Pronectin Promotes Calcium Signaling by Interferon-γ Human Neutrophils via G-Protein and Sphingosine Kinase-Dependent Mechanisms

A common intracellular signal activating polymorphonuclear leukocytes (PMN) in inflammation is a change in cytosolic calcium concentration. Previously, we have shown that interferon-γ (IFN-γ) induces transient calcium signals in PMN, but only after intracellular calcium store depletion. Using a digital imaging system, we show that adhesion of PMN is critical for IFN-γ-induced calcium signals, and with PMN attached to the optimal coating, the calcium signals are evoked even in presence of extracellular calcium, that is, non-depleted calcium stores. Adhesion to fibronectin, pure or extracted from plasma by gelatin, improved the IFN-γ responses compared with serum, plasma, or vitronectin coats. In accordance with previous observations, IFN-γ-induced calcium signals in fibronectin adherent cells were totally abolished by the G-protein inhibitor pertussis toxin and were also inhibited by the sphingosine kinase inhibitors dimethylsphingosine (DMS) and N-acetylsphingosine (N-Ac-Sp). PMN contact with fibronectin alone, measured in cells sedimenting onto a fibronectin-coated surface or by addition of fibronectin to glass-adherent cells, evoked transient calcium signals. However, PMN in suspension did not respond to the addition of fibronectin or arginine-glycine-aspartate (RGD). The fibronectin-induced calcium signals were also clearly depressed by pertussis toxin and by the sphingosine kinase inhibitors DMS, dihydrosphingosine (DHS), and N-Ac-Sp. When the product of sphingosine kinase activity, sphingosine I-phosphate (S1-P), was added to the cells, similar calcium signals were induced, which were dependent on a pertussis toxin-sensitive G-protein activity. Finally, addition of S1-P to the cells prior to stimulation with IFN-γ partly mimicked the priming effect of fibronectin. In conclusion, fibronectin contact evokes by itself a calcium signal in PMN and further promotes calcium signaling by IFN-γ. We suggest that fibronectin might activate sphingosine kinase, and that the sphingosine 1-phosphate thereby generated induces a calcium signal via a G-protein-dependent mechanism. Apparently, sphingosine kinase activity is also involved in IFN-γ induced calcium signals.     

Protein kinase C is crucial in signal transduction during IFN-γ induction in endothelial cells

We have demonstrated that IFN-γ, a potent peptide mediator in inflammatory responses, operates via the protein kinase C dependent transduction pathway in the induction of class II MHC antigens on rat microvascular endothelial cells. Stimulators of protein kinase C, like PMA, replaced IFN-γ in the induction of MHC class II on endothelial cells in a dose-dependent manner. Selective enzyme inhibitors of protein kinase C, H-7 as well as sphingosine down-regulated the IFN-γ induced class II expression in a dose-dependent manner. Addition of cAMP or cGMP in the culture, had no effect on the class II expression on the endothelial cells. Transient rise of cytosolic Ca²⁺ by calcium ionophore A23187, or a calmodulin antagonist W-7, had no effect on the IFN-γ induced class II expression.     

Functional characterization of sphingosine 1-phosphate receptor agonist in human endothelial cells

Sphingosine 1-phosphate (S1P) is a pleiotropic lysophospholipid mediator involved in many cellular responses, including transient calcium mobilization, activation of MAP kinase signaling, inhibition of adenylyl cyclase and increased cell migration. S1P has been shown to be an effective activator of vascular endothelial cells via the interaction with cell surface G protein-coupled receptors (GPCRs), namely S1P-R (formerly EDG-R). The potent immunomodulator, FTY720, is phosphorylated by sphingosine kinase (SK) to FTY720-P. Recently it was shown that FTY720-P, not FTY720, can bind to four out of five of the S1P-R. In the present study, we evaluated the effects of FTY720, FTY720-P, and analogues of FTY720-P: an active (R)-enantiomer [AFD(R)] and an inactive (S)-enantiomer [AFD(S)], on endothelial cell functions. Treatment of HUVEC with FTY720-P, but not FTY720, lead to a robust transient increase in calcium mobilization, detected using the fluorometric imaging plate reader (FLIPR) assay. Additionally, only the phosphorylated derivative (FTY720-P) stimulated MAPK activation. We also observed complementary activities of S1P and FTY720-P in an established in vitro endothelial morphogenesis (Matrigel tube formation) assay and an in vitro endothelial cell migration assay. Using a potent inhibitor of sphingosine kinase, N,N-dimethylsphingosine (DMS), FTY720's effects were inhibited in the migration assay, suggesting that FTY720-P is the active mediator. The effects of FTY720-P in these assays were inhibited by pre-treatment with PTx (pertussis toxin), indicating the requirement of a Gi-coupled S1P receptor. These findings suggest that agonist of S1P-R are able to regulate important endothelial cell properties, which may lead to a greater insight into vascular functions.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Sphingosine kinase regulates voltage operated calcium channels in GH4C1 rat pituitary cells

