C2-ceramide and reactive oxygen species ... - Wiley Online Library

Journal of Neurochemistry, 2001, 76, 778±788

C2-ceramide and reactive oxygen species inhibit pituitary adenylate cyclase activating polypeptide (PACAP)-induced cyclic-AMP-dependent signalling pathway V. SeÂe, B. Koch and J. P. Loef¯er UniversiteÂ Louis Pasteur, UMR 7519 CNRS, Strasbourg Cedex, France

Abstract The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor, a seven-domain transmembrane receptor, is positively coupled to both adenylate cyclase and phospholipase C. PACAP exerts neurotrophic effects which are mainly mediated through the cAMP/protein kinase A pathway. Here we show that the cell-permeable C2-ceramide selectively blocks PACAP-activated cAMP production, without affecting phosphoinositide breakdown. Thus by blocking the neuroprotective cAMP signalling pathway, C2-ceramide will reinforce its direct death-inducing signalling. We found that a reactive oxygen species scavenger reversed the C2-ceramide effect and that H2O2 mimicked it. Together these data indicate

that reactive oxygen species (ROS) mediates C2-ceramideinduced cAMP pathway uncoupling. This uncoupling did not involve ATP supply or Gas protein function but rather adenylate cyclase function per se. Further, the tyrosine phophatases inhibitors, but not the serine/threonine phosphatases inhibitors, prevent inhibition of cAMP production by ROS. This suggests that H2O2 requires a functional tyrosine phopsphatase(s) to block PACAP-dependent cAMP production. Keywords: ATP, cAMP, C2-ceramide, PACAP, reactive oxygen species, tyrosine phosphatase. J. Neurochem. (2001) 76, 778±788.

Ceramide has emerged recently as an important mediator of several agents that affect cell growth, viability and differentiation (Jayadev et al. 1995; Hannun 1996; Prinetti et al. 1997). This lipid second messenger is the breakdown products of membrane sphingomyelins; a reaction catalysed by acidic or neutral sphingomyelinases. Agonists of the sphingomyelin-ceramide pathway include membrane receptors ligands such as tumour necrosis factor a (TNFa) (Kim et al. 1991; Dressler et al. 1992), interleukin-1b (Ballou et al. 1992; Mathias and Kolesnick 1993), nerve growth factor (NGF)/p75 (Ito and Horigome 1995; Casaccia et al. 1996), as well as stress-inducing agents including ultra-violet (UV), ionizing radiations or hydrogen peroxide (H2O2) (Verheij et al. 1996) (for review see Hannun 1996). Activation of these pathways can be mimicked by direct treatment with cell permeant ceramides such as the C2-ceramide. Depending on the cell type, this compound has been shown to exert a wide range of biological effects, including mitogenic signalling, survival promotion, growth inhibition and apoptosis. In neurones, there is now strong evidence of ceramide-induced neuronal apoptosis (Centeno

et al. 1998; Brann et al. 1999; Yu et al. 1999; Craighead et al. 2000). Such a multiplicity of biological activities suggests that ceramide recruits several down-stream targets, which in turn activate distinct intracellular pathways. These targets include a Mg21-dependent protein kinase termed ceramide-activated protein kinase (CAPK) (Mathias et al. 1993), and a cytosolic protein phosphatase termed ceramideactivated protein phosphatase (CAPP) (Dobrowsky and Hannun 1992). Ceramide has also been described to activate

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Received June 5, 2000; revised manuscript received September 6, 2000; accepted September 11, 2000. Address correspondence and reprint requests to J. P. Loef¯er, UniversiteÂ Louis Pasteur, UMR 7519 CNRS 21, rue ReneÂ Descartes, 67084 Strasbourg Cedex ± France. E-Mail: loef¯[email protected] Abbreviations used: BSA, bovine serum albumin; CAPK, ceramideactivated protein kinase; CAPP, ceramide-activated protein phosphatase; DMEM, Dulbecco's modi®ed Eagle's medium; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NGF, nerve growth factor; PACAP, pituitary adenylate cyclase activating polypeptide; ROS, reactive oxygen species; TNFa, tumour necrosis factor a; VIP, vasoactive intestinal peptide.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 778±788

