- Email: [email protected]
Characteristics of dual specificity phosphatases mRNA regulation by 3,4-methylenedioxymethamphetamine acute treatment in mice striatum
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Characteristics of dual specificity phosphatases mRNA regulation by 3,4-methylenedioxymethamphetamine acute treatment in mice striatum Cynthia Marie-Claire, Nadia Benturquia, Ann Lundqvist, Cindie Courtin, Florence Noble⁎ Laboratoire de Neuropsychopharmacologie des addictions (UMR CNRS 7157, INSERM U705) Université Paris Descartes, Faculté de Pharmacie, Paris, France
A R T I C LE I N FO
AB S T R A C T
Article history:
3,4-methylenedioxymethamphetamine (MDMA) is a popular recreational drug that has
Accepted 18 August 2008
rewarding properties in rodents but little is known about its effects at the cellular and
Available online 28 August 2008
molecular levels. We have previously shown that the ERK pathway is important for the regulation in gene expression observed in mice striatum after acute treatment with MDMA.
Keywords:
Interestingly, three dual specificity phosphatases were found among the genes modulated
MDMA
by MDMA acute treatment. In this study we investigated the signalling pathways leading to
Gene expression
the up-regulation of these three mRNAs and the kinetics of their regulation. We found that
Dual specificity phosphatases
the increase in Dusp14 mRNA depends on the activation of ERK and lasts longer than those
Mice
of Dusp1 and Dusp5. The modulation of the three studied Dusps depends partially on the
Striatum
activation of D1 receptors but is independent of the activation of D2 receptors. These results suggest that at least two separate signalling cascades lead to the up-regulation of MAPK phosphatase mRNAs. The increase of Dusp1 and Dusp5 mRNAs is not controlled by ERK activation while that of Dusp14 is a direct negative-feedback mechanism of MDMA-induced ERK signalling. Both mechanisms converge to increase the expression levels of phosphatases able to inactivate ERK. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
MDMA (3-4-methylenedioxymethamphetamine) is the psychoactive compound of the widely abused drug ecstasy. The mechanism of action of MDMA is complex and not wellknown, most of its effects can be attributed to the increase of dopamine and serotonin observed and the subsequent interaction of the neurotransmitters with their respective pre- and post-synaptic receptors (review in Colado et al., 2004). MDMA inhibits the serotonin and dopamine transporters but also
displays a moderate affinity to a broad variety of receptors whose activation could be at the origin of certain effects of the drug (Battaglia et al., 1988). At the intracellular level we have shown, in mice, that ERK (Extracellular signal-Regulated Kinase) signaling is involved in several MDMA behavioral and transcriptional effects, by using a specific inhibitor of ERK activation, SL327 (Salzmann et al., 2003). ERK is an important regulator of neuronal functions, and is involved in various neurobiological events such as synaptic plasticity and memory (Sweatt, 2001). It has been suggested that ERK may play a
⁎ Corresponding author. Fax: +33 1 53 73 97 19. E-mail address: [email protected] (F. Noble). Abbreviations: PCR, polymerase chain reaction; DMSO, dimethyl sulfoxide; MDMA, 3,4-methylenedioxymethamphetamine; ERK, extracellular signal-regulated kinase 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.050
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role in the rewarding properties of several drugs of abuse (Valjent et al., 2004). In addition, it has been shown that D-amphetamine- and cocaine-activated ERK pathway was restricted within a subpopulation of dynorphin and D1 receptor-positive striatal medium-sized spiny neurons in mouse (Valjent et al., 2005). Moreover, Acquas et al. (2007) have shown that activated ERK may represent a post-synaptic correlate of the stimulant effect of MDMA on D1-dependent dopamine transmission in the ventral striatum of rat. Furthermore, it has been shown that D1 receptors play a key role in the acute MDMA-induced hyperlocomotion and that the activation of the ERK pathway is partially under D1 receptor control (Benturquia et al., 2008). Since MAPK signaling pathway is involved in numerous biological functions, dysregulation of this pathway can have widespread consequences on cellular functions. The specificity of the cellular effects induced by a stimulus depends on the magnitude and duration of the activation of the MAPKs (Ebisuya et al., 2005). One of the major negative regulatory mechanisms consists of the dephosphorylation of the activated protein by phosphatases. In mammalian cells, MAPK inactivation is controlled mainly by a family of phosphatases, the dual specificity (Thr/Tyr) phosphatases (Dusp) (Owens and Keyse, 2007). The Dusp family is subdivided into four groups (I–IV) according to their functional domains, mechanism of substrate recognition and enzymatic regulation (Farooq and Zhou, 2004). Using oligonucleotide arrays, we identified several genes regulated by acute administration of MDMA within mice dorsal striatum (Marie-Claire et al., 2007, 2008; Salzmann et al., 2006). Three dual specificity phosphatases were found among the 27 genes modulated: Dusp1, 5 and 14. Since the role of these phosphatases is to regulate MAPK activation, it's interesting that they were up-regulated by the same drug that could activate ERK. Of the three Dusps regulated by MDMA, one (Dusp14) belongs to subgroup I and its modulation was partially dependent on ERK activation while the two others (Dusp1 and 5) which belong to subgroup II did not depend on this pathway for MDMA-induced up-regulation (Salzmann et al., 2006). We therefore investigated the regulation of the three Dusp genes by MDMA using real time quantitative RT-PCR, in order to validate the modulation observed in the microarray study and the potential implication of the ERK pathway. A time course of the regulation of the Dusps in mice striatum after acute MDMA injection was performed. We also used D1 and D2 receptor antagonists (SCH23390 and eticlopride respectively) to study the involvement of these receptors in the MDMAinduced regulation of these phosphatases.
2.
