Báo cáo khoa học: Regulation of ERK1/2 phosphorylation by acute and chronic morphine – implications for the role of cAMP-responsive element binding factor (CREB)-dependent and Ets-like protein-1 (Elk-1)-dependent transcription; small interfering RNA-based strategy

Regulation of ERK1/2 phosphorylation by acute and chronic morphine – implications for the role of cAMP-responsive element binding factor (CREB)-dependent and Ets-like protein-1 (Elk-1)-dependent transcription; small interfering RNA-based strategy Agnieszka Ligeza, Agnieszka Wawrzczak-Bargiela, Dorota Kaminska, Michal Korostynski and Ryszard Przewlocki

Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland

Keywords ERK; opioids; protein kinases; protein phosphatases; RNA interference

Correspondence R. Przewlocki, Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland Fax: +48 12 637 4500 Tel: +48 12 662 3218 E-mail: nfprzewl@cyf-kr.edu.pl

(Received 11 March 2008, revised 23 May 2008, accepted 2 June 2008)

doi:10.1111/j.1742-4658.2008.06531.x

level of activated ERKs

Extracellular signal-regulated kinases (ERKs) have been shown to be acti- vated by opioids and functionally linked to addiction. Morphine-associated changes in ERK activity seem to be the characteristic features of opioid action. In this study, we observed a rapid and severe increase in ERK1 ⁄ 2 activity after a 5 min morphine treatment of HEK-MOR cells (transfected with the rat l-opioid receptor MOR1) expressing l-opioid receptor. Cellu- lar adaptations to chronic (72 h) morphine treatment were manifested by a slight and sustained increase in ERK1 ⁄ 2 activity. Withdrawal caused by an opioid receptor antagonist – naloxone – attenuated phosphorylation of ERK1 ⁄ 2. Little information is available on the precise mechanism of ERK activity regulation. Using RNA interference technology, we generated stably transfected cells with silenced expression of cAMP-responsive element binding factor (CREB) and Ets-like protein-1 (Elk-1) transcription factors, which are known targets for activated ERK1 ⁄ 2. In these cells, ERK1 ⁄ 2 activity regulation was altered. Silencing of CREB or Elk-1 signif- icantly increased ERK activation observed after 5 min of morphine stimu- in these cells was also lation. The initial augmented. Moreover, the cellular response to withdrawal signals and chronic opioid treatment was diminished. These differences suggest that both CREB-dependent and Elk-1-dependent transcription contribute to the expression of proteins regulating morphine-induced ERK activity (particu- lar phosphatases, upstream kinases or their activatory proteins).

signal-regulated kinase

and, more recently, also the mitogen-activated protein kinase ⁄ extracellular (MAP- K ⁄ ERK) pathway [6–8] have been shown to be involved in the cellular response to opioids. The MAP- K ⁄ ERK signaling cascade mediates a variety of cellu- lar functions. Initially implicated in cell differentiation and proliferation, it has also been shown to be vital

It has been shown that opioids, via speciﬁc G-protein- coupled receptors, activate several intracellular path- ways. As a result, a multipart network of cross-talks is created, increasing the complexity and regulatory potential of the ﬁnal response. Protein kinase A [1,2], protein kinase C [3], glycogen synthase kinase-3b [4], [5] calcium ⁄ calmodulin-dependent protein kinase-II

Abbreviations CRE, cAMP-responsive element; CREB, cAMP-responsive element binding factor; Elk-1, Ets-like protein-1; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MKP, mitogen-activated protein kinase phosphatase; p-ERK, phosphorylated extracellular signal-regulated kinase; PW, precipitated withdrawal; RNAi, RNA interference; siRNA, small interfering RNA; SRE, serum response element.

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subfamily of

for synaptic plasticity [9], memory formation [10–13], and long-term gene expression changes underlying drug tolerance and addiction [7,14]. Moreover, the role of ERK1 ⁄ 2 in the rewarding effects of drugs of abuse has been well documented. Blockade of the MAPK ⁄ ERK pathway in the nucleus accumbens with speciﬁc inhibitors prevented the induction of conditioned place preference by cocaine [15]. An increase in ERK2 sig- naling in ERK1 mutant mice caused hypersensitivity to the rewarding properties of morphine [12]. Acute administration of drugs commonly abused by humans, including morphine, generated a distinct regional pat- tern of ERK activation in mouse brain [16]. Also, chronic morphine treatment has been shown to regu- late ERK1 ⁄ 2 catalytic activity in a region-speciﬁc manner [17,18].

nuclear targets for activated ERK1 ⁄ 2. cAMP-respon- sive element binding factor (CREB) and Ets-like protein-1 (Elk-1) transcription factors were chosen because both of them are effectors of the Raf ⁄ MEK ⁄ ERK1 ⁄ 2 pathway [13,23,29] and, what is more, both have been implicated in the cellular response to opioids. Additionally, in several brain structures, CREB has been shown to play important roles in many aspects of opioid addiction [30]. Elk-1 is a member of the ternary complex factor the Erythroblastoma Twenty-Six domain transcription factors. Elk-1 forms ternary complexes on target promoter elements [serum response elements (SREs)] with serum response factor. Ternary complex factors typically activate immediate early genes, such as c-fos [31]. MAPK ⁄ ERK pathway activation results in Elk-1 phosphorylation at multiple including phosphorylation at serine 383, which sites, is crucial for the transcriptional activation. CREB, initially described as a target of a protein kinase A, is also activated by ERK1 ⁄ 2. Activation of CREB occurs via phosphorylation at serine 133. Activated CREB forms dimers that bind to cAMP-responsive elements (CREs) within the promoter of the target gene.

