Download Protein Kinase A Activation Down-Regulates, Whereas Extracellular

0022-3565/07/3201-202–210
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 320:202–210, 2007
Vol. 320, No. 1
108415/3164862
Printed in U.S.A.
Protein Kinase A Activation Down-Regulates, Whereas
Extracellular Signal-Regulated Kinase Activation Up-Regulates
␴-1 Receptors in B-104 Cells: Implication for Neuroplasticity
Gianfrancesco Cormaci, Tomohisa Mori, Teruo Hayashi, and Tsung-Ping Su
Cellular Pathobiology Unit/Development and Plasticity Section/Cellular Neurobiology Research Branch, Intramural Research
Program/National Institute on Drug Abuse/National Institutes of Health/Department of Health and Human Services,
Baltimore, Maryland
Received May 24, 2006; accepted October 17, 2006
The ␴-1 receptor (Sig-1R) is a unique intracellular nonopioid binding site with nano- to low-micromolar affinities for
diverse classes of drugs including benzomorphans, antidepressants, neuroleptics, steroid hormones including progesterone, and psychostimulants such as cocaine and methamphetamine (Sharkey et al., 1988; Su et al., 1988; Snyder and
Largent, 1989; Quirion et al., 1992; Nguyen et al., 2005).
This work was supported by the Intramural Research Program of National
Institute on Drug Abuse, National Institutes of Health, Department of Health
and Human Services.
G.C., T.M., and T.H. contributed equally to this work.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.108415.
sulfonamide dihydrochloride (H-89). However, dB-cAMP, when
used at 2 mM, caused an up-regulation of Sig-1Rs that was
insensitive to the H-89 blockade but was partially blocked by an
extracellular signal-regulated kinase (ERK)/mitogen-activated
protein kinase inhibitor PD98059 (2⬘-amino-3⬘-methoxyflavone). Furthermore, 2 mM dB-cAMP induced an ERK phosphorylation lasting at least 90 min, at which time the phosphorylation caused by 0.5 mM dB-cAMP had already diminished.
PD98059, applied 90 min after addition of 2 mM dB-cAMP,
attenuated the Sig-1R up-regulation. Our results indicate that
cAMP is bimodal in regulating Sig-1R expression: a downregulation via PKA and an up-regulation via ERK. Results also
suggest that psychostimulants may manipulate the cAMPPKA-Sig-1R and/or the cAMP-ERK-Sig-1R pathways to
achieve a neuroplasticity that favors addictive behaviors.
Sig-1Rs exist in the central and peripheral nervous systems
where they modulate neurotransmitter release and cell excitability via ion channels (Monnet et al., 1990; Hayashi and
Su, 2001; Aydar et al., 2002; Nuwayhid and Werling, 2003).
Behavioral studies implicated Sig-1Rs in higher ordered
brain functions including mood and learning and memory
(Maurice et al., 2001; Meunier et al., 2004).
Recently, a number of studies suggested that psychostimulant-induced behaviors involve an up-regulation of Sig-1Rs.
Ujike et al. (1996) demonstrated that cocaine-induced locomotor sensitization was cross-sensitized to the Sig-1R agonist 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and that the
sensitization could be suppressed by a Sig-1R antagonist,
ABBREVIATIONS: Sig-1R, ␴-1 receptor; BMY 14802, ␣-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine; DA, dopamine; PKA, protein
kinase A; ERK, extracellular signal-regulated kinase; dB-cAMP, N6,2⬘-O-dibutyryl-cAMP; NE-100, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride; PD98059, 2⬘-amino-3⬘-methoxyflavone; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s
medium; MS-377, ((R)-(⫹)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartate; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tris-buffered saline plus 0.05%
Tween 20; ANOVA, analysis of variance; SCH23390, R-(⫹)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SpcBIMP, Sp isomer of cyclic-5,6-dichloro-benzimidazol-riboside thiophosphate.