Ceramides/ROS and cAMP signalling cross talk 779

stress-activated protein kinases (SAPK/JNK), the activation of which led to apoptosis (Westwick et al. 1995; Verheij et al. 1996; Jarvis et al. 1997). It is now well documented that some highly reactive molecules derived from oxygen (reactive oxygen species: ROS) are implicated in programmed cell death (reviewed by Jacobson 1996; Jabs 1999). The link between oxidative stress, ceramides and ROS has been extensively investigated. Interestingly, oxidative stress stimulates ceramide production (Verheij et al. 1996; Mansat-de Mas et al. 1999) and, in turn, ceramide stimulates the production of mitochondrial hydrogen peroxide (Quillet et al. 1997) and ROS (France et al. 1997; Garcia et al. 1997), suggesting the existence of a mutual up-regulation mechanism between the ROS and ceramide signalling pathway. Since pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic effects in primary neurones by activating the cAMP pathway (Kienlen-Campard et al. 1997), we were interested in analysing the possible interactions between ceramide/ROS and PACAP signalling pathways in neuronal cells. To this end, we used a cathecolaminergic neurone-like CATH.a cell line (Suri et al. 1993). We have previously demonstrated that these cells do possess PACAP type I receptors (PR1) coupled to both adenylate-cyclase and phospholipase C pathways (Muller et al. 1997). The PACAP gene encodes a PACAP precursor, which gives rise to biologically active PACAP with either 38 or 27 amino acid residues (PACAP 38 and PACAP 27). These peptides were originally isolated from ovine hypothalami and are members of the secretin/ glucagon/vasoactive intestinal peptide (VIP) family (reviewed by Arimura et al. 1994; Rawlings and Hezareh 1996). The effects of PACAPs are mediated through at least two types of receptors with multiple splice variants, which are mainly distinguished by their af®nity for VIP. Type I receptors have higher af®nities for PACAP than VIP, whereas type II (PR2) receptors bind PACAP and VIP with similar af®nities. Both forms of receptors stimulate adenylate cyclase activity (Spengler et al. 1993), whereas type I and some new isoform of type II, in addition, are also positively coupled to phospholipase C-b and phosphoinositides. The aim of this work is to understand the molecular links between the pro-apoptotic ceramide/ROS pathway and membrane receptors that promote survival through activation of the G protein transduction pathway. In this study we investigate the effects of both C2-ceramide and ROS on the PACAP-stimulated second messengers in CATH.a cells. We ®rst demonstrate that C2-ceramide and H2O2 (used to generate ROS) inhibit PACAP-stimulated cAMP production, but have no effect on PACAP-stimulated phosphoinositides breakdown. Neither ATP levels nor Gas protein levels seem to be involved in this selectively uncoupling mechanism. However, our results show that adenylate

cyclase activity takes part in this inhibition and that tyrosine phosphatases mediate ROS effects on cAMP production.

Materials and methods Materials PACAP 38 was from Bachem (Bachem Biochimie SARL, France). C2-Ceramide was from Biomol (Plymouth, PA, USA); vanadate and dephostatin from Calbiochem (La Jolla, CA, USA). 2,7-Dichloro¯uorescin was from Molecular Probes (Eugene, OR, USA). The ATP bioluminescence assay kit was purchased from Boerhinger (Mannheim, Germany). myo-[3H] Inositol (102 Ci/mmol) and Amprep (SAX,RPN 1908) minicolumns were from Amersham (Uppsala, Sweden). Other products and reagents were from Sigma (St Louis, MO, USA). Culture of CATH.a cells CATH.a cells were generously donated by D. M. Chikaraishi (Boston, MA, USA). Cells were seeded in 24-wells cluster plates for measurements of cAMP and ATP; and in 10-cm dishes for both western blot analysis and adenylate-cyclase assay. Cells were maintained in Dulbecco's modi®ed Eagle's medium (DMEM)/F12 supplemented with 10% fetal calf serum, 60 mg/mL penicillin and 100 mg/mL streptomycin, in a humidi®ed atmosphere of 5% CO2 in air for 1±2 days before experiments. Experiments were performed in serum-free medium supplemented with 0.1% fatty acid-free bovine serum albumin (BSA). Colorimetric MTT assay Cells were cultured in 96-well culture dishes (Costar) and treated with C2-ceramide. A modi®ed procedure of the original method (Mossmann 1983) was used to measure mitochondrial activity (MTT assay). Brie¯y, at the end of C2-ceramide treatment, cultures were incubated for 1 h at 378C with freshly prepared culture medium containing 0.5 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide; Sigma). Medium was then removed and dark blue crystals formed during reaction were dissolved by adding 100 mL/well of 0.04 N HCl in isopropanol. Plates were stirred at room temperature to ensure that all crystals were dissolved and read on a Metertech S960 micro-ELISA platereader, using a test wavelength of 490 nm and a non-speci®c wavelength of 650 nm for background absorbency. Results are given as a percentage of survival, taking culture without ceramide treatment as 100%. Hoechst staining Condensed and fragmented nuclei were evaluated in situ in the cells (Brugg et al. 1996), by intercalation into nuclear DNA of the ¯uorescent probe bisbenzimide: Hoechst 33342 (Sigma, St Louis, MO, USA). Brie¯y, after ®xation with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, cells were incubated with the Hoechst dye 33342 at 1 mg/mL for 45 min at room temperature. Hoechst is visualized with AMCA ®lter (excitation 350 nm, emission 450 nm), is cell-permeant and labels both intact and apoptotic nuclei. Apoptosis was observed as small, brightstaining nuclei, often very rounded and usually fragmented into distinct sections.


