized and biologically characterized. Biochemical assay results indicate that K145 is a selective SphK2 inhibitor. Molecular modeling studies also support this notion. In vitro studies using human leukemia U937 cells demonstrated that K145 accumulates in U937 cells, suppresses the S1P level, and inhibits SphK2. K145 also exhibited inhibitory effects on the growth of U937 cells as well as apoptotic effects in U937 cells, and that these effects may be through the inhibition of down-stream ERK and Akt signaling pathways. K145 also significantly inhibited the growth of U937 tumors in nude mice by both intraperitoneal and oral administration, thus demonstrating its in vivo efficacy as a potential lead anticancer agent. The antitumor activity of K145 was also confirmed in a syngeneic mouse model by implanting murine breast cancer JC cells in BALB/c mice. Collectively, these results strongly encourage further optimization of K145 as a novel lead compound for development of more potent and selective SphK2 inhibitors.
The class I histone deacetylases HDAC1 and HDAC2 belong to a family of 11 zinc-dependent human HDACs and are overexpressed in many cancers. Inhibitors of these HDACs now in clinical trials show activity against several types of cancers. This review is focuse on recent advances in both clinical and preclinical efforts to understand the basis for HDACi actions, with an emphasis on implications for rational combinations with conventional or other targeted agents. We will address new perspectives on the molecular mechanisms by which HDACs act and how these actions relate to cancer. We will also review new evidence demonstrating that HDACs are direct intracellular targets of the potent sphingolipid mediator sphingosine-1-phosphate (S1P), the first identified endogenous nuclear regulator of these enzymes, linking sphingolipid metabolism in the nucleus to remodeling of chromatin and epigenetic regulation of gene expression. Understanding how endogenous molecules regulate HDAC activity in vivo may facilitate the search for safer and more effective anti-cancer drugs capable of interfering with HDAC functions in a highly specific manner.
histone deacetylase; histone deacetylase inhibitor; apoptosis; sphingosine-1-phosphate; cancer
Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacological evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared to age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining Directed In Vivo Angiogenesis Assays (DIVAA) with Fluorescence Activated Cell Sorting (DIVAA/FACS), thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I-treated animals. Taken together, our findings demonstrate that SphK1-produced S1P is a crucial mediator of breast cancer-induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer.
sphingosine kinase 1; sphingosine-1-phosphate; lymphangiogenesis; angiogenesis; lymph node metastasis
Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW), a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P) (100 µg/kg BW). Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of sphingosine kinase 1; as a consequence, S1P salvages fenretinide induced emphysema in rat lungs.
Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including the extracellular regulated kinase (ERK)1/2 and AKT as well as the G-protein coupled receptor (GPCR), TGR5/M-BAR. Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be pertussis toxin (PTX) and dominant negative Gαi sensitive in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid activated phylogenetic family, expressed in HEK293 cells, identified sphingosine 1-phosphate receptor 2 (S1P2) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P-induced activation of ERK1/2 and AKT were significantly inhibited by JTE-013, a S1P2 antagonist, in primary rat hepatocytes. JTE-013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knock down of the expression of S1P2 by a recombinant lentivirus encoding S1P2 shRNA, markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P2 knock out (S1P2−/−) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P2 can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily via S1P2 in primary rodent hepatocytes.
G protein coupled receptor; homology modeling; S1P receptor 2 knockout mice; cell signaling
The bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), is now recognized as a critical regulator of many physiological and pathophysiological processes, including cancer, atherosclerosis, diabetes and osteoporosis. S1P is produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2. Many cells secrete S1P, which can then act in an autocrine or paracrine manner. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. More recently, it was shown that S1P also has important intracellular targets involved in inflammation, cancer and Alzheimer’s disease. This suggests that S1P actions are much more complex than previously thought, with important ramifications for development of therapeutics. This review highlights recent advances in our understanding of mechanisms of action of S1P and its roles in disease.