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ABSTRACT
The ␴-1 receptor (Sig-1R) can bind psychostimulants and was
shown to be up-regulated in the brain of methamphetamine
self-administering rats. Up-regulation of Sig-1Rs has been
implicated in neuroplasticity. However, the mechanism(s)
whereby Sig-1Rs are up-regulated by psychostimulants is unknown. Here, we employed a neuroblastoma cell line B-104,
devoid of dopamine receptors and transporter, and examined
the effects of psychostimulants as well as cAMP on the expression of Sig-1Rs in this cell line, with a specific goal to identify
signal transduction pathway(s) that may regulate Sig-1R expression. Chronic treatments of B-104 cells with physiological
concentrations of cocaine or methamphetamine failed to alter
the expression of Sig-1Rs. N6,2⬘-O-Dibutyryl-cAMP (dBcAMP), when used at 0.5 mM, caused a down-regulation of
Sig-1Rs that could be blocked by a protein kinase A (PKA)
inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-
ERK Up-Regulates ␴-1 Receptors
postsynaptic regulation of Sig-1Rs. B-104 cells were chosen
in this study as a postsynaptic model because B-104 cells are
presumably devoid of DA receptors, at least the D2 receptor
(Severson et al., 1983). These cells thus allow us to examine
whether cocaine or methamphetamine may up-regulate
Sig-1R expression in a manner independent of presynaptic
DA interferences. Furthermore, B-104 cells contain cAMP,
thus serving as a tool for investigating cAMP-related signals
that may regulate the expression of Sig-1Rs. The results are
presented in this report.
Materials and Methods
Materials. Cocaine hydrochloride, S-(⫹)-methamphetamine hydrochloride, and (⫹)-pentazocine were obtained from National Institute on Drug Abuse (Rockville, MD). NE-100 was a kind donation
from Dr. Okuyama (Taisho Pharmaceutical Co., Saitama, Japan).
N6,2⬘-O-dibutyryl-cAMP and the mitogen-activated protein kinase
kinase inhibitor PD98059 were purchased from Sigma (St. Louis,
MO), whereas the PKA inhibitor H-89 was from Calbiochem (La
Jolla, CA). The polyclonal antibody against phospho-ERK was from
Cell Signaling (Beverly, MA), and the anti-ERK antibody was from
Santa Cruz Inc. (Santa Cruz, CA). Culture medium, antibiotics, and
fetal calf serum were from Gibco BRL (Carlsbad, CA). Antibodies
directed against dopamine receptors (D1, D2, D3, D4, and D5) and
dopamine transporters were purchased from Chemicon (Temecula,
CA).
Sig-1R Polyclonal Antibody. The polyclonal anti-Sig-1R antibody was produced by immunizing rabbits with synthetic antigenic
peptides. The antigenic peptide corresponds to the second hydrophilic domain of the rat Sig-1R (52-CLDHELAFSRRLIVELRRLH69). Anti-Sig-1R IgGs were purified from serum by using affinity
column chromatography.
Cell Cultures and Drug Treatments. B-104 cell cultures were
maintained on a 175-cm2 flask (Falcon; BD Biosciences Discovery
Labware, Bedford, MA) in 10% FCS-containing Dulbecco’s modified
Eagle’s medium (DMEM), with 2 mM glutamine, 50 U/ml penicillin,
and 50 ␮g/ml streptomycin until use. Cells were cultured at 37°C
with 5% CO2 under saturating humidity. The medium was changed
every 2 to 3 days. Cells were seeded in 12-well plates (Falcon) in 1%
FCS-containing DMEM (1 ml/well). Cells were treated with drugs in
the following fashion. When a test drug was required, a half-amount
of the culture medium was replaced daily with a fresh medium
containing the test drug. dB-cAMP, however, was prepared as a 50 or
200 mM stock solution in deionized water, 10-␮l aliquots of which
were then directly applied to cultured cells to reach the desired final
concentration. PD98059 was dissolved in dimethyl sulfoxide as a 10
mM stock solution that, when needed, was further diluted to reach
final concentrations. Cocaine, methamphetamine, (⫹)-pentazocine,
and dihydrexidine were dissolved in distilled H2O. Controls received
the same amount of water or dimethyl sulfoxide.
Western Blotting. Cells were seeded at 5 or 10 ⫻ 104 cells/well in
12-well plates. After treatments with drugs, cells were placed on ice,
rapidly washed once with ice-cold phosphate-buffered saline, and
then scraped into 1 ml of phosphate-buffered saline with a rubber
policeman. After transferring into Eppendorf tubes, cell suspensions
were centrifuged at 3000 rpm for 5 min at 4°C. The supernatants
were discarded and pellets dissolved with 50 ␮l of 2⫻ Laemmli
sample buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS).
Samples were sonicated and assayed for proteins using a BCA kit
(Pierce Chemicals, Rockford, IL). After addition of ␤-mercaptoethanol and bromphenol blue, samples were boiled for 2 min, and proteins were resolved with a 13% polyacrylamide SDS-PAGE. For
Sig-1R detection, 15 ␮g of proteins was loaded into each lane. For
detection of other proteins, proteins were loaded at 25 to 30 ␮g per
lane.
