The Growth-related Gene Product β Induces Sphingomyelin Hydrolysis and Activation of c-Jun N-terminal Kinase in Rat Cerebellar Granule Neurones*

The growth-related gene product β (GROβ) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GROβ receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GROβ receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase C, GROβ activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase 1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GROβ-induced JNK1 activation is an event probably independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity. This is the first report on the activation of the SMase pathway by chemokines.

The growth-related gene product ␤ (GRO␤) 1 is a small cytokine, with chemoattractant properties, that belongs to the CXC chemokine family (1). GRO␤ binds to the high affinity interleukin-8 receptor type B (IL8RB/CXCR2) and to the Duffy antigen/chemokine receptor. While Duffy antigen/chemokine receptor has probably a "chemokine clearance" function, because it does not transduce in response to chemokine binding (2), CXCR2 acts through a pertussis toxin-sensitive G protein coupled to phospholipase C (PLC) ␤ and raises cytosolic Ca 2ϩ (3,4). CXCR2 also induces activation of the mitogen-activated protein kinase, the extracellular signal-regulated kinase (ERK) 2 and phosphorylation on tyrosine residues of Crk-associated substrate (5,6). GRO␤ is a potent chemotactic factor for neutrophils, and has growth regulatory activities on alveolar epithelial, capillary endothelial cells, and melanocytes (7)(8)(9).
Moreover, chemokines and their receptors are expressed in different regions of the central nervous system: human cerebellar neurones stain positively for CXCR2 and Duffy antigen/ chemokine receptor, which are expressed mainly in Purkinje cells and in neurite processes projecting to Purkinje cells (10) and we have recently shown that agonists of the CXCR2 have neuromodulatory roles in mouse and rat cerebellum, increasing the frequency of postsynaptic currents and impairing the induction of long-term depression (11,12). Chemokine levels in the central nervous system increase in several neurological pathologies (reviewed in Ref. 13), and deletion of chemokine receptor CXCR4 has severe consequence on the correct cerebellar development (14). For all these reasons, primary cultures of cerebellar granules have been chosen to study the biochemical pathways activated by GRO␤ treatment of homogeneous populations of cerebellar neurones. While previous reports demonstrated that, in addition to PLC, chemokine signaling can occur via phospholipase D and phospholipase A 2 activation (15,16), in this paper we describe for the first time that a chemokine induces the activation of sphingomyelin hydrolysis. We also report that GRO␤ mediates the activation of the stress-activated protein kinase c-Jun N-terminal kinase 1 (JNK1) and that this effect is not influenced by A-SMase inhibitors.
Granule Neurone Cultures-Cerebellar granule cells were obtained from 8-day-old Wistar rats (17), and cultured in basal Eagle's medium containing 25 mM KCl and 10% heat-inactivated fetal calf serum. Cells were used after 8 days of culture when they had fully differentiated. Most of the experiments were comparably carried out in Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO 3 , 2.3 mM CaCl 2 , 5.6 mM glucose, buffered with 5 mM Hepes, pH 7.4) or in phosphate-buffered saline (PBS) containing 5 mM glucose.
Immunofluorescence-Rat granule cells were plated on coverslips coated with poly-L-lysine (Sigma). Cells were rinsed in PBS, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 in PBS * This work was supported in part by grants from ISS Progetto Sclerosi Multipla (to F. E.) and from MURST (to F. E. and A. S.). 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  for 5 min. After extensive washing, primary antibody was applied as indicated by the supplier and incubated at 4 -8°C in PBS. Cells were washed and incubated with fluorescein isothiocyanate-conjugated goat affinity purified secondary antibody (Sigma). The samples were routinely examined with a microscope (Dialux, Leica, Northvale, NJ) equipped with a ϫ50 objective. Confocal analysis was carried out with a Leica TCS 4D system, equipped with a 100 ϫ 1.3-0.6 oil immersion lense.
Western Blot Analysis of CXCR2 Expression-Cerebellar granule cells were scraped off the plate in a buffer containing 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5 mM EDTA, 1% Nonidet P-40, 10 M leupeptin, 10 M aprotinin, and 1 mM PMSF, sonicated for 5 s on ice with a probe sonicator (Bandelin, Germany), and centrifuged for 15 min at 15,000 rpm in a microcentrifuge. Proteins were analyzed on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose paper at 4°C for 2 h. Blots were incubated for 1 h with 5% non-fat dry milk to block nonspecific binding sites and then incubated with affinity purified rabbit antibody to CXCR2 (K-19). The immunoreactivity was detected with a chemiluminescent substrate (ECL).