To investigate the molecular aspects of opioid- induced ERK regulation, we used HEK 293 cells transfected to express l-opioid receptor. These cells are easy to transfect, and they express high level of receptor. HEK 293 cells have been used previously in a number of studies as an in vitro model to examine opioid action at the receptor level [32–37] as well as downstream of the receptor [33,38–43]. Prolonged mor- phine exposure of these cells resulted in an adaptive cellular response considered to be a cellular correlate of tolerance and dependence [38,41].

important because of

The data presented here conﬁrm an association between Elk-1 and CREB downregulation and impaired opioid-induced ERK1 ⁄ 2 activity regulation, suggesting strongly that gene transcription mediated by Elk-1 and CREB plays a role in the deactivation of ERK1 ⁄ 2 in this system.

The MAPK ⁄ ERK pathway operates through sequen- tial phosphorylation events to ﬁnally phosphorylate either cytosolic targets (thus regulating intracellular events) or transcription factors (thus regulating gene expression). ERK1 ⁄ 2 catalytic activity is regulated by dual phosphorylation on speciﬁc tyrosine and threo- nine residues by the upstream kinase MAPK ⁄ ERK kinase. This two-site phosphorylation is required for full activation, and consequently, two-site dephosphor- ylation is required for full deactivation. The precise mechanism of MAPK activity regulation is of particu- lar importance because the intensity and the duration of signaling through the MAPK pathway is critical for the cellular response [19–21]. Negative control of ERKs may involve alternative mechanisms. Several classes of phosphatases have been suggested to play a role in MAPK deactivation [22–24] and in the mechanism of the tyro- morphine tolerance [25]. Among them, sine ⁄ threonine dual-speciﬁcity phosphatases, known also as MAP kinase phosphatases (MKPs), seem to be the most their speciﬁcity for ERK1 ⁄ 2. Additionally, MKPs have been shown to be induced by ERK1 ⁄ 2 activity and attenuate MAPK events in an inhibitory feedback loop [26,27]. Although morphine-associated changes

Results

Small interfering RNA (siRNA)-based Elk-1 and CREB silencing

We employed RNAi technology in order to knock down the expression of selected transcription factors: CREB and Elk-1. Using an siRNA expression vector (pSilencer 2.1-U6 hygro, Ambion, Austin, TX, USA), we transfected HEK-MOR cells (transfected with the rat l-opioid receptor MOR1) expressing l-opioid receptor. After hygromycin selection, we obtained

in ERK activity seem to be the characteristic features of opioid action and can be considered as good markers of cellu- lar adaptations to opioids, little information is avail- able on the precise mechanism of ERK activity regulation. In our previous studies [28], we observed strong morphine-associated ERK activity changes regulated by protein kinase C and calcium ⁄ calmodulin- dependent protein kinase. In the current study, we interrupted signal transduction downstream of ERKs to check the role of feedback-based regulation in ERK activity. We employed RNA interference (RNAi) tech- nology to shut off transcription factors that are known

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stably transfected cell lines expressing proper silencing constructs capable of degradation of targeted tran- scription factor mRNA. RT-PCR analysis revealed diverse efﬁciency of silencing for different clones, vary- ing from about 20–60% for both transcription factors (Fig. 1A). For further experiments, stably transfected clones with the most pronounced downregulation (about 60%) of the expression of Elk-1 mRNA (clone 3) or CREB mRNA (clone 2) were chosen. Sta- bly transfected cells proliferated properly, and no signs of any abnormalities or morphological changes were visible under the optical microscope.

For the chosen clones, decreased protein expression

n ot tra n sfecte d

of Elk-1 and CREB was examined (Fig. 1B).

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In the case of Elk-1, the effectiveness of protein silencing calculated versus cells transfected with scram- bled-type siRNA was about 50–60%, and in the case of CREB, apparent protein silencing was less pronounced, although mRNA silencing was similar in both cases.

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Two-hour time course of morphine-induced ERK1/2 activation

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Morphine-induced ERK1 ⁄ 2 phosphorylation increased very rapidly, reaching a maximum at 5 min poststimu- lation, and declined rapidly thereafter, remaining at 30% of the maximum when measured only 15 min poststimulation (Fig. 2). A further decrease developed slowly over the next 105 min, and reached 20% of the maximum at 120 min.

B-actin

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Effect of Elk-1 or CREB silencing on basal ERK1/2 phosphorylation level

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Silencing of Elk-1 or CREB transcription factors in HEK-MOR cells caused a signiﬁcant increase in the phosphorylated ERK (p-ERK) (Fig. 3A). The observed level of p-ERK in these cells was about 3–4- fold higher than that observed in reference HEK- MOR cells without siRNA transfection or transfected with scrambled-type siRNA.

