From the
The recent isolation and cloning of the G protein-coupled
central cannabinoid receptor (CB1) from brain tissue has provided a
molecular basis to elucidate how cannabinoid compounds may mediate
their psychoactive effects. Here we report the high expression of
cannabinoid receptors in human astrocytoma tumors of different grades,
in the astrocytoma cell lines U373 MG and GL-15, as well as in normal
astrocytes. From an analysis of the coupling mechanisms of functional
CB1 receptors in U373 MG, we show that, in addition to the inhibition
of adenylyl cyclase, activation by the cannabinoid agonist CP-55940
induces the expression of the immediate-early gene krox-24,
also known as NGFI-A, zif/268, egr-1, and TIS8. The
amount of Krox-24 protein and the level of Krox-24 DNA binding
activity, as measured by Western blot and electrophoretic mobility
shift assay, respectively, were also increased by the addition of
CP-55940. These effects were blocked by incubation with pertussis toxin
but not by treatment with hydrolysis-resistant cAMP analogues,
suggesting that the transduction pathway between the cannabinoid
receptor and krox-24 involves a pertussis toxin-sensitive
GTP-binding protein and is independent of cAMP metabolism. The specific
involvement of CB1 in Krox-24 induction was demonstrated in Chinese
hamster ovary cells transfected with the human CB1 receptor and also in
experiments using the CB1-selective cannabinoid antagonist SR 141716A.
Although the psychoactive and medicinal properties of marijuana
have been well known for centuries, the mechanism of action of
Astrocytes have long been considered
to be supportive rather than transmissive in the nervous system.
However, recent studies have challenged this assumption by
demonstrating that astrocytes (i) possess functional neurotransmitter
receptors and (ii) directly modulate free cytosolic calcium ions,
suggesting they affect the transmission characteristics of neighboring
neurons (Nedergaard, 1994).
The objectives of the present study were
2-fold: first, the evaluation of the expression of central cannabinoid
receptors in astrocytes; and second, the exploration of the biological
functions associated with these receptors.
We show here that
astrocytoma cell lines, biopsies of astrocytoma tumors of different
grades, and normal astrocytes all express mRNA for the central
cannabinoid receptor CB1. Using the human astrocytoma cell U373 MG,
which expresses a particularly high level of CB1, we investigated the
effects of cannabinoid agonists on cAMP accumulation and also on the
regulation of gene expression. Candidate genes selected to be
potentially regulated by cannabinoids were immediate-early genes (IEG)
from the leucine zipper family (c-fos, c-jun, and
jun-B) and the zinc finger family (Krox-20 and
krox-24). We observed that cannabinoid agonists, in addition
to the inhibition of the cAMP accumulation, activate gene expression of
several transcription factors. To investigate the mechanism of action
by which cannabinoid agonists produce this latter effect and to
establish that it is specifically mediated by CB1, experiments were
also performed on CHO cells stably expressing the transfected human
cannabinoid receptor CB1. Finally, the specificity of these cannabinoid
actions was determined using the recently described selective CB1
receptor antagonist, SR 141716A (Rinaldi-Carmona et al.,
1994). The data presented here raise the possibility that the
cannabinoid receptor and its corresponding endogenous ligand might
influence astrocyte functions and support a regulatory role for CB1
receptors in astrocyte physiology in vivo.
Comparison of Fig. 3B and
5A shows that the dose-response curves for the inhibition of
adenylyl cyclase correlate well with that of krox-24 induction. This could suggest that krox-24 activation is
the result of G
We examined whether the activation
of the cannabinoid receptor was coupled to IEG expression in U373 MG
cells. By quantitative RT-PCR, we showed that CP-55940 treatment
resulted in rapid and transient increases in Krox-20, krox-24,
and jun-B but not c-fos mRNA levels. Because the mRNA
expression levels of Krox-20 and jun-B are below the
sensitivity limit of the Northern blot method currently used in this
study, the effect of CP-55940 on IEG expression was examined in detail
only for krox-24, which is readily detectable and thereby
particularly useful for monitoring the cannabinoid effect on IEG
expression.