In Sig-1R Western blottings, gels were electroblotted onto polyvi-
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BMY 14802. Likewise, methamphetamine-induced locomotor
sensitization was also blocked by a Sig-1R antagonist, MS377 (Takahashi et al., 2000). Up-regulation of Sig-1Rs thus
has been demonstrated in specific brain areas of methamphetamine- or cocaine-sensitized rats (Itzhak, 1993; Liu et
al., 2005). We found that Sig-1Rs were up-regulated by 50%
in the midbrain of rats self-administering methamphetamine
in a response-contingent manner (Stefanski et al., 2004).
Up-regulation of Sig-1Rs promotes cellular differentiation in
PC-12 cells (Takebayashi, 2002) and in oligodendrocytes (Hayashi and Su, 2004), causes the reconstitution of plasma
membrane lipid rafts (Hayashi and Su, 2005), and potentiates the brain-derived neurotrophic factor signaling for glutamate release (Yagasaki et al., 2006). These findings suggest that Sig-1Rs may promote addictive processes by
altering the structure or morphology of neurons (Hayashi
and Su, 2005). Although cocaine or methamphetamine binds
with low micromolar affinity to Sig-1Rs, exactly how these
psychostimulants may up-regulate the expression of Sig-1Rs
is unknown. Other factors in addition to binding may also be
involved.
Monoaminergic systems are related to the action of methamphetamine and cocaine. Of these, the dopaminergic system plays an important role in the methamphetamine- or
cocaine-induced sensitization in animals. There are two superfamilies of dopamine (DA) receptors, designated D1- and
D2-like receptors. D1 and D2 receptors are coupled to Gs and
Gi proteins, respectively, thereby modulating the production
of cAMP. cAMP is well known to activate protein kinase A
(PKA), which, in turn, controls numerous cellular functions.
However, cAMP has also been demonstrated to induce the
activation of the extracellular signal-regulated kinase (ERK)
(Stork and Schmitt, 2002). ERK phosphorylates several transcription-regulating molecules related to cellular differentiation and apoptosis. The ERK pathway is also known to be
crucial for the establishment of behavioral responses to psychostimulants (Valjent et al., 2005). Thus, the ERK activation plays a role in the relapse to cocaine self-administration
(Lu et al., 2005), the retrieval of cocaine-induced conditioned
place preference (Miller and Marshall, 2005; Valjent et al.,
2006a), and psychostimulant-induced sensitization (Valjent
et al., 2006b). However, the cellular biological processes
downstream of ERK activation leading to altered neural plasticity are not totally understood.
We suggested recently a link between cAMP and the regulation of Sig-1R expression. Subchronic treatment of NG108 cells with a stable cAMP analog, dB-cAMP, induced an
up-regulation of Sig-1Rs in the cell (Stefanski et al., 2004).
However, the exact signaling pathway downstream of cAMP
leading to the Sig-1R up-regulation was not identified. The
present study, therefore, was conducted to identify the potential signal transduction pathways downstream of cAMP
that may participate in the regulation of Sig-1R expression.
We also aimed to understand how psychostimulants may
up-regulate Sig-1Rs. Cocaine and methamphetamine might
up-regulate Sig-1Rs by a direct binding to Sig-1Rs. Alternatively, psychostimulants might up-regulate Sig-1Rs through
the drugs’ ability to increase DA at the synaptic cleft, which
subsequently interacts with postsynaptic DA receptors. Sig1Rs were also shown to be able to regulate DA release in a
model system simulating a presynaptic mechanism (Ault and
Werling, 2000). The present study, however, focused on the
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ethanol; 50°C) before being reprobed for the total ERK. The degree of
ERK phosphorylation was calculated by dividing the intensity of
p-ERK by that from the total ERK.
cAMP Assays. cAMP was extracted from cell lysates with 0.1 N
HCl and then measured by an immunoassay method (Assay Designs;
Sigma) according to the manufacturer’s instruction. Cells were stimulated for 3 to 20 min with DA receptor ligands. Samples stimulated
with 1 ␮M forskolin for 3 to 20 min served as positive controls.
Statistical Analyses. Data shown in this study are expressed as
mean ⫾ S.E.M. Statistical significance was analyzed by one-way
analysis of variance (ANOVA) followed by Tukey test with the significance level set at p ⬍ 0.05.