ERK1/2 phosphorylation after acute morphine treatment

Fig. 1. siRNA-based Elk-1 and CREB silencing. (A) Real-time RT-PCR measurements of Elk-1 and CREB mRNA levels in sepa- rated clones of HEK-MOR cells after stable transfection with an siRNA expression vector targeting either Elk-1 or CREB (see Experi- mental procedures). All data are expressed as the means ± SD of three independent experiments. (B) Western blot analysis of Elk-1 and CREB protein expression in chosen clones of stably transfect- ed HEK-MOR cells: scr, cells transfected with scrambled-type siRNA; creb or elk, cells transfected with Elk-1-directed or CREB-directed siRNA respectively.

(not homologous to any known gene) siRNA did not inﬂuence in any way the observed ERK1 ⁄ 2 activation (Fig. 3B, upper right panel). Apparent activation of

As in our previous studies in SH-SY5Y cells [28], western blot analysis of total cellular protein extracts revealed strong temporal activation of the MAP- K ⁄ ERK pathway after acute l-receptor agonist admin- istration. Incubation of HEK-MOR cells with 1 lm morphine for 5 min (Fig. 3B, upper left panel) caused a rapid and severe (about ninefold) increase in p-ERK level. Transfection of these cells with scrambled-type

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***

10.0

7.5

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transfected with scrambled-type

treatment (Fig. 4, upper left panel), suggesting a long- term character of this activation. Full return to the basal level was ﬁnally observed after 4–6 h of morphine stimulation. No remarkable difference in response between HEK-MOR cells and HEK-MOR cells siRNA was noticeable (Fig. 4, upper right panel).

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Using HEK-MOR cells with silenced expression of Elk-1 or CREB transcription factors, we did not observe any particular differences in the cellular response to consecutive hours of morphine treatment as assessed by ERK1 ⁄ 2 activation (Fig. 4, lower pan- els). Similarly, the observed ERK activation did not return to the level of control even after 2 h of mor- phine it ﬁnally remaining returned to the control value after 4–6 h.

Total ERK expression after hours of morphine treatment

Fig. 2. The 2 h time course of ERK1 ⁄ 2 activation after morphine treatment in HEK-MOR cells transfected with scrambled-type siRNA. The cells were incubated with 1 lM morphine for the indi- cated time period and then subjected to western blot analysis. For each sample, a relative level of p-ERK1 ⁄ 2 versus total ERK1 ⁄ 2 was calculated. All data are expressed as the means ± SD of three independent experiments. ANOVA followed by Tukey’s multiple comparison test was performed; ***P < 0.01 as compared with the control group.

Total ERK immunoreactivity after up to 6 h of mor- phine treatment (Fig. 5) did not show any changes in the expression level of ERK1 ⁄ 2.

ERK1 ⁄ 2 was much stronger than recently reported in HEK cells by Rios et al. [44].

ERK1/2 phosphorylation during prolonged morphine treatment and withdrawal

Effect of Elk-1 or CREB silencing on ERK1/2 phos- phorylation after acute morphine treatment

In cells with silenced expression of Elk-1 or CREB, the relative to control fold change of ERK activation was about half of the fold change observed in reference cells (Fig. 3B; compare lower left and right panels with upper panels). However, as we summarized data (Fig. 3C) using absolute values of controls for each group of cells, the strong and massive increase in levels of ERK phosphorylation after 5 min of MAPK stimulation with 1 lm morphine was noticeable. Fur- thermore, Fig. 3C clearly demonstrates that morphine- induced levels of p-ERK are additionally increased in HEK-MOR cells with silenced expression of Elk-1 or CREB.

ERK1/2 phosphorylation after hours of morphine treatment

To assess the duration of ERK activation, p-ERK immunoreactivity was observed within a time course of hours. Surprisingly, a signiﬁcantly elevated level of p-ERK1 ⁄ 2 was still observed after a 2 h morphine

To study the inﬂuence of chronic morphine treatment on ERK1 ⁄ 2 phosphorylation, the experiment was ﬁrst performed on reference HEK-MOR cells, both not transfected with siRNA and stably transfected with scrambled siRNA. Prolonged (72 h) treatment of these cells with 1 lm morphine slightly but signiﬁcantly increased ERK1 ⁄ 2 phosphorylation above the levels seen in control cells (Fig. 6B). Precipitated withdrawal (PW) achieved by administration of 3 lm naloxone to cells formerly exposed to morphine for 72 h attenuated phosphorylation of ERK1 ⁄ 2 (Fig. 6A,B). At 15 min after the addition of this opioid receptor antagonist (PW15), the ERK1 ⁄ 2 phosphorylation decreased by 25–50%, and it decreased further, reaching a level of about 30–60% of the initial value, when measured at 180 min after naloxone administration. That ﬁnal level of p-ERK1 ⁄ 2 was also signiﬁcantly lower than that seen in the control group and in the PW15 group. This time point of 180 min of withdrawal was of particular interest because it enabled us to observe the effects of proteins synthesized de novo in response to withdrawal signals.

