C2-ceramide and reactive oxygen species inhibit pituitary adenylate cyclase activating polypeptide...

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-ceramide-

induced 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 phospha-

tases inhibitors, prevent inhibition of cAMP production by

ROS. This suggests that H2O2 requires a functional tyrosine

phopsphatase(s) to block PACAP-dependent cAMP produc-

tion.

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 ceramide-

activated protein phosphatase (CAPP) (Dobrowsky and

Hannun 1992). Ceramide has also been described to activate

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

Received June 5, 2000; revised manuscript received September 6, 2000;

accepted September 11, 2000.

Address correspondence and reprint requests to J. P. Loef¯er, Uni-

versiteÂ 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, ceramide-

activated protein kinase; CAPP, ceramide-activated protein phosphatase;

DMEM, Dulbecco's modi®ed Eagle's medium; MTT, (3-[4,5-dimethyl-

thiazol-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.

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 pro-

grammed 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 hydro-

gen 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 activa-

tion 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 produc-

tion, but have no effect on PACAP-stimulated phospho-

inositides 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 streptomy-

cin, 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, bright-

staining nuclei, often very rounded and usually fragmented into

distinct sections.

Ceramides/ROS and cAMP signalling cross talk 779

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

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 suspen-

sions 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 mono-

phosphate (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 homo-

genate 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 (Amer-

sham), followed by three additional washes and speci®c bands were

then detected by ECL. Blots were exposed for 1 min to BIOMAX-

MR 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 measure-

ment 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).

780 V. SeÂe et al.


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 signal-

ling 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 time-

dependent 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 con-

ditions, 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 pro-

duction 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 isobutylmethyl-

xanthine (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 produc-

tion 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

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 con-

densation 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.



inhibits the PACAP-induced cAMP production in a dose-

dependent manner (Fig. 3c) but leaves PI breakdown

unaffected (Fig. 3d). Further, as already shown with C2-

ceramide, 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.

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

on the transduction process, adenylate cyclase activity was

assessed `in 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

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 concentra-

tions were measured by radioimmunoassay as described under

`experimental procedures'. (b) C2-ceramide does not affect PACAP-

stimulated 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 indi-

cated, 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 inhibi-

tor 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).

782 V. SeÂe et al.


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 con-

tribution. 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 counter-

act 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 `in vitro' assay, which

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 pre-

treated (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.



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.

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. 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 `in vitro'

adenylate cyclase activity. Cells were pre-

treated 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 repre-

sent 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) Western-

blot of Gas, Gaq, Gai and actin in cells pre-

treated 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 soft-

ware (molecular analyst).

Fig. 5 ATP levels are not affected by C2-ceramide or H2O2

treatment. Cath.a cells were treated with 50 mM C2-ceramide at indi-

cated 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.

784 V. SeÂe et al.


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

Fig. 6 Impairment of cAMP production by

H2O2 is mediated by tyrosine phospha-

tases. 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 statisti-

cal differences (p , 0.01) compared to con-

trols without phosphatase inhibitors.



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 forskolin-

induced 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 PACAP-

stimulated 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/cera-

mide-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 ceramide-

activated 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

786 V. SeÂe et al.


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 signal-

ling 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 mech-

anism may have important biological functions since

blockade of the cAMP pathway will reinforce the proapop-

totic 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 `Association pour la recherche contre le

cancer', ARC (n89821).

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