Confocal Microscopy-Fluorescence determinations were made using a real time confocal laser microscope (Odyssey, Noran Instruments, Redwood City, CA) equipped with an argon laser and interfaced to an upright microscope (Axioscope, Zeiss, Germany). The unit was driven by Image-1 software (Universal Imaging Corp., West Chester, PA). An excitation wavelength () of 488 nm was used and emission was monitored at Ͼ 515 nm, with a confocal slit of 100 m. The laser beam was set at 30% intensity, with minimum power. Real time acquisition was performed at an effective resolution of 20 Hz. Granule cells were incubated at 37°C for 30 -60 min with the dye Ca 2ϩ -Green-1 AM (4 -5 M), extensively washed with medium and transferred to the stage of the confocal microscope for fluorescence recording. Images were captured in a video recorder (Sony VO 9600P, Japan) for further analysis. The cytosolic Ca 2ϩ concentration induced by GRO␤ was expressed as the ratio of fluorescence increase after treatment/basal fluorescence (⌬F/F). Cells were continuously superfused with external medium. For this and the following assays the concentration of GRO␤ was 60 nM, which gave the most reproducible results. Agonists were applied by a gravitydriven perfusion system using independent external pipettes connected to a fast exchanger system (RSC-100, BioLogic, France).
Determination of Cellular Inositol 1,4,5-Trisphosphate (InsP 3 )-Primary cultures of rat granule cells (8-day-old) were incubated for 2-3 h in PBS and stimulated with GRO␤. To investigate the effect of the phospholipase inhibitor U73122, this compound was given (10 -30 M) to cells for 30 min before GRO␤ stimulation. At various times, the reaction was stopped by adding 15% ice-cold trichloroacetic acid (1:1 v/v). Inositol phosphates were extracted as described (18) and InsP 3 mass determination was performed following the manufacturer's instructions (Amersham Pharmacia Biotech).
Diacylglycerol (DAG) Accumulation-Cells labeled for 4 h with 250 nCi of [1-14 C]arachidonic acid were washed and reincubated for 1 h in PBS. When necessary, U73122 was applied for 30 -60 min before GRO␤ stimulation. Labeled cells were stimulated with GRO␤ and, at fixed times, the reaction was blocked by the addition of ice-cold methanol. Lipids were extracted and analyzed by high performance thin layer chromatography as described (18). Spots comigrating with authentic DAG standard were scraped off the plates and quantified by scintillation counting.
SM Hydrolysis-Rat granule cells were incubated with 1 Ci/ml of L-[3- 14 C]serine for 48 -72 h in the culture medium. After this time, cells were washed and starved for 2 h in PBS, and stimulated with GRO␤ for different times from 2 min to 2 h. Reactions were blocked with ice-cold methanol, and lipids were extracted and analyzed as described (19).
Assay for Neutral and Acidic SMase-Granule cells were incubated for 1-2 h in PBS and stimulated with GRO␤. To study the effect of inhibitors on SMase activation, cells were preincubated for different times (from 30 min to 2 h) and stimulated still in their presence. At fixed times, GRO␤ was removed and the reactions were stopped by addition of an ice-cold buffer containing 20 mM HEPES, pH 7.4, 10 mM MgCl 2 , 2 mM EDTA, 5 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 0.1 mM NaMoO 4 , 30 mM p-nitrophenyl phosphate, 10 mM ␤-glycerophosphate, 750 M ATP, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.2% Triton X-100 for N-SMase and 0.2% Triton X-100 for A-SMase. After a short incubation on ice, cells were scraped off the plates and sonicated for 5 s with a probe sonicator. Proteins were quantified by a BCA assay and the same amounts of proteins (50 g) were incubated (37°C/30 min) with 2 l of N-[methyl- 14 C]sphingomyelin (56 mCi/mmol) in 20 mM Hepes, 1 mM MgCl 2 , pH 7.4, for N-SMase and in 250 mM sodium acetate, 1 mM EDTA, pH 5.5, for A-SMase. The reaction was stopped by adding 5 volumes of chloroform: methanol:acetic acid (4:2:1); phospholipids were extracted and [ 14 C]SM levels were quantified by scintillation counting after analysis on high performance thin layer chromatography plates.
