Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors.

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).     

Sphingosine-1-phosphate induces a PDGFR-dependent cell detachment via inhibiting beta1 integrin in HEK293 cells

Several different types of interactions between sphingosine-1-phosphate (S1P) receptors and platelet-derived growth factor receptor (PDGFR) have been revealed recently. In this work, we used HEK293 cells to further investigate the potential crosstalk. Interestingly, we observed that S1P specifically induced a PDGFR-dependent cell detachment in HEK293 cells, which could be inhibited by AG1296, a specific inhibitor for PDGFR. EGFR on the other hand, did not have any effect on cell detachment. The detachment was extracellular matrix (ECM) protein specific, suggesting the involvement of specific integrin molecules. When beta(1) integrin was engaged into an active state, S1P-induced cell detachment was blocked, suggesting that S1P induced an inside-out inhibitory effect on beta(1) integrin. G(i) protein and ERK activation were required for the cell detachment induced by S1P, suggesting an endogenous receptor for S1P is likely to be involved.     

Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that has recently been shown to bind cell surface S1P receptors (previously called endothelial differentiation gene (Edg) receptors), which are members of the G-protein-coupled family of receptors. Signaling via S1P is a complex process, as cells usually express a number of these receptors on their surfaces. Many of the S1P receptors share common G-proteins, invoking the question of how these receptors are specific in their actions. This review describes the coupling pathways of S1P receptors, and highlights the in vitro and in vivo evidence for the "uniqueness" of each receptor in activating downstream signaling pathways, taking the effect of S1P on migration as an example.     

Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells

Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.     

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with G?6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.     

Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis

Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors.     

Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts.

Polyclonal antibodies to the mannose 6-phosphate specific receptor from human liver inhibited the endocytosis of lysosomal enzymes in fibroblasts by greater than 95% and enhanced 3-20-fold the secretion of precursors of lysosomal enzymes in these cells. Exposing fibroblasts for 4 h to antibody resulted in loss of greater than 90% of the membrane-bound receptors. If fibroblasts were treated with the antibody in the presence of CBZ-Phe-Ala-CHN2, an inhibitor of lysosomal cysteine proteinases, the receptor and smaller degradation products are recovered in dense lysosomes. In treated cells 18-58% of total receptor-related polypeptides were recovered in dense lysosomes. In control cells less than 4% of the receptor was found in the lysosomal fraction. We conclude from these results that normally the receptor is spared from lysosomal degradation. When tagged with antibody, however, the receptor is transported into lysosomes and degraded. The loss of intracellular receptors involved in segregation of newly synthesized lysosomal enzymes indicates an exchange between the former and the plasma membrane-bound receptors.     

Sphingosine-1-phosphate: a potential therapeutic target for rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, which has as its primary target, the synovial tissues and articular cartilage. The current pharmacological treatment of RA includes non-steroidal anti-inflammatory drugs, corticosteroids, and disease-modifying anti-rheumatic drugs. Newer biological agents that work by inactivation of proinflammatory cytokines are available for treatment of RA. Sphingosine-1-phosphate (S1P) is a bioactive lipid that is generated from phosphorylation of sphingosine by activation of sphingosine kinase, and has been implicated as an important mediator in pathophysiological processes, including cell growth, differentiation, migration and survival, and angiogenesis. Several studies have explored the role of S1P in the pathogenesis of RA. The aim of this article was to review the biology and distribution of S1P, together with its role in RA, and to discuss its potential as a therapeutic target for RA.     

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P₁R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P₁ receptor desensitization caused by synthetic S1P₁ receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P₁ receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P₁ receptors. In recombinant S1P₁ receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P₁ receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P₁ receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase – by facilitating S1P1 receptor recycling – is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates.     