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Fig. 3. Western blot analysis of ERK1 ⁄ 2 phosphorylation in HEK-MOR cell lines with- out siRNA transfection (C) and transfected with three different types of siRNA: scram- bled type (scr), Elk-1-directed (elk) and CREB-directed (creb). (A) ERK1 ⁄ 2 phosphor- ylation levels observed in controls (not treated with morphine) of different cell lines. The data are expressed as the means ± SD of three to six independent experiments. ANOVA followed by Tukey’s multiple comparison test was performed; *P < 0.05, ***P < 0.001, as compared with cell line C; #P < 0.05 as compared with Celk cell line. (B) The effect of 5 min of morphine treat- ment (1 lM) on ERK1 ⁄ 2 phosphorylation. All data are expressed as the means ± SD of six independent experiments. A t-test was performed: ***P < 0.001, as compared with the control group. (C) The overall compari- son of ERK1 ⁄ 2 phosphorylation after mor- phine treatment (1 lM) between different cell lines. The data were recalculated against the Cscr group. The data are expressed as the means ± SD of three to six independent experiments. ANOVA followed by Tukey’s multiple comparison test was performed; ***P < 0.001, as compared with the mor- phine-stimulated group of HEK-MOR cells without siRNA plasmids or with scrambled siRNA; #P < 0.05 as compared with mor- phine-stimulated HEK-MOR cells transfected with siRNA against CREB.

Effect of CREB or Elk-1 silencing on ERK1/2 phosphorylation during prolonged morphine treatment and withdrawal

transcription

factors

these

are

transcription contributes

To study the transcriptional dependence of ERK1 ⁄ 2 the experiment was performed activity regulation, on HEK-MOR cells stably transfected with siRNA targeting either Elk-1 or CREB transcription factors. Both direct nuclear targets for the activated MAPK ⁄ ERK cas- treatment with 1 lm cade. After prolonged (72 h) morphine of HEK-MOR cells with silenced CREB,

we observed a slight increase in ERK1 ⁄ 2 phosphory- lation, although with no statistical signiﬁcance in an applied post hoc test (Fig. 6C). In comparison to the reference HEK-MOR cells (compare Fig. 6A,B), the most remarkable difference was observed after 180 min of naloxone-precipitated withdrawal. At that time point, in contrast to the observations in refer- ence HEK-MOR cells, no decrease in p-ERK1 ⁄ 2 that level was observed. This difference suggests to the CREB-dependent expression of proteins regulating the MAPK ⁄ ERK pathway.

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Fig. 4. The time course of morphine- induced ERK1 ⁄ 2 phosphorylation in HEK- MOR cells (upper left) and HEK-MOR cells transfected with scrambled-type siRNA (upper right). The time courses of morphine- induced ERK1 ⁄ 2 phosphorylation in the same cells transfected with siRNA against CREB or Elk-1 transcription factors are shown in the bottom panels. The cells were incubated with 1 lM morphine for 2, 4 or 6 h and then subjected to western blot analysis. All data are expressed as the means ± SD of three independent experiments. ANOVA followed by Tukey’s multiple comparison test was performed; *P < 0.05, **P < 0.01 as compared with the control group.

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pare Fig. 6A,B), these notable differences suggest that the cellular response to withdrawal signals and chronic opioid treatment is affected by Elk-1 silencing. Pro- teins expressed de novo with SRE-driven transcription seem to be engaged in the regulation of p-ERK1 ⁄ 2 level during withdrawal as well as during chronic morphine administration.

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Fig. 5. Western blot analysis of total ERK1 ⁄ 2 expression during 6 h of morphine treatment in HEK-MOR cells. No changes in total ERK1 ⁄ 2 expression were noticed. All data are expressed as the means ± SD of three independent experiments.

The data presented in Fig. 6 suggest that chronic morphine- and withdrawal-mediated ERK1 ⁄ 2 activity regulation was tightly regulated under the conditions used in our experiments, thus emphasizing the role of Elk-1- and CREB-dependent protein expression in the above regulation. The question of whether these two pools of proteins (CRE-regulated and SRE-regulated) are at least partially the same or completely different remains to be tested, as well as the question of which particular protein expression was cut off by the above described genetic manipulation.

Total ERK expression during chronic morphine treatment

Similarly, using HEK-MOR cells stably transfected with siRNA targeting Elk-1, after 180 min of nalox- one-precipitated withdrawal, no decrease in p-ERK1 ⁄ 2 level was noticeable (Fig. 6D). Moreover, after chronic morphine administration, activation of ERK1 ⁄ 2 was not visible. In comparison with reference cells (com-

Total ERK immunoreactivity after chronic morphine treatment (Fig. 7A,B) did not show any changes in the expression level of ERK1 ⁄ 2 during the whole experi- mental period (72 h).

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Fig. 6. Western blot analysis of ERK1 ⁄ 2 phosphorylation after prolonged morphine treatment and during morphine withdrawal in HEK-MOR cells (A) and HEK-MOR cells transfected with scrambled-type siRNA (B). The effects of CREB or Elk-1 silencing on ERK1 ⁄ 2 phosphorylation after prolonged morphine treatment and during morphine withdrawal in HEK-MOR cells are shown in (C) and (D) respectively. The cells were incubated with 1 lM morphine for 72 h [chronic morphine (CM)] and after that period the withdrawal was precipitated with 3 lM naloxone for 15 min (PW15) or 180 min (PW180). For each sample, a relative level of p-ERK1 ⁄ 2 versus total ERK1 ⁄ 2 was calculated.All data are expressed as the means ± SD of three independent experiments. ANOVA followed by Tukey’s multiple comparison test was performed; *P < 0.05, **P < 0.01, ***P < 0.001, as compared with the CM group; ##P < 0.01, ###P < 0.001 as compared with the control group (C); $P < 0.05, as compared with the PW15 group.