43
treatment with the ERK activation inhibitor. On the contrary, Dusp14 up-regulation was significantly reduced (36%) by a pretreatment with SL327.
2.2. Time course of dual specificity phosphatases regulation by MDMA In order to determine whether the differences observed with Dusp14 could be attributed to its subgroup belonging, we
Results
Effect of SL327 on the MDMA-induced up-regulation of Dusp1, Dusp5 and Dusp14 2.1.
In order to study the involvement of the ERK pathway in the modulation observed, we studied the impact of a pre-treatment with the ERK activation inhibitor SL327 on mRNA levels of the three regulated Dusps in mice striatum (Fig. 1). Dusp1 and Dusp5 mRNA levels increase were not affected by a pre-
Fig. 1 – Real time quantitative PCR study of the effect of SL327 pre-treatment on the regulation of Dusp1, Dusp5 and Dusp14 in mice striatum. SL327 (50 mg/kg) was administered 1 h before MDMA (9 mg/kg) and mice were killed 2 h after MDMA injection. Results represent the means ± SEM (10–12 animals per group). Statistical analysis was done by ANOVA followed by Bonferroni test. **p < 0.01, ***p < 0.001 as compared to the control group; #p < 0.05 as compared to the SL327/MDMA group.
44
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Fig. 2 – Kinetics of the effects of MDMA treatment on Dusp3, Dusp1, Dusp5 and Dusp14 transcription. Mice were killed 0.5, 1, 2, 4, 6 and 12 h after MDMA injection. The mRNA levels were measured as fluorescent intensities using quantitative real time PCR and normalized to Hprt mRNA levels (see Experimental procedures for details). Values represent fold change as compared to saline-treated animals at each time point (n = 12/group). Statistical analysis was done by Student's test. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to control.
included a fourth Dusp (Dusp3) in our studies which belongs to the same subgroup as Dusp14. We examined the time course of the regulation of the four dual specificity phosphatases 30 min, 1 h, 2 h, 4 h, 6 h and 12 h after the injection of MDMA (Fig. 2). In the case of Dusp3 no regulation by MDMA was observed throughout the kinetics study. Dusp1 mRNA was significantly increased in mice striatum 30 min after the MDMA injection, the up-regulation was at its maximum at 1 h after the treatment and decreased slowly to reach the basal level at 6 h post-injection. The Dusp5 and Dusp14 mRNA increase in the striatum was slightly delayed as compared to Dusp1, with a maximum at 2 h post-injection. In the case of Dusp5 the mRNA level returned to the basal level 6 h after the MDMA injection while that of Dusp14 was still significantly up-regulated as compared to controlled mice (Fig 2). The mRNA levels of the four Dusps were similar in the saline- and MDMA-treated mice groups 12 h after the injection.
2.3. Effect of eticlopride on the MDMA-induced up-regulation of Dusp1, Dusp5 and Dusp14 To examine the role of dopamine D2 receptors in the regulation of the three Dusps in the striatum we used the D2 antagonist eticlopride (Fig 3). The pre-injection of the antago-
nist had no significant effect on the MDMA-induced increase in mRNA levels of the three tested Dusp.
2.4. Effect of SCH23390 on the MDMA-induced up-regulation of Dusp1, Dusp5 and Dusp14 To examine the role of dopamine D1 receptors in the regulation of the three Dusps in mice striatum we used the D1 antagonist SCH23390 (Fig. 4). Conversely to SL327 the preinjection of the D1 receptor antagonist partially, but significantly, blocked the increased transcription of the three Dusps modulated by MDMA treatment: Dusp1, Dusp5, and Dusp14 (26%, 24%, and 30% respectively).
3.
Discussion
We have previously shown that acute MDMA treatment activates the ERK pathway and induces the modulation of 27 genes in mice striatum (Salzmann et al., 2003, 2006). Interestingly, 3 genes up-regulated by acute MDMA (Dusp1, 5 and 14) belong to the dual specificity phosphatase family involved in the negative regulation of MAPK signalling. In this study we showed that among these genes Dusp14 is particular. Its mRNA increase partially depends on the
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45
However, the modulation of the three Dusp mRNAs by acute MDMA partially and similarly depends on the activation of D1 receptors.
Fig. 3 – Real time quantitative PCR study of the effect of eticlopride pre-treatment on the regulation of Dusp1, Dusp5 and Dusp14 in mice striatum. Eticlopride (0.05 mg/kg) was administered 30 min before MDMA (9 mg/kg) and mice were killed 2 h after MDMA injection. Results represent the means ± SEM (10–12 animals per group). Statistical analysis was done by ANOVA followed by Bonferroni test. ***p < 0.001 as compared to the control group; ###p < 0.001 as compared to the MDMA group.
activation of ERK and lasts longer than those of Dusp1 and Dusp5. The acute MDMA-induced increase in mRNA levels of the three Dusps is not regulated by D2 receptor activation.
Fig. 4 – Real time quantitative PCR study of the effect of SCH23390 pre-treatment on the regulation of Dusp1, Dusp5 and Dusp14 in mice striatum. SCH23390 (0.05 mg/kg) was administered 30 min before MDMA (9 mg/kg) and mice were killed 2 h after MDMA injection. Results represent the means ± SEM (10–12 animals per group). Statistical analysis was done by ANOVA followed by Bonferroni test. ***p < 0.001 as compared to the control group; ##p < 0.01 and ###p < 0.001 as compared to the MDMA group.