Kinase Assay for JNK1-To study JNK1 activation, granule cells were stimulated in Locke's buffer with GRO␤, C6-ceramide, or bacterial SMase for different times (from 15 to 30 min), after which the cells were lysed in ice-cold lysis buffer containing: 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5 mM EDTA, 1% Nonidet P-40, 0.1 mM dithiothreitol, 1 mM PMSF, 10 M leupeptin, 10 M aprotinin. Cell lysates, typically containing 100 g of protein, were incubated at 4°C for 16 h with 0.5 g of an affinity purified anti-JNK1 polyclonal antibody previously conjugated to Protein A-Sepharose. Immunoprecipitates were washed twice in lysis buffer and twice in kinase buffer containing: 35 mM Tris-HCl, pH 7.5, 15 mM MgCl 2 , 0.5 mM EGTA, 0.1 mM CaCl 2 , 10 mM p-nitrophenyl phosphate, and 1 mM dithiothreitol, and incubated in this same buffer in the presence of 5 g of GST-c-Jun (1-223), 3 Ci of [␥-32 P]ATP (10 M), at 25°C for 30 min. The reaction was blocked with hot Laemmli buffer and the samples boiled for 5 min at 100°C. Samples were analyzed on 10% SDS-polyacrylamide gel electrophoresis and the phosphorylated GST-c-Jun was visualized by autoradiography. The relative band intensity was determined by densitometry (Scan Jet 4C HP) and analyzed by computer (Sigma Gel software, Jandel Scientific).
Western Blot Analysis of JNK, ERK and p38 Activation-To study ERK phosphorylation, cells were preincubated for 1 h in Locke's buffer and then stimulated with GRO␤ for different times (from 5 to 60 min). For JNKs and p38 cells were stimulated as described above. Cells were then lysed in buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.5% Nonidet P-40, 0.5% Triton X-100, 10 M leupeptin, 10 M aprotinin, 1 mM PMSF, 1 mM Na 3 VO 4 , and 10 mM sodium fluoride. Proteins were analyzed on SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose paper at 4°C for 2 h. Blots were incubated for 1 h with 3% bovine serum albumin to block nonspecific binding sites and then for 2 h with the corresponding phospho-specific antibodies. The immunoreactivity was detected with a chemiluminescent substrate (ECL).

RESULTS
Rat Granule Cells Express CXCR2-We investigated the expression of CXCR2 in rat cerebellar granules by immunocytochemistry and Western blot. Fig. 1 (A and B) indicates that granule cells positively stain for CXCR2, revealing the presence of the receptor over the whole cell surface. The specificity of the staining was verified by labeling the cultures with antibodies to CXCR2 preincubated with the CXCR2 peptide used as immunogen (Fig. 1, C and D). CXCR2 expression was further confirmed by Western blot analysis, that revealed the presence of a major protein band (40 kDa) recognized by a specific CXCR2 antibody, as shown in Fig. 2. Normal rabbit IgG was used for controls (not shown).
GRO␤ Activates PIP 2 -PLC Generating Cellular InsP 3 and DAG- Fig. 3A shows the time course of InsP 3 accumulation that peaks at 1 min after cell stimulation, reaching 197.5 Ϯ 20.5% of control values, and declining thereafter. We have also investigated the accumulation of DAG in cells prelabeled with [ 14 C]arachidonic acid. A similar kinetics of [ 14 C]DAG accumulation was observed upon GRO␤ stimulation (Fig. 3B) ]DAG accumulation over basal levels, and greatly reduced InsP 3 formation after 15 s with GRO␤ (54% inhibition). When U73122 was used at a concentration of 20 M, InsP 3 accumulation was even more reduced (70% inhibition). The basal levels of InsP 3 were comparable both in control and U73122-treated cells.
GRO␤ Mobilizes Intracellular Ca 2ϩ -We investigated the Ca 2ϩ -mobilizing properties of GRO␤ on cerebellar granule cells. When bath applied to neurones loaded with Ca 2ϩ -Green 1 AM fluorescent dye, GRO␤ produced an increase of basal fluorescence with ⌬F/F ϭ 0.3 Ϯ 0.01 (mean Ϯ S.E.; 107 out of total 245 cells examined, 107/245). The fluorescence increase had a delay of 9 Ϯ 0.5 s, and a time-to-peak, defined as the time from the baseline to peak amplitude, of 69 Ϯ 4 s; the increase in [Ca 2ϩ ] i was sustained for more than 500 s after chemokine withdrawal. A comparably long delay between CXCR2 stimulation and onset of the Ca 2ϩ response was elsewhere described (20). Representative examples of the fluorescence transient induced by GRO␤ are shown in Fig. 4.