MKP-1 expression during prolonged morphine treatment and withdrawal

glyceraldehyde-3-phosphate

(PW180)

further conﬁrmed in real-time RT-PCR studies. MKP-1 mRNA appeared to be quite abundant – the observed threshold cycle for this gene suggested that it was only about 30 times less abundant than the common house- dehydrogenase keeping gene and about 60 times more abundant than the FOS B gene given as an Immediate Early gene example (data not shown).

Discussion

The data presented here provide evidence that l-opioid receptor stimulation in HEK-MOR cells differentially modulates ERK1 ⁄ 2 activity after acute or chronic opioid treatment and during opioid withdrawal.

We did not observe any particular MKP-1 expression changes in cells not transfected with siRNA or stably transfected with scrambled siRNA HEK-MOR cells, either during prolonged morphine treatment or during naloxone-precipitated withdrawal (Fig. 8, upper panels). Moreover, the results obtained in HEK- MOR cells with silenced expression of Elk-1 or CREB were similar (Fig. 8, lower panels). These data suggest that MKP-1 expression is not altered by CREB or Elk-1 deﬁciency. Surprisingly, in all the cell types, we observed a relatively high basal level of MKP-1 expres- sion, which was visible during immunoblotting and

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Fig. 7. Western blot analysis of total ERK1 ⁄ 2 expression during chronic morphine treatment of 72 h (CM) and naloxone-precipitated withdrawal of 15 min (PW15) and of 180 min (PW180) in HEK- MOR cells (A) and the same cells transfected with scrambled-type siRNA (B). No changes in total ERK1 ⁄ 2 expression were noticed within the whole experimental period. All data are expressed as the means ± SD of three experiments.

regardless of

time

l-opioid receptor in HEK-MOR transfected cells is much higher than in SH-SY5Y cells with endogenous l-opioid receptor expression. What is more, in contrast to our previous observation [28], the ERK activation level observed in HEK-MOR cells remained elevated and did not return to the control level over hours of observation. A precise time course (Fig. 2) conﬁrmed that the elevated level of p-ERK1 ⁄ 2 observed after 2 h of morphine treatment was not a result of the second phase of ERK phosphorylation. Previously, such a sec- ond peak of ERK activation has been reported in the hippocampus after acute electroconvulsive seizures those [46]. The complex action of phosphatases, expressed at basal levels and those induced in response to opioid signaling, is responsible for this two-stage (early and late) ERK phosphorylation [46]. Addition- inhibition of protein phosphatase 1 ⁄ 2A with ally, okadaic acid has been shown to activate ERK1 ⁄ 2 in rat striatum [23]. The impact of other ERK-directed phosphatases on ERK1 ⁄ 2 activation in a neuronal cell line has been documented [47]. In HEK-MOR cells, silencing of CREB or Elk-1 with RNAi caused the initial (without morphine treatment) level of p-ERK to be raised, which suggests the involvement of phospha- tases with CRE-driven or SRE-driven expression in ERK1 ⁄ 2 dephosphorylation. Abrogation of CREB or Elk-1 caused attenuation of the expression of particu- lar phosphatases, which resulted in an elevated p-ERK1 ⁄ 2 level. Consequently, the MAPK activation observed after acute agonist exposure was more promi- nent in these cells. In contrast, the response to mor- phine treatment within hours was not affected. The mechanisms that underlie morphine-induced responses include both intracellular signaling and genomic mech- anisms, the latter being important for longer durations of morphine exposure. In our experiment, we could not detect any inﬂuence of CREB or Elk-1 silencing on MAPK ⁄ ERK activity even after hours of morphine suggesting that dephosphorylation events exposure, within this the regime occur impaired CRE-driven and SRE-driven transcription.

the activation observed by us

the magnitude of

Morphine, whether given acutely or chronically, acti- vates ERK1 ⁄ 2, whereas morphine withdrawal leads to ERK1 ⁄ 2 dephosphorylation. In accordance with previ- ous reports on cellular systems [28,42,44,45] after acute morphine treatment, we observed a robust induction of p-ERK. Within the ﬁrst 5 min of opioid receptor agonist exposure, a strong activation was detected, which then declined into the second phase, which was sustained for up to 4 h. This temporal activation of the MAPK ⁄ ERK pathway in HEK-MOR cells resem- bles recently in SH-SY5Y cells [28] acutely treated with morphine, although several differences are notable. In the current the ERK activation was studies, more than three-fold that observed in SH-SY5Y cells, which might be due to the fact that the density of

After chronic (72 h) morphine stimulation of l-opi- oid receptors in HEK-MOR cells, we observed a slightly but signiﬁcantly elevated level of p-ERK1 ⁄ 2. The understanding of the meaning of this sustained MAPK signaling activation during chronic drug expo- sure might be relevant for the understanding of addic- tion mechanisms. Chronic morphine-induced increases of ERK1 ⁄ 2 activity have been previously reported in different brain regions of chronically opioid-treated animals. In the ventral tegmental area, a brain region involved in the rewarding properties of morphine, chronic administration of morphine was reported to