46
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In a previous study using oligonucleotide arrays we have shown that acute MDMA treatment increases the mRNA levels of Dusp1, 5 and 14 (Salzmann et al., 2006). In this work we not only confirmed the up-regulation of these three phosphatases by MDMA but also that Dusp14 modulation was partially prevented by SL327 pre-treatment. This suggests that the modulations of the three Dusp depend on distinct pathways and that only Dusp14 is under the control of ERK activation albeit partially. Acute MDMA treatment induces a rapid and transient increase in the mRNA levels of Dusp1, Dusp5 and Dusp14. This time course is consistent with the increase of Dusp1 and Dusp6 mRNA observed in rat striatum after acute methamphetamine treatment (Takaki et al., 2001). However in this study Dusp14 mRNA level was still increased 6 h after the injection. This effect is specific to acute MDMA treatment since the mRNA levels of the studied Dusps were not affected after chronic MDMA treatment in mice striatum (data not shown). These results show that the longer effect on mRNA levels was observed on Dusp14, and that only this Dusp is under the control of the ERK pathway. Dual specificity phosphatases have been grouped into four categories on the basis of the structural and functional characteristics (Farooq and Zhou, 2004). It is interesting to notice that Dusp1 and Dusp5 belong to the same subgroup (type II). They are both able to dephosphorylate ERK but this MAPK is not their preferential substrate. On the other hand Dusp14 belongs to the type I subgroup and ERK is one of its favourite substrate (review in Farooq and Zhou, 2004). The differences observed between Dusp14 and Dusp1 and Dusp5 expression level modulation could be attributed to the Dusp14's better efficiency towards activated ERK, to its structural subgroup belonging or to its specific cellular functions. We therefore studied the modulation by MDMA acute treatment of another type I Dusp displaying a good selectivity for ERK (Farooq and Zhou, 2004). Dusp3 was not modulated by MDMA indicating that the possibility for a particular Dusp to be modulated by MDMA could not be linked to its substrate specificity or to its belonging to a specific subgroup but rather to its physiological functions in the cell. Due to the large number of Dusps with overlapping substrate specificities few studies have focussed on their cellular functions. The generation of mouse gene knockouts allowed the demonstration of a role of these enzymes in immune function, stress responses and metabolic regulation (review in Dickinson and Keyse, 2006). Unfortunately, only few data on Dusp14 are available in the literature. As a member of the MAPK phosphatase family, Dusp14 down-modulates cellular responses by dephosphorylating and inactivating MAP kinases. This enzyme is important in immune functions and Dusp14 has been shown to be a major negative-feedback regulator of CD28 co-stimulation in T-cells (Marti et al., 2001). Nakano has identified Dusp14 as a non specific suppressor factor of contact hypersensitivity secreted by macrophageslike cells (Nakano, 2007). It has been recently shown that Dusp14 plays a critical role in the control of the proliferation of pancreatic β-cells (Klinger et al., 2008). Dusp14 mRNA increase was partially blocked by SL327 suggesting a partial dependence upon the ERK pathway. To test whether D1 or D2 receptors were involved in the observed
MDMA-induced mRNA increases of the three Dusps we first examine the effect of a D2 receptor antagonist. The MDMAinduced regulation of the three Dusps was not affected by a D2 receptor blockade. This is consistent with previous results showing that Dusp1 enzymatic activity was not regulated by D2 dopamine receptors in the striatum of control rats (Zhen et al., 2002). It has been shown that the ERK pathway is activated in D1 receptors expressing neurons in the striatum (Acquas et al., 2007; Valjent et al., 2005), we therefore examined the role of D1 receptors in the MDMA-induced transcriptional effects on Dusps. We used SCH23390 a D1 receptor antagonist but also an agonist of 5-HT2C receptors (Millan et al., 2001). A 5-HT2C effect of this molecule has been reported in MDMA sensitized rats (Ramos et al., 2005). However in our study we used naïve animals to assess the acute effects of MDMA, and these authors have shown that in these conditions the main effect of SCH23390 is the blockade of D1 receptors (Ramos et al., 2005). We found that D1 receptor blockade with SCH23390 only partially inhibits the up-regulation of Dusp14. This is in accordance with previous studies showing that in mice striatum the ERK pathway is partially under D1 receptor control to induce immediate early gene regulation by acute MDMA treatment (Benturquia et al., 2008). However, the MDMA-induced increases in mRNA levels of Dusp1 and Dusp5 were also partially blocked by SCH23390 suggesting a separate pathway for these genes involving D1 receptors but not ERK signalling. Among other signalling effects, MDMA initiates a cascade of events, regulated by phosphorylation/dephosphorylation reactions and gene expression, crucial for the maintenance of cellular homeostasis. MDMA-induced ERK activation and MAPK phosphatase up-regulation are two components of the current understanding of the phosphoregulatory mechanisms that mediate the complex processes of signal transduction secondary to MDMA exposure. Together these results suggest a complex signalling cascade specific for each gene induced by MDMA in mice striatum. The actual molecular target of MDMA is still not known; moreover it seems that the metabolites may also have molecular and behavioural functions (Colado et al., 2004; Escobedo et al., 2004; Monks et al., 2004). In addition to dopaminergic receptors, NMDA receptors, serotonin transporters and serotonin receptors have been shown to be implicated in MDMA-induced transcriptional effects (Shirayama et al., 2000). Therefore, the administration of MDMA leads to multiple cascades of events involving several neuromediators and further studies will be needed to understand the very sophisticated pattern of regulation induced by MDMA in mice striatum.
4.
Experimental procedures
4.1.
Animals and drugs
Male CD-1 mice (Charles River, France) weighing 22–24 g were housed in a room with 12 h alternating light/dark cycle and controlled temperature (21 ± 1 °C). Food and water were available ad libitum. All drugs were injected intraperitoneally (i.p.). MDMA (Lipomed, Switzerland), eticlopride and SCH23390
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(Sigma, France) were dissolved in saline solution (0.9% NaCl). The MEK inhibitor SL327, a generous gift of Bristol-Myers Squibb (Wilmington) was dissolved in 100% DMSO, as previously described (Salzmann et al., 2003). Volumes of injection were 0.1 ml per 10 g of body weight for MDMA, SCH23390, eticlopride (or saline) and 0.02 ml per 10 g of body weight for SL327 (or vehicle).