780 V. SeÂe et al.

Measurement of cyclic AMP production CATH.a cells were pretreated or not with either C2-ceramide or H2O2 and then stimulated with 1029 M PACAP 38 for 15 min. After completion of the incubation period, the reaction was arrested by addition of 1 volume of ice-cold 0.2 m HCl. After a freeze-thaw cycle, cells were further disrupted by sonication and the suspensions were spun at 10 000 g for 10 min. The resulting supernatants were stored at 2 208C for measurement of cyclic AMP by radioimmunoassay (Koch and Lutz 1992). Measurement of inositol phosphate accumulation After 2 days in culture, CATH.a cells were cultured for 2 additional days in the presence of myo-[3H] inositol (4 mCi/mL) in myo-inositol-free DMEM/F12 culture medium supplemented with 2% fetal calf serum. After being pretreated with C2-ceramide or H2O2, cells were washed and incubated for 10 min in 10 mm LiCl in HEPES buffer composed of 150 mm NaCl, 5 mm KCl, 0.8 mm MgSO4, 1 mm CaCl2, 5 mm HEPES, 5.5 mm glucose and 0.1% BSA, at pH 7.4. They were then exposed to 1028 M PACAP 38 for 20 min in the same medium. At the completion of the incubation period, cells were recovered in ice-cold 5% perchloric acid and homogenized. After centrifugation of the homogenate at 10 000 g for 15 min, the supernatant was recovered and neutralized with 10 mm KOH. The clear supernatant obtained after a ®nal centrifugation was applied to Dowex AG-1 mini-columns. Columns were washed with water to remove free [3H] inositol, and glycerophosphoinositol was washed out with a mixture of 60 mm ammonium formate and 5 mm sodium tetraborate. Inositol monophosphate (Ins P1), Inositol diphosphate (Ins P2) and Inositol triphosphate (Ins P3) were then eluted by means of a stepwise gradient of 0.1 m formic acid in 0.7 m ammonium formate (Berridge et al. 1982). Western blot analysis Cells cultured in 10-cm dishes were washed with PBS, harvested by scraping and homogenized with 20 strokes of a Dounce homogenizer (type B) in 5 mm HEPES, pH 8, containing 1 mm EDTA and protease inhibitors (0.5 mm dithiotreitol, 0.5 mm phenylmethylsulphonyl¯uoride, 2 mg/mL leupeptin). The homogenate was spun at 700 g for 5 min at 48C to remove the nuclei; the supernatant containing the cytosolic and the membrane fraction was collected. The protein concentration was measured by the Bradford assay (Biorad, Hercules, CA, USA) then diluted twice in sample buffer 2X (125 mm Tris±HCl pH 6.8, 20% glycerol, 2% sodium dodecyl sulphate (SDS), 2% b-mercaptoethanol, 0.2% bromophenol blue) and boiled (5 min). One hundred micrograms of protein were loaded on a 10% SDS-acrylamide gel. Proteins were blotted onto a pure nitrocellulose membrane (Biorad 0.45 mm). Unspeci®c labelling was blocked in 50 mm Tris±HCl pH 7.4, 150 mm NaCl and 0.05% Tween-20 supplemented with 5% non-fat dry milk, for 1 h and membranes were incubated overnight at 48C with the rabbit antiserum against G protein (Ohlmann et al. 1995) or against actin diluted in 50 mm Tris±HCl pH 8, 150 mm NaCl, 0.05% Tween-20 and 3% milk. Antisera against Gas (AS 348) Gaq (AS 369) and Gai (AS266) were generously donated by Dr NuÈrnberg (InstituÈt for Pharmakologie, Berlin, Germany) (Offermanns et al. 1994; Ohlmann et al. 1995). Monoclonal antibody against actin was a generous gift of Dr Ciesielski-Treska (Strasbourg, France) (Goetschy et al. 1987). After three washes,

membranes were incubated for 2 h at room temperature with 1/2000 dilution of antirabbit IgG, HRP-conjugated (Interchim) or with a 1/2000 dilution of anti-mouse, HRP-conjugated (Amersham), followed by three additional washes and speci®c bands were then detected by ECL. Blots were exposed for 1 min to BIOMAXMR KODAK ®lms. They were further quanti®ed with the Molecular Analyst software (Biorad).

Adenylate cyclase activity CATH.a cells were cultured to near con¯uency in 10 cm dishes. Following treatments with C2-ceramide or H2O2, cells were washed in PBS and homogenized in ice-cold 20 mm Tris±HCl buffer (pH 7.4), containing 5 mm MgCl2, 1 mm EGTA and 0.01% bacitracin, using a Dounce homogenizer. The homogenate was then spun at 37 000 g for 10 min and the resulting crude membrane fraction was resuspended in the same buffer. The adenylate cyclase activity was assayed in 30 ml aliquots of membrane fractions (corresponding to 30±50 mg proteins) in a ®nal volume of 80 ml of the precedent Tris±HCl buffer supplemented with 0.25% BSA, 1 mm adenosine 5 0 -triphosphate, 5 mm phosphocreatine, 0.5 mg/mL creatine phosphokinase. Ten microliters of PACAP 38 (1028 M) or of forskolin (5.1025 M) were then added to initiate the reaction, after 10 min, the reaction was stopped by the addition of 10 mL HCl and the sample were then spun at 14 500 r.p.m. for 5 min. The resulting supernatants were frozen at 2 208C until measurement of cAMP content.

