Formyl peptide receptor signaling in HL-60 cells through sphingosine kinase.

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.

During the last few years, it has become clear that sphingolipids, in addition to being structural constituents of cell membranes, are sources of important signaling molecules. Particularly, the sphingolipid metabolites, ceramide and sphingosine-1-phosphate (SPP), 1 have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (1)(2)(3)(4). Interest in SPP focused recently on two distinct cellular actions of this lipid, namely its function as extracellular ligand activating specific G protein-coupled membrane receptors and its role as intracellular second messenger (5). Important clues to a specific intracellular action of SPP were the following findings. First, activation of various plasma membrane receptors, such as the platelet-derived growth factor receptor (6,7), the Fc⑀RI (8), and the Fc␥RI antigen receptors (9), was found to rapidly increase intracellular SPP production through stimulation of sphingosine kinase. Second, inhibition of sphingosine kinase stimulation strongly reduced or even prevented cellular events triggered by these tyrosine kinase-linked receptors, such as receptor-stimulated DNA synthesis, Ca 2ϩ mobilization, and vesicular trafficking (6,8,9). Finally, intracellular SPP was found to mimic the receptor responses, i.e. it stimulated DNA synthesis and mobilized Ca 2ϩ from internal stores (10 -14). We recently reported that the G protein-coupled muscarinic acetylcholine receptor subtypes m2 and m3 expressed in HEK-293 cells also induce a rapid and transient SPP production by sphingosine kinase. Furthermore, intracellular injection of SPP rapidly mobilized Ca 2ϩ in intact HEK-293 cells and inhibition of sphingosine kinase markedly inhibited Ca 2ϩ signaling by these and other G protein-coupled receptors (14).
To characterize the mechanisms leading to sphingosine kinase activation by G protein-coupled receptors and the cellular role of this pathway, we investigated whether the formyl peptide receptor stimulates sphingosine kinase in myeloid differentiated human leukemia (HL-60) cells, whether G proteins participate in this process, and, finally, whether sphingosine kinase activation is involved in specific cellular responses to receptor stimulation in HL-60 cells. It is well established that most, if not all, cellular responses to formyl peptide receptors in HL-60 cells and neutrophils, including phospholipase C (PLC) stimulation, Ca 2ϩ mobilization, superoxide production, and enzyme release, are mediated by pertussis toxin (PTX)-sensitive G i -type G proteins (15). Here, we report that formyl peptide receptor activation rapidly stimulates sphingosine kinase in differentiated HL-60 cells and that G proteins are involved in this process. Moreover, it is shown that inhibition of sphingosine kinase by DL-threo-dihydrosphingosine (DHS) or N,N-dimethylsphingosine (DMS) does not affect PLC stimulation and superoxide production but largely inhibits receptor-stimulated Ca 2ϩ mobilization and enzyme release.
HL-60 Cell Culture-HL-60 cells were grown in RPMI 1640 medium * This work was supported by Grant 0310493A from Bayer AG and the Bundesministerium fü r Bildung und Wissenschaft, Forschung, und Technologie and by a fellowship of the Ministerio de Educacion y Ciencia, Madrid, Spain (to R. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
supplemented with 10% fetal calf serum, 150 units/ml penicillin, and 150 g/ml streptomycin in 5% CO 2 . Differentiation into neutrophil-like cells was induced by culturing HL-60 cells for 48 h in the presence of 0.5 mM dibutyryl cAMP. For PTX treatment, cells were incubated for 20 h with 50 ng/ml PTX (16,17).
Assay of Sphingosine Kinase in Intact HL-60 Cells-Sphingosine kinase-catalyzed formation of SPP was measured as described before (14). In brief, differentiated HL-60 cells (1.8 ϫ 10 6 cells) equilibrated in Hanks' balanced salt solution/bovine serum albumin for 5 min at 37°C were incubated with [ 3 H]sphingosine (ϳ10 5 cpm; final concentration, 30 nM) for 30 s to 10 min at 37°C in a total volume of 200 l. The reactions were stopped by addition of 2 ml of ice-cold methanol, followed by 1 ml of chloroform. After vigorous vortexing and incubation for 1 h at 37°C, particulate matter was pelleted by centrifugation, and the supernatant was evaporated to dryness in a SpeedVac centrifuge. After redissolving in 20 l of methanol, the samples were spotted onto Silica gel 60 TLC plates (Merck), together with authentic unlabeled sphingosine and SPP. Separation of the products was achieved by TLC in 1-butanol: acetic acid:water (3:1:1) as a solvent system. Sphingosine and SPP spots were visualized by staining with ninhydrin spray. After scraping, radioactivity in the spots was measured by liquid scintillation counting. Formation of [ 3 H]SPP is expressed as cpm/1.8 ϫ 10 6 cells and corrected for time 0 count rates, amounting to 80 -100 cpm.