We have previously shown that sphingosine inhibits depolarisation-induced calcium influx through voltage-operated calcium channels (VOCCs) in GH(4)C(1) cells, whereas sphingosine-1-phosphate (S1P) does not. In the present study we investigated whether sphingosine kinase modulates VOCC activity in GH(4)C(1) cells by removing inhibitory sphingosine. Sphingosine and the structurally similar sphingosine kinase inhibitor dimethylsphingosine (DMS) both rapidly attenuated the calcium influx evoked by depolarisation. The inhibitory effect declined over time to a greater extent in cells treated with sphingosine than in cells treated with DMS, indicating that sphingosine is being metabolised more rapidly. When the specific sphingosine kinase inhibitor 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKi) was added to the cells after depolarisation there was likewise a reduction of the calcium response. This inhibitory effect was slow and reached a plateau about 3 min after application. In contrast, the sphingosine-mediated inhibition was immediate, suggesting that the SKi-induced inhibition was due to build-up of cellular sphingosine. In experiments on cells overexpressing sphingosine kinase, the inhibitory effect of sphingosine was reversed faster than in control cells. The effect was not due to the produced S1P, since S1P did not have any effect on VOCCs even at concentrations as high as 50 microM. In patch-clamp experiments the calcium entry through VOCCs was attenuated in GH(4)C(1) cells overexpressing a kinase-dead sphingosine kinase, compared with cells overexpressing the wild type sphingosine kinase. In addition, in cells treated with SKi the calcium entry through VOCCs was attenuated compared with control cells. Our results provide compelling evidence that sphingosine kinase regulates the function of voltage-operated calcium channels in GH(4)C(1) cells, not through its catalytic product, but by removal of the substrate sphingosine.     

Phosphorylation and action of the immunomodulator FTY720 inhibits vascular endothelial cell growth factor-induced vascular permeability

FTY720, a potent immunosuppressive agent, is phosphorylated in vivo into FTY720-P, a high affinity agonist for sphingosine 1-phosphate (S1P) receptors. The effects of FTY720 on vascular cells, a major target of S1P action, have not been addressed. We now report the metabolic activation of FTY720 by sphingosine kinase-2 and potent activation of vascular endothelial cell functions in vitro and in vivo by phosphorylated FTY720 (FTY720-P). Incubation of endothelial cells with FTY720 resulted in phosphorylation by sphingosine kinase activity and formation of FTY720-P. Sphingosine kinase-2 effectively phosphorylated FTY720 in the human embryonic kidney 293T heterologous expression system. FTY720-P treatment of endothelial cells stimulated extracellular signal-activated kinase and Akt phosphorylation and adherens junction assembly and promoted cell survival. The effects of FTY720-P were inhibited by pertussis toxin, suggesting the requirement for Gi-coupled S1P receptors. Indeed, transmonolayer permeability induced by vascular endothelial cell growth factor was potently reversed by FTY720-P. Furthermore, oral FTY720 administration in mice potently blocked VEGF-induced vascular permeability in vivo. These findings suggest that FTY720 or its analogs may find utility in the therapeutic regulation of vascular permeability, an important process in angiogenesis, inflammation, and pathological conditions such as sepsis, hypoxia, and solid tumor growth.     

Enhancement of intracellular sphingosine-1-phosphate production by inositol 1,4,5-trisphosphate-evoked calcium mobilisation in HEK-293 cells: endogenous sphingosine-1-phosphate as a modulator of the calcium response