4.2.
Drug treatment and dissection
47
detection (usually greater than 40 cycles), indicating that primer–dimer formation and genomic DNA contamination effects were negligible. The primer nucleotide sequences used for Hprt (hypoxanthine guanine phosphoribosyl transferase) have been previously described (Salzmann et al., 2006). Fluorescent PCR reactions were performed on a Light-Cycler® instrument (Roche Diagnostics, Meylan, France) using the LCFastStart® DNA Master SYBR Green I kit (Roche Diagnostics, Meylan, France). The cDNAs were diluted 200-fold and 5 μl were added to the PCR reaction mix to yield a total volume of 10 μl. The reaction buffer contained 0.5 μM of each primer. The PCR reactions were performed with 10–12 samples/drug treatment, each sample being prepared with bilateral structures from one mouse. Quantification was made on the basis of a calibration curve using cDNA from an untreated mouse brain. As previously described, in addition to the genes of interest, the Hprt transcript was also quantified and each sample was normalized on the basis of its Hprt content (Salzmann et al., 2006). Results are expressed as gene of interest transcript/Hprt transcript.
The dose of MDMA (9 mg/kg) was chosen based on previous studies showing an activation of ERK, hyperlocomotion and place preference in mice (Salzmann et al., 2003). SL327 (50 mg/ kg) and SCH23390 (0.05 mg/kg) were injected 1 h and 30 min before MDMA as previously described (Benturquia et al., 2008; Salzmann et al., 2003). Locomotor activity was used to perform a dose response curve of eticlopride (0.015–0.2 mg/kg) and an inactive dose of the D2 antagonist (0.05 mg/kg) able to block MDMA-induced hyperlocomotion (data not shown) was injected 30 min before MDMA. For kinetics studies mice were killed at the indicated time points by cervical dislocation. For the inhibition studies mice were killed 2 h after the MDMA injection. The brain was quickly removed, frozen in isopentane at−50 °C, and placed in an acrylic matrice (David Kopf Instruments, Phymep, France) allowing the reproducible slicing of 1 mm coronal sections. Dorsal striatum were then dissected free-hand on ice within the slices, and stored at −80 °C until processing.
All series of data were analysed with GraphPad Prism® 4.0 software. Statistical analyses were performed using Student's t test or one-way ANOVA between subjects, followed by Bonferroni tests for post-hoc comparisons. The level of significance was set at p < 0.05.
4.3. RNA isolation and reverse transcription for quantitative PCR
Acknowledgment
Total RNA used for quantitative PCR experiments were extracted by a modified acid–phenol guanidinium method, following the manufacturer's protocol (RNABle ®, Eurobio, France). The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide. Quantification of total RNA was assessed using a NanoDrop® ND-1000 spectrophotometer (NanoDrop® Technologies, USA). Reverse transcription of RNA was performed in a final volume of 20 μl containing 1× first strand buffer (Invitrogen, France), 500 μM each dNTP, 20 U of Rnasin ribonuclease inhibitor (Promega, France), 10 mM dithiothreitol, 100 U of Superscript II Rnase H− reverse transcriptase (Invitrogen, France), 1.5 μM random hexanucleotide primers (Amersham Biosciences, France) and 1 μg of total RNA.
4.4.
Real time quantitative RT-PCR
PCR primers were chosen with the assistance of Oligo 6.42 software (MedProbe, Norway). Sequences of the primers used were as follow: Dusp1 fwd 5′-AACAGGGCAGAAGAGAAAGG-3′; Dusp1 rev 5′-TCATCGGGAATGGTTAATACTG-3′; Dusp3 fwd 5′-TTAGCCATCCTTTCCTGTAAT-3′ and Dusp3 rev 5′-CCTGGGTCACTGAGATGTAT-3′; Dusp5 fwd 5′-AGGAGGAGCGTGGTCTCTC-3′ and Dusp5 rev 5′-GTGGAGGGCAGGATCTCA-3′; Dusp14 fwd 5′-ATTGGGGGATTTAGTGTTGC-3′ and Dusp14 rev 5′-CTCGGGAGAACCAGAGAGAG-3′. For each pair of primers, we performed no-template control and no-reverse transcriptase control assays, which produced negligible signal
4.5.
Statistical analysis
The authors thank Didier Fauconnier for helpful technical assistance.