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2

1 n i t c a - B / 1 - P K M

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Fig. 8. Western blot analysis of MKP-1 expression during chronic morphine treat- ment and naloxone-precipitated morphine withdrawal in HEK-MOR cells (upper left) and HEK-MOR cells transfected with scram- bled-type siRNA (upper right). The same cells transfected with siRNA against Elk-1 or CREB transcription factors are shown in the bottom panels. The cells were incubated with 1 lM morphine for 72 h [chonic mor- phine (CM)], and after that period the with- drawal was precipitated with 3 lM naloxone for 180 min (PW180). For each sample, a relative level of MKP-1 versus b-actin was calculated. All data are expressed as the means ± SD of three independent experiments. ANOVA followed by Tukey’s multiple comparison test was performed; *P < 0.05, as compared with the control group (C).

in the increase ERK catalytic activity [17]. Also, pons ⁄ medulla, p-ERK immunoreactivity increased remarkably after 7 days of repeated morphine injec- tions [18]. Another addiction-related issue concerns the changes in MAPK signaling during the process of withdrawal. In our model system, after naloxone- precipitated morphine withdrawal, we observed a decrease in ERK1 ⁄ 2 activity. We measured the with- drawal-induced ERK activity changes at two different time points: 15 min was chosen to observe the fast signal transduction pathway response, and a 180 min time point was chosen to verify whether the fast response would be sustained, decreased or elevated with time. The long-term intracellular signaling adap- tations are of particular interest because in brain neuronal circuits they are responsible for overall changes in gene expression and ﬁnal cellular responses that underlie the complex behavior of the animal and withdrawal syndromes. Next, we studied whether the activation of chronic morphine-related or withdrawal- related MAPK signaling was regulated by a feedback relied on new protein regulatory mechanism that induction. Using siRNA directed against CREB and Elk-1 transcription factors, we veriﬁed the putative role of genes with CRE-driven and SRE-driven expres- sion in ERK1 ⁄ 2 regulation. Both these transcription factors are effectors of the Raf ⁄ MEK ⁄ ERK1 ⁄ 2 path- way [13,29] and mediate the effects of opioids. Using

an siRNA-based strategy directed against transcription factors, we were able to turn down a set of genes controlled by this transcription factor in response to opioids. Previously, disruption of Elk-1 activity by use of mutant Elk-1 alleles was shown to repress SRE- dependent gene expression in a cell culture [48]. Also in brains of Elk-1-deﬁcient mice, kainate-induced expression of some SRE-dependent Immediate Early genes was reduced (c-fos) but not that of others (Egr- 1) [49]. Pretreatment of cultured hippocampal neurons with CREB siRNA completely blocked the estrogen- induced spinophilin (dendritic spines marker protein) expression [50]. Our current results demonstrate that gene expression mediated by Elk-1 and CREB plays a role in ERK1 ⁄ 2 deactivation after long-term precipi- tated withdrawal. Silencing of Elk-1 or CREB com- pletely reversed dephosphorylation of ERK1 ⁄ 2 caused by long-term withdrawal. Possibly, in reference cells, chronic morphine-activated and ⁄ or withdrawal-acti- vated pathways stimulate transcriptional induction of numerous CRE-driven and SRE-driven genes (via the activation of CREB and Elk-1), which in turn directly or indirectly downregulate the activity of the MAPK pathway by deactivation of ERK1 ⁄ 2. At this stage, we are unable to name these proteins precisely, but on the basis of literature data, our ﬁrst considerations were focused on phosphatases. Inhibition of ERK-directed phosphatases has been shown to be a mechanism that

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tested are induced and responsible for the observed ERK1 ⁄ 2 inactivation after 180 min of naloxone-precip- itated withdrawal. In any case, future studies on the role of MKPs in the development of opioid depen- dence and tolerance might be of great importance.

Interestingly, our results showed that Elk-1, but not CREB, silencing impaired ERK1 ⁄ 2 regulation not only after long-term withdrawal but also during chronic morphine and short-term withdrawal. This result sug- gests that Elk-1 activation, probably by MAPK signal- ing, leads to transcription of target genes that are directly or indirectly important for proper regulation of ERK1 ⁄ 2. Such a system constitutes a feedback loop, a common biological regulatory mechanism. Silencing of Elk-1 caused attenuation of all characteris- tic chronic morphine-associated and withdrawal-associ- ated ERK1 ⁄ 2 activity changes, although ERK1 ⁄ 2 activation after acute morphine remained unchanged. As the established p-ERK level observed during chronic opioid exposure and withdrawal, regulated by the coordinated action of many kinases, phosphatases is important for the long-term and their regulators, development of various forms of neural plasticity asso- ciated with opiate addiction, Elk-1 silencing seem to be an interesting approach to change that regulation.