The induction of krox-24 mRNA by CP-55940 was
associated with an enhancement of synthesis of active Krox-24 protein.
This information was obtained from Western blot analysis using
anti-Krox-24 antibody and by measuring the Krox-24 DNA binding activity
by EMSA.
At this point, two hypotheses can be
formulated: either (i) two different cannabinoid receptors (CB1 and
CBx) are each coupled to a single type of effector system or
(ii) one receptor (CB1) is coupled to two separate effector systems.
Our data support the latter possibility since, in CB1-expressing CHO
cells, CB1 is functionally coupled not only to the inhibition of
adenylyl cyclase but also to the activation of krox-24 gene
expression with similar EC
The molecular mechanisms
located downstream from the G protein and leading to krox-24 activation by CP-55940 still remain unclear. We have ruled out the
possibility that the activation of krox-24 could be ascribed
to known PTX-sensitive G protein pathways, including those involving
phospholipases A
The phosphorylation of proteins is
often involved in cellular signal transduction. Preliminary experiments
show that induction of Krox 24 by CP-55940 failed to be abolished by
treatment with the PKC inhibitor GF109203X, whereas it was inhibited by
the tyrosine kinase inhibitor herbimycin A (data not shown). On this
basis, we propose that a protein tyrosine kinase could lie on the route
between G
We are grateful to Dr. M. Tardy for having provided us
with RNAs from astrocytoma tumors of different grades and Dr. M.
Portier for critical review of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-tetrahydrocannabinol (THC),
(
)
the main active principle of marijuana, began to be
described only a few years ago. It is now known that THC and other
potent synthetic cannabinoid agonists as well as anandamide, the
putative endogenous ligand (Devane et al., 1992), bind to
specific cannabinoid receptors and inhibit both adenylyl cyclase
(Howlett, 1985, 1987) and N-type calcium channels via a pertussis
toxin-sensitive guanine nucleotide-binding protein (G protein) (Mackie
and Hille, 1992). The cannabinoid receptor (CB1) cDNA has been cloned
from both rat and human, it codes for a protein belonging to the G
protein-coupled receptor superfamily (Matsuda et al., 1990;
Gérard et al., 1991). CB1 is expressed primarily in
brain tissue (Herkenham et al., 1991; Matsuda et al.,
1993), but CB1 mRNA has also been found in testis (Gérard et
al., 1991), spleen (Kaminski et al., 1992), and
leukocytes (Bouaboula et al., 1993). A second cannabinoid
receptor (CB2) has been cloned and characterized recently. CB2 is not
expressed in brain but in macrophages from the marginal zone of spleen
(Munro et al., 1993).
Reagents
CP-55940 was obtained from Pfizer.
[H]CP-55940 was purchased from Du Pont NEN.
-THC, 8-bromoadenosine 3`,5`-cyclic monophosphate, and
staurosporin were from Sigma. WIN 55212-2 and pertussis toxin (PTX)
were obtained from Research Biochemicals Inc. The bisindolylmaleimide
(GF 109203X) was from Calbiochem. Phorbol ester (PMA) and herbimycin A
were from Life Technologies, Inc. Dibutyryl cAMP and
3-isobutyl-1-methylxanthine were from Boehringer Mannheim.
Anti-krox-24 antibody was from Santa Cruz Biotechnology.
Oligonucleotides were synthesized on a Biosearch 8750 automated
synthesizer (Millipore). SR 141716A
(N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
hydrochloride) was synthesized at the Chemistry Department, Sanofi
Recherche (Montpellier, France) as described (Rinaldi-Carmona et
al., 1994). The structure of SR 141716A is given in Fig. 1.
Figure 1:
Chemical
structure of SR 141716A.
