Activation Of S1P1 Receptors Research Articles

Fingolimod inhibits glutamate release through activation of S1P1 receptors and the G protein βγ subunit-dependent pathway in rat cerebrocortical nerve terminals

Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator approved for treating multiple sclerosis, is reported to prevent excitotoxic insult. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the effect of fingolimod on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) was investigated in the current study. Fingolimod decreased 4-aminopyridine (4-AP)-stimulated glutamate release and calcium concentration elevation. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was dependent on extracellular calcium, persisted in the presence of the glutamate transporter inhibitor DL-TBOA or intracellular Ca2+-releasing inhibitors dantrolene and CGP37157, and was prevented by blocking vesicular transporters or N- and P/Q-type channels. Western blot and immunocytochemical analysis revealed the presence of S1P1 receptor proteins in presynaptic terminals. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was also abolished by the sphingosine kinase inhibitor DMS, selective S1P1 receptor antagonist W146, Gi/o protein inhibitor pertussis toxin, and G protein βγ subunit inhibitor gallein; however, it was unaffected by the adenylyl cyclase inhibitor SQ22536, protein kinase A inhibitor H89, and phospholipase C inhibitor U73122. These data indicate that fingolimod decreases glutamate release from rat cerebrocortical synaptosomes by suppressing N- and P/Q-type Ca2+ channel activity; additionally, the activation of presynaptic S1P1 receptors and the G protein βγ subunit participates in achieving the effect.

Activation of S1P₁ receptor regulates PI3K/Akt/FoxO3a pathway in response to oxidative stress in PC12 cells.

FTY720 (fingolimod) is a sphingosine analogue that, when phosphorylated, becomes a prototypical sphingosine-1-phosphate (S1P) receptor modulator. It can enter the CNS and act on S1PRs expressed by most neural lineages. Recently, FTY720 neuroprotective and regenerative actions in the CNS have been demonstrated. In the present study, we have investigated whether the PI3K-Akt-FoxO3a axis is downstream to the S1P1 receptor modulation and involved in the cytoprotective effect of FTY720 in PC12 cells exposed to hydrogen peroxide (H2O2). The data showed that oxidative stress induces cell death in parallel with a significant decrease in PI3K, Akt and Akt target, and FoxO3a phosphorylation. FTY720 pretreatment increased cell survival which can be attributed to enhanced levels of inactive phosphorylated FoxO3a, a transcription factor playing critical role in oxidative stress-induced cell death. FTY720-phosphate (p-FTY720), a pan agonist of S1P receptors, as well as SEW2871, a selective S1P1 receptor agonist, similarly exerted cytoprotective effects. W123, a S1P1 receptor antagonist, abolished the effects of all three drugs, and concomitant application of DMS, a sphingosine kinase inhibitor, prevented the protective effects of FTY720. The data suggests that S1P1 receptor activation in the context of oxidative stress maintains PI3K/Akt signaling to prevent activation of FoxO3a, thereby promoting PC12 cell survival.

S1P1 receptor modulation preserves vascular function in mesenteric and coronary arteries after CPB in the rat independent of depletion of lymphocytes.

BackgroundCardiopulmonary bypass (CPB) may induce systemic inflammation and vascular dysfunction. Sphingosine 1-phosphate (S1P) modulates various vascular and immune responses. Here we explored whether agonists of the S1P receptors, FTY720 and SEW2871 improve vascular reactivity after CPB in the rat.MethodsExperiments were done in male Wistar rats (total n = 127). Anesthesia was induced by isoflurane (2.5–3%) and maintained by fentanyl and midazolam during CPB. After catheterization of the left femoral artery, carotid artery and the right atrium, normothermic extracorporeal circulation was instituted for 60 minutes. In the first part of the study animals were euthanized after either 1 hour, 1 day, 2 or 5 days of the recovery period. In second part of the study animals were euthanized after 1 day of postoperative period. We evaluated the contractile response to phenylephrine (mesenteric arteries) or to serotonin (coronary artery) and vasodilatory response to acethylcholine (both arteries).ResultsContractile responses to phenylephrine were reduced at 1 day recovery after CPB and Sham as compared to healthy control animals (Emax, mN: 7.9±1.9, 6.5±1.5, and 11.3±1.3, respectively). Mainly FTY720, but not SEW2871, caused lymphopenia in both Sham and CPB groups. In coronary and mesenteric arteries, both FTY720 and SEW2871 normalized serotonin and phenylephrine-mediated vascular reactivity after CPB (p<0.05) and FTY720 increased relaxation to acetylcholine as compared with untreated rats that underwent CPB.ConclusionPretreatment with FTY720 or SEW2871 preserves vascular function in mesenteric and coronary artery after CPB. Therefore, pharmacological activation of S1P1 receptors may provide a promising therapeutic intervention to prevent CPB-related vascular dysfunction in patients.