Results
Quantitative Western Blotting of Sig-1Rs in B-104
Cells. Affinity-purified Sig-1R antibodies against rat Sig-1R
(52– 69) recognized Sig-1Rs in B-104 cells as a 25-kDa band
in Western blotting (Fig. 1A). B-104 cells thus constitutively
express Sig-1Rs. The linear range for a quantitative detection of Sig-1Rs in the Western blotting was established by
loading different amounts of proteins into each lane. As
shown in Fig. 1B, Sig-1R intensities were linearly correlated
with amounts of loaded proteins (r2 ⫽ 0.9866, p ⬍ 0.001)
when 5 to 25 ␮g of proteins was used in the assay. Based on
these results, 15 ␮g of protein/lane was used in this study for
quantitative measurements of Sig-1Rs.
Lack of Dopamine Receptors and Dopamine Transporter in B-104 Cells. As mentioned in the introduction, to
exclude psychostimulant effects via dopaminergic signaling,
B-104 cells, presumably devoid of DA receptors (Severson et
al., 1983), were used in this study. Using Western analyses,
Fig. 1. Characterization of Sig-1R expression in B104 cells. A, representative Western blotting using an affinity-purified anti-Sig-1R antibody against
the rat Sig-1R (52-CLDHELAFSRRLIVELRRLH-69). B-104 cell lysates separated by 13% SDS-PAGE were immunoblotted with either normal rabbit
IgG (left) or anti-Sig-1R IgG (right). Arrowhead, position of Sig-1Rs. B, quantitative detection of Sig-1R protein levels by using Western blotting. B-104
proteins ranging from 0 to 50 ␮g per lane were loaded (x-axis). y-axis, densitometrically analyzed intensities of Sig-1R bands. Each point represents
mean ⫾ S.E.M. of six samples. Note the significantly linear regression within the range of 5 to 25 ␮g/lane. C, dopamine receptors, tyrosine hydroxylase
(TH), and dopamine transporter (DAT) are below detection levels in B-104 cell (rat brain samples serve as positive controls). Total proteins from B-104
cells (100 ␮g/lane) and rat brains (30 ␮g/lane) were loaded for the Western blotting. D, dopamine; R, receptor.
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nylidene difluoride membranes (Bio-Rad, Hercules, CA) in Towbeen
buffer (25 mM Tris-base, 192 mM glycine) without methanol. Membranes were blocked with 10% nonfat dry milk (Bio-Rad) in Trisbuffered saline plus 0.05% Tween 20 (TBS-T) for 1 h at room temperature. Membranes were incubated for 2 h at room temperature
with anti-Sig-1R polyclonal antibodies (1:1500) in TBS-T and probed
with the secondary anti-rabbit IgG horseradish peroxidase conjugate
for 1 h at room temperature (1:15000 dilution). Protein bands were
visualized with an ECL chemiluminescence enhancer (GE Healthcare, Little Chalfont, Buckinghamshire, UK) in a Kodak Image Station 440 CF (Kodak, New Haven, CT). For phospho-ERK western
blottings, membranes were blocked in TBS-T plus 5% bovine serum
albumin, then incubated overnight with polyclonal anti-pERK antibodies at a 1:1000 dilution in TBS-T plus 5% bovine serum albumin.
For the detection of dopamine receptors and dopamine transporter, the Wistar rat brain was used: the striatum for D1, D2, D3,
D5, and dopamine transporter; and the midbrain for D4 and tyrosine
hydroxylase. The brain was quickly removed after decapitation, and
brain regions were dissected out on ice. Fresh tissues were homogenized with a Daunce homogenizer in 2⫻ Laemmli sample buffer (10
v/w). After sonication and centrifugation, supernatants were subjected to SDS-PAGE. Trans-blotted membranes were incubated overnight with respective specific antibodies (1:300 dilution; Chemicon).
Analysis of Protein Levels. Western blotting digital images
were analyzed by using the Kodak 1D Image Analysis Software. The
background intensity was subtracted from the total intensity for
each band. In analyzing the Sig-1R protein levels, we did not use
tubulin or actin as internal controls because cAMP or methamphetamine treatments seemed to affect these proteins. Sig-1R levels were
therefore calculated based on intensities of Sig-1Rs on the same film
obtained from blots from various treatment conditions. Each experiment was thus repeated at least five times. For determination of the
total ERK, the membrane was first probed for p-ERK, and then the
same membrane was stripped of p-ERK (5% SDS, 0.5% ␤-mercapto-
ERK Up-Regulates ␴-1 Receptors
We decided therefore to repeat the cAMP experiment using
NG-108 cells and established the full dose-response curve for
dB-cAMP on its regulation on the expression of Sig-1Rs.