Preparation of Astrocytes
Purified astrocytes were
prepared from newborn Sprague-Dawley rat hippocampi using a
modification of the method previously described (Robbins et
al., 1987). Briefly, hippocampi were dissected from newborn rats
and the meningeal tissue was stripped off. Hippocampi were mechanically
dissociated, and cells were passed through a sterile 70-µm nylon
sieve into nutrient medium. The medium consisted of Dulbecco's
modified Eagle's medium (Seromed)/Ham's F-12 medium
(Boehringer) (1/1) containing 10% fetal calf serum, 2 mM
glutamine, 0.1% gentamycin. After washing, cells were layered on a
Percoll density gradient (Pharmacia Biotech Inc.) consisting of
50%-30%-10% Percoll solution diluted in phosphate-buffered saline
(PBS). After centrifugation for 15 min at 5000 g,
cells separated from red blood cells and cellular debris were carefully
pipetted out, washed once in PBS, and washed once in nutrient medium.
The hippocampal cells were plated at 4
10
cells/100-mm Petri dish and incubated at 37 °C in a
humidified 5% CO
, 95% air atmosphere. The total RNA was
isolated when astrocytes reached confluence (21 days in
vitro). The purity of the astrocytes was 97% as determined by
staining for glial fibrillary acidic protein as described (Eng, 1985).
Expression of Human CB1 Receptor in CHO Cells
CB1
cDNA from IM-9 (Shire et al., 1995) was amplified with a sense
primer bearing a HindIII site and a Kozak consensus sequence
(5`-CCACACAAGCTTGCCACCATGGAGGAATGCTGGGTG) and an antisense primer
bearing an EcoRI site (5`-CCACTCGGATCCTCAGCAATCAGAGAGGTCTAG).
The amplicon was digested with HindIII/EcoRI and
inserted into p658, an expression plasmid derived from p7055 (Miloux
and Lupker, 1994) in which the interleukin-2 coding sequence was
replaced by a polylinker. The CB1 expression vector was transfected
into CHO dihydrofolate reductase-negative cells by a modified
CaPO precipitation method (Graham and Van der Eb, 1973).
CHO-transfected cells are referred as CHO-CB1 cells.
Cell Lines
The human lymphoblastoid cell line IM9
(Van Boxel and Buell, 1974) and the human promyelomonocytic cell line
HL60 (Collins et al., 1977) were maintained in suspension and
grown at 37 °C in a humidified 5% CO, 95% air
atmosphere in culture medium consisting of RPMI 1640 supplemented with
10% heat-inactivated fetal calf serum, 2 mM glutamine, and
streptomycin (100 µg/ml) and penicillin (100 units/ml). U373 MG
astrocytoma cells (Preissig et al., 1979) from ATCC were grown
as monolayers in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2 mM glutamine,
penicillin (100 units/ml) and streptomycin (100 µg/ml), 1%
vitamins, and 1 mM sodium pyruvate. U373 MG cells were
synchronized for 24 h in serum-free medium prior to CP-55940 treatment.
CHO cells stably transfected with CB1 (CHO-CB1) and the wild-type CHO
cells were grown as monolayers in minimal essential medium supplemented
with 10% dialyzed fetal calf serum, 2 mM glutamine, 40
µg/ml L-proline, 60 µg/ml Tylocine, 1 mM
sodium pyruvate, and 5 µg/ml gentamicin. CHO cells and CHO-CB1 were
maintained in 0.5% fetal calf serum medium for 24 h before CP-55940
treatment.
Radioligand-binding Assays
For binding
experiments, U373 MG and CHO-CB1 cells were seeded in 225-cm culture flasks and grown to confluence. Cells were collected by
scraping and spun at 1000
g for 10 min at 25 °C.
The cell pellet was then resuspended in binding buffer (0.05 M
Tris-HCl, pH 7.7). Cells (0.5
10
cells) were
incubated at 30 °C with [
H]CP-55940 in 1 ml
of binding buffer for 1 h. A rapid filtration technique using Whatman
GF/C filters (pretreated with 0.5% polyethyleneimine (w/v) and a
48-well filtration apparatus (Brandel) was used to harvest and rinse
labeled cells (three consecutive washes with 5 ml of cold buffer (0.05
M Tris-HCl, pH 7.7, containing 0.25% bovine serum albumin).