Sphingosine-1-phosphate acts as a developmental stage specific inhibitor of platelet-derived growth factor-induced chemotaxis of osteoblasts

The development and maintenance of a healthy skeleton depends on the migration of cells to areas of new bone formation. Osteoblasts, the bone forming cells of the body, mature from mesenchymal stem cells under the influence of bone morphogenetic protein. It is unclear at what developmental stage the osteoblasts start to migrate to their functional location. We have studied migration of immature pre-osteoblasts and of mature osteoblasts in response to Platelet-derived growth factor (PDGF) and sphingosine-1-phosphate (S1P). PDGF is a growth factor involved in bone remodeling and fracture healing whereas S1P is a circulating sphingolipid known to control cell trafficking. Our data indicate that PDGF acts as a chemotactic cue for pre-osteoblasts. In contrast, S1P is a chemorepellent to these cells. Upon Bone Morphogenetic Protein 2-induced conversion to the osteoblast phenotype, the chemotaxis response to PDGF is retained whereas the sensitivity to S1P is lost. By RNA interference and overexpression experiments we showed that the expression level of the S1P2 receptor is the sole determinant controlling responsiveness to S1P. The combined data indicate that migration of osteoblasts is controlled by the balance between PDGF, S1P and the differentiation state of the cells. We propose that this mechanism preserves the osteoprogenitor pool in the bone marrow, only allowing the more differentiated cell to travel to sites of bone formation.     

Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers.

The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane-domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E-115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes similar responses, but the cell-rounding and neurite-retracting activity of serum is not attributable to thrombin. Neural cell rounding is transient, subsiding after 10-15 min, and subject to homologous desensitization, whereas retracted neurites rapidly degenerate. Thrombin action is inhibited by cytochalasin, but not colchicine. A novel 14-amino acid peptide agonist of the thrombin receptor fully mimics thrombin's morphoregulatory activity, indicating that thrombin-induced shape changes are receptor-mediated and not secondary to extracellular matrix degradation. Although thrombin receptors couple to phosphoinositide hydrolysis and Ca2+ mobilization, thrombin-induced shape changes appear to depend neither on the Ca2+/protein kinase C- nor the cyclic nucleotide-mediated signal transduction pathways; however, the morphological response to thrombin is blocked by pervanadate, an inhibitor of tyrosine phosphatases, and by broad-specificity kinase inhibitors. Our results suggest that the thrombin receptor communicates to an as-yet-uncharacterized effector to reorganize the actin cytoskeleton and to reverse the differentiated phenotype of neural cells.     

Zinc induces ERK-dependent cell death through a specific Ras isoform

The effect of Zn on p53-independent cell death was examined in IIC9 embryonic fibroblasts. Despite the fact that these cells are p53-minus, Zn-mediated death occurs via an apoptotic mechanism. Death is facilitated by the presence of the Zn ionophore, pyrithione, indicating that intracellular Zn initiates the death response. Our investigations of the mechanism of Zn action demonstrate that Zn induces the death of IIC9 cells in a manner that is ERK-dependent. Expression of dn-(dominant negative)Ras attenuates ERK1/2 activation by Zn, and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments confirm that Zn treatment activates Ras and identified H-Ras as the specific isoform activated. This contrasts the activation of N-Ras that occurs when IIC9 cells are stimulated with thrombin. Thus, although the prolonged activation of the Ras/ERK pathway by Zn is similar to that seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation. This work was supported by National Institute of Health Grants DK-52194 and AI-44458 (to J.A.C). P.L Blackwell was supported by an American Heart Association Grant in Aid 0555574Z (to C.K), K.J. Hughes was supported by the National Institute of Health Training Grant (GM008306) and Kimberly Creach was supported by St. Louis University Medical School Summer Research Fellowship.     

Sphingosine-1-phosphate activates phospholipase D in human airway epithelial cells via a G protein-coupled receptor

Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o(-)), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8-10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8-10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G(i/o) protein-coupled receptor.     