Sphingosine 1-phosphate to p38 signaling via S1P1 receptor and Gαi/o evokes augmentation of capsaicin-induced ionic currents in mouse sensory neurons.

The perception of painful thermal stimuli by sensory neurons is largely mediated by TRPV1. Upon tissue injury or inflammation, S1P is secreted by thrombocytes as part of an inflammatory cocktail, which sensitizes nociceptive neurons towards thermal stimuli. S1P acts on G-protein coupled receptors that are expressed in sensory neurons and sensitize TRPV1 channels towards thermal stimuli. In this study, the S1P mediated signaling pathway required for sensitization of TRPV1 channels was explored.The capsaicin induced peak inward current (ICAPS) of sensory neurons was significantly increased after S1P stimulation within minutes after application. The potentiation of ICAPS resulted from activation of Gαi through G-protein coupled receptors for S1P. Consequently, Gαi led to a signaling cascade, involving phosphoinositide-3-kinase (PI3K) and protein kinase C, which augmented ICAPS in nociceptive neurons. The S1P1 receptor agonist SEW2871 resulted in activation of the same signaling pathway and potentiation of ICAPS. Furthermore, the mitogen-activated protein kinase p38 was phosphorylated after S1P stimulation and inhibition of p38 signaling by SB203580 prevented the S1P-induced ICAPS potentiation. The current data suggest that S1P sensitized ICAPS through G-protein coupled S1P1 receptor activation of Gαi-PI3K-PKC-p38 signaling pathway in sensory neurons.Electronic supplementary materialThe online version of this article (doi:10.1186/1744-8069-10-74) contains supplementary material, which is available to authorized users.

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P1) phosphorylation was studied. Activation of S1P1 receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P1 phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P1 phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P1 phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P1 immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P1 phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P1.

The conundrum of protection from AKI by adenosine in rodent clamp ischemia models

Kim et al. show that isoflurane uses a tubule-based transforming growth factor-β/CD73-dependent process that generates adenosine to protect mice from ischemic acute kidney injury (AKI) with effects to prevent the 'no-reflow phenomenon' and decrease inflammation. While direct cytoprotection occurred in culture, extensive research suggests that in vivo adenosine protection from rodent ischemic AKI is mediated by a mutually cooperative mechanism involving blood flow, inflammation, and innate immunity through multiple adenosine receptors with promiscuous actions on diverse cell types.

A polysaccharides MDG-1 augments survival in the ischemic heart by inducing S1P release and S1P1 expression

Ophiopogon japonicus is a traditional Chinese medicine used to treat cardiovascular disease. Recent studies have confirmed the anti-ischemic properties of a water-soluble β-d-fructan (MDG-1) from O. japonicus. The sphingosine 1-phosphate (S1P) signaling pathway is involved in its cytoprotective effects. Herein, we explore the role of the S1P signaling pathway in the anti-ischemic effect of MDG-1 and assess one possible mechanism by which it induces S1P release and sphingosine 1-phosphate receptor 1 (S1P1) expression in human microvascular endothelial cells (HMEC-1) and cardiomyocytes. Our evidence demonstrates that MDG-1 promotes sphingosine kinase (SPHK) activity in HMEC-1 cells. An analytical method for measuring the mass of S1P using ESI/MS/MS was developed and we found that MDG-1 increases intracellular S1P levels. Meanwhile, MDG-1 is protective during hypoxia and ischemia through mechanisms that require S1P1 receptor activation, which was confirmed both in oxygen glucose deprivation (OGD) and coronary artery ligation models by using transfection of cloned human S1P1 receptor and RNA interference. These data indicate that the increase of intracellular S1P generation, particularly by activation of the SPHK enzyme, coupled with the autocrine and paracrine stimulation of cell surface S1P receptors, is a potential mechanism in the anti-ischemic and cell protective effect of MDG-1.