The pleiotropic sphingolipid mediator, sphingosine-1-phosphate, produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2, regulates many cellular and physiological processes important for homeostasis and development and pathophysiology. Many of the actions of S1P are mediated by a family of five specific cell surface receptors that are ubiquitously and specifically expressed, although important direct intracellular targets of S1P have also recently been identified. S1P, SphK1, and or S1P receptors have been linked to onset and progression of numerous diseases, including many types of cancer, and especially inflammatory disorders, such as multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel disease, and sepsis. S1P formation and signaling are attractive targets for development of new therapeutics. The effects of a number of inhibitors of SphKs and S1PRs have been examined in animal models of human diseases. The effectiveness of the immunosuppressant FTY720 (known as Fingolomod or Gilenya), recently approved for the treatment of multiple sclerosis, whose actions are mediated by downregulation of S1PR1, has become the gold standard for S1P-centric drugs. Here, we review S1P biology and signaling with an emphasis on potential therapeutic benefits of specific interventions and discuss recent development of small molecule antagonists and agonists that target specific subtypes of S1P receptors as well as inhibitors of SphKs.
sphingosine-1-phosphate; sphingosine kinase; inhibitors; antagonists; cancer; multiple sclerosis; asthma; rheumatoid arthritis; inflammatory bowel disease; sepsis; apoptosis; S1P receptors
Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that regulates several processes important for hematologic cancer progression. S1P is generated by two sphingosine kinases, SphK1 and SphK2, and is exported outside the cell, where it activates specific cell surface S1P G-protein coupled receptors in autocrine/paracrine manner, coined “inside-out signaling”. In this review, we highlight the importance of SphK1 and inside-out signaling by S1P in hematologic malignancy. We also summarize the results of studies targeting the SphK1/S1P/S1P receptor axis and the effects of the S1P receptor modulator, FTY720, in hematologic malignancy.
sphingosine-1-phosphate; sphingosine kinase; sphingosine-1-phosphate receptors; leukemia; lymphoma; hematologic malignancies
Although mitochondria are key determinants of myocardial injury during ischemia–reperfusion (I/R), their interaction with critical cytoprotective signaling systems is not fully understood. Sphingosine-1-phosphate (S1P) produced by sphingosine kinase-1 protects the heart from I/R damage. Recently a new role for mitochondrial S1P produced by a second isoform of sphingosine kinase, SphK2, was described to regulate complex IV assembly and respiration via interaction with mitochondrial prohibitin-2. Here we investigated the role of SphK2 in cardioprotection by preconditioning. Littermate (WT) and sphk2−/− mice underwent 45 min of in vivo ischemia and 24 h reperfusion. Mice received no intervention (I/R) or preconditioning (PC) via 5 min I/R before the index ischemia. Despite the activation of PC-cytoprotective signaling pathways in both groups, infarct size in sphk2−/− mice was not reduced by PC (42 ± 3% PC vs. 43 ± 4% I/R, p = ns) versus WT (24 ± 3% PC vs. 43 ± 3% I/R, p < 0.05). sphk2−/−mitochondria exhibited decreased oxidative phosphorylation and increased susceptibility to permeability transition (PTP). Unlike WT, PC did not prevent ischemic damage to electron transport or the increased susceptibility to PTP. To evaluate the direct contribution to the resistance of mitochondria to cytoprotection, SphK2, PHB2 or cytochrome oxidase subunit IV was depleted in cardiomyoblasts. PC protection was abolished by each knockdown concomitant with decreased PTP resistance. These results point to a new action of S1P in cardioprotection and suggest that the mitochondrial S1P produced by SphK2 is required for the downstream protective modulation of PTP as an effector of preconditioning protection.
Mitochondria; Permeability transition pore; Sphingosine kinase-2; Mitochondrial S1P; Oxidative phosphorylation; Ischemic preconditioning
The potent lipid mediator sphingosine-1-phosphate (S1P) is produced inside cells by two closely related kinases, sphingosine kinase 1 (SPHK1) and SPHK2, and has emerged as a crucial regulator of immunity. Many of the actions of S1P in innate and adaptive immunity are mediated by its binding to five G protein-coupled receptors, designated S1PR1–5, but recent findings have also identified important roles for S1P as a second messenger during inflammation. In this Review, we discuss recent advances in our understanding of the roles of S1P receptors and describe the newly identified intracellular targets of S1P that are crucial for immune responses. Finally, we discuss the therapeutic potential of new drugs that target S1P signalling and functions.
Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-β1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-β1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-β1 had balancing effects on mast cell survival, migration, and FcεRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-β1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-β1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that correlated with inactivation of ERK1/2 and activation of JNK1-3. Activation of JNK1-3 was dependent on protein kinase R–like endoplasmic reticulum kinase (PERK), and GST-MDA-7 lethality was suppressed in PERK−/− cells. JNK1-3 signaling activated BAX, whereas inhibition of JNK1-3, deletion of BAX, or expression of dominant-negative caspase-9 suppressed lethality. GST-MDA-7 also promoted a PERK-, JNK-, and cathepsin B–dependent cleavage of BID; loss of BID function promoted survival. GST-MDA-7 suppressed BAD and BIM phosphorylation and heat shock protein 70 (HSP70) expression. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methylade-nine, expression of HSP70 or BiP/GRP78, or knockdown of ATG5 or Beclin-1 expression but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin-1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data show that GST-MDA-7 induces an endoplasmic reticulum stress response that is causal in the activation of multiple proapoptotic pathways, which converge on the mitochondrion and highlight the complexity of signaling pathways altered by mda-7/IL-24 in glioma cells that ultimately culminate in decreased tumor cell survival.
In MKN1 gastric cancer cells, lysophosphatidic acid (LPA) upregulates expression of sphingosine kinase 1 (SphK1) and its downregulation or inhibition suppresses LPA-mediated proliferation. Although LPA activates numerous signaling pathways downstream of its receptors, including ERK1/2, p38, JNK, and Akt, and the transactivation of the EGF receptor, pharmacological and molecular approaches demonstrated that only activation of ERK1, in addition to the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor, is involved in transcriptional upregulation of SphK1 by LPA. Our data implicate ERK1 as an important mediator of LPA signaling leading to upregulation of SphK1 and point to SphK1 and S1P production as potential therapeutic targets in gastric cancer.
Lysophosphatidic Acid; Sphingosine Kinase; Gastric Cancer; Proliferation
The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat but not the individual drugs rapidly increased ROS, Ca2+ and ceramide levels in GI tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation and cell killing was also blocked by quenching of induced Ca2+ levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS however combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca2+. Quenching of ROS did not impact on drug-induced ceramide/dihydro-ceramide levels whereas quenching of Ca2+ reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knock down of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings demonstrate that induction of cytosolic Ca2+ by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.
Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors. In this study, we defined the proximal mechanisms by which Ad-mda-7 kills glioma cells. Key factors implicated included activation of the endoplasmic reticulum stress kinase protein kinase R–like endoplasmic reticulum kinase (PERK), Ca++ elevation, ceramide generation and reactive oxygen species (ROS) production. PERK inhibition blocked ceramide or dihydroceramide generation, which were critical for Ca++ induction and subsequent ROS formation. Activation of autophagy and cell death relied upon ROS formation, the inhibition of which ablated Ad.mda-7–killing activity. In contrast, inhibiting TRX induced by Ad.MDA-7 enhanced tumor cytotoxicity and improved animal survival in an orthotopic tumor model. Our findings indicate that mda-7/IL-24 induces an endoplasmic reticulum stress response that triggers production of ceramide, Ca2+, and ROS, which in turn promote glioma cell autophagy and cell death.
Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator with crucial roles in a wide variety of cellular functions across a broad range of organisms. Though a simple molecule in structure, S1P functions are complex. The formation of S1P is catalyzed by one of two sphingosine kinases that have differential cellular distributions as well as both overlapping and opposing functions and which are activated by many different stimuli. S1P can act on a family of G protein-coupled receptors (S1PRs) that are also differentially expressed in different cell types, which influences the cellular responses to S1P. In addition to acting on receptors located on the plasma membrane, S1P can also function inside the cell, independently of S1PRs. It also appears that both the intracellular location and the isotype of sphingosine kinase involved are major determinants of inside-out signaling of S1P in response to many extracellular stimuli. This chapter is focused on the current literature on extracellular and intracellular actions of S1P
sphingosine kinase; sphingosine-1-phosphate; inside-out signaling; nucleus; sphingosine; dihydrosphingosine; S1PR
TNF receptor-associated factor 2 (TRAF2) is a key component in NF-κB signaling triggered by TNF–α 1,2. Genetic evidence indicates that TRAF2 is necessary for polyubiquitination of receptor interacting protein 1 (RIP1) 3 that then serves as a platform for recruitment and stimulation of IκB kinase (IKK) leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyzes the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1) 4, one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and production of S1P is necessary for Lys 63-linked polyubiquitination of RIP1, phosphorylation of IKK and IκBα, and IκBα degradation, leading to NF-κB activation. Surprisingly, these responses were mediated by intracellular S1P independently of its cell surface G protein-coupled receptors. S1P specifically binds to TRAF2 at the N-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, dramatically increased recombinant TRAF2-catalyzed Lys 63- but not Lys 48-linked polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data reveal that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing co-factor for TRAF2 E3 ubiquitin ligase activity, suggesting a new paradigm for regulation of Lys 63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signaling and the canonical NF-κB activation pathway important in inflammatory, anti-apoptotic, and immune processes.
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on clarifying the mechanism(s) by which glutathione S-transferase (GST)-MDA-7 altered cell survival of human renal carcinoma cells in vitro. GST-MDA-7 caused plasma membrane clustering of CD95 and the association of CD95 with procaspase-8. GST-MDA-7 lethality was suppressed by inhibition of caspase-8 or by overexpression of short-form cellular FLICE inhibitory protein, but only weakly by inhibition of cathepsin proteases. GST-MDA-7–induced CD95 clustering (and apoptosis) was blocked by knockdown of acidic sphingomyelinase or, to a greater extent, ceramide synthase-6 expression. GST-MDA-7 killing was, in parallel, dependent on inactivation of extracellular signal–regulated kinase 1/2 and on CD95-induced p38 mitogen-activated protein kinase and c-jun NH2-terminal kinase-1/2 signaling. Knockdown of CD95 expression abolished GST-MDA-7–induced phosphorylation of protein kinase R–like endoplasmic reticulum kinase. GST-MDA-7 lethality was suppressed by knockout or expression of a dominant negative protein kinase R–like endoplasmic reticulum kinase that correlated with reduced c-jun NH2-terminal kinase-1/2 and p38 mitogen-activated protein kinase signaling and maintained extracellular signal–regulated kinase-1/2 phosphorylation. GST-MDA-7 caused vacuolization of LC3 through a mechanism that was largely CD95 dependent and whose formation was suppressed by knockdown of ATG5 expression. Knockdown of ATG5 suppressed GST-MDA-7 toxicity. Our data show that in kidney cancer cells GST-MDA-7 induces ceramide-dependent activation of CD95, which is causal in promoting an endoplasmic reticulum stress response that activates multiple proapoptotic pathways to decrease survival.
Systemic exacerbation of allergic responses, in which mast cells play a critical role, results in life-threatening anaphylactic shock. Sphingosine-1–phosphate (S1P), a ligand for a family of G protein–coupled receptors, is a new addition to the repertoire of bioactive lipids secreted by activated mast cells. Yet little is known of its role in human mast cell functions and in anaphylaxis. We show that S1P2 receptors play a critical role in regulating human mast cell functions, including degranulation and cytokine and chemokine release. Immunoglobulin E–triggered anaphylactic responses, including elevation of circulating histamine and associated pulmonary edema in mice, were significantly attenuated by the S1P2 antagonist JTE-013 and in S1P2-deficient mice, in contrast to anaphylaxis induced by administration of histamine or platelet-activating factor. Hence, S1P and S1P2 on mast cells are determinants of systemic anaphylaxis and associated pulmonary edema and might be beneficial targets for anaphylaxis attenuation and prophylaxis.