S1P 信号通路：心血管疾病治疗的新靶点

1- 磷酸鞘氨醇是一种有生物活性的脂质代谢产物,具有调节细胞增殖、再生、迁移,细胞内钙离子移动,黏附分子表达以及激活单核细胞黏附内皮细胞等功效,在血管生理性再生及动脉粥样硬化斑块发生发展中发挥重要作用。1- 磷酸鞘氨醇在高密度脂蛋白中含量在所有脂蛋白中最高,其参与调节高密度脂蛋白的抗氧化、抗血栓、抗炎等效应,而这些反应与1- 磷酸鞘氨醇的生物学功能如血管发生、内皮保护、抑制平滑肌细胞迁移、心肌缺血再灌注损伤的保护等密切相关。对1- 磷酸鞘氨醇信号通路在心血管系统中的作用及以该通路为靶点的相关药物研究进展进行综述,为今后研究提供参考。     

Sphingosine-1-phosphate and oligodendrocytes: From cell development to the treatment of multiple sclerosis

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide variety of biological effects in different cells and tissues. This review discusses the effects of S1P signaling in oligodendrocytes, the myelin making cells of the central nervous system (CNS). Results from different laboratories have uncovered direct actions of S1P at different maturational stages along the oligodendroglial lineage. There is also evidence for the existence in oligodendrocytes of interactions between S1P and signaling by factors which, like neurotrophin-3 (NT-3) and platelet-derived growth factor (PDGF), have profound effects on oligodendrocyte development and myelination. Moreover, S1P signaling in oligodendrocytes may not only play an important role during normal CNS development but also offer new therapeutic avenues to stimulate remyelination in demyelinating diseases like multiple sclerosis.     

G-protein-coupled receptor activation induces the membrane translocation and activation of phosphatidylinositol-4-phosphate 5-kinase I alpha by a Rac- and Rho-dependent pathway.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K I alpha activation following the addition of thrombin. Under resting condition expressed PIP5K I alpha was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of G alpha(q), PIP5K I alpha translocated to the plasma membrane. Movement of PIP5K I alpha to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active G alpha(q), 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K I alpha translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K I alpha. In addition, a dominant negative variant of Rho blocked the PIP5K I alpha membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K I alpha membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K I alpha by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K I alpha, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K I alpha correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K I alpha with either constitutively active G alpha(q), Rac, or Rho led to a 5- to 7-fold increase in PIP5K I alpha activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of G alpha(q), Rac, Rho, and PIP5K I alpha. Once activated and translocated to the cell membrane, PIP5K I alpha becomes available to phosphorylate PI4P to generate PI4,5P(2) on the plasma membrane.     

Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices

Endothelial cell invasion is a key step in angiogenic blood vessel formation. Sphingosine-1-phosphate (S1P) has been previously reported to play a role in endothelial cell proliferation, survival, migration, and angiogenesis. Here, we examine the ability of S1P to regulate human endothelial cell invasion into three-dimensional collagen or fibrin matrices. We show that S1P potently stimulated human endothelial cell invasion, lumen formation, and branching morphogenesis in collagen, and fibrin matrices, (5- and 15-fold increases in invasion were observed, respectively). The S1P-induced invasion response was pertussis-toxin sensitive and completely dependent on integrins. Addition of integrin blocking reagents revealed that the alpha2beta1 integrin regulated invasion in collagen matrices, while a combination of alphavbeta3 and alpha5beta1 integrins regulated invasion in fibrin. Additionally, the S1P-induced invasion response was dependent on matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinase-3 (TIMP-3) was the only physiologic inhibitor of metalloproteinases that completely inhibited the potent stimulation of invasion induced by S1P. In contrast, TIMP-1 had no blocking effect on invasion or morphogenesis, while TIMP-2 and TIMP-4 partially reduced invasion but completely blocked lumen formation events. Collectively, these data reveal a marked ability of S1P to induce metalloproteinase- and integrin-dependent human endothelial cell invasion and morphogenesis in both collagen and fibrin three-dimensional matrices, the two most physiologically relevant matrices for angiogenesis.