Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate

Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P(1) knock-out and the unavailability of selective agonists or antagonists. A potent, S1P(1)-receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5'-3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P(1)- but not S1P(3)-expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P(3) receptor reveals that agonism of S1P(1) receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P(1) receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P(3), and not S1P(1), is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor non-selective immunosuppressive agonists in wild-type mice is abolished in S1P(3)-/- mice, whereas S1P(1)-selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P(1) receptor agonists with an enhanced therapeutic window. S1P(1)-selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P(1) suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.     

Marked perinatal lethality and cellular signaling deficits in mice null for the two sphingosine 1-phosphate (S1P) receptors,S1P(2)/LP(B2)/EDG-5 and S1P(3)/LP(B3)/EDG-3

Five cognate G protein-coupled receptors (S1P(1-5)) have been shown to mediate various cellular effects of sphingosine 1-phosphate (S1P). Here we report the generation of mice null for S1P(2) and for both S1P(2) and S1P(3). S1P(2)-null mice were viable and fertile and developed normally. The litter sizes from S1P(2)S1P(3) double-null crosses were remarkably reduced compared with controls, and double-null pups often did not survive through infancy, although double-null survivors lacked any obvious phenotype. Mouse embryonic fibroblasts (MEFs) were examined for the effects of receptor deletions on S1P signaling pathways. Wild-type MEFs were responsive to S1P in activation of Rho and phospholipase C (PLC), intracellular calcium mobilization, and inhibition of forskolin-activated adenylyl cyclase. S1P(2)-null MEFs showed a significant decrease in Rho activation, but no effect on PLC activation, calcium mobilization, or adenylyl cyclase inhibition. Double-null MEFs displayed a complete loss of Rho activation and a significant decrease in PLC activation and calcium mobilization, with no effect on adenylyl cyclase inhibition. These data extend our previous findings on S1P(3)-null mice and indicate preferential coupling of the S1P(2) and S1P(3) receptors to Rho and PLC/Ca(2+) pathways, respectively. Although either receptor subtype supports embryonic development, deletion of both produces marked perinatal lethality, demonstrating an essential role for combined S1P signaling by these receptors.     

Memo Has a Novel Role in S1P Signaling and Crucial for Vascular Development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.     

Structural and functional characteristics of S1P receptors

The sphingosine-1-phosphate (S1P) family of G protein-coupled receptors (GPCR) regulates essential cellular processes such as proliferation, migration, cytoskeletal organization, adherens junction assembly, and morphogenesis. S1P, a product from the breakdown of sphingomyelin, binds to the five members of this receptor family, S1P(1), S1P(2), S1P(3), S1P(4), and S1P(5), previously referred to as endothelial differentiation gene (EDG)-1, -5, -3, -6, and -8. S1P receptors are widely expressed in different tissues, so it is not surprising that the S1P receptor family regulates many physiological processes, such as vascular maturation, cardiac development, lymphocyte trafficking, and vascular permeability. FTY720, a new S1P receptor agonist, is undergoing clinical trials as an immunosuppressor. Understanding the physiological role of these receptors and the basics of the ligand-receptor interaction will potentially provide new therapies to control a variety of diseases.     

Platelet-derived growth factor (PDGF)-induced chemotaxis does not require the G protein-coupled receptor S1P1 in murine embryonic fibroblasts and vascular smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, signals via G protein-coupled receptors (GPCR). The prototypical S1P receptor, S1P1 (also known as EDG-1), a Gi-linked receptor, is critical for vascular maturation during development. Recent work suggested that platelet-derived growth factor (PDGF)-induced cell migration required the S1P1 receptor, representing a novel mechanism for cross-talk between receptor tyrosine kinases and GPCRs. Since both S1P and PDGF are implicated in vascular smooth muscle cell (VSMC) pathobiology and development, we investigated this issue in rat VSMC and in embryonic fibroblasts derived from S1P1 null mice. Our data suggest that the S1P1 receptor is critical for S1P-induced, Gi-dependent migration but not for PDGF-BB-induced, receptor tyrosine kinase-dependent chemotaxis in VSMC. In addition, lack of S1P1 receptor in mouse embryonic fibroblasts did not significantly affect PDGF-induced cell migration. These data question the generality of the concept that S1P1 GPCR is a critical downstream component of PDGF-induced chemotaxis.     