Assay of Sphingosine Kinase Activity in HL-60 Cytosol-Differentiated HL-60 cells were suspended in ice-cold lysis buffer (1.2 ϫ 10 6 cells/ml) containing 100 mM potassium phosphate buffer (pH 7.4), 1 mM EDTA, 10 mM MgCl 2 , and 1 mM 2-mercaptoethanol and disrupted on ice by 50 -100 strokes in a pre-cooled Dounce homogenizer. After centrifugation for 20 min at 50,000 ϫ g and 4°C, the supernatant (cytosolic fraction) was collected and used directly in sphingosine kinase assays. Enzyme activity was determined for 20 min at 37°C in a total volume of 200 l, containing 10 M [ 3 H]sphingosine (ϳ10 5 cpm), 1 mM ATP, 10 mM MgCl 2 , 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mg/ml fatty acidfree bovine serum albumin, 100 mM potassium phosphate buffer (pH 7.4), and HL-60 cytosol (120 -200 g of protein). Reactions were terminated by addition of 2 ml of ice-cold methanol. Extraction, TLC separation, and quantification of [ 3 H]SPP were carried out as described above. Sphingosine kinase activity is expressed as pmol SPP formed per mg protein and 20 min.
[Ca 2ϩ ] i Measurements-Intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) was determined with the fluorescent Ca 2ϩ indicator dye Fura-2 in a Hitachi spectrofluorimeter as described before (18). In some experiments, Ca 2ϩ was omitted from the medium, and in addition 5 mM EGTA was added 2 min before stimulus exposure.
Assay of PLC-Measurement of PLC-catalyzed inositol phosphate formation was performed as described in detail previously (19). In brief, after labeling for 48 h with myo-[ 3 H]inositol (2.5 Ci/ml) in inositol-free medium, differentiated HL-60 cells were equilibrated for 10 min at 37°C in Hanks' balanced salt solution/bovine serum albumin with or without the indicated agents. Cells were then incubated with 10 M fMLP at 37°C in the presence of 10 mM LiCl for 20 s and 10 min to measure formation of [ 3 H]inositol 1,4,5-trisphosphate (IP 3 ) and total [ 3 H]inositol phosphates, respectively, as reported before (19).
Measurement of Superoxide Anion Generation-Generation of superoxide anions in differentiated HL-60 cells was measured with the lucigenin assay (20) as described previously (18).
Measurement of Enzyme Release-Release of ␤-glucuronidase and N-acetyl-␤-glucosaminidase enzymes from HL-60 cells was assessed as described (21). Briefly, differentiated HL-60 cells (3.5-5 ϫ 10 6 cells) were suspended in 500 l of Hanks' balanced salt solution and incubated for 5 min at 37°C in the presence of cytochalasin B (5 g/ml). Reactions initiated by the addition of fMLP were conducted for 5 min at 37°C and terminated by placement of the tubes on ice. After cell pelleting, activities of ␤-glucuronidase and N-acetyl-␤-glucosaminidase enzymes in supernatant fluids and cell lysates were determined as described (22). Release of the enzymes is given as a percentage of the total cellular content.
Data Presentation and Analysis-Unless otherwise stated, results are presented as the means Ϯ S.E. of three independent experiments, each done in duplicates or triplicates. Statistical analysis was performed by Student's two-tailed t test for unpaired data.  (Fig. 1A). Activation of the formyl peptide receptor by 10 M fMLP induced a rapid and transient increase in
To investigate the signaling pathway leading to sphingosine kinase activation, we studied first whether G i -type G proteins are involved in the formyl peptide receptor action. Thus, formation of [ 3 H]SPP was measured in control and PTX-treated HL-60 cells. Stimulation of control cells for 2 min with 10 M fMLP increased [ 3 H]SPP levels by 67 Ϯ 17% over unstimulated cells ( Fig. 2A). PTX treatment (50 ng/ml, 20 h) had no effect on basal [ 3 H]SPP production but completely blocked fMLP-induced stimulation of sphingosine kinase, indicating that activation of sphingosine kinase by the formyl peptide receptor is mediated by G i -type G proteins. To directly assess the role of G proteins in sphingosine kinase stimulation, we studied the effects of direct G protein activation on [ 3 H]SPP formation in intact cells and in a cell-free system. Direct activation of heterotrimeric G proteins by AlF 4 Ϫ in intact HL-60 cells led to a rapid and transient production of [ 3 H]SPP, in a manner similar to the receptor agonist fMLP (Fig. 2B). [ 3 H]SPP levels in cells stimulated for 1, 2, and 5 min with AlF 4 Ϫ amounted to 117, 142, and 46%, respectively, above unstimulated control cells.