Measurement of ROS production Reactive oxygen species were detected with 2 0 ,7 0 -dichlorodihydro¯uoresceine diacetate (H2DCFDA, Molecular Probes, Eugene, OR, USA), which produces a green ¯uorescence when oxidized (Schwarz et al. 1994). Cells were loaded 30 min at 37 8C with 10 mm DCFDA and rinsed with fresh culture medium. They were then treated for indicated periods of time with C2-ceramide in presence or not of lipoic-acid. Cells were rinsed twice with PBS prior to sonication. Fluorescence (excitation 486 nm/emission 534 nm) was measured in a Perkin Elmer HTS7000 microplate ¯uorimeter (Foster City, CA, USA).

ATP measurement Cells grown in a 24-wells cluster plates were treated with ceramide or H2O2, washed in PBS and harvested by scraping. ATP levels were assessed according to manufacturer's instructions with a kit purchased from Boehringer (Mannheim, Germany). The measurement is based on the reaction of ATP with luciferine that leads to luciferase and chimioluminescence production. Light emission was measured with a Tropix luminometer.

Statistics Statistical signi®cance of data was assessed by means of analysis of variance (one-way anova), followed by the Dunnet test for comparisons of all values versus control using the Graphpad's in Stat2 software. The half-maximum value (EC50) for dose±response curve was calculated with the Graphpad Prism software (San Diego, CA, USA).



Fig. 1 C2-ceramide-induced apoptosis of Cath.a cells. (a) Time course of C2-ceramide-induced cell death. Cath.a cells were treated with 50 mM C2-ceramide for the indicated period of time. Cell survival was assessed by MTT assay. The hatched bar represents a 24-h treatment with an inactive form of C2-ceramide (50 mM). Results are mean ^ SEM of eight independent values. Each experiment was performed at least three times. *Indicates a statistical difference with p , 0.05 compared to control. (b) C2-ceramide induces nuclear condensation and fragmentation. Apoptotic cells were monitored by chromatin condensation using Hoechst 33342 (1 mg/mL). (a) Nuclei of Cath.a cells without any treatment; (b) nuclei of Cath.a cells treated with 50 mM of C2-ceramide during 16 h.

Results C2-ceramide selectively inhibits PACAP-stimulated cAMP production The second messenger ceramide activates numerous cellular responses. In our experimental model of Cath.a cells, we ®rst show that 50 mm of the cell permeant C2-ceramide induces cell death. Fig. 1(a) shows that after 12 h of C2-ceramide treatment, cell survival progressively declines. In contrast the inactive ceramide analogue which lacks the trans double bound at C4±5 of the sphingoid base backbone

(C2-dihydro ceramide) is ineffective at 24 h. This cell death presents apoptotic features as shown on Fig. 1(b), where nuclei from C2-ceramide treated cells appear condensed and fragmented. Cell death is only detectable after a 12-h period of treatment. Consequently, all signal transduction studies were performed at earlier time points. To test whether C2-ceramide modulates PACAP signalling pathways induced through PRI, both cAMP formation and phosphoinositides (PI) breakdown were measured in CATH.a cells. We show that C2-ceramide (50 mm) strongly inhibits PACAP-stimulated cAMP accumulation in a timedependent manner (Fig. 2a), with a maximum inhibition of 70% at 12 h of ceramide treatment. The basal levels of cAMP, in the absence of PACAP, are constant whatever the ceramide treatment. Under similar experimental conditions, PACAP-induced inositols phosphate production was unaffected by a ceramide treatment (Fig. 2b), indicating a selective effect of C2-ceramide on the cAMP signalling pathway. The ceramide effects on cyclic nucleotide production is dose-dependent with an IC50 around 40 mm after 12 h of ceramide pretreatment (Fig. 2c). Fig. 2(c) also shows that the inactive ceramide analogue is ineffective at 100 mm. To investigate whether the C2-ceramide effects on modulating cAMP levels impinge on cAMP production, experiments with C2-ceramide were performed in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). As shown in Fig. 2(d), 0.5 mm IBMX signi®cantly increased the cAMP levels evoked by PACAP, but did not abolish the inhibitory effect of C2-ceramide pretreatment. This suggests that C2-ceramide acts at the level of cAMP production, rather than on cAMP breakdown. C2-ceramide modulates the cAMP transduction pathway by generating ROS Ceramide has been shown to generate ROS in various cell types (France et al. 1997; Garcia et al. 1997). To test whether this occurs in CATH.a cells, cells were treated with C2-ceramide and ROS production was measured with the ¯uorescent dye (2 0 -7 0 dichloro¯uoresein). Fig. 3(a) shows a signi®cant, sixfold increase of ¯uorescence after 6 h of ceramide treatment. This increase of ¯uorescence, indicative of ROS production, is maximal at 8 h of C2-ceramide treatment (eightfold increase). This increased ROS production is inhibited by the ROS scavenger, lipoic acid. Assuming that ROS is an effector by which C2-ceramide modulates cAMP production, the ceramide effect should be reversed by a potent ROS scavenger. Fig. 3(b) shows that when cells were pretreated with the ROS scavenger, lipoic acid, it inhibits 60% of the effects of C2 on the cAMP production. This suggests that C2 modulates the PR1/cAMP transduction pathway mainly via ROS. Furthermore, ROS generated by a 15-min H2O2 treatment (0.25 mm) appear to mimic the effects of 50 mm C2-ceramide on signal transduction (Fig. 3c and d). Indeed, H2O2 signi®cantly