GRO␤ Induces SM Hydrolysis in Granule Cells-To investigate the possibility that GRO␤ induces the activation of SM hydrolysis, granule cells were prelabeled for 48 h with [ 14 C]serine, and then exposed to GRO␤ (60 nM). The results are illustrated in Fig. 5. The hydrolysis of cellular [ 14 C]SM begins within the first minutes of cell stimulation, and proceeds for about 90 min, diminishing thereafter (Fig. 5A). Accordingly, upon GRO␤ stimulation there was an increase in [ 14 C]ceramide. Cellular [ 14 C]ceramide reached 125.6 Ϯ 3.7% of control values after 15 min of GRO␤ and returned to control levels after about 90 min (Fig. 5B). The levels of [ 14 C]ceramide increase did not fully parallel the decrease in [ 14 C]SM, probably because of its metabolism. When vehicle alone was given to cells neither SM nor ceramide levels were altered, for the time observed.
Activation of N-and A-SMases by GRO␤-To assess whether the hydrolysis of SM induced by GRO␤ was carried out by more than one type of SMase, we measured the activity of both Nand A-SMases, with an in vitro assay that makes use of the exogenous substrate [ 14 C]SM. As shown in Fig. 6, the N-SMase activity is maximal after 15 min stimulation with GRO␤, persists for the following 15 min, and returns to basal levels within 60 min. The activity of the A-SMase was more prolonged, lasting for about 60 min of cell stimulation, and decreased thereafter. Since the A-SMase can be activated by DAG (21,22), we hypothesized that the mechanism responsible for A-SMase activation upon GRO␤ stimulation is due to the accumulation of DAG that occurs during GRO␤ stimulation of rat granules. Accordingly, at concentrations between 20 and 30 M, the PIP 2 -PLC inhibitor U73122 abolished GRO␤-mediated A-SMase activation (Fig. 7), leaving unaffected the activation of the N-SMase. The polyether antibiotic monensin, an inhibitor of A-SMase because of its ability to alter the pH of endolysosomal compartments (22,23), blocked completely GRO␤-induced A-SMase activation at concentrations 0.5-1 M (Fig. 7). Cell pretreatment for 2 h with imipramine (40 M) or desipramine (20 M), both inducing the degradation of cellular acid sphingomyelinase (24,25), also inhibited the activation of A-SMase (Fig. 7). These effects were specific, because N-SMase was not affected by inhibitor treatment (not shown). In addition, neither monensin nor U73122 were effective on constitutive A-SMase activity ( GRO␤ Activates JNK1 and ERKs-JNK1 can be efficiently activated by exposing cells to a variety of perturbing agents like ionizing and ultraviolet radiations, peroxides, heat and osmotic shocks, as well as chemokines and inflammatory cytokines (26). We investigated JNK1 activation upon GRO␤ cell stimulation by following the phosphorylation of the fusion proteinspecific substrate, GST-c- Jun (1-223). Fig. 8 shows that cell   FIG. 1. Rat granule neurones express CXCR2. Immunofluorescence analysis of CXCR2 expression in cultured rat granule neurones. Confocal micrographs of cultured granules labeled with polyclonal antibody to CXCR2 preincubated (C and D) or not (A and B) with control CXCR2 peptide, and revealed with tetramethylrhodamine B isothiocyanateconjugated goat anti-rabbit antibodies. A and B and C and D show, respectively, two optical sections of the same field, through the dendrites (A and C) and through the cell bodies (B and D). The images were recorded with the same laser intensity and amplification parameters to allow direct comparison of the fluorescence signal. Bar: 10 m. Recent evidence indicates that ceramide generated by different stressing signals can mediate JNK activation (27,28). Accordingly, treating the granule cells for 30 min with 30 M C6ceramide induced JNK1 activation: 128 Ϯ 6% (mean of five different experiments Ϯ S.E., Student's t test p Ͻ 0.01) of control value. When the cells were stimulated by 50 units/ml bacterial SMase for 30 min, the phosphorylation of GST-c-Jun was again increased over the control. These data would suggest that the effect of GRO␤ on JNK1 may be mediated by ceramide. Interestingly, we observed that neither U73122 (not shown) nor imipramine or desipramine treatment (Fig. 9A) could block GRO␤-induced JNK1 activation, suggesting that the activation of A-SMase is not necessary for this activation. The study with monensin was hampered by its effect on the basal kinase activity, while U73122, imipramine, and desipramine did not significantly alter basal JNK activity (data not shown). The observed GRO␤-induced JNK1 activation was comparable when granule neurones were stimulated in Locke's medium or in basal Eagle's medium containing 25 mM KCl and 10% fetal calf serum, further indicating that the observed kinase activation was not due to the stress of nutrient withdrawal (data not shown). The activation of JNKs was further confirmed following GRO␤-induced JNK phosphorylation with a phospho-specific Ab (Fig. 9A). With the same approach we have also investigated the activation of ERKs and p38, and the results are shown in Fig. 9B. GRO␤-induced ERK activation in rat granule cells had been already shown in a previous paper (12) with a kinase assay. Fig. 9B also shows that p38 is not activated by 15 min GRO␤ treatment. The same results were obtained when cells were stimulated for different times (from 5 to 60 min, data not shown).