S1P2 receptor regulation of sphingosine-1-phosphate effects on conventional outflow physiology

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P₁ receptor on the inner wall of SC, we hypothesized that S1P₁ receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P₁-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P₂ antagonist JTE-013, but not the S1P₁ or S1P₁,₃ antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P₂ , and not S1P₁ or S1P₃, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.

Role for peroxynitrite in sphingosine-1-phosphate-induced hyperalgesia in rats

Sphingosine-1-phosphate (S1P) is an important mediator of inflammation recently shown in in vitro studies to increase the excitability of small-diameter sensory neurons, at least in part, via activation of the S1P1 receptor subtype. Activation of S1PR1 has been reported to increase the formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide (O2·−) and nitric oxide synthase (NOS)-derived nitric oxide (NO). This process favors the formation of peroxynitrite (ONOO− [PN]), a potent mediator of hyperalgesia associated with peripheral and central sensitization. The aims of our study were to determine whether S1P causes peripheral sensitization and thermal hyperalgesia via S1PR1 activation and PN formation. Intraplantar injection of S1P in rats led to a time-dependent development of thermal hyperalgesia that was blocked by the S1PR1 antagonist W146, but not its inactive enantiomer W140. The hyperalgesic effects of S1P were mimicked by intraplantar injection of the well-characterized S1PR1 agonist SEW2871. The development of S1P-induced hyperalgesia was blocked by apocynin, a NADPH oxidase inhibitor; NG-nitro-l-arginine methyl ester, a nonselective NOS inhibitor; and by the potent PN decomposition catalysts (FeTM-4-PyP5+ and MnTE-2-PyP5+). Our findings provide mechanistic insight into the signaling pathways engaged by S1P in the development of hyperalgesia and highlight the contribution of the S1P1 receptor-to-PN signaling in this process.Sphingosine-1-phosphate (S1P)-induced hyperalgesia is mediated by S1P1 receptor activation and mitigated by inhibition or decomposition of peroxynitrite, providing a target pathway for novel pain management strategies.

Sphinganine-1-phosphate protects kidney and liver after hepatic ischemia and reperfusion in mice through S1P1 receptor activation

Liver failure due to ischemia and reperfusion (IR) and subsequent acute kidney injury are significant clinical problems. We showed previously that liver IR selectively reduced plasma sphinganine-1-phosphate levels without affecting sphingosine-1-phosphate (S1P) levels. Furthermore, exogenous sphinganine-1-phosphate protected against both liver and kidney injury induced by liver IR. In this study, we elucidated the signaling mechanisms of sphinganine-1-phosphate-mediated renal and hepatic protection. A selective S1P1 receptor antagonist blocked the hepatic and renal protective effects of sphinganine-1-phosphate, whereas a selective S1P2 or S1P3 receptor antagonist was without effect. Moreover, a selective S1P1 receptor agonist, SEW-2871, provided similar degree of liver and kidney protection compared with sphinganine-1-phosphate. Furthermore, in vivo gene knockdown of S1P1 receptors with small interfering RNA abolished the hepatic and renal protective effects of sphinganine-1-phosphate. In contrast to sphinganine-1-phosphate, S1P's hepatic protection was enhanced with an S1P3 receptor antagonist. Inhibition of extracellular signal-regulated kinase, Akt or pertussis toxin-sensitive G-proteins blocked sphinganine-1-phosphate-mediated liver and kidney protection in vivo. Taken together, our results show that sphinganine-1-phosphate provided renal and hepatic protection after liver IR injury in mice through selective activation of S1P1 receptors and pertussis toxin-sensitive G-proteins with subsequent activation of ERK and Akt.