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Fig. 2 Effects of C2-ceramide on PACAP-stimulated transduction pathways. (a) Time-course of PACAP-stimulated cAMP inhibition by C2-ceramide. Cath.a cells were pretreated (W) or not (X) with 50 mM C2-ceramide for the indicated periods of time, and then stimulated with 1029 M PACAP 38 for 15 min. Dotted line (K) represents basal levels of cAMP without any PACAP stimulation. cAMP concentrations were measured by radioimmunoassay as described under èxperimental procedures'. (b) C2-ceramide does not affect PACAPstimulated PIs breakdown. Cath.a cells were pretreated (solid bars) or not (open bars) with 50 mM C2-ceramide for 4, 8 or 16 h as indicated, and stimulated with 1028 M PACAP 38 during 20 min in the presence of 10 mM LiCl to inhibit inositol-1-P degradation. The hatched bar represents basal levels of PIs breakdown, without any

PACAP stimulation. (c) Dose±response of C2-ceramide treatment on cAMP levels. Cells are pretreated during 12 h with C2-ceramide (X) at indicated concentrations, and then stimulated with 1029 M PACAP 38 during 15 min. An inactive form of C2-ceramide (B) is used as control (50 mM). (d) In¯uence of a phosphodiesterase inhibitor on the cAMP response. Cells were pretreated (solid bars) or not (open bars) with 50 mM C2-ceramide, and then treated or not with 0.5 mM IBMX as indicated before PACAP 38 stimulation (1029 M, 15 min). The hatched bar represents basal levels of cAMP, without any PACAP stimulation. Results are mean ^ SEM of quadruplicate values. Each experiment was performed at least three times. **Indicates a statistical difference with p , 0.01 compared to control (ct).

inhibits the PACAP-induced cAMP production in a dosedependent manner (Fig. 3c) but leaves PI breakdown unaffected (Fig. 3d). Further, as already shown with C2ceramide, pretreatment with 0.5 mm IBMX does not abrogate the inhibitory effect of H2O2 (Fig. 3c, insert). Taken together, these data (Figs 2 and 3) suggest that C2-ceramide selectively uncouples the PACAP-induced cAMP signalling pathway by generating ROS.

on the transduction process, adenylate cyclase activity was assessed ìn vitro' on isolated membranes from cells that had been pretreated with C2-ceramide or H2O2. As shown in Fig. 4(b), 15 min of H2O2 treatment signi®cantly inhibits 50% of the adenylate cyclase activity, whatever the type of stimulation (PACAP or forskolin). When adenylate cyclase was stimulated by PACAP, C2-ceramide initially increases its activity and then inhibits it at later time point (Fig. 4c). When adenylate cyclase is activated by forskolin, the initial activity was not increased by the C2-ceramide treatment. Adenylate cyclase inhibition by C2-ceramide was observed at a 8-h C2-ceramide treatment (Fig. 4c). One mean to increase adenylate cyclase function could be speci®c changes in G-protein levels. To test whether such changes do occur, levels of speci®c G-proteins were measured by western-blot in C2-ceramide treated cells. As shown in Fig. 4(d), levels of Gas progressively increased with C2 treatment (8 h, 50 mm); in contrast, no signi®cant changes

C2-Ceramide and ROS act downstream of the PR1/G-protein/adenylate cyclase transduction complex To test whether C2-ceramide and ROS act directly on the PR1 or further downstream of the receptor, we analysed their effects when adenylate cyclase was directly activated by forskolin. As shown on Fig. 4(a), both C2-ceramide and H2O2 strongly blunt the response to forskolin, suggesting an intracellular effect downstream of the PACAP receptor. To further investigate the level of C2-ceramide and ROS action



Fig. 3 Oxidative stress mimics C2-ceramide effect on PACAP signal transduction. (a) C2-ceramide induces ROS production. Cath.a cells were loaded for 30 min with the ¯uorescent dye H2-DCFDA and then treated for the indicated periods of time with 50 mM C2-ceramide in the presence (W) or in absence (X) of 1026 M of lipoic acid. (b) Effects of 1026 M of lipoic acid on cAMP level inhibition induced by C2-ceramide. Cells were treated 10 h with 50 mM C2-ceramide (black bars) in the presence or in absence of 1026 M of lipoic acid, and stimulated with 1029 M PACAP 38 for 15 min. The hatched bar represents basal levels of cAMP, without any PACAP stimulation. (c) Effects of H2O2 on PACAP-induced cAMP levels. Cells were pretreated (solid bars) or not (open bar) for 15 min with H2O2 at

increasing concentrations, and exposed to 1029 M PACAP 38 for 15 min. The hatched bar represents basal levels without any PACAP stimulation. Insert: cells were pretreated (solid bars) or not (open bars) with 0.25 mM H2O2, and treated or not with 0.5 mM IBMX as indicated before PACAP 38 stimulation (1029 M, 15 min). (d) Effects of H2O2 on PACAP-induced PIs breakdown. Cells were pretreated (solid bars) or not (open bar) for 15 min with H2O2 at various concentrations, and with 1028 M PACAP 38 for 20 min. The hatched bar represents basal levels of PIs breakdown, without any PACAP stimulation. Results are mean ^ SEM of quadruplicate values. Each experiment was performed at least three times. *p , 0.05; **p , 0.01, ***p , 0.001 versus control.