Likewise, a dose-response curve was also established for
dB-cAMP on its effect on the Sig-1R expression in B-104 cells.
The two curves were then compared. cAMP increased Sig1Rs in NG-108 cells in a monophasic manner (Fig. 3B, upper
curve). However, cAMP affected the level of Sig-1Rs in B-104
cells apparently in a biphasic manner; firstly, with a downward trend that maximized at approximately 0.5 mM cAMP,
and then with an upward trend causing an approximately
175% increase of Sig-1Rs at 2 mM cAMP (Fig. 3B, lower
curve).
These results from the dose-response curves suggested,
among other possibilities, that cAMP in B-104 cells, unlike
that seen with NG-108 cells, might have activated two different intracellular pathways that act in opposite directions
in affecting the expression of Sig-1Rs. Because cAMP is
known to activate mainly the PKA pathway and the ERK
pathway (see Introduction), we hypothesized that the activation of one of these cAMP-activated pathways, the PKA pathway or the ERK pathway, may cause an alteration of Sig-1Rs.
The following experiments were thus designed to test this
possibility.
Firstly, we examined whether the direct activation of PKA
by an Sp isomer of cyclic-5,6-dichloro-benzimidazol-riboside
thiophosphate (Sp-cBIMPS) might cause an alteration of Sig1Rs. Sp-cBIMPS dose-dependently caused a down-regulation
of Sig-1Rs (Fig. 3C). To further confirm the involvement of
PKA in the down-regulation of Sig-1Rs, we treated the cells
with a PKA inhibitor, H-89. H-89 at 200 nM has been shown
to be a highly selective PKA inhibitor (Robin et al., 1998).
H-89 alone (treatment time, 24 h) was able to increase Sig-1R
expression in a dose-dependent manner (Fig. 3D). The H-89
effect disappeared on day 3 (Fig. 3E). In the presence of H-89,
Fig. 2. Effects of cocaine (COC), methamphetamine (METH), or Sig-1R ligands on the expression of Sig-1Rs. Sig-1R levels were measured by Western
blotting as specified in Fig. 1. Data represent means ⫾ S.E.M. of six to eight independent experiments. A, effects of duration of treatment (1–3 days)
with cocaine or methamphetamine (10 ␮M) on Sig-1R levels in B-104 cells. B, effects of different concentrations of cocaine or methamphetamine on
Sig-1R levels in B-104 cells. Cells were treated with psychostimulants for 3 days. C, effects of Sig-1R agonist (⫹) pentazocine [(⫹) PTZ] or antagonist
NE-100 (NE) on the Sig-1R level in B-104 cells. (⫹) Pentazocine at 300 nM, NE-100 at 1 ␮M. No significant differences were seen between any treated
groups versus controls (one-way ANOVA).
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we confirmed the lack of tyrosine hydroxylase, DA receptors,
and DA transporter in B-104 cells (Fig. 1C). Because the
detection of DA receptors using Western blotting is not always consistent and reliable using the commercial antibodies, we decided to confirm the absence of DA receptors in
B-104 cells by employing functional assays detecting the
formation of cAMP. We found that neither DA receptor agonists nor DA antagonists could alter cAMP levels in B-104
cells, whereas forskolin positively increased cAMP (base, ⬍1
pM; forskolin at 1 ␮M for 10 min, 62.4 pM; dihydrexidine at
1 ␮M for 10 min, ⬍1 pM; SCH23390 at 1 ␮M for 10 min, ⬍1
pM; quinpirole at 1 ␮M for 10 min, ⬍1 pM; sulpiride at 1 ␮M
for 10 min, ⬍1 pM; n ⫽ 3 for each determination).
Effect of Cocaine or Methamphetamine on Sig-1R
Expression. We next treated B-104 cells with cocaine or
methamphetamine. B-104 cells were seeded in 1% FCS-containing DMEM and treated for 1 to 3 days with 300 nM to 10
␮M methamphetamine or cocaine. Sig-1R expression was
unaltered under these conditions (Fig. 2, A and B). We also
treated cells with the selective Sig-1R agonist (⫹)-pentazocine (300 nM) in the presence or absence of selective Sig-1R
antagonist NE-100 (1 ␮M). The agonist or the antagonist or
the combination of them could not alter Sig-1R levels over a
course of 3 days (Fig. 2C). None of the tendencies, if any,
reached statistical significance.
cAMP Down-Regulates Sig-1Rs in B-104 Cells
through the Activation of PKA. In our previous study
(Stefanski et al., 2004), we used a single dose of 0.5 mM
cAMP and found that dB-cAMP caused an increase of Sig1Rs in NG-108 cells. In the same manner, we treated B-104
cells in the present study with 0.5 mM dB-cAMP for 1 to 3
days, with an anticipation to see an increase of Sig-1Rs.