Sphingosine kinase activity has been found to be present in the cytosol of various cell types (4), and purification of a cytosolic sphingosine kinase from rat kidney has recently been reported (26). Therefore, to study G protein regulation of sphingosine kinase in a cell-free system, the effects of various guanine nucleotides on sphingosine kinase activity were investigated in cytosol prepared from differentiated HL-60 cells. Addition of the stable GTP analog GTP␥S stimulated SPP production by sphingosine kinase in HL-60 cell cytosol by about 50% in a concentration-dependent manner, with an EC 50 value of less than 1 M and a maximal effect at 10 -100 M (Fig. 3A).
On the other hand, addition of GTP (100 M) and the stable GDP analog GDP␤S (500 M) did not alter basal sphingosine kinase activity (Fig. 3B). However, GDP␤S (500 M) almost fully blocked sphingosine kinase stimulation by GTP␥S (100 M). Together, these results indicate that G proteins are signal transduction components in the pathway leading to sphingosine kinase activation in myeloid differentiated HL-60 cells.

Role of Sphingosine Kinase in Formyl Peptide
Receptor Signaling in HL-60 Cells-Previous studies in other cell types suggested that sphingosine kinase activation represents a Ca 2ϩ signaling pathway for some plasma membrane receptors (8,9,14). Because fMLP stimulated sphingosine kinase, we investigated the role of this pathway in Ca 2ϩ mobilization in  (Table I). Furthermore, release of 45 Ca 2ϩ induced by IP 3 (0.3-20 M) from 45 Ca 2ϩ -preloaded saponin-permeabilized cells, measured as described before (27) Finally, the role of sphingosine kinase in two well characterized functional responses of myeloid differentiated HL-60 cells to formyl peptide receptor activation was examined, namely production of superoxide anions by NADPH oxidase and enzyme exocytosis. Pretreatment of differentiated HL-60 cells for 1 min with 30 M DHS did not affect the superoxide production induced either by a maximally effective concentration of fMLP (1 M) or by a half-maximally effective one (10 nM) (Fig. 5). On the other hand, staurosporine (100 nM) strongly inhibited (by 70 Ϯ 0.5%) the fMLP (1 M)-stimulated production of superoxide anions (data not shown).
In contrast to superoxide production, formyl peptide receptor-stimulated enzyme release was strongly affected by the sphingosine kinase inhibitors. As illustrated in Fig. 6A, pretreatment of HL-60 cells for 5 min with 30 M DHS or 15 M DMS inhibited, by 45-50%, the fMLP (1 M)-stimulated release of ␤-glucuronidase. A similar inhibition was observed when the effect of DMS on fMLP (1 M)-stimulated release of N-acetyl-␤-glucosaminidase was examined (Fig. 6B). Pretreatment with DMS inhibited receptor-stimulated release of this enzyme halfmaximally and maximally (about 85% inhibition) at 12 and 20 M DMS, respectively. Basal enzyme release was not affected by the sphingosine kinase inhibitors. In contrast to DHS and DMS, pretreatment of HL-60 cells with staurosporine (100 nM, 3 min) did not affect fMLP-stimulated ␤-glucuronidase release (data not shown). DISCUSSION In the present study, we demonstrate that sphingosine kinase catalyzing formation of SPP from sphingosine is rapidly activated by formyl peptide receptors in myeloid differentiated HL-60 cells, a process apparently mediated by G i -type G proteins, and that sphingosine kinase activity is also stimulated by direct G protein activation, both in intact cells and in a cell-free system. Furthermore, by using the two sphingosine kinase inhibitors DHS and DMS, evidence is provided suggesting that fMLP-stimulated Sphingosine Kinase the sphingosine kinase pathway plays an important role in functional responses of HL-60 cells triggered by activation of the formyl peptide receptor. The time course and the magnitude of fMLP-stimulated SPP production in HL-60 cells were comparable with those reported for the G protein-coupled muscarinic receptors expressed in HEK-293 cells and for various tyrosine kinase-linked receptors in other cellular systems (6 -9, 14). The sphingosine kinase inhibitors DHS and DMS effectively suppressed basal and fMLP-stimulated SPP formation, indicating that sphingosine kinase is in fact responsible for SPP production in HL-60 cells.