Deafness and stria vascularis defects in S1P2 receptor-null mice

The S1P(2) receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P(2) receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P(2) receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P(2) receptor-null mice showed markedly disturbed structures. JTE013, an S1P(2) receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P(2) receptor-null mice.     

Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice

There is growing evidence that sphingosine 1-phosphate (S1P) plays an important role in regulating the development, morphology, and function of the cardiovascular system. There is little data, however, regarding the relative contribution of endogenous S1P and its cognate receptors (referred to as S1P(1-5)) to cardiovascular homeostasis. We used S1P(2) receptor knockout mice (S1P(2)(-/-)) to evaluate the role of S1P(2) in heart and vascular function. There were no significant differences in blood pressure between wild-type and S1P(2)(-/-) mice, measured in awake mice. Cardiac function, evaluated in situ by using a Millar catheter, was also not different in S1P(2)(-/-) mice under baseline or stimulated conditions. In vivo analysis of vascular function by flowmetry revealed decreases in mesenteric and renal resistance in S1P(2)(-/-) mice, especially during vasoconstriction with phenylephrine. In intact aortic rings, the concentration-force relations for both KCl and phenylephrine were right shifted in S1P(2)(-/-) mice, whereas the maximal isometric forces were not different. By contrast, in deendothelialized rings the concentration-force relations were not different but the maximal force was significantly greater in S1P(2)(-/-) aorta. Histologically, there were no apparent differences in vascular morphology. These data suggest that the S1P(2) receptor plays an important role in the function of the vasculature and is an important mediator of normal hemodynamics. This is mediated, at least in part, through an effect on the endothelium, but direct effects on vascular smooth muscle cannot be ruled out and require further investigation.     

High-fat/high-cholesterol diet promotes a S1P receptor-mediated antiapoptotic activity for VLDL

Withdrawing growth factors or serum from endothelial cells leads to the activation of effector caspases 3 and 7, resulting in apoptotic cell death. HDL protects against caspase induction through sphingosine-1-phosphate (S1P) receptors. This anti-caspase activity of HDL is antagonized by VLDL from apolipoprotein E4 (apoE4) (genotype, APOE4/4; apolipoprotein, apoE) targeted replacement (TR) mice, but not by VLDL from TR APOE3/3 mice, and requires the binding of apoE4-VLDL to an LDL receptor family member. In the absence of HDL, apoE4-VLDL and apoE3-VLDL from TR mice have limited antiapoptotic activity. In contrast, we show here that a high-fat/high-cholesterol/cholate diet (HFD) radically alters this biological activity of VLDL. On HFD, both apoE3-VLDL and apoE4-VLDL (HFD VLDL) inhibit caspase 3/7 activation initiated by serum withdrawal. This activity of HFD VLDL is independent of an LDL receptor family member but requires the activation of S1P(3) receptors, as shown by the ability of pharmacological block of S1P receptors by VPC 23019 and by small interfering RNA-mediated downregulation of S1P(3) receptors to inhibit HFD VLDL anticaspase activity.     

Changes in S1P1 and S1P2 expression during embryonal development and primitive endoderm differentiation of F9 cells

Sphingosine 1-phosphate (S1P) is a ligand for S1P family receptors (S1P(1)-S1P(5)). Of these receptors, S1P(1), S1P(2), and S1P(3) are ubiquitously expressed in adult mice, while S1P(4) and S1P(5) are tissue specific. However, little is known of their expression during embryonal development. We performed Northern blot analyses in mouse embryonal tissue and found that such expression is developmentally regulated. We also examined the expression of these receptors during primitive endoderm (PrE) differentiation of mouse F9 embryonal carcinoma (EC) cells, a well-known in vitro endoderm differentiation system. S1P(2) mRNA was abundantly expressed in F9 EC cells, but little S1P(1) and no S1P(3), S1P(4), or S1P(5) mRNA was detectable. However, S1P(1) mRNA expression was induced during EC-to-PrE differentiation. Studies using small interference RNA of S1P(1) indicated that increased S1P(1) expression is required for PrE differentiation. Thus, S1P(1) may play an important function in PrE differentiation that is not substituted for by S1P(2).     