are observed in the levels of Gaq and Gai. a-Actin is used as an internal control for gel loading. However several reasons argue against a major contribution of changes in G-protein levels as a mechanism by which C2-ceramide modulates cAMP production. An increase in Gas would be expected to increase cAMP production rather than decrease it as observed here (see Fig. 2). Moreover, the variations in Gai levels appear too weak to have any signi®cant contribution. This interpretation is further strengthened by control experiments that revealed that pertussis toxin treatment (an irreversible inhibitor of Gai and Gao) did not modify the inhibitory effect of both C2-ceramide and H2O2 (data not shown). Finally if ROS represent the major effector of C2-ceramide, as suggested by our experiments, their rapid effects (15 min) can not correlate

with any changes in G-proteins levels (not shown). Although the increase in Gas content after 8 h of C2 treatment are compatible with the increase of adenylate cyclase activity observed in isolated membranes, they can not account for the inhibition of cAMP production observed in whole cells. These results suggest the presence of a compensation mechanism that could take place during the time of ceramide treatment (4±8 h). Indeed, cells seem to counteract the C2-ceramide effect on cAMP production by increasing Gas, which itself increase the adenylate cyclase activity. This will be detected on isolated membranes. However, in the whole cell, the compensation mechanism will be overriden, and the diminution of cAMP levels is predominant. This suggests a major contribution of an intracellular signal, not present in the ìn vitro' assay, which


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Fig. 4 Effects of C2-ceramide and H2O2 on G-proteins and adenylate cyclase activity. (a) Effects of C2-ceramide and H2O2 on forskolin-stimulated cAMP levels. Cath.a cells were pretreated with 50 mM C2-ceramide for 12 h, or with 0.25 mM H2O2 for 15 min and stimulated with 50 mM forskolin during 15 min. Open bars represent cAMP levels without any pretreatment. (b,c) Effects of H2O2 (b) and C2-ceramide (c) on ìn vitro' adenylate cyclase activity. Cells were pretreated with either 0.25 mM H2O2 during 15 min or with 50 mM C2-ceramide for 4 h, 8 h and 15 h as indicated. Open bars represent the control adenylate-cyclase activity with PACAP or forskolin stimulation only, relative to non-treated cells (hatched bars). Membranes were collected and adenylate cyclase activity was assessed by cAMP measurement after PACAP 38 (1029 M) or forskolin (5 mM) stimulation. *p , 0.05, **p , 0.01 versus control (ct). (d) Westernblot of Gas, Gaq, Gai and actin in cells pretreated or not (ct) with 50 mM C2-ceramide for 4 or 8 h. Numbers below each band represent relative changes (ct 1), as quanti®ed by Biorad image analysis software (molecular analyst).

participates at inhibiting the adenylate cyclase in the intact cells. To verify that the ceramide/ROS effects on cAMP production are not due to ATP depletion, we monitored ATP levels in the cells. Fig. 5 shows that a treatment of

cells with 50 mm C2 or 0.5 mm H2O2 for up to 12 h and 15 min, respectively, does not alter signi®cantly ATP levels. This suggests that the uncoupling of the cAMP pathway by ceramides or ROS does not involve ATP stocks depletion.

Fig. 5 ATP levels are not affected by C2-ceramide or H2O2 treatment. Cath.a cells were treated with 50 mM C2-ceramide at indicated times (solid bars) or with 0.25 mM H2O2 for 15 min (hatched bar); the open bar represents the control without any treatment. Cells were collected in PBS and the intracellular levels of ATP were measured with the kit purchased from Boehringer. Results are mean ^ SEM of quadruplicate values. Each experiment was performed at least twice.

Tyrosine phosphatases are involved in the uncoupling of the PACAP-induced cAMP pathway To further investigate the mechanisms by which ROS uncouple the cAMP pathway from PACAP stimulation, we checked whether H2O2 induces protein phosphorylation modi®cation. We therefore tested the effects of several protein phosphatase inhibitors on H2O2 treatment. Fig. 6(a) shows that calyculin, an inhibitor of the serine/threonine phosphatases PP2A and PP1, as well as okadaic acid (Fig. 6b) even at a high dose (1027 M), do not signi®cantly affect H2O2 inhibition of cAMP production. These results suggest that the state of serine/threonine phosphorylation is not obviously implicated in ROS effects on the cAMP production. In contrast, Fig. 6(c) shows that 1026 M or 1027 M of the tyrosine phosphatase inhibitor dephostatin reduces the effects of H2O2 on PACAP-induced cAMP production by 80%. Vanadate (1023 M) another tyrosine phosphatase inhibitor also completely reversed H2O2 effects (Fig. 6d). These results suggest that H2O2 selectively uncouples the cAMP pathway by recruiting a tyrosine phosphatase(s).