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Escobedo, I., O'Shea, E., Orio, L., Sanchez, V., Segura, M., de la Torre, R., Farre, M., Green, A.R., Colado, M.I., 2004. A comparative study on the acute and long-term effects of MDMA and 3,4-dihydroxymethamphetamine (HHMA) on brain monoamine levels after i.p. or striatal administration in mice. 144, 231–241. Farooq, A., Zhou, M. -M., 2004. Structure and regulation of MAPK phosphatases. Cell. Signal. 16, 769–779. Klinger, S., Poussin, C., Debril, M. -B., Dolci, W., Halban, P.A., Thorens, B., 2008. Increasing GLP-1-induced {beta}-cell proliferation by silencing the negative regulators of signaling cAMP response element modulator-{alpha} and DUSP14. Diabetes 57, 584–593. Marie-Claire, C., Salzmann, J., David, A., Courtin, C., Canestrelli, C., Noble, F., 2007. Rnd family genes are differentially regulated by 3,4-methylenedioxymethamphetamine and cocaine acute treatment in mice brain. Brain Res. 1134, 12–17. Marie-Claire, C., Palminteri, S., Romualdi, P., Noble, F., 2008. Effects of the selective neurotensin antagonist SR 142948A on 3,4-methylenedioxymethamphetamine-induced behaviours in mice. Neuropharmacology 54, 1107–1111. Marti, F., Krause, A., Post, N.H., Lyddane, C., Dupont, B., Sadelain, M., King, P.D., 2001. Negative-feedback regulation of CD28 costimulation by a novel mitogen-activated protein kinase phosphatase, MKP6. J. Immunol. 166, 197–206. Millan, M.J., Newman-Tancredi, A., Quentric, Y., Cussac, D., 2001. The ‘selective’ dopamine D1 receptor antagonist, SCH23390, is a potent and high efficacy agonist at cloned human serotonin2C receptors. Psychopharmacology (Berlin) 156, 58–62. Monks, T.J., Jones, D.C., Bai, F., Lau, S.S., 2004. The role of metabolism in 3,4-(+)-methylenedioxyamphetamine and 3,4-(+)-methylenedioxymethamphetamine (ecstasy) toxicity. Ther. Drug Monit. 26, 132–136. Nakano, Y., 2007. Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type hypersensitivity. Br. J. Dermatol. 156, 848–860. Owens, D.M., Keyse, S.M., 2007. Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene 26, 3203–3213. Ramos, M., Goni-Allo, B., Aguirre, N., 2005. Administration of SCH 23390 into the medial prefrontal cortex blocks the
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Characteristics of dual specificity phosphatases mRNA regulation by 3,4-methylenedioxymethamphetamine acute treatment in mice striatum Cynthia Marie-Claire, Nadia Benturquia, Ann Lundqvist, Cindie Courtin, Florence Noble⁎ Laboratoire de Neuropsychopharmacologie des addictions (UMR CNRS 7157, INSERM U705) Université Paris Descartes, Faculté de Pharmacie, Paris, France
A R T I C LE I N FO
AB S T R A C T
Article history:
3,4-methylenedioxymethamphetamine (MDMA) is a popular recreational drug that has
Accepted 18 August 2008
rewarding properties in rodents but little is known about its effects at the cellular and
Available online 28 August 2008
molecular levels. We have previously shown that the ERK pathway is important for the regulation in gene expression observed in mice striatum after acute treatment with MDMA.
Keywords:
Interestingly, three dual specificity phosphatases were found among the genes modulated
MDMA
by MDMA acute treatment. In this study we investigated the signalling pathways leading to
Gene expression
the up-regulation of these three mRNAs and the kinetics of their regulation. We found that
Dual specificity phosphatases
the increase in Dusp14 mRNA depends on the activation of ERK and lasts longer than those
Mice
of Dusp1 and Dusp5. The modulation of the three studied Dusps depends partially on the
Striatum
activation of D1 receptors but is independent of the activation of D2 receptors. These results suggest that at least two separate signalling cascades lead to the up-regulation of MAPK phosphatase mRNAs. The increase of Dusp1 and Dusp5 mRNAs is not controlled by ERK activation while that of Dusp14 is a direct negative-feedback mechanism of MDMA-induced ERK signalling. Both mechanisms converge to increase the expression levels of phosphatases able to inactivate ERK. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
MDMA (3-4-methylenedioxymethamphetamine) is the psychoactive compound of the widely abused drug ecstasy. The mechanism of action of MDMA is complex and not wellknown, most of its effects can be attributed to the increase of dopamine and serotonin observed and the subsequent interaction of the neurotransmitters with their respective pre- and post-synaptic receptors (review in Colado et al., 2004). MDMA inhibits the serotonin and dopamine transporters but also
displays a moderate affinity to a broad variety of receptors whose activation could be at the origin of certain effects of the drug (Battaglia et al., 1988). At the intracellular level we have shown, in mice, that ERK (Extracellular signal-Regulated Kinase) signaling is involved in several MDMA behavioral and transcriptional effects, by using a specific inhibitor of ERK activation, SL327 (Salzmann et al., 2003). ERK is an important regulator of neuronal functions, and is involved in various neurobiological events such as synaptic plasticity and memory (Sweatt, 2001). It has been suggested that ERK may play a
⁎ Corresponding author. Fax: +33 1 53 73 97 19. E-mail address: [email protected] (F. Noble). Abbreviations: PCR, polymerase chain reaction; DMSO, dimethyl sulfoxide; MDMA, 3,4-methylenedioxymethamphetamine; ERK, extracellular signal-regulated kinase 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.050
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role in the rewarding properties of several drugs of abuse (Valjent et al., 2004). In addition, it has been shown that D-amphetamine- and cocaine-activated ERK pathway was restricted within a subpopulation of dynorphin and D1 receptor-positive striatal medium-sized spiny neurons in mouse (Valjent et al., 2005). Moreover, Acquas et al. (2007) have shown that activated ERK may represent a post-synaptic correlate of the stimulant effect of MDMA on D1-dependent dopamine transmission in the ventral striatum of rat. Furthermore, it has been shown that D1 receptors play a key role in the acute MDMA-induced hyperlocomotion and that the activation of the ERK pathway is partially under D1 receptor control (Benturquia et al., 2008). Since MAPK signaling pathway is involved in numerous biological functions, dysregulation of this pathway can have widespread consequences on cellular functions. The specificity of the cellular effects induced by a stimulus depends on the magnitude and duration of the activation of the MAPKs (Ebisuya et al., 2005). One of the major negative regulatory mechanisms consists of the dephosphorylation of the activated protein by phosphatases. In mammalian cells, MAPK inactivation is controlled mainly by a family of phosphatases, the dual specificity (Thr/Tyr) phosphatases (Dusp) (Owens and Keyse, 2007). The Dusp family is subdivided into four groups (I–IV) according to their functional domains, mechanism of substrate recognition and enzymatic regulation (Farooq and Zhou, 2004). Using oligonucleotide arrays, we identified several genes regulated by acute administration of MDMA within mice dorsal striatum (Marie-Claire et al., 2007, 2008; Salzmann et al., 2006). Three dual specificity phosphatases were found among the 27 genes modulated: Dusp1, 5 and 14. Since the role of these phosphatases is to regulate MAPK activation, it's interesting that they were up-regulated by the same drug that could activate ERK. Of the three Dusps regulated by MDMA, one (Dusp14) belongs to subgroup I and its modulation was partially dependent on ERK activation while the two others (Dusp1 and 5) which belong to subgroup II did not depend on this pathway for MDMA-induced up-regulation (Salzmann et al., 2006). We therefore investigated the regulation of the three Dusp genes by MDMA using real time quantitative RT-PCR, in order to validate the modulation observed in the microarray study and the potential implication of the ERK pathway. A time course of the regulation of the Dusps in mice striatum after acute MDMA injection was performed. We also used D1 and D2 receptor antagonists (SCH23390 and eticlopride respectively) to study the involvement of these receptors in the MDMAinduced regulation of these phosphatases.