Unexpectedly, 0.5 mM dB-cAMP caused a down-regulation of
Sig-1Rs in B-104 cells with an apparent peak down-regulation on day 1 (Fig. 3A).
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dB-cAMP failed to affect the level of Sig-1Rs (Fig. 3F). These
data indicate that the activation of the cAMP/PKA pathway
causes the down-regulation of Sig-1Rs in B-104 cells.
ERK Signaling up-Regulates Sig-1Rs. Inasmuch as the
other main pathway regulated by cAMP in the living system
is the ERK pathway, we examined the possibility that the
increase of Sig-1Rs caused by 2 mM cAMP might have been
through the activation of the cAMP/ERK pathway. The possibility was not far-fetched because we have found that the
nerve growth factor could activate ERK and concomitantly
caused the up-regulation of Sig-1Rs in PC-12 cells (Takebayashi et al., 2004).
Thus, we examined whether cAMP might activate ERK in
B-104 cells, specifically in a PKA-independent manner. In-
deed, dB-cAMP at either 0.5 or 2 mM tended (in four determinations) to cause the activation of ERK in B104 cells in a
time-dependent fashion (Fig. 4A, two left figures). dB-cAMP
at 0.5 mM caused a transient ERK activation that diminished to the control level at 90 min (Fig. 4A, left). However, 2
mM dB-cAMP promoted a long-lasting ERK phosphorylation
that lasted at least 90 min (Fig. 4A, middle). To confirm the
potential differences, specifically at the 90-min time point,
we decided to replicate the experiment at 90 min in eight
determinations. Data indeed show that 2 but not 0.5 mM
dB-cAMP significantly caused an increase of ERK phosphorylation at the 90-min time point (Fig. 4A, bar graph).
To demonstrate that the ERK activation caused by cAMP
is independent of the PKA, we used the PKA inhibitor H-89
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Fig. 3. Involvement of PKA in cAMP-induced Sig-1R down-regulation. A, effects of chronic treatment (1–3 days) of B-104 cells with 0.5 mM dB-cAMP
on the expression of Sig-1R expression. B, dose-response curves of cAMP on the expression of Sig-1Rs in NG-108 cells (upper curve) and in B-104 cells
(lower curve). C, effects of a specific PKA activator Sp-cBIMPS on the expression of Sig-1Rs. B-104 cells were treated with Sp-cBIMPS for 24 h. D,
dose-dependent effect of a PKA inhibitor H-89 (200 nM), per se, on the expression of Sig-1Rs. E, time course of the effect of H-89 per se on the
expression of Sig-1Rs. F, effect of H-89 on cAMP-induced Sig-1R down-regulation. B-104 cells were treated with dB-cAMP (0.5 mM) and/or H-89 (200
nM) for 24 h. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.0001 compared with controls by one-way ANOVA followed by the Tukey test. Data represents mean ⫾
S.E.M. of five to nine independent determinations. Sig-1R levels in A to E were measured by Western blotting as specified in Fig. 1.
ERK Up-Regulates ␴-1 Receptors
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and tested if it may block the ERK activation caused by
cAMP. Figure 4B shows that the ERK activation, caused
either by 0.5 mM dB-cAMP at the 30-min time point (Fig. 4B,
middle) or by 2 mM dB-cAMP at the 90-min time point (Fig.
4B, bottom), was not blocked by the PKA inhibitor H-89.
Results are consistent with those found by Iacovelli et al.
(2001).
We also used PD98095, a well known inhibitor of the ERK
kinase MEK, and tested if the inhibition of ERK activation
might cause a down-regulation of Sig-1Rs. Indeed, PD98059
dose-dependently suppressed the ERK phosphorylation (Fig.
4C, upper) and concomitantly caused a down-regulation of
Sig-1Rs (Fig. 4C, lower).