Fig. 6 Impairment of cAMP production by H2O2 is mediated by tyrosine phosphatases. In all experiments, CATH.a cells, maintained in F12 medium supplemented with bovine serum albumin, were treated for 15 min with phosphatases inhibitors at the indicated concentration (a: calyculin, b: okadaic acid, c: sodium orthovanadate added up with 0.1 M H2O2 to catalyse its transformation into pervanadate and d: dephostatin). Cells were then treated (black bars) or not (empty bars) with 0.25 mM H2O2 during 15 min before stimulation with 1029 M PACAP 38 during 15 additional minutes. Hatched bars represent basal levels of cAMP (no PACAP treatment). Histograms represent means ^ SEM of quadruplicate values. Each experiment was performed at least twice. **Indicates statistical differences (p , 0.01) compared to controls without phosphatase inhibitors.

C2-ceramide effects develop slowly (. 8 h). As all antagonists used here induce cell death upon earlier periods of time (3±4 h), treatment with C2-ceramide could not be tested. Since we have shown, that C2-ceramide blocks cAMP production through a ROS-dependent mechanism, it is likely that the effect of C2-ceramide also rests on the recruitment of tyrosine phosphatases. Discussion In a physiological setting, the functioning and fate of each individual component must be tightly correlated to the activities of its neighbours. In the particular case of a highly specialized neuronal network, two signal pathways of prime importance affecting neuronal survival or demize will be cAMP and Ca21. The integration of the information from these two pathways will bring together signalling from many inputs including much synaptic and growth factors activity. Indeed the consequences of modulating each of these inputs on neuronal survival is well documented for a number of neuronal types (Walton et al. 1996; Obrietan and van den Pol 1997; Tanaka et al. 1997). In the series of experiments reported here we analysed the consequence of ceramide activation on the PACAP signalling pathway. This was of interest for two main reasons. First, the neuroprotective effect of PACAP through activation of this receptor exerts neurotrophic and neuroprotective effects in a variety of neuronal cell types (Arimura et al. 1994; Morio et al. 1996; Tanaka et al. 1996; Villalba et al. 1997). In addition, several

experimental data suggest that these neuroprotective effects are primarily mediated by the cAMP-dependent signalling pathway (Kienlen-Campard et al. 1997). and second, ceramide, a well-documented second messenger that mediates the biological activity of several cell death promoting receptors (e.g. TNFa, Mathias et al. 1991) interferes with PACAP-dependent signalling. Here we used Cath.a cells, to test whether C2-ceramide may interfere with the neuroprotective signalling initiated by PACAP. This cell line was generated from locus coeruleus neurones by targeted expression of the SV40 large antigen (Suri et al. 1993) and the cells express the PACAP receptor type 1 that transduces intracellular signals through both the cAMP and IP signalling pathways (Muller et al. 1997). The major ®nding of this study is that C2-ceramide selectively blocks PACAP receptor-mediated cAMP production, without impairing PI breakdown. This result clearly shows the speci®city of the C2-ceramide effects on the neuroprotective cAMP-signalling pathway. Indeed, since at time points up to 16 h, where cAMP production is severely blunted, we still observe the same PI breakdown in response to PACAP indicating that the drop in cAMP response is not due to cell death, and that cells can still transduce intracellular signals. Ceramides do induce cell death through apoptosis in the CATH.a cell line (see Fig. 1b). However, a measurable decrease in mitochondrial activity, as followed by the MTT assay, is only observed after 16 h of treatment (Fig. 1a). Thus, uncoupling of the cAMP pathway represents an early step in apoptosis in this cell line. Our hypothesis is that the


786 V. SeÂe et al.

blockade of the cAMP pathway by ceramides has important physiological implications. According to Kolesnick and Hannun (1999), ceramide functions as a signal transducer in a generalized stress-response pathway. The result presented here show that one of such pathway is the suppression of survival signals mediated by cAMP. A similar mechanism of C2-ceramide-induced neuroprotective pathway blockade has also been reported in PC12 cells by Salinas et al. (2000). They show that C2-ceramide inhibits the neuroprotective PKB/Akt pathway. The cellular mechanisms by which C2-ceramide inhibits cAMP production needed to be elucidated. A likely candidate for relaying the ceramide signal is ROS and more especially H2O2 (Garcia et al. 1997). Our data show that ceramide does produce ROS. This is demonstrated here by the use of redox-sensitive ¯uorescent dye (Fig. 3a), and was in line with data reported for several other cell types (France et al. 1997; Lambeng et al. 1999). Second, ROS scavenging with the antioxidant compound lipoic acid blocks both ROS production (Fig. 3a) by C2-ceramide and their inhibitory effects on cAMP production (Fig. 3b). Third, direct chemical production of ROS with H2O2 mimics the inhibition of cAMP production, and like the action of C2-ceramide, is without effect on IP breakdown. However, in contrast to C2-ceramide that needs several hours to develop its biological effects, H2O2 blocks cAMP production rapidly. Maximal effects are seen within minutes, and this leads us to suggest that ROS operate at a later stage of the ceramide-signalling cascade. A major issue in the deciphering of the mechanism by which C2-ceramide and ROS decrease cAMP levels in response to PACAP, is the level at which these two signalling pathways cross-talk: cAMP production or cAMP breakdown. The fact that both C2-ceramide and H2O2 remain ef®cient inhibitors under experimental conditions where cAMP degradation is essentially suppressed by the phosphodiesterase inhibitor IBMX suggest that C2-ceramide and H2O2 inhibit cAMP production rather than increase its rates of degradation. Thus C2-ceramide and ROS appear to operate primarily at the level of the cell membrane on the functioning of the PACAP receptor/adenylate cyclase transduction system. It is unlikely that the receptor itself is profoundly altered (e.g. number of available receptors), since, as discussed above, the IP response to PACAP remains constant. However, we cannot exclude subtle modi®cation that would alter the coupling to Gas but not Gaq. A strong argument for adenylate cyclase being the main target of these agents is the observation that cAMP stimulation by forskolin, a direct activator of adenylate cyclase, is also blunted by C2-ceramide and H2O2 (Fig. 4a). However, when the same agents are tested on isolated membranes from pretreated cells, the mechanisms that come into play appear more complex. In this model the effects of ceramides and H2O2 are clearly different. On membranes, a short pretreatment

