Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain.
ABSTRACT: P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-?) and sphingolipid-based signaling. Critical to this pathway was signaling through sphingosine-1-phosphate receptor 1 (S1PR1). In brain capillaries, S1P acted through S1PR1 to rapidly and reversibly reduce P-glycoprotein transport activity. Sphingosine reduced transport by a sphingosine kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S1P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, (3)H-verapamil (threefold increase), (3)H-loperamide (fivefold increase), and (3)H-paclitaxel (fivefold increase); blocking S1PR1 abolished this effect. Tight junctional permeability, measured as brain (14)C-sucrose accumulation, was not altered. Therefore, targeting signaling through S1PR1 at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain.
Project description:At the blood-brain and blood-spinal cord barriers, P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to central nervous system (CNS) pharmacotherapy. Recently, we showed that signaling through tumor necrosis factor-α (TNF-α), sphingolipids, and sphingosine-1-phosphate receptor 1 (S1PR1) rapidly and reversibly reduced basal P-glycoprotein transport activity in the rat blood-brain barrier. The present study extends those findings to the mouse blood-brain and blood-spinal cord barriers and, importantly, identifies multidrug resistance-associated protein 1 (Mrp1, Abcc1) as the transporter that mediates S1P efflux from brain and spinal cord endothelial cells. In brain and spinal cord capillaries isolated from wild-type mice, TNF-α, sphingosine, S1P, the S1PR agonist fingolimod (FTY720), and its active, phosphorylated metabolite, FTY720P, reduced P-glycoprotein transport activity; these effects were abolished by a specific S1PR1 antagonist. In brain and spinal cord capillaries isolated from Mrp1-null mice, neither TNF-α nor sphingosine nor FTY720 reduced P-glycoprotein transport activity. However, S1P and FTY720P had the same S1PR1-dependent effects on transport activity as in capillaries from wild-type mice. Thus, deletion of Mrp1 alone terminated endogenous signaling to S1PR1. These results identify Mrp1 as the transporter essential for S1P efflux from the endothelial cells and thus for inside-out S1P signaling to P-glycoprotein at the blood-brain and blood-spinal cord barriers.
Project description:The development of chemotherapy-induced painful peripheral neuropathy is a major dose-limiting side effect of many chemotherapeutics, including bortezomib, but the mechanisms remain poorly understood. We now report that bortezomib causes the dysregulation of de novo sphingolipid metabolism in the spinal cord dorsal horn to increase the levels of sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) ligands, S1P and dihydro-S1P. Accordingly, genetic and pharmacological disruption of S1PR1 with multiple S1PR1 antagonists, including FTY720, blocked and reversed neuropathic pain. Mice with astrocyte-specific alterations of S1pr1 did not develop neuropathic pain and lost their ability to respond to S1PR1 inhibition, strongly implicating astrocytes as a primary cellular substrate for S1PR1 activity. At the molecular level, S1PR1 engaged astrocyte-driven neuroinflammation and altered glutamatergic homeostasis, processes blocked by S1PR1 antagonism. Our findings establish S1PR1 as a target for therapeutic intervention and provide insight into cellular and molecular pathways. As FTY720 also shows promising anticancer potential and is FDA approved, rapid clinical translation of our findings is anticipated.
Project description:Nitric oxide is one of the major endothelial-derived vasoactive factors that regulate blood pressure (BP), and the bioactive lipid mediator S1P (sphingosine-1-phosphate) is a potent activator of endothelial nitric oxide synthase through G protein-coupled receptors. Endothelial-derived S1P and the autocrine/paracrine activation of S1PR (S1P receptors) play an important role in preserving vascular functions and BP homeostasis. Furthermore, FTY720 (fingolimod), binding to 4 out of 5 S1PRs recently approved by the Food and Drug Administration to treat autoimmune conditions, induces a modest and transient decrease in heart rate in both animals and humans, suggesting that drugs targeting sphingolipid signaling affect cardiovascular functions in vivo. However, the role of specific S1P receptors in BP homeostasis remains unknown. The aim of this study is to determine the role of the key vascular S1P receptors, namely, S1PR1 and S1PR3, in BP regulation in physiological and hypertensive conditions. The specific loss of endothelial S1PR1 decreases basal and stimulated endothelial-derived nitric oxide and resets BP to a higher-than-normal value. Interestingly, we identified a novel and important role for S1PR1 signaling in flow-mediated mechanotransduction. FTY720, acting as functional antagonist of S1PR1, markedly decreases endothelial S1PR1, increases BP in control mice, and exacerbates hypertension in angiotensin II mouse model, underlining the antihypertensive functions of S1PR1 signaling. Our study identifies S1P-S1PR1-nitric oxide signaling as a new regulatory pathway in vivo of vascular relaxation to flow and BP homeostasis, providing a novel therapeutic target for the treatment of hypertension.