Sphingosine-1-phosphate (S1P) regulates many cellular functions, such as migration, differentiation and growth. The effects of S1P are thought to be primarily mediated by G-protein coupled receptors, but an intracellular function as a calcium releasing second messenger has also been proposed. Here we show that in HEK-293 cells, exogenous S1P mobilises sequestered calcium by a mechanism primarily dependent on the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) pathway, and secondarily on the subsequent synthesis of intracellular S1P. Stimulating HEK-293 cells exogenously with S1P increased the production of both inositol phosphates and intracellular S1P. The calcium response was inhibited in cells treated with 2-APB, caffeine or U73122, showing that the PLC/IP3 pathway for calcium release is activated in response to exogenous S1P. The calcium response was partially inhibited in cells treated with the sphingosine kinase inhibitor DMS and in cells expressing a catalytically inactive sphingosine kinase, showing that endogenously produced S1P is also involved. Importantly, 2-APB and U73122 inhibited the S1P-evoked production of intracellular S1P. S1P is therefore not likely a major calcium releasing second messenger in HEK-293 cells, but rather a secondary regulator of calcium mobilisation.     

Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins

Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Gα or Gβγ subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gβγ subunits are likely to be involved since the expression of the C terminus of β-adrenergic receptor kinase 1, a Gβγ subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gβγ dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gβγ subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gβγ subunits and activation of MAPKs to repress skeletal muscle differentiation.     

Tethering of the platelet-derived growth factor beta receptor to G-protein-coupled receptors. A novel platform for integrative signaling by these receptor classes in mammalian cells

Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.     

Anaphylatoxin signaling in human neutrophils. A key role for sphingosine kinase

Anaphylatoxins activate immune cells to trigger the release of proinflammatory mediators that can lead to the pathology of several immune-inflammatory diseases. However, the intracellular signaling pathways triggered by anaphylatoxins are not well understood. Here we report for the first time that sphingosine kinase (SPHK) plays a key role in C5a-triggered signaling, leading to physiological responses of human neutrophils. We demonstrate that C5a rapidly stimulates SPHK activity in neutrophils and differentiated HL-60 cells. Using the SPHK inhibitor N,N-dimethylsphingosine (DMS), we show that inhibition of SPHK abolishes the Ca2+ release from internal stores without inhibiting phospholipase C or protein kinase C activation triggered by C5a but has no effect on calcium signals triggered by other stimuli (FcgammaRII). We also show that DMS inhibits degranulation, activation of the NADPH oxidase, and chemotaxis triggered by C5a. Moreover, an antisense oligonucleotide against SPHK1, in neutrophil-differentiated HL-60 cells, had similar inhibitory properties as DMS, suggesting that the SPHK utilized by C5a is SPHK1. Our data indicate that C5a stimulation decreases cellular sphingosine levels and increases the formation of sphingosine-1-phosphate. Exogenously added sphingosine has a dual effect on C5a-stimulated oxidative burst: it has a priming effect at lower concentrations but a dose-dependent inhibitory effect at higher concentrations; however, C5a-triggered protein kinase C activity was only reduced at high concentration of sphingosine. In contrast, C5a-triggered Ca2+ signals, chemotaxis, and degranulation were not affected by sphingosine at all. Exogenous sphingosine-1-phosphate, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and oxidative burst and had a priming effect, enhancing all the C5a-triggered responses. Taken together, these results suggest that SPHK plays an important role in the immune-inflammatory pathologies triggered by anaphylatoxins in human neutrophils and point out SPHK as a potential therapeutic target for the treatment of diseases associated with neutrophil hyperactivation.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

Enhancement by sphingosine 1-phosphate in vasopressin-induced phosphoinositide hydrolysis in aortic smooth-muscle cells: involvement of p38 MAP kinase

We previously reported that sphingosine 1-phosphate (S-1-P), a sphingomyelin metabolite, activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in aortic smooth-muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C-catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C(2)-ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S-1-P, which alone slightly stimulated the IPs formation, dose-dependently amplified the AVP-induced formation of IPs. Tumor necrosis factor-alpha enhanced the AVP-induced formation of IPs. However, S-1-P did not enhance the formation of IPs by NaF, a heterotrimeric GTP-binding protein activator. Pertussis toxin inhibited the effect of S-1-P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S-1-P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S-1-P on the formation of IPs by AVP. SB203580 inhibited the AVP-induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S-1-P. These results indicate that S-1-P amplifies AVP-induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth-muscle cells.     