The S1P2 receptor negatively regulates platelet-derived growth factor-induced motility and proliferation

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G protein-coupled receptors, named S1P(1) to S1P(5). In this study, we found that cross-communication between platelet-derived growth factor receptor and S1P(2) serves as a negative damper of PDGF functions. Deletion of the S1P(2) receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P(1) and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P(2) deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P(2)-null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P(2) reduced DNA synthesis and expression of SphK1. Thus, S1P(2) serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.     

Inhibition of Ca(2+) signalling by the sphingosine 1-phosphate receptor S1P(1)

The lysophospholipid, sphingosine 1-phosphate (S1P), regulates a multitude of cellular functions by activating specific G protein-coupled receptors (GPCRs) (S1P(1-5), plus three newly identified S1P receptors). The G(i)-coupled S1P(1) receptor inhibits adenylyl cyclase, stimulates mitogen-activated protein kinases (MAP kinases) and cell migration, and is required for blood vessel maturation. Here, we report that S1P(1) inhibits Ca(2+) signalling in a number of cell types. In HEK-293 cells, which endogenously express S1P(1-3), overexpression of S1P(1) reduced intracellular free Ca(2+) concentration ([Ca(2+)](i)) increases induced by various receptor agonists as well as thapsigargin. The inhibitory Ca(2+) signalling of S1P(1) was blocked by pertussis toxin (PTX) and the protein kinase C (PKC) inhibitor, G?6976, and imitated by phorbol ester and overexpression of classical PKC isoforms. Activation of S1P(1) stably expressed in RH7777 cells, which endogenously do not express S1P receptors, also inhibited Ca(2+) signalling, without mediating Ca(2+) mobilization on its own. It is concluded that the widely expressed S1P receptor S1P(1) inhibits Ca(2+) signalling, most likely via G(i) proteins and classical PKC isoforms. Co-expression of S1P(1) with S1P(3), but not S1P(2), reversed the inhibitory effect of S1P(1), furthermore suggesting a specific interplay of S1P receptor subtypes usually found within a single cell type.     

The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis

Nur77 and its family members, Nor-1 and Nurr1, are orphan steroid receptors implicated in a wide variety of biological processes, including apoptosis and dopamine neuron agenesis. Expression of these family members can be detected at low levels in many tissues but they are expressed at very high levels when cells are stimulated by outside signals, including serum, nerve growth factor, and receptor engagement. Introduction of a dominant negative Nur77 protein that blocks the activities of all family members led to inhibition of apoptosis in T cells. Nur77-deficient mice, however, exhibit no phenotype, and a line of Nor-1 mutant mice was reported to exhibit a mild ear development phenotype but no other gross abnormalities. Here, we report the generation of Nor-1-deficient mice with a block in early embryonic development. Nor-1 is expressed early during embryogenesis, and its loss leads to embryonic lethality around embryonic day 8.5 of gestation. The mutant embryos fail to complete gastrulation and display distinct morphological abnormalities, including a decrease in overall size, developmental delay and an accumulation of mesoderm in the primitive streak during gastrulation. Abnormal expression of a number of early developmental markers and defects in growth or distribution of emerging mesoderm cells were also detected. These data suggest that Nor-1 plays a crucial role in gastrulation.     

Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1-/-tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.     

Tie2Cre-mediated inactivation of plexinD1 results in congenital heart, vascular and skeletal defects

PlexinD1 is a membrane-bound receptor that mediates signals derived from class 3 secreted semaphorins. Although semaphorin signaling in axon guidance in the nervous system has been extensively studied, functions outside the nervous system including important roles in vascular patterning have also been demonstrated. Inactivation of plexinD1 leads to neo-natal lethality, structural defects of the cardiac outflow tract, peripheral vascular abnormalities, and axial skeletal morphogenesis defects. PlexinD1 is expressed by vascular endothelial cells, but additional domains of expression have also been demonstrated including in lymphocytes, osteoblasts, neural crest and the central nervous system. Hence, the cell-type specific functions of plexinD1 have remained unclear. Here, we describe the results of tissue-specific gene inactivation of plexinD1 in Tie2 expressing precursors, which recapitulates the null phenotype with respect to congenital heart, vascular, and skeletal abnormalities resulting in neonatal lethality. Interestingly, these mutants also have myocardial defects not previously reported. In addition, we demonstrate functions for plexinD1 in post-natal retinal vasculogenesis and adult angiogenesis through the use of inducible cre-mediated deletion. These results demonstrate an important role for PlexinD1 in embryonic and adult vasculature.