Project description:The immunomodulatory prodrug 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), which acts as an agonist for sphingosine-1-phosphate (S1P) receptors (S1PR) when phosphorylated, is proposed as a novel pain therapeutic. In this study, we assessed FTY720-mediated antinociception in the radiant heat tail-flick test and in the chronic constriction injury (CCI) model of neuropathic pain in mice. FTY720 produced antinociception and antiallodynia, respectively, and these effects were dose-dependent and mimicked by the S1PR1-selective agonist CYM-5442. Repeated administration of FTY720 for 1 week produced tolerance to acute thermal antinociception, but not to antiallodynia in the CCI model. S1PR-stimulated [35S]GTP?S autoradiography revealed apparent desensitization of G protein activation by S1P or the S1PR1 agonist 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole (SEW-2871) throughout the brain. Similar results were seen in spinal cord membranes, whereby the Emax value of S1PR-stimulated [35S]GTP?S binding was greatly reduced in repeated FTY720-treated mice. These results suggest that S1PR1 is a primary target of FTY720 in alleviating both acute thermal nociception and chronic neuropathic nociception. Furthermore, the finding that tolerance develops to antinociception in the tail-flick test but not in chronic neuropathic pain suggests a differential mechanism of FTY720 action between these models. The observation that repeated FTY720 administration led to desensitized S1PR1 signaling throughout the central nervous system suggests the possibility that S1PR1 activation drives the acute thermal antinociceptive effects, whereas S1PR1 desensitization mediates the following: 1) tolerance to thermal antinociceptive actions of FTY720 and 2) the persistent antiallodynic effects of FTY720 in neuropathic pain by producing functional antagonism of pronociceptive S1PR1 signaling.
Project description:Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1-5) and plays a key role in regulating the inflammatory response. Our previous studies demonstrated that rat sensory neurons express the mRNAs for all five S1PRs and that S1P increases neuronal excitability primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability.Isolated sensory neurons were treated with either short-interfering RNAs (siRNAs) or a variety of pharmacological agents targeted to S1PR1/R2/R3 to determine the role(s) of these receptors in regulating neuronal excitability. The excitability of isolated sensory neurons was assessed by using whole-cell patch-clamp recording to measure the capacity of these cells to fire action potentials (APs).After siRNA treatment, exposure to S1P failed to augment the excitability. Pooled siRNA targeted to S1PR1 and R3 also blocked the enhanced excitability produced by S1P. Consistent with the siRNA results, pretreatment with W146 and CAY10444, selective antagonists for S1PR1 and S1PR3, respectively, prevented the S1P-induced increase in neuronal excitability. Similarly, S1P failed to augment excitability after pretreatment with either VPC 23019, which is a S1PR1 and R3 antagonist, or VPC 44116, the phosphonate analog of VPC 23019. Acute exposure (10 to 15 min) to either of the well-established functional antagonists, FTY720 or CYM-5442, produced a significant increase in the excitability. Moreover, after a 1-h pretreatment with FTY720 (an agonist for S1PR1/R3/R4/R5), neither SEW2871 (S1PR1 selective agonist) nor S1P augmented the excitability. However, after pretreatment with CYM-5442 (selective for S1PR1), SEW2871 was ineffective, but S1P increased the excitability of some, but not all, sensory neurons.These results demonstrate that the enhanced excitability produced by S1P is mediated by activation of S1PR1 and/or S1PR3.
Project description:HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI's C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.
Project description:The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.
Project description:The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein-coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both ?-arrestin 1 and ?-arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G-protein coupled receptor's (GPCR)'s.
Project description:Classical Hodgkin lymphoma (CHL), a neoplasm of abnormal B lymphocytes (Hodgkin-Reed-Sternberg (HRS) cells), has been described to have a typical pattern of clinical presentation and dissemination often involving functionally contiguous lymph nodes. Despite the progress made in understanding CHL pathophysiology, the factors that regulate the spread of lymphoma cells in CHL are poorly understood. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid present at high concentrations in the plasma and lymphatic fluid, is known to have a critical role in regulating lymphocyte trafficking mainly through sphingosine-1-phosphate receptor 1 (S1PR1). In this study, we explore the role of the S1P-S1PR1 axis in Hodgkin lymphoma cell migration and the expression of S1PR1 in CHL cell lines and clinical cases. We found that S1PR1 is present in the KM-H2 and SUP-HD1 Hodgkin lymphoma cell lines at the mRNA and protein level. In addition, functionally, S1P potently stimulated migration of both cell lines. S1P-induced migration was inhibited by the S1PR1 antagonist, VPC44116, and the S1PR1 functional antagonist, FTY720-P, but was potentiated by the S1PR2-specific antagonist, JTE013. We also determined that S1PR1 induced migration in the KM-H2 and SUP-HD1 cells via the heterotrimeric G-protein Gi and the phosphatidylinositol-3-kinase pathway. Immunohistochemical assessment of the tissue from CHL samples revealed that a subset of cases (7/57; 12%) show strong, membranous staining for S1PR1 in HRS cells. Altogether, our data indicate that S1PR1 is a functional receptor on HRS cells, which governs tumor cell migration and is expressed in a subset of CHL cases. Given the availability of S1PR1 antagonists, some of which are used clinically for modulation of the immune system, these results suggest that S1PR1 could be a future therapeutic target in the treatment of those cases of S1PR1-positive, refractory/recurrent CHL.