2.
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treatment with the ERK activation inhibitor. On the contrary, Dusp14 up-regulation was significantly reduced (36%) by a pretreatment with SL327.
2.2. Time course of dual specificity phosphatases regulation by MDMA In order to determine whether the differences observed with Dusp14 could be attributed to its subgroup belonging, we
Results
Effect of SL327 on the MDMA-induced up-regulation of Dusp1, Dusp5 and Dusp14 2.1.
In order to study the involvement of the ERK pathway in the modulation observed, we studied the impact of a pre-treatment with the ERK activation inhibitor SL327 on mRNA levels of the three regulated Dusps in mice striatum (Fig. 1). Dusp1 and Dusp5 mRNA levels increase were not affected by a pre-
Fig. 1 – Real time quantitative PCR study of the effect of SL327 pre-treatment on the regulation of Dusp1, Dusp5 and Dusp14 in mice striatum. SL327 (50 mg/kg) was administered 1 h before MDMA (9 mg/kg) and mice were killed 2 h after MDMA injection. Results represent the means ± SEM (10–12 animals per group). Statistical analysis was done by ANOVA followed by Bonferroni test. **p < 0.01, ***p < 0.001 as compared to the control group; #p < 0.05 as compared to the SL327/MDMA group.
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Fig. 2 – Kinetics of the effects of MDMA treatment on Dusp3, Dusp1, Dusp5 and Dusp14 transcription. Mice were killed 0.5, 1, 2, 4, 6 and 12 h after MDMA injection. The mRNA levels were measured as fluorescent intensities using quantitative real time PCR and normalized to Hprt mRNA levels (see Experimental procedures for details). Values represent fold change as compared to saline-treated animals at each time point (n = 12/group). Statistical analysis was done by Student's test. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to control.
included a fourth Dusp (Dusp3) in our studies which belongs to the same subgroup as Dusp14. We examined the time course of the regulation of the four dual specificity phosphatases 30 min, 1 h, 2 h, 4 h, 6 h and 12 h after the injection of MDMA (Fig. 2). In the case of Dusp3 no regulation by MDMA was observed throughout the kinetics study. Dusp1 mRNA was significantly increased in mice striatum 30 min after the MDMA injection, the up-regulation was at its maximum at 1 h after the treatment and decreased slowly to reach the basal level at 6 h post-injection. The Dusp5 and Dusp14 mRNA increase in the striatum was slightly delayed as compared to Dusp1, with a maximum at 2 h post-injection. In the case of Dusp5 the mRNA level returned to the basal level 6 h after the MDMA injection while that of Dusp14 was still significantly up-regulated as compared to controlled mice (Fig 2). The mRNA levels of the four Dusps were similar in the saline- and MDMA-treated mice groups 12 h after the injection.
2.3. Effect of eticlopride on the MDMA-induced up-regulation of Dusp1, Dusp5 and Dusp14 To examine the role of dopamine D2 receptors in the regulation of the three Dusps in the striatum we used the D2 antagonist eticlopride (Fig 3). The pre-injection of the antago-
nist had no significant effect on the MDMA-induced increase in mRNA levels of the three tested Dusp.
2.4. Effect of SCH23390 on the MDMA-induced up-regulation of Dusp1, Dusp5 and Dusp14 To examine the role of dopamine D1 receptors in the regulation of the three Dusps in mice striatum we used the D1 antagonist SCH23390 (Fig. 4). Conversely to SL327 the preinjection of the D1 receptor antagonist partially, but significantly, blocked the increased transcription of the three Dusps modulated by MDMA treatment: Dusp1, Dusp5, and Dusp14 (26%, 24%, and 30% respectively).
3.
Discussion
We have previously shown that acute MDMA treatment activates the ERK pathway and induces the modulation of 27 genes in mice striatum (Salzmann et al., 2003, 2006). Interestingly, 3 genes up-regulated by acute MDMA (Dusp1, 5 and 14) belong to the dual specificity phosphatase family involved in the negative regulation of MAPK signalling. In this study we showed that among these genes Dusp14 is particular. Its mRNA increase partially depends on the
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However, the modulation of the three Dusp mRNAs by acute MDMA partially and similarly depends on the activation of D1 receptors.
Fig. 3 – Real time quantitative PCR study of the effect of eticlopride pre-treatment on the regulation of Dusp1, Dusp5 and Dusp14 in mice striatum. Eticlopride (0.05 mg/kg) was administered 30 min before MDMA (9 mg/kg) and mice were killed 2 h after MDMA injection. Results represent the means ± SEM (10–12 animals per group). Statistical analysis was done by ANOVA followed by Bonferroni test. ***p < 0.001 as compared to the control group; ###p < 0.001 as compared to the MDMA group.
activation of ERK and lasts longer than those of Dusp1 and Dusp5. The acute MDMA-induced increase in mRNA levels of the three Dusps is not regulated by D2 receptor activation.