with H2O2 (15 min) inhibits the PACAP and the forskolininduced cAMP response, i.e. decrease adenylate cyclase activity, and this effect may account for the result seen in whole cells. Surprisingly, C2-ceramide effects on PACAPstimulated adenylate cyclase activity develop slowly over time and result in a clear-cut stimulation of adenylate cyclase activity. Such an induction of adenylate cyclase activity by ceramide has also been reported by Bosel (Bosel and Pfeuffer 1998). We have shown here that C2-ceramide treatment produces a gradual increase in membrane Gas content, Gaq or Gai staying more or less constant. This increase in Gas could well account for the stimulation of adenylate cyclase activity in isolated membranes. This interpretation is further in line with the ®nding that forskolin-stimulated adenylate cyclase activity (a direct effect on adenylate cyclase that bypasses G proteins) is not increased by C2-ceramide treatment (Fig. 4c). Thus, during the build up of the ceramide response, neurones appear to recruit compensatory mechanisms that blunt and override the direct inhibitory mechanisms of ROS, even in isolated membranes. Most importantly, this set of data shows that an intracellular component, probably partially lost during membrane puri®cation, does contribute to the ROS/ceramide-dependent adenylate cyclase inhibition in whole cells. Our results exclude the most trivial possibility: depletion of the adenylate cyclase substrate, ATP. This ®nding is consistent with a previous report (France et al. 1997) showing that C2-ceramide treatment in PC12 cells does not alter ATP concentrations until cells actually die after 24 h of ceramide treatment. We next addressed the problem of phosphatase activity, as some reports have produced apparently con¯icting results. Ceramides have been shown to activate a ceramideactivated protein phosphatase (Wolff et al. 1994; Prinetti et al. 1997), whereas ROS have been shown to inhibit protein phosphatases (Sullivan et al. 1994; Robinson et al. 1999). These events could alter the state of phosphorylation and subsequent transduction properties of the PR1/Gas/ adenylate cyclase complex. Our results show that tyrosine, but not serine/threonine phosphatase inhibitors are able to prevent the inhibitory effects of ROS on PACAP-dependent cAMP production, indicating that at one point of the regulatory cascade, ROS recruit a tyrosine phosphatase to inhibit the adenylate cyclase coupled PACAP transduction system. These phosphatases might represent the ®nal effector, since phosphatases have been shown to control membrane located transduction units. For example, tyrosine phosphorylation of Gas enhances Gas coupling with adenylate cyclase (Poppleton et al. 1996). One could thus speculate that activation of tyrosine phosphatase by ceramides and ROS will speci®cally decrease the af®nity of Gas for adenylate cyclase by reducing the state of Gas phophorylation, and thereby produce an inhibition of adenylate cyclase activity. Although such data are not yet



available for the PACAP receptor, such a mechanism may also represent, at least in part, one molecular basis by which C2-ceramide and ROS control PACAP receptors. Further, since direct activation of cAMP production by forskolin is also strongly modulated by C2-ceramide and H2O2, it is conceivable that adenylate cyclase activity is directly regulated by phosphorylation mechanisms. In summary, our data show a novel mechanism by which ceramide and ROS selectively uncouple the cAMP signalling pathway, within a transduction unit that operates through both adenylate cyclase and phospholipase C. Further this study favours a model where C2-ceramide, by altering the cellular redox state ultimately recruits a tyrosine phosphatase(s) to exert its biological effects. Such a mechanism may have important biological functions since blockade of the cAMP pathway will reinforce the proapoptotic properties of ceramide and receptors that signal though this second messenger. Acknowledgements The technical assistance of F. Herzog, L. Le Personic and C. Nelson are acknowledged. We are grateful to Dr NuÈrnberg (Berlin, Germany), for the generous gift of Ga proteins directed antibodies. We also thank Dr Ciesielki-Treska (Strasbourg, France), for the generous gift of anti-actin antibody. This work was supported by the Àssociation pour la recherche contre le cancer', ARC (n89821).

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