Pertussis toxin B-subunit-induced Ca2(+)-fluxes in Jurkat human lymphoma cells: the action of long-term pre-treatment with cholera and pertussis holotoxins

The exotoxins of Bordetella pertussis and Vibrio cholera have been used to investigate signal transduction in the human T-cell lymphoma Jurkat. Stimulation of the cells, leading to an increase in cytoplasmic free calcium, could be achieved by the anti-T-cell receptor complex antibody OKT3 and by pertussis holotoxin (PTHT), or its B-subunit (PTB), but not by cholera holotoxin (CTHT) or its B-subunit (CTB). Both holotoxins ADP-ribosylated specifically G-proteins in the plasma membrane of intact cells, while their B-subunits had no ADP-ribosyltransferase activity. Incubation of the cells with CTHT led to a state of unresponsiveness to all stimulants. CTB was without any effect, indicating that the ADP-ribosyltransferase activity of cholera toxin (located in the A-subunit of the holotoxin) was necessary for the inhibition of cellular signalling. The inhibitory effect of cholera toxin on the pertussis toxin action was not due to a blockade of pertussis toxin interaction with the cell surface, because pertussis toxin was still able to ADP-ribosylate membrane proteins in cholera toxin treated intact cells. In addition, the cholera toxin mediated inhibition was not due to elevated levels of cyclic-AMP, as forskolin (a direct activator of the adenylate cyclase) and no inhibitory effect. The stimulating effect of PTHT was independent of its ADP-ribosyltransferase activity, because it could also be obtained by the B-subunit alone. In addition, the increase of cytoplasmic free calcium after stimulation by PTHT clearly preceded the ADP-ribosylation. Pre-treatment with PTHT, PTB or OKT3, led to a long lasting increase in the level of intracellular Ca2+ in Jurkat cells, which could not, therefore, be stimulated further. Inhibition by cholera holotoxin of the stimulation by OKT3 and pertussis toxin (PTHT and PTB) imply that the mitogenic effect of pertussis toxin is perhaps mediated via the T-cell antigen receptor signalling cascade. The presented data do not support the idea that a pertussis toxin-sensitive G-protein is involved in coupling the T-cell antigen receptor to the phospholipase C.     

Pertussis but not cholera toxin inhibits the stimulated increase in actin association with the cytoskeleton in rabbit neutrophils: role of the "G proteins" in stimulus-response coupling

Treatment of rabbit neutrophils with pertussis toxin, but not cholera toxin, inhibits the increases produced by formylmethionyl-leucyl-phenylalanine, leukotriene B4 and the calcium ionophore A23187 in the amounts of actin associated with the cytoskeletons. The increase in the cytoskeletal actin produced by phorbol 12-myristate, 13-acetate on the other hand is not affected by pertussis toxin. Incubation of the neutrophils with cholera toxin, unlike pertussis toxin, did not inhibit the fMet-Leu-Phe induced rise in the intracellular concentration of free calcium, and caused only a shift to the right of the dose-response curve of N-acetyl-beta-glucosaminidase release. This shift was more marked in the presence of 1-methyl-3-isobutylxanthine. In addition, the stimulated breakdown of phosphatidylinositol 4,5 bis-phosphate was inhibited by pertussis toxin. These results suggest that pertussis toxin acts at an early step in the signal transduction and does not affect the sequence of reactions initiated by the activation of the protein kinase C. Furthermore, the guanine nucleotide regulatory protein Gi, but not Gs, is closely involved in signal transduction in these cells.     

Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth

We present evidence that direct activation of neuronal second messenger pathways in PC12 cells by opening voltage-dependent calcium channels mimics cell adhesion molecule (CAM)-induced differentiation of these cells. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin in the presence of KCl or a calcium channel agonist Bay K 8644. Both potassium depolarization and agonist-induced activation of calcium channels promoted substantial neurite outgrowth from PC12 cells cultured on control 3T3 monolayers and increased neurite outgrowth from those cultured on N-cadherin-expressing 3T3 monolayers. The potassium-induced response could be inhibited by L- and N-type calcium channel antagonists and by kinase inhibitor K-252b but was unaffected by pertussis toxin. In contrast activators of protein kinase C did not stimulate neurite outgrowth, and the neurite outgrowth response induced by activation of protein kinase A was not inhibited by calcium channel antagonists or pertussis toxin. These studies support the postulate that CAM-induced neuronal differentiation involves a specific transmembrane signaling pathway and suggest that activation of this pathway after CAM binding may be more important for the neurite outgrowth response than CAM-dependent adhesion per se.     