Although, to date, much effort has been focused on examining different aspects of ERK responses to opioids, the whole picture is still far from complete. Little is known about the MAPK regulatory mecha- nisms, although the duration and intensity of ERK activation are considered to be the crucial factors deter- mining the ﬁnal cellular response. These two factors appear to be a result of the dynamic balance between the activities of different kinases and phosphatases. Our ﬁndings provide new insights into intracellular mechanisms underlying the response to opioids in terms of MAPK regulation. First, we measured MAPK act- ivity at the level of ERK in response to different times of opioid exposure and withdrawal. Second, we have begun to investigate the role of a set of genes with CRE-driven and SRE-driven transcription in ERK activity regulation after opioid exposure. We found that Elk-1 and CREB are important for ERK1 ⁄ 2 activ- ity regulation, especially during the opioid withdrawal.

Experimental procedures

Materials

leads to aberrant ERK1 ⁄ 2 activation [47]. As MKPs are known to precisely dephosphorylate both phospho- threonine and phosphotyrosine of activated MAPKs, they seemed to be good candidates. MKP-3 seemed to is very speciﬁc be of particular interest because it towards ERKs and possesses CRE and SRE [51] sequences within its promoter. MKP-1 is less speciﬁc towards ERKs, and has been reported to have CRE but not SRE sequences within its promoter [52]. Previ- ously, in vivo induction of MKP-1 and MKP-3 expres- sion in different brain structures in association with MAPK ⁄ ERK activation have been shown by several authors. Electroconvulsive seizures induced MKP-1 and MKP-3 in limbic regions of the rat brain [46], methamphetamine treatment activated MKP-1 and MKP-3 in several cortices, the striatum and thalamus or hippocampus respectively [53], MKP-1 induction was controlled by ERK activation on corticostriatal stimulation [29], and MKP-3 was one of the genes upregulated in the CA1 area during long-term memory consolidation in response to MAPK ⁄ ERK signaling (1 h after fear conditioning) [51]. In cellular studies, MKP-3 has been shown to be induced by nerve growth factor in differentiating PC12 cells [54] and to be neuroprotective in primary neurons [47]. When the ERK1 ⁄ 2-dependent transcriptome in mammary epithe- lial cells was analyzed, MKP-3 was shown to be the most upregulated (143-fold mRNA induction at 24 h) of all upregulated genes analyzed from seven time points over 24 h [55]. We tested whether the MKP-1 or MKP-3 mRNA level is upregulated after chronic morphine (72 h) or withdrawal (30 min) in HEK cells. Using real-time PCR analysis, we did observe mRNA induction of MKP-1 after prolonged morphine expo- sure which was further augmented during withdrawal (data not shown), although this regulation seemed to be neither CRE-dependent nor SRE-dependent. Unfor- tunately, in the case of MKP-3, the level of mRNA was so low that it was hard to detect. To make sure that in our cellular system the MKP-3 protein level is not relevant, we studied the possible morphine-associ- ated MKP-3 protein induction in HEK-MOR cells. Using western blotting, we could not detect MKP-3 protein; this might be a speciﬁc feature of HEK cells. MKP-1 protein was easy to detect at a basal level, but we did not observe MKP-1 protein induction. More- over, expression of MKP-1 was not altered in cells with silenced Elk-1 or CREB. It was shown previously that synergistic action of multiple transcription factors was required to induce MKP-1 and that transcription was maintained when a single transcription factor in isolation was eliminated [56]. But still we cannot exclude the possibility that MKPs other than those

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from GIBCO-Invitrogen purchased Morphine hydrochloride was from Polfa Kutno, Poland; DMEM, fetal bovine serum, hygromycin B and Lipofectin were (Karlsruhe, Germany); naloxone was obtained from RBI; the Quanti-

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Regulatory action of opioids on ERKs

RNA extraction

RNAi-based silencing of a particular mRNA was conﬁrmed by RT-PCR.

Cell culture

Tect SYBR-Green RT-PCR kit and plasmid puriﬁcation QIAﬁlter Plasmid Midi kit were from Qiagen (Hilden, Germany); the plasmid vector pSilencer 2.1-U6 hygro was from Ambion; for antibodies, see Western blotting below; all other reagents were from Sigma-Aldrich (St Louis, MO, USA). according to

Real-time RT-PCR

Total RNA was isolated by acid guanidinium thiocya- nate ⁄ phenol ⁄ chloroform extraction the method described by Chomczynski & Sacchi [57]. The qual- ity of the total RNA was assessed by the intensity of 28S and 18S bands after denaturing agarose electrophoresis. The RNA concentration was determined by UV spectro- photometry.

HEK-MOR cells were a kind gift from V. Hollt [35,36]. The cells were maintained at 37 (cid:2)C in a humidiﬁed CO2 incubator in DMEM supplemented with 10% fetal bovine serum and standard penicillin ⁄ streptomycin antibiotics. When needed, selection antibiotics were added: hygromy- cin B (150 lgÆmL)1) and puromycin (1 lgÆmL)1). Approxi- mately 10 h before the experiment, complete medium was substituted by serum-reduced DMEM containing 0.5% fetal bovine serum. Cells were kept in the serum-reduced medium until the end of each experiment to avoid activa- tion of MAPK ⁄ ERK kinases by growth factors. Morphine and naloxone (diluted in NaCl ⁄ Pi) were added directly to the culture medium on the plate. Experiments were carried out on cells at approximately 70–80% of conﬂuency.