Fig. 4 – Real time quantitative PCR study of the effect of SCH23390 pre-treatment on the regulation of Dusp1, Dusp5 and Dusp14 in mice striatum. SCH23390 (0.05 mg/kg) was administered 30 min before MDMA (9 mg/kg) and mice were killed 2 h after MDMA injection. Results represent the means ± SEM (10–12 animals per group). Statistical analysis was done by ANOVA followed by Bonferroni test. ***p < 0.001 as compared to the control group; ##p < 0.01 and ###p < 0.001 as compared to the MDMA group.
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In a previous study using oligonucleotide arrays we have shown that acute MDMA treatment increases the mRNA levels of Dusp1, 5 and 14 (Salzmann et al., 2006). In this work we not only confirmed the up-regulation of these three phosphatases by MDMA but also that Dusp14 modulation was partially prevented by SL327 pre-treatment. This suggests that the modulations of the three Dusp depend on distinct pathways and that only Dusp14 is under the control of ERK activation albeit partially. Acute MDMA treatment induces a rapid and transient increase in the mRNA levels of Dusp1, Dusp5 and Dusp14. This time course is consistent with the increase of Dusp1 and Dusp6 mRNA observed in rat striatum after acute methamphetamine treatment (Takaki et al., 2001). However in this study Dusp14 mRNA level was still increased 6 h after the injection. This effect is specific to acute MDMA treatment since the mRNA levels of the studied Dusps were not affected after chronic MDMA treatment in mice striatum (data not shown). These results show that the longer effect on mRNA levels was observed on Dusp14, and that only this Dusp is under the control of the ERK pathway. Dual specificity phosphatases have been grouped into four categories on the basis of the structural and functional characteristics (Farooq and Zhou, 2004). It is interesting to notice that Dusp1 and Dusp5 belong to the same subgroup (type II). They are both able to dephosphorylate ERK but this MAPK is not their preferential substrate. On the other hand Dusp14 belongs to the type I subgroup and ERK is one of its favourite substrate (review in Farooq and Zhou, 2004). The differences observed between Dusp14 and Dusp1 and Dusp5 expression level modulation could be attributed to the Dusp14's better efficiency towards activated ERK, to its structural subgroup belonging or to its specific cellular functions. We therefore studied the modulation by MDMA acute treatment of another type I Dusp displaying a good selectivity for ERK (Farooq and Zhou, 2004). Dusp3 was not modulated by MDMA indicating that the possibility for a particular Dusp to be modulated by MDMA could not be linked to its substrate specificity or to its belonging to a specific subgroup but rather to its physiological functions in the cell. Due to the large number of Dusps with overlapping substrate specificities few studies have focussed on their cellular functions. The generation of mouse gene knockouts allowed the demonstration of a role of these enzymes in immune function, stress responses and metabolic regulation (review in Dickinson and Keyse, 2006). Unfortunately, only few data on Dusp14 are available in the literature. As a member of the MAPK phosphatase family, Dusp14 down-modulates cellular responses by dephosphorylating and inactivating MAP kinases. This enzyme is important in immune functions and Dusp14 has been shown to be a major negative-feedback regulator of CD28 co-stimulation in T-cells (Marti et al., 2001). Nakano has identified Dusp14 as a non specific suppressor factor of contact hypersensitivity secreted by macrophageslike cells (Nakano, 2007). It has been recently shown that Dusp14 plays a critical role in the control of the proliferation of pancreatic β-cells (Klinger et al., 2008). Dusp14 mRNA increase was partially blocked by SL327 suggesting a partial dependence upon the ERK pathway. To test whether D1 or D2 receptors were involved in the observed
MDMA-induced mRNA increases of the three Dusps we first examine the effect of a D2 receptor antagonist. The MDMAinduced regulation of the three Dusps was not affected by a D2 receptor blockade. This is consistent with previous results showing that Dusp1 enzymatic activity was not regulated by D2 dopamine receptors in the striatum of control rats (Zhen et al., 2002). It has been shown that the ERK pathway is activated in D1 receptors expressing neurons in the striatum (Acquas et al., 2007; Valjent et al., 2005), we therefore examined the role of D1 receptors in the MDMA-induced transcriptional effects on Dusps. We used SCH23390 a D1 receptor antagonist but also an agonist of 5-HT2C receptors (Millan et al., 2001). A 5-HT2C effect of this molecule has been reported in MDMA sensitized rats (Ramos et al., 2005). However in our study we used naïve animals to assess the acute effects of MDMA, and these authors have shown that in these conditions the main effect of SCH23390 is the blockade of D1 receptors (Ramos et al., 2005). We found that D1 receptor blockade with SCH23390 only partially inhibits the up-regulation of Dusp14. This is in accordance with previous studies showing that in mice striatum the ERK pathway is partially under D1 receptor control to induce immediate early gene regulation by acute MDMA treatment (Benturquia et al., 2008). However, the MDMA-induced increases in mRNA levels of Dusp1 and Dusp5 were also partially blocked by SCH23390 suggesting a separate pathway for these genes involving D1 receptors but not ERK signalling. Among other signalling effects, MDMA initiates a cascade of events, regulated by phosphorylation/dephosphorylation reactions and gene expression, crucial for the maintenance of cellular homeostasis. MDMA-induced ERK activation and MAPK phosphatase up-regulation are two components of the current understanding of the phosphoregulatory mechanisms that mediate the complex processes of signal transduction secondary to MDMA exposure. Together these results suggest a complex signalling cascade specific for each gene induced by MDMA in mice striatum. The actual molecular target of MDMA is still not known; moreover it seems that the metabolites may also have molecular and behavioural functions (Colado et al., 2004; Escobedo et al., 2004; Monks et al., 2004). In addition to dopaminergic receptors, NMDA receptors, serotonin transporters and serotonin receptors have been shown to be implicated in MDMA-induced transcriptional effects (Shirayama et al., 2000). Therefore, the administration of MDMA leads to multiple cascades of events involving several neuromediators and further studies will be needed to understand the very sophisticated pattern of regulation induced by MDMA in mice striatum.