DISCUSSION
Chemokines are an expanding family of leukocyte chemoattractants that act through specific receptors selectively expressed on well defined lymphoid cell populations (1). Furthermore, chemokines, and their receptors, have been found also in other tissues including the central nervous system (10,29,30). The role of chemokines in the central nervous system during normal and pathological conditions is only recently emerging, but already some close correlations between their expression levels and clinical signs have been traced (31). Studies with transgenic animals expressing N51/KC, a neutrophil-specific chemokine, established that chemokine expression in the central nervous system is responsible for leukocyte trafficking from the vascular compartment (32), and that CXCR4 expression is essential for a functional cerebellar development (14). In neural cells, CXCR4 also mediates human immunodeficiency virus type-1-induced apoptosis (33). In addition, we have re- cently suggested additional functions for the CXC chemokines IL-8 and GRO␣ in the central nervous system, namely, the modulation of the synaptic functions of cerebellar Purkinje neurones (11).
To better understand the effects of chemokines on neuronal function and development, it is crucial to describe the set of biochemical pathways activated by these molecules. Here we show that cerebellar granule cells express functional CXCR2 and we demonstrate, to our knowledge for the first time, that a chemokine, namely GRO␤, activates the SM pathway, with the involvement of both neutral and acid sphingomyelinases. This double activation, even though generating the same lipid second messenger ceramide, may subserve different cellular functions, as proposed for tumor necrosis factor-␣ and IL-1 receptor signaling (34,35). Since we have found CXCR2, a GRO␤ receptor, on granule neurones, we speculate that this receptor mediates the activation of the SMases. Although SM hydrolysis has been recently described for endothelin-1-stimulated rat small arteries (36), the activation of the SM cycle has not been shown to be mediated by G protein-coupled receptors, and the mechanisms involved in SMases activation are not known. We also show here that the activation of the A-SMase is dependent on the accumulation of cellular DAG, as reported for tumor necrosis factor-␣ receptor (22), because it is inhibited by U73122, that blocks PIP 2 -PLC activation. The ceramide produced by SMase activation plays important roles in the regulation of cell cycle arrest, growth, and differentiation (37), and some of its intracellular targets have been identified, including a ceramide-activated protein phosphatase (38), the atypical PKC (39), the retinoblastoma protein (40), a 97-kDa protein kinase suppressor of Ras (41), pp60 (Src), and the phosphoinositide 3 kinase (42) and JNK (27,28). Ceramide produced in granule neurones during GRO␤ stimulation could be involved in JNK1 activation, since both exogenous C6-ceramide and bacterial SMase induce JNK1 activation. Nevertheless, the observation that GRO␤ induces JNK1 activation even upon U73122, imipramine, and desipramine treatment, when A-SMase activity is inhibited, indicates that ceramide produced by this enzyme is not necessary to activate JNK1. Although the coordinated activation of the sphingomyelin pathway and JNK1 by stress induces apoptosis in a number of proliferating cell types (27,28,43), the effects of ceramide generation in primary cultures of postmitotic neurones seem to be rather distinct. Primary cultures of cortical neurones (44) are resistant to ceramide-induced apoptosis, and ceramide can be partially cytoprotective on sympathetic neurones (45), while both protective (46) and toxic (47) effects have been reported for cerebellar granule neurones. At low doses, ceramide potentiates elongation of neural processes (48,49) and inhibitors of sphingolipid metabolism disrupt axon growth (50). Furthermore, JNK1 activation has been described in mature neuronal cells following injury, suggesting that JNK1 could play a role in the organization of a strong response to cell damage (51). In contrast, the activation of JNK and c-Jun phosphorylation have been shown to be essential for apoptosis of neurones deprived of growth factors (52)(53)(54). The effects of GRO␤-induced SMase and JNK1 activation in rat granules is currently under investigation, since we observe that chemokine treatment modulates the survival of cerebellar granule neurones from cell death induced by potassium deprivation. 2 Since recently ceramide has been also reported to modulate, in cultured oligodendrocytes, K ϩ channel, in a ras-dependent way (55), and since glial cells express several chemokine receptors, among which CXCR2, chemokine-mediated ceramide generation could also be relevant in mediating the regulation of synaptic transmission. Data reported in these paper describe a novel signal transduction pathway for chemokines, and offer new perspectives to understand the role played by chemokines in the central nervous system during development, and in the response to local inflammatory conditions, when their level can increase severalfold (30).