Genetic constructs for the expression of hairpin siRNA targeting Elk-1 and CREB

Western blotting

theoretically according the to For quantiﬁcation of RNA targets, the Qiagen QuantiTect SYBR Green RT-PCR kit was used, and real-time RT-PCR one-step reactions were run on the iCycler device (Bio-Rad, Hercules, CA, USA). PCR primers were designed with primer3 software. The quality of products was con- ﬁrmed by checking melting curves. For each reaction, 200 ng of total RNA was used, and primers were added to a ﬁnal concentration of 0.5 lm. Threshold cycle values were calculated automatically. The abundance of RNA was calculated equation: abundance = 2) (threshold cycle).

leupeptin aprotinin 2.5 lgÆmL)1, Vector inserts targeting human Elk-1 and CREB were designed by Ambion according to the cenix algorithm. The targeting sequence for Elk-1 (GenBank accession no. is 5¢-GGUGCACAUCCCUUCUAUCTT-3¢ NM_005229) and that for CREB (transcription variants A and B, Gen- Bank accession nos NM_134442 and NM_004379) is 5¢-GGAGGCCUUCCUACAGGAATT-3¢. Two types of

Generation of stable cell clones

expression plasmid encoding hairpin siRNA were obtained after ligation of designed inserts with pSilencer 2.1-U6 hygro plasmid. Plasmids were ampliﬁed in Escherichia coli, and puriﬁed with the QIAﬁlter Plasmid Midi kit. The plasmid expressing a scrambled (nonhomolo- gous to any known gene) hairpin siRNA was used for the generation of stable clones as a control.

antibodies

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Cells were washed with ice-cold NaCl ⁄ Pi, and immediately lysed in a RIPA buffer containing 1 mm orthovanadate, 1 mm sodium ﬂuoride, and pepstatin at 5 lgÆmL)1, and 1 mm phen- ylmethanesulfonyl ﬂuoride. The protein concentration was (Sigma- determined using the BCA Protein Assay Kit Aldrich). Aliquots of crude extracts (containing 6–11 lg of protein) were then subjected to electrophoresis on a 10% SDS ⁄ PAGE gel, and proteins were electroblotted onto nitrocellulose (Bio-Rad Laboratories) or poly(vinylidene (Roche, Mannheim, Germany) membranes. diﬂuoride) The membranes were blocked overnight, and incubated for 1 h with primary antibodies at room temperature. For p-ERK1 ⁄ 2 detection, mouse monoclonal antibodies raised against a peptide corresponding to amino acids 196–209 of ERK1 ⁄ 2 of human origin phosphorylated at tyrosine 204 were used (sc-7383; Santa Cruz Biotechnology, Santa Cruz, CA, USA). For control of total ERK1 ⁄ 2 expression, rabbit polyclonal antibodies were used (sc-93; Santa Cruz). Elk-1 was detected using rabbit monoclonal antibodies (ab32106; Abcam, Cambridge, UK), and CREB was detected using rabbit polyclonal (ab47781; Abcam). For MKP-1 detection, rabbit polyclonal antibodies were used (sc-1199; Santa Cruz), and for b-actin detection, mouse monoclonal antibodies (Sigma) were used. Subsequently, HEK-MOR cells were transfected with the above-listed RNAi plasmids for stable integration, using Lipofectin as a transfection reagent according to the manufacturer’s instructions. Transfected cells were selected using culture medium containing hygromycin B (150 lgÆmL)1), starting 72 h after transfection. Selection of resistant clones lasted for a period of 14 days. Subsequently, hygromycin-resistant clones were transferred to separate wells on the plate and propagated under continuous selection using 150 lgÆmL)1 hygromycin B. In each obtained clonal line, the degree of

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10 Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM & Swe- att JD (1998) The MAPK cascade is required for mam- malian associative learning. Nat Neurosci 1, 602–609. 11 Kelly A, Laroche S & Davis S (2003) Activation of

horseradish peroxidase-conjugated secondary antibodies were applied for 30 min, and immunocomplexes were detected using a buffered mixture of luminol and coumaric acid with hydrogen peroxide. Levels of immunoreactivity were visualized and quantiﬁed with a Fujiﬁlm LAS-1000 ﬂuoroimager system and Fujiﬁlm software (image gauge). The two bands representing ERK1 and ERK2 were analyzed together. mitogen-activated protein kinase ⁄ extracellular signal- regulated kinase in hippocampal circuitry is required for consolidation and reconsolidation of recognition memory. J Neurosci 23, 5354–5360.

Acknowledgements

We would like to thank Professor Volker Hollt from the Department of Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg for support- ing us with HEK-MOR cells transfected with l-opioid receptor. These studies were supported by statutory activity of the Institute of Pharmacology, Polish Acad- emy of Sciences (IF PAN), Krakow, Poland.

12 Mazzucchelli C, Vantaggiato C, Ciamei A, Fasano S, Pakhotin P, Krezel W, Welzl H, Wolfer DP, Pages G, Valverde O et al. (2002) Knockout of ERK1 MAP kinase enhances synaptic plasticity in the striatum and facilitates striatal-mediated learning and memory. Neuron 34, 807–820.

13 Davis S, Vanhoutte P, Pages C, Caboche J & Laroche S (2000) The MAPK ⁄ ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J Neurosci 20, 4563–4572.

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