Sphingosine-1-Phosphate Induces an Antiinflammatory Phenotype in Macrophages

Activated macrophages acquire a proinflammatory (classic) or antiinflammatory (alternative) phenotype that influences atherosclerosis. The present study investigated whether sphingosine-1-phosphate (S1P), with its known antiinflammatory effects, could regulate the inflammatory phenotype of lipopolysaccharide (LPS)-stimulated mouse macrophages. Activation of macrophages by LPS significantly increases proinflammatory cytokine secretion. Pretreatment of macrophages with 500 nmol/L S1P markedly reduced LPS-mediated secretion of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and interleukin-12. Such antiinflammatory actions were also evident in LPS-stimulated macrophages treated with the S1P1 receptor-specific agonist SEW2871. Pharmacological antagonism of the S1P1 receptor on macrophages using the S1P1-specific antagonist VPC44116 also blocked proinflammatory cytokine secretion in response to LPS. Studies using bone marrow-derived macrophages from S1P2-deficient mice revealed that the S1P2 receptor did not play a pivotal role in this process. Thus, activation of the S1P1 receptor in mouse macrophages limits the expression of proinflammatory cytokines. Furthermore, we demonstrated that S1P increased arginase I activity and inhibited LPS-induced inducible NO synthase activity in LPS-treated macrophages, again through S1P1 receptor activation on macrophages. Analysis of a 1.7-kb region of the murine inducible NO synthase promoter revealed the presence of putative nuclear factor kappaB, activator protein-1, and STAT-1 response elements. Using inducible NO synthase promoter-reporter constructs, we found that S1P significantly reduced the nuclear factor kappaB-mediated induction of inducible NO synthase. These findings demonstrate an important role for S1P in the regulation of macrophage phenotypic switching. Therefore, we conclude that S1P promotes the production of an alternative antiinflammatory macrophage phenotype through activation of the macrophage S1P1 receptor.

Phosphorylated FTY720 promotes astrocyte migration through sphingosine‐1‐phosphate receptors

Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.

Involvement of Sphingosine-1-Phosphate in Glutamate Secretion in Hippocampal Neurons

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P(1) receptor at the puncta was activated during depolarization and that depolarization-induced S1P(1) receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na(+)-channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.

Phosphorylated FTY720 stimulates ERK phosphorylation in astrocytes via S1P receptors

Sphingosine-1-phosphate receptors (S1P1-5) are activated by the endogenous agonist S1P and are expressed in the central nervous system. In astrocytes, activation of S1P receptors leads to phosphorylation of extracellular-signal regulated kinase (ERK), a signaling cascade which plays intimate roles in cell proliferation. Fingolimod (FTY720) is in phase III clinical trials for the treatment of multiple sclerosis and its phosphorylated version (FTY720P) activates S1P receptors. We examined the effects of FTY720P on ERK phosphorylation and determined which S1P receptor subtype(s) mediated this signaling event. FTY720P augmented ERK phosphorylation in cortical cultures prepared from embryonic day 18 rat brains and was blocked by an MEK inhibitor or by pertussis toxin. Co-localisation of phosphorylated ERK occurred in glial fibrillary acidic protein (GFAP) positive astrocytes but not neurons or oligodendrocytes. Furthermore, FTY720P stimulated ERK phosphorylation in highly enriched astrocyte cultures made from postnatal day 2 rat cortices. The effects of FTY720P were mimicked by selective S1P1 receptor agonists and blocked by S1P1 receptor antagonists. Collectively, these results demonstrate that FTY720P mediates ERK phosphorylation in astrocytes via the activation of S1P1 receptors.

Rac1 Modulates Sphingosine 1-Phosphate-mediated Activation of Phosphoinositide 3-Kinase/Akt Signaling Pathways in Vascular Endothelial Cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that activates G protein-coupled S1P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac1 is implicated in diverse S1P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S1P-mediated Rac1 activation are incompletely understood. We studied the pathways involved in S1P-mediated Rac1 activation in bovine aortic endothelial cells (BAEC) and found that S1P-induced Rac1 activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S1P-mediated Rac1 activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac1 activation while completely blocking S1P-induced Akt phosphorylation. We used Rac1-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac1 expression and found that siRNA-mediated Rac1 knockdown significantly impaired basal as well as S1P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S1P-induced phosphorylation of the mitogen-activated protein kinases ERK1/2 was unperturbed by siRNA-mediated Rac1 knockdown. We found that overexpression of the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 markedly enhanced Rac1 activity, whereas a dominant negative Tiam1 mutant significantly attenuated S1P-mediated Rac1 activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam1 as upstream modulators of S1P-mediated Rac1 activation, and establish a central role for Rac1 in S1P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.