Sphingosine kinase: a point of convergence in the action of diverse neutrophil priming agents

Neutrophils are a vital component of the early acute inflammatory response, but can cause profound tissue damage when activated to excess or prevented from undergoing apoptosis. However, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. The structurally diverse neutrophil-priming agents platelet-activating factor, TNF-alpha, and the substance P analog [D-Arg(6), D-Trp(7,9),N(me)Phe(8)]-substance P(6-11) (SP-G) stimulated a rapid increase in sphingosine kinase activity in freshly isolated human neutrophils. This activity was blocked by preincubation with the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS). DMS also inhibited the increase in intracellular calcium concentration stimulated by platelet-activating factor, fMLP, and SP-G. This suggests that the increase in intracellular calcium concentration by these agents is dependent on sphingosine kinase activation and the generation of sphingosine-1-phosphate. Changes in cell polarization and the augmentation of the fMLP-induced superoxide anion generation, by all priming agents were also inhibited by DMS, while only the superoxide anion release was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Moreover, SP-G and GM-CSF inhibited constitutive neutrophil apoptosis which was completely blocked by DMS. These results suggest a novel role for sphingosine kinase in the regulation of neutrophil priming.     

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.     

FTY720-phosphate is dephosphorylated by lipid phosphate phosphatase 3

FTY720 is a novel immunomodulatory drug efficacious in the treatment of multiple sclerosis. The drug is converted in vivo to the monophosphate, FTY720-P, by sphingosine kinase 2. This conversion is incomplete, suggesting opposing actions of kinase and phosphatase activities. To address which of the known lipid phosphatases might dephosphorylate FTY720-P, we overexpressed the broad specificity lipid phosphatases LPP1-3, and the specific S1P phosphatases (SPP1 and 2) in HEK293 cells, and performed in vitro assays using lysates of transfected cells. Among LPPs, only LPP3 was able to dephosphorylate FTY720-P; among SPPs, only SPP1 showed activity against FTY720-P. On intact cells, LPP3 acted as an ecto-phosphatase or FTY720-P, thus representing the major phosphatase involved in the equilibrium between FTY720 and FTY720-P observed in vivo.     

Rho-mediated phosphorylation of focal adhesion kinase and myosin light chain in human endothelial cells stimulated with sphingosine 1-phosphate, a bioactive lysophospholipid released from activated platelets

Since sphingosine 1-phosphate (Sph-1-P) is stored in abundant amounts in blood platelets and released extracellularly upon stimulation, it is important to clarify the effects of this bioactive lysophospholipid on vascular endothelial cells from the viewpoint of platelet-endothelial cell interactions. In this study, we investigated the effects of Sph-1-P on the cytoskeletal remodeling of human umbilical vein endothelial cells (HUVECs). Of a focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases, HUVECs were found to express FAK, but scarcely proline-rich tyrosine kinase 2. Sph-1-P induced FAK tyrosine phosphorylation, myosin light chain phosphorylation, and the formation of stress fibers in HUVECs. The specific Rho inactivator C3 transferase from Clostridium botulinum abolished all of these cytoskeletal responses induced by Sph-1-P, while pertussis toxin only partly inhibited FAK tyrosine phosphorylation, and hardly affected myosin light chain phosphorylation and stress fiber formation. In contrast, Sph-1-P-induced intracellular Ca(2)(+) mobilization was suppressed by pertussis toxin, but not at all by C3 exoenzyme. Our results suggest that Sph-1-P, a bioactive lipid released from activated platelets, induces endothelial cell cytoskeletal reorganization, mainly through Rho-mediated signaling pathways.     

Rac-1-mediated O2- secretion requires Ca2+ influx in neutrophil-like HL-60 cells

Neutrophil-like HL-60 cells reacted to N -formyl- l -Methionyl- l -Leucyl- l -P henylalanine (f MLP) with a rise in the intracellular calcium concentration ([Ca2]i), NADPH oxidase activation, and increased superoxide anion (O2-) production. [Ca2+]i mobilization and superoxide production were largely dependent on extracellular calcium (Ca2+]e) and a capacitative calcium entry. The monomeric G-protein, Rac-1, regulates NADPH oxidase activity. We tested the effect of removal of Ca2+]e on Rac-1 plasma membrane sequestration and activation of NADPH oxidase using immunodetection and a double labelling fluorescent method. Results showed that Rac-1 activation is mediated via a pertussis toxin (PTX)-sensitive heteromeric G-protein pathway, and that Rac-1 membrane sequestration was preceded by [Ca2+]i mobilization following entry of Ca2+ e. Therefore, we propose that O2- production is dependent on activation of PTX-sensitive G-proteins and sequestration of Rac-1 in the plasma membrane, following entry of Ca2+ e.