The present study demonstrates for the first time that G proteins are involved in sphingosine kinase activation. First, similar to most other cellular responses of formyl peptide receptors in HL-60 cells and neutrophils (15), the fMLP-induced sphingosine kinase stimulation was completely abolished in PTX-treated cells, indicating participation of G i -type G proteins. Second, direct activation of heterotrimeric G proteins by AlF 4 Ϫ also markedly increased SPP production in intact HL-60 cells, with a time course similar to that observed for the formyl peptide receptor agonist. Third, the stable GTP analog GTP␥S stimulated sphingosine kinase activity in HL-60 cytosolic fractions with a concentration-response curve in the M range, and this stimulation was prevented by the stable GDP analog, GDP␤S. The identity of the GTP-binding protein mediating the stimulatory effect of GTP␥S, which can activate both heterotrimeric and low molecular weight GTP-binding proteins, on HL-60 cytosolic sphingosine kinase activity is presently under investigation.
In agreement with our previous findings on muscarinic re-ceptors in HEK-293 cells (14), we provide evidence that the sphingosine kinase pathway is involved in the formyl peptide receptor-mediated Ca 2ϩ mobilization in HL-60 cells. Shortterm pretreatment of the cells with the sphingosine kinase inhibitors DHS and DMS potently reduced the fMLP-induced increases in [Ca 2ϩ ] i , with IC 50 values (5-6 M) in the ranges of those described for inhibition of sphingosine kinase activity (6,8,(23)(24)(25) (29). Thus, the absence of SPP after DHS treatment may explain the delay in Ca 2ϩ release induced by thapsigargin. Our data thus suggest that although the formyl peptide receptor strongly stimulates PLC-catalyzed IP 3 formation, an intact sphingosine kinase/SPP pathway is required for the receptor-elicited Ca 2ϩ mobilization, similar to the results reported before for other receptors in other cell types (8,9,14).
Recently, Ca 2ϩ mobilization in Chinese hamster ovary cells by the G protein-coupled receptors for endothelin-1 and thrombin, both strongly stimulating the PLC/IP 3 pathway, has been reported to be independent of the IP 3 receptor (30). Thus, an alternative or additional mechanism, which may be the sphingosine kinase/SPP pathway, appears to be involved in Ca 2ϩ signaling by these receptors, similar to the results shown here for the formyl peptide receptor in HL-60 cells. At present, the connection between the two pathways in Ca 2ϩ signaling is not clear.
To investigate the physiological role of the sphingosine kinase in chemoattractant signaling, we studied the effects of the sphingosine kinase inhibitors on superoxide production and enzyme release. Production of superoxide anions by the NADPH oxidase in phagocytic cells involves several membrane and cytosolic proteins and can be turned on by various distinct mechanisms, including increase in [Ca 2ϩ ] i and PKC activation but also by Ca 2ϩ -and PKC-independent pathways (31)(32)(33). Treatment of HL-60 cells with the sphingosine kinase inhibitor, DHS (30 M), causing marked inhibition of fMLP-induced [Ca 2ϩ ] i increase, had no effect on superoxide production induced by fMLP, both at maximally and half-maximally effective concentrations of this stimulus, whereas the PKC inhibitor staurosporine strongly inhibited the fMLP response. These results indicate that sphingosine kinase is apparently not involved in the signaling pathway to NADPH oxidase. In contrast to superoxide production, there is a tight correlation between rises in [Ca 2ϩ ] i and enzyme exocytosis in HL-60 granulocytes (34). Pretreatment of differentiated HL-60 cells with DHS and DMS strongly inhibited the fMLP-stimulated release of ␤-glucuronidase and N-acetyl-␤-glucosaminidase. Moreover, the inhibition of fMLP-induced N-acetyl-␤-glucosaminidase release and [Ca 2ϩ ] i increase by DMS exhibited a similar concentration dependence, strongly suggesting that inhibition of enzyme release is, at least to a major extent, due to blockade of Ca 2ϩ mobilization. Because the fMLP-stimulated enzyme release was not completely inhibited by DHS or DMS at concentrations effectively blocking fMLP-induced [Ca 2ϩ ] i rises, it appears that the activated receptor generates additional signals required for exocytosis, e.g. stimulation of phosphatidylinositol 3-kinase (34). Finally, the differential sensitivity of the cellular events triggered by the formyl peptide receptor to DHS and DMS (i.e. no effect on PLC stimulation and superoxide production, but marked inhibition of Ca 2ϩ mobilization and enzyme release) underlines the specificity of the sphingosine kinase inhibitors.
In conclusion, this study demonstrates for the first time that formyl peptide receptors stimulate through PTX-sensitive G itype G proteins SPP production by sphingosine kinase in myeloid differentiated HL-60 cells and that this enzyme is also stimulated by direct G protein activation. Furthermore, evidence is provided that the sphingosine kinase/SPP pathway is involved in Ca 2ϩ and exocytosis signaling pathways but apparently not in superoxide production in phagocytic cells.