Filter-bound radioactivity was counted with 4 ml of Biofluor liquid
scintillor. Nonspecific binding was determined in the presence of 1
µM CP-55940. In competition experiments, the drug
concentration producing 50% inhibition (IC
) of radioligand
binding and the Hill coefficient (n
) values were
determined from Hill plots of log((B
-
B)/B) versus log (concentration) of test
drug, where B
and B are specific binding
in the absence and presence of competitor, respectively. Inhibition
constant (K
) values were calculated from
IC
values using the Cheng and Prusoff equation (Cheng and
Prusoff, 1973).
Extraction of Cellular RNA, Reverse Transcriptase
(RT)-PCR, and Southern Blot Analysis
Total RNA was isolated from
astrocytoma cells (U373 MG), human lymphoblastoid cells (IM9), human
monocytic cell line (HL-60), rat astrocytes, and rat cortex by lysis in
guanidinium isothiocyanate and purified by CsCl gradient
ultracentrifugation. Human brain RNA was obtained from Clontech. RNAs
from GL-15 and from astrocytoma tumors of different grades
(Daumas-Duport et al., 1988) were gifts from Dr. M. Tardy. For
RT-PCR analysis, total RNAs were treated with DNase I for 30 min,
purified by two phenol-chloroform extraction steps, and then
ethanol-precipitated. RNAs were resuspended in reverse transcriptase
buffer with oligo(dT) primers and Moloney murine
leukemia virus reverse transcriptase Superscript II (Life Technologies,
Inc.) and incubated for 60 min at 45 °C. PCR amplification was
carried out using the primer pair 5`-TATATTCTCTGGAAGGCTCACAGCC-3` and
5`-GAGCATACTGCAGAATGCAAACACC-3` (for amplification of a 270-bp product
for human and rat CB1) or 5`-TTTCCCACTGATCCCCAATG-3` and
5`-ATGGAGTTGATGAGGCACAG-3` (for human CB2, 333-bp product) or
5`-TTTCACGGTGTGGACTCC-3` and 5`-TAGGTAGGAGATCAAGCG-3` (for rat CB2,
214-bp product). The steps of amplification were 95 °C, 20 s, 60
°C, 30 s, 75 °C, 40 s per cycle and performed with a 9600
cycler according to the manufacturer's instructions (Perkin
Elmer). For rat CB2, PCR amplification conditions were 94 °C, 1
min, 54 °C, 1 min, and 72 °C, 1 min. Southern blot analysis was
performed as described (Bouaboula et al. 1992) using the
horseradish peroxidase-linked oligonucleotide probes
5`-TGATCAACACCACCAGGATC-3` and 5`-AGCAAAGGCCTTCTTGACCT-3` for CB1 and
CB2, respectively. No PCR product was detected when reverse
transcriptase was omitted, indicating that the PCR products derived
specifically from cDNA (data not shown). Comparative quantification of
CB1 mRNA level was performed in the exponential phase of amplification
using
-microglobulin as external control (Dornand
et al., 1992).