4.
Experimental procedures
4.1.
Animals and drugs
Male CD-1 mice (Charles River, France) weighing 22–24 g were housed in a room with 12 h alternating light/dark cycle and controlled temperature (21 ± 1 °C). Food and water were available ad libitum. All drugs were injected intraperitoneally (i.p.). MDMA (Lipomed, Switzerland), eticlopride and SCH23390
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(Sigma, France) were dissolved in saline solution (0.9% NaCl). The MEK inhibitor SL327, a generous gift of Bristol-Myers Squibb (Wilmington) was dissolved in 100% DMSO, as previously described (Salzmann et al., 2003). Volumes of injection were 0.1 ml per 10 g of body weight for MDMA, SCH23390, eticlopride (or saline) and 0.02 ml per 10 g of body weight for SL327 (or vehicle).
4.2.
Drug treatment and dissection
47
detection (usually greater than 40 cycles), indicating that primer–dimer formation and genomic DNA contamination effects were negligible. The primer nucleotide sequences used for Hprt (hypoxanthine guanine phosphoribosyl transferase) have been previously described (Salzmann et al., 2006). Fluorescent PCR reactions were performed on a Light-Cycler® instrument (Roche Diagnostics, Meylan, France) using the LCFastStart® DNA Master SYBR Green I kit (Roche Diagnostics, Meylan, France). The cDNAs were diluted 200-fold and 5 μl were added to the PCR reaction mix to yield a total volume of 10 μl. The reaction buffer contained 0.5 μM of each primer. The PCR reactions were performed with 10–12 samples/drug treatment, each sample being prepared with bilateral structures from one mouse. Quantification was made on the basis of a calibration curve using cDNA from an untreated mouse brain. As previously described, in addition to the genes of interest, the Hprt transcript was also quantified and each sample was normalized on the basis of its Hprt content (Salzmann et al., 2006). Results are expressed as gene of interest transcript/Hprt transcript.
The dose of MDMA (9 mg/kg) was chosen based on previous studies showing an activation of ERK, hyperlocomotion and place preference in mice (Salzmann et al., 2003). SL327 (50 mg/ kg) and SCH23390 (0.05 mg/kg) were injected 1 h and 30 min before MDMA as previously described (Benturquia et al., 2008; Salzmann et al., 2003). Locomotor activity was used to perform a dose response curve of eticlopride (0.015–0.2 mg/kg) and an inactive dose of the D2 antagonist (0.05 mg/kg) able to block MDMA-induced hyperlocomotion (data not shown) was injected 30 min before MDMA. For kinetics studies mice were killed at the indicated time points by cervical dislocation. For the inhibition studies mice were killed 2 h after the MDMA injection. The brain was quickly removed, frozen in isopentane at−50 °C, and placed in an acrylic matrice (David Kopf Instruments, Phymep, France) allowing the reproducible slicing of 1 mm coronal sections. Dorsal striatum were then dissected free-hand on ice within the slices, and stored at −80 °C until processing.
All series of data were analysed with GraphPad Prism® 4.0 software. Statistical analyses were performed using Student's t test or one-way ANOVA between subjects, followed by Bonferroni tests for post-hoc comparisons. The level of significance was set at p < 0.05.
4.3. RNA isolation and reverse transcription for quantitative PCR
Acknowledgment
Total RNA used for quantitative PCR experiments were extracted by a modified acid–phenol guanidinium method, following the manufacturer's protocol (RNABle ®, Eurobio, France). The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide. Quantification of total RNA was assessed using a NanoDrop® ND-1000 spectrophotometer (NanoDrop® Technologies, USA). Reverse transcription of RNA was performed in a final volume of 20 μl containing 1× first strand buffer (Invitrogen, France), 500 μM each dNTP, 20 U of Rnasin ribonuclease inhibitor (Promega, France), 10 mM dithiothreitol, 100 U of Superscript II Rnase H− reverse transcriptase (Invitrogen, France), 1.5 μM random hexanucleotide primers (Amersham Biosciences, France) and 1 μg of total RNA.
4.4.
Real time quantitative RT-PCR
PCR primers were chosen with the assistance of Oligo 6.42 software (MedProbe, Norway). Sequences of the primers used were as follow: Dusp1 fwd 5′-AACAGGGCAGAAGAGAAAGG-3′; Dusp1 rev 5′-TCATCGGGAATGGTTAATACTG-3′; Dusp3 fwd 5′-TTAGCCATCCTTTCCTGTAAT-3′ and Dusp3 rev 5′-CCTGGGTCACTGAGATGTAT-3′; Dusp5 fwd 5′-AGGAGGAGCGTGGTCTCTC-3′ and Dusp5 rev 5′-GTGGAGGGCAGGATCTCA-3′; Dusp14 fwd 5′-ATTGGGGGATTTAGTGTTGC-3′ and Dusp14 rev 5′-CTCGGGAGAACCAGAGAGAG-3′. For each pair of primers, we performed no-template control and no-reverse transcriptase control assays, which produced negligible signal
4.5.
Statistical analysis
The authors thank Didier Fauconnier for helpful technical assistance.
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