Sphingosine-1-phosphate (S1P) is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce S1P, is upregulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and non-established human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of ERK1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the JNK pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease.
sphingosine-1-phosphate; sphingosine kinase type 1; glioblastoma; Akt
Sphingosine-1-phosphate is a ubiquitous, lipophilic cellular mediator that acts in part by activation of G-protein-coupled receptors. Modulation of S1P signaling is an emerging pharmacotherapeutic target for immunomodulatory drugs. Although multiple S1P receptor types exist in the CNS, little is known about their function. Here we report that S1P stimulated G- protein activity in the CNS, and results from [35S]GTPγS autoradiography using the S1P1-selective agonist SEW2871 and the S1P1/3-selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P1 receptors. S1P receptor activation inhibited glutamatergic neurotransmission as determined by electrophysiological recordings in cortical neurons in vitro, and this effect was mimicked by SEW2871 and inhibited by VPC44116. Moreover, central administration of S1P produced in vivo effects resembling the actions of cannabinoids, including thermal antinociception, hypothermia, catalepsy and hypolocomotion, but these actions were independent of CB1 receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P1 receptor mediated because it was produced by SEW2871 and attenuated by VPC44116. These results indicate that CNS S1P receptors are part of a physiologically relevant and widespread neuromodulatory system, and that the S1P1 receptor contributes to S1P-mediated antinociception.
S1P receptor; GPCR; Glutamate; Analgesia; Hypothermia; Autoradiography
The sphingolipid metabolite, sphingosine-1-phosphate (S1P), has emerged as a critical player in a number of fundamental biological processes and is important in cancer, angiogenesis, wound healing, cardiovascular function, atherosclerosis, immunity and asthma, among others. Activation of sphingosine kinases, enzymes that catalyze the phosphorylation of sphingosine to S1P, by a variety of agonists, including growth factors, cytokines, hormones, and antigen, increases intracellular S1P. Many of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface in an autocrine and/or paracrine manner. Therefore, understanding the mechanism by which intracellularly generated S1P is released out of cells is both interesting and important. In this review, we will discuss how S1P is formed and released. We will focus particularly on the current knowledge of how the S1P gradient between tissues and blood is maintained, and the role of ABC transporters in S1P release.
ABC transporter; Sphingosine-1-phosphate; Sphingosine kinase; Release
The pleiotropic lipid mediator sphingosine-1-phosphate (S1P) can act intracellularly independently of its cell surface receptors through unknown mechanisms. Sphingosine kinase 2 (SphK2), one of the isoenzymes that generates S1P, was associated with histone H3 and produced S1P that regulated histone acetylation. S1P specifically bound to the histone deacetylases HDAC1 and HDAC2 and inhibited their enzymatic activity, preventing the removal of acetyl groups from lysine residues within histone tails. SphK2 associated with HDAC1 and HDAC2 in repressor complexes and was selectively enriched at the promoters of the genes encoding the cyclin-dependent kinase inhibitor p21 or the transcriptional regulator c-fos, where it enhanced local histone H3 acetylation and transcription. Thus, HDACs are direct intracellular targets of S1P and link nuclear S1P to epigenetic regulation of gene expression.
A hypothesis is presented that asthma is not only an airway disease, but that the disease involves the entire lung, and that the chronicity of asthma and asthma exacerbations can perhaps be explained if one considers asthma as a systemic disease. Increased lung—not only airway—vascularity may be the result of the action of angiogenesis factors, such as vascular endothelial growth factor (VEGF) and sphingosine-1-phosphate (S1P). A bone-marrow lung axis can be postulated as one element of the systemic nature of the asthma syndrome, in which the inflamed lung emits chemotactic signals, which the bone marrow responds to by releasing cells that contribute to lung angiogenesis. A molecular model of the pathobiology of asthma can be built by connecting hypoxia-inducible transcription factor-1 alpha, VEGF S1P, and bone-marrow precursor cell mobilization and acknowledging that angiogenesis is part of the inflammatory response.
angiogenesis; asthma; bone marrow; HIF-1α; systems disease; VEGF
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA1 receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA1 receptor. An LPA1 receptor antagonist or down-regulation of its expression prevented SphK1 and S1P3 receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P3 expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P3, but not S1P1, also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.