Selective sphingosine 1-phosphate 1 receptor activation reduces ischemia-reperfusion injury in mouse kidney

The mechanisms involved in renal ischemia-reperfusion injury (IRI) are complex and appear to involve the early participation of bone marrow-derived cells. T lymphocytes participate in the pathogenesis of IRI. Sphingosine 1-phosphate (S1P) induces peripheral T cell depletion. Therefore, we hypothesized that S1P1 receptor activation protects kidney from IRI. FTY-720, a non-receptor-selective sphingosine analog, was given intraperitoneally to C57BL/6 mice, and animals were subjected to ischemia for 32 min followed by reperfusion for 24 h. Plasma creatinine, blood count, myeloperoxidase (MPO) activity, and renal histology were determined. IRI led to a marked increase in plasma creatinine, MPO activity, leukocyte infiltration, and vascular permeability. FTY-720 significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of approximately 73 and approximately 69% with doses of 240 and 48 microg/kg, respectively. MPO, leukocyte infiltration, vascular permeability, and peripheral blood lymphocyte counts were markedly decreased with FTY-720 treatment. The protective effect of FTY-720 was reversed with VPC-44116, a selective S1P1 receptor antagonist. Furthermore, SEW-2871, a selective S1P1 agonist, significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of approximately 70% with a dose of 10 mg/kg. Analysis of kidneys by light microscopy revealed minimal histological signs of ischemic injury with FTY-720 or SEW-2871 treatment compared with the vehicle group. Using RT-PCR, we found a time-dependent increase in the S1P1 mRNA expression following IRI that begins after 2 h with the maximum expression at approximately 4 h. We conclude that the protective effect of FTY-720 is due primarily to activation of S1P1 receptors. The mechanism of protection is not known but may be related to peripheral lymphocyte depletion or direct effects on kidney cells expressing S1P1 receptor.

Activated Protein C Mediates Novel Lung Endothelial Barrier Enhancement: ROLE OF SPHINGOSINE 1-PHOSPHATE RECEPTOR TRANSACTIVATION

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.

Sphingosine 1-phosphate receptor regulation of N-cadherin mediates vascular stabilization.

Vascular stabilization, a process by which nascent vessels are invested with mural cells, is important in angiogenesis. Here we describe the molecular basis of vascular stabilization regulated by sphingosine 1-phosphate (S1P), a platelet-derived lipid mediator. S1P1 receptor-dependent cell-surface trafficking and activation of the cell-cell adhesion molecule N-cadherin is essential for interactions between endothelial and mural cells. Endothelial cell S1P1/Gi/Rac pathway induces microtubule polymerization, resulting in trafficking of N-cadherin to polarized plasma membrane domains. S1P treatment modulated the phosphorylation of N-cadherin as well as p120-catenin and induced the formation of cadherin/catenin/actin complexes containing novel regulatory and trafficking factors. The net result of endothelial cell S1P1 receptor activation is the proper trafficking and strengthening of N-cadherin-dependent cell-cell adhesion with mural cells. Perturbation of N-cadherin expression with small interfering RNA profoundly attenuated vascular stabilization in vitro and in vivo. S1P-induced trafficking and activation of N-cadherin provides a novel mechanism for the stabilization of nascent blood vessels by mural cells and may be exploited to control angiogenesis and vascular diseases.

Receptor tyrosine kinase-GPCR signal complexes.

The formation of complexes between growth factor receptors and members of a family of G-protein-coupled receptors whose natural ligands are S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) represents a new signalling entity. This receptor complex allows for integrated signalling in response to growth factor and/or S1P/LPA and provides a mechanism for more efficient activation (due to integrated close-proximity signalling from both receptor classes) of the p42/p44 MAPK (mitogen-activated protein kinase) pathway. This article provides information on the molecular events at the interface between receptor tyrosine kinases and S1P/LPA receptors. Examples include the PDGF (platelet-derived growth factor)-induced tyrosine phosphorylation of G(i)alpha, released upon S1P(1) receptor activation, which is required for initiation of the p42/p44 MAPK pathway. Critical to this event is the formation of endocytic vesicles containing functionally active PDGFbeta receptor-S1P(1) receptor complexes, which are internalized and relocated with components of the p42/p44 MAPK pathway. We also report examples of cross-talk signal integration between the Trk A (tropomyosin receptor kinase A) receptor and the LPA(1) receptor in terms of the NGF (nerve growth factor)-dependent regulation of the p42/p44 MAPK pathway. NGF induces recruitment of the LPA(1) receptor to the nucleus (delivery might be Trk A-dependent), whereupon the LPA(1) receptor may govern gene expression via novel nuclear signalling processes.