Because 2 mM dB-cAMP caused a long-lasting activation of
ERK, at least for 90 min (Fig. 4A, middle), the possibility
exists that the long-lasting phase of ERK activation could be
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Fig. 4. ERK signaling pathway and the up-regulation of Sig-1Rs by cAMP. A, time-dependent activation of ERK by 0.5 (left) and 2 (right) mM
dB-cAMP. Phosphorylated and total ERK were measured by Western blotting using specific respective antibodies. “ERK activation” was calculated by
dividing intensities of phosphorylated ERK by those from total ERK. Note that a sustained activation of ERK at 90 min is seen only in 2 mM
cAMP-treated B-104 cells. Data represent means ⫾ S.E.M. of four independent determinations. A, bar graph, data from a repeat of eight independent
determinations specifically on the 90-min time point. B, effects of H-89 on the cAMP-induced ERK activation caused by 0.5 (upper bar graph; at 30
min) or 2 (lower bar graph; at 90 min) mM cAMP. Data represent means ⫾ S.E.M. of six independent determinations. C, dose-dependent
down-regulation of Sig-1R expression by PD98059. Data represent mean ⫾ S.E.M. of four to six independent determinations. D, effects of PD98059,
added to cells at different time points, on the up-regulation of Sig-1R induced by 2.0 mM dB-cAMP. The addition of PD98059 90 min after the addition
of 2 mM dB-cAMP (column 5) caused an inhibition of Sig-1R expression equivalent to that obtained from the coadministration of cAMP and PD98059
at time 0 (column 4). cAMP, db-cAMP; PD, PD98059 at 50 ␮M. Sig-1R protein levels were assessed by Western blotting 24 h after the application of
cAMP. Data represent mean ⫾ S.E.M. of six independent determinations. Sig-1Rs were measured as indicated in Fig. 1. For all panels: ⴱ, p ⬍ 0.05;
and ⴱⴱ, p ⬍ 0.01, compared with controls by one-way ANOVA followed by Tukey test. For D: ⴱ, p ⬍ 0.05 compared with control; ##, p ⬍ 0.01 compared
with 2 mM cAMP.
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mainly responsible for the up-regulation of Sig-1Rs caused by
2 mM cAMP in B-104 cells. An approach to test this possibility, although not a perfect one, was to examine whether
PD98059, after the establishment of the long-lasting ERK
activation (i.e., 90 min after the cAMP treatment), can block
the Sig-1R up-regulation. We decided to examine this possibility therefore by first allowing 2 mM cAMP to activate ERK
for 90 min and then adding PD98059 to the system. The
Sig-1R level was examined 24 h after the addition of cAMP.
PD98059 by itself, again like that seen in Fig. 4C, attenuated
the Sig-1R level (Fig. 4D, third bar from left). When coadministrated with cAMP at time 0, PD98059 blocked the
Sig-1R expression (Fig. 4D, fourth bar from left). When cAMP
was allowed to activate ERK for 90 min and then PD98059
was added to the system, PD98059 blocked the Sig-1R expression (bar on the right).
The understanding of the basic function of Sig-lRs largely
derives from testing Sig-1R ligands in physiological/pathological systems or behavioral models. Sig-1Rs were shown to be
up-regulated in the brain of rats that were sensitized to
methamphetamine (Takahashi et al., 2000), self-administering methamphetamine (Stefanski et al., 2004), or conditioned
to cocaine-induced place preference (Romieu et al., 2004). In
addition, the overexpression of Sig-1Rs in cultured cells
caused morphological and functional changes of biological
membranes (Hayashi and Su, 2005). However, information is
very limited so far on the potential signal transduction pathways that may lead to the up- or down-regulation of Sig-1Rs.
The present study, therefore, constitutes the first report describing that specific kinases downstream of cAMP, i.e., PKA
and ERK, can regulate Sig-1R expression in a biological
system. However, other signaling pathways may also exist to
regulate the expression of Sig-1Rs in the biological system.
For example, cocaine has been shown to concomitantly upregulate a transcription factor, Fra-2, and Sig-1Rs in the rat
brain (e.g., Liu et al., 2005).
The binding of ligands to receptors, as seen in a number of
hormone and neurotransmitter systems, often causes receptor internalization, receptor degradation, as well as the activation of transcriptions, which consequently lead to the alteration of receptor expression. The intracellular distribution
of Sig-1Rs has been defined recently. Sig-1Rs localize at
lipid-enriched globular structures associated with endoplasmic reticulum as reported in NG-108 –15 cells (Hayashi and
Su, 2003a), primary hippocampal neurons (Hayashi and Su,
2004), or oligodendrocytes (Hayashi and Su, 2004). Upon
stimulation with agonists like (⫹)-pentazocine and cocaine,
Sig-1Rs translocate on the ER toward perinuclear areas and
the tip of neurites (Hayashi and Su, 2001, 2003b). However,
cocaine, methamphetamine, or even a selective Sig-1R ligand
(⫹)pentazocine did not change the Sig-1R expression as reported in this study. Cocaine levels in the post-mortem brain
of cocaine addicts were estimated to be between 0.1 and 4 ␮M
(Kalasinsky et al., 2000), which is close to the Ki value of
cocaine for Sig-1Rs (Sharkey et al., 1988). In this study, we
used the concentrations of cocaine and methamphetamine
that are close to the physiological concentrations of those
drugs in human brains and found that cocaine, methamphetamine, or the selective Sig-1R agonist, (⫹)pentazocine, can-
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 9, 2017
Discussion
not change the level of Sig-1Rs. One possible explanation
might be that these drugs are difficult to pass the plasma
membrane. However, it is known that (⫹)pentazocine is actively up-taken in neuronal cells in a time-, energy-, and
temperature-dependent manner (Yamamoto et al., 2001).
Therefore, the treatment of B-104 cells with these compounds at concentrations at least 10-fold higher than their Ki
values for Sig-1Rs for 1 to 3 days at 37°C should be theoretically sufficient for ligands to bind to Sig-1Rs and alter the
expression of Sig-1Rs, if any. Our results indicated otherwise. Thus, direct interaction of Sig-1R ligands with Sig-1Rs
per se, even though ensuing a Sig-1R translocation (Hayashi
and Su, 2003b), may not lead to transcriptional activities
and/or protein degradation that may change Sig-1R levels in
B-104 cells.
cAMP affects a number of biological processes like cellular
proliferation, differentiation, embryogenesis, and neural
plasticity. The main mechanism by which cAMP exerts its
cellular effects involves its activation of PKA. In our test
model using B-104 cells, both db-cAMP (0.5 mM) and a PKA
agonist, Sp-cBIMPS, caused a down-regulation of Sig-1Rs.
These results indicate that the activation of the cAMP/PKA
pathway negatively modulates the Sig-1R expression. Furthermore, our finding that H-89 alone is able to increase
Sig-1Rs implicates a tonic inhibition of Sig-1R expression by
PKA.
The repeated treatment of psychostimulants induces
sensitization in rodents and humans. The behavioral alterations induced by repeated administration of abused
drugs are thought to be mediated by molecular and cellular
adaptations of neurons including those in the mesolimbic
dopaminergic system. In dopaminergic systems, cAMP is
the primary second messenger system associated with DA
receptors. Alterations in the functions of the cAMP system
have been hypothesized to initiate stable neural changes
associating with abuse of psychostimulants (Lu et al.,
2003; Schroeder et al., 2004). As such, it is worth noting
that repeated administrations of amphetamine or cocaine
induce a decrease of PKA in the nucleus accumbens (Crawford et al., 2004). Inasmuch as our present data indicate
that a decreased PKA activity leads to Sig-1R up-regulation, we speculate that Sig1Rs may increase in the nucleus
accumbens of animals in those studies. We showed previously that Sig-1Rs are up-regulated in the methamphetamine self-administering rats, specifically in the midbrain.
We did not, however, examine the PKA activity in the
midbrain of those rats, nor did we examine the nucleus
accumbens for Sig-1R levels.
In the present study, we demonstrated that dB-cAMP
can stimulate ERK in B-104 cells. cAMP has been shown to
activate the ERK signaling in a PKA-dependent or -independent mechanism. Previous reports also showed that
ERK could be activated by cAMP in a cell-specific manner
(Stork and Schmitt, 2002). In the present study, dB-cAMPinduced ERK phosphorylation was not blocked by a PKA
antagonist, H-89. This result indicates that in B-104 cells,
cAMP activates ERK in a PKA-independent manner. ERK
has been considered a central player in neuronal plasticity.
Activation of ERK by psychostimulants could not be
achieved in mice lacking dopamine- and cAMP-regulated
phosphoprotein DARPP-32 (Valjent et al., 2005). Furthermore, incapacitating the ERK blocks psychostimulant-in-
ERK Up-Regulates ␴-1 Receptors
expression and that up-regulation of Sig-1R seen in the brain
of psychostimulant self-administering or sensitized animals
may involve DA and the activation of DA receptors and
associated signaling pathways in a manner that need to be
totally clarified. Psychostimulants like cocaine can affect
other biological systems such as voltage-gated ion channels
(Nasif et al., 2005). The contribution of other biological systems, in addition to those described here, to the regulation of
Sig-1R expression is unknown at present.
Acknowledgments
We thank F. Cambi for the generous gift of B-104 cells.
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Address correspondence to: Dr. Tsung-Ping Su, Cellular Pathobiology
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E-mail: [email protected]
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