Quantitative RT-PCR Using a Multispecific cRNA
Standard
Quantitative RT-PCR analysis using a competitive
multispecific cRNA standard was performed as described previously
(Bouaboula et al., 1992; Legoux et al., 1992). In
brief, constant amounts of cellular RNA (1 µg) were spiked with
known variable amounts of standard cRNA (pQB-2) using serial 1:2.5
dilutions (from 10 to 6.7
10
molecules), then were co-reverse-transcribed using
oligo(dT)
and Moloney murine leukemia virus
reverse transcriptase Superscript II. The appropriate primer pairs were
added to 1/10 of the cDNA mixture, after which 35 cycles of PCR were
carried out in the presence of 1 µCi of
[
-
P]dCTP (3000 Ci/mmol). The primer pairs
used are: 5`-GAGCTGACTGATACACTCCA-3` and 5`-GCTCTTGACAGGTTCCACTG-3`
(c-fos), 5`-CCAAGAACGTGACAGATGAG-3` and
5`-AGTTGCTGAGGTTTGCGTAG-3` ( jun-B),
5`-GCTGTCGACAACATCTACC-3` and 5`-ACTGTGGGTCAAGGAGAAC-3` (Krox-20)
5`-ATTGTGAGGGACATGCTCAC-3` and 5`-ACAAAAATCGCCGCCTACTC-3`
(krox-24); 5`-CCAGCAGAGAATGGAAAGTC-3` and
5`-GATGCTGCTTACATGTCTCG-3` (
-microglobulin). Standard
and cellular amplicons were separated by polyacrylamide gel
electrophoresis and visualized by ethidium bromide staining, the bands
were excised, and the radioactivity was determined by scintillation
counting. The ratio of standard amplicon/cellular amplicon intensities
was plotted against the quantity of standard cRNA on a log-log scale
(not shown). The quantity of cellular RNA was obtained by reading from
the graph the quantity of standard cRNA for which the ratio standard
amplicon/cellular amplicon was unity.
Northern Blot
Northern blot was performed
following standard procedures (Church and Gilbert, 1984). The
krox-24 and -microglobulin probes were
prepared from a purified radiolabeled PCR product obtained using the
primers specific for krox-24 and
-microglobulin described above.
Cyclic AMP Analysis
U373 MG cells were grown to
confluence, washed, and incubated for 30 min in serum-free medium
containing Ro-201724 (0.25 mM), IBMX (0.1 mM), and
forskolin (5 µM), supplemented or not with cannabinoids.
The reaction was ended by the addition of 0.1 M HCl.
Determination of cAMP levels was performed by radioimmunoassay
according to the manufacturer's instructions (Pharmacia). In some
experiments, cells were cultured in complete medium in the presence of
pertussis toxin for 18 h prior to forskolin treatment. Each data point
is the mean of triplicates, and experiments were repeated twice.
Electrophoretic Mobility Shift Assay (EMSA)
U373
MG cells were stimulated for 90 min with the various agents and washed
with ice-cold PBS, and the nuclei were isolated by treatment with 0.1%
Nonidet P-40. Nuclear extracts were prepared as described by Dignam
et al.(1983) with minor modifications (Cao et al.,
1993). Synthetic oligonucleotides containing a single copy of Krox-24
binding site (5`-CCCGGCGCGGGGGCGATTTCGAGTCA-3`) (Cao et al.,
1993) or Oct-1 binding site
(5`-AATTGCATGCCTGCAGGTCGACTCTAGAGGATCCATGCAAATGGATCCCCGGGTACCGAGCTC-3`)
(Schöler et al., 1989) were 5`-end-labeled with T4
polynucleotide kinase and [-
P]ATP (3000
Ci/mmol). The binding reaction was performed by preincubating 10 µg
of nuclear protein with 0.8 µg of double-stranded poly(dI-dC)
(Pharmacia) for 10 min on ice in a buffer containing 20 mM
Hepes, pH 7.9, 70 mM KCl, 5 mM MgCl
, 10
µM ZnCl
, 2 mM dithiothreitol, and 12%
glycerol. 0.5 ng of the labeled probe (about 40,000 cpm) was added to
the mixture containing the nuclear extract, and the reaction was
incubated for 30 min at room temperature. The samples were loaded on a
6% polyacrylamide gel in 0.25
TBE buffer (1
TBE
consists of 0.089 mM Tris borate, 0.089 mM boric
acid, and 2 mM EDTA), and the gel was run at 120 V at 4
°C. For competition experiments, an excess of cold oligonucleotides
was added to the reaction mixture 10 min prior to the addition of the
radiolabeled probe. Similarly, in some experiments, 0.5 µg of
anti-Krox-24 antibody was added to the reaction mixture and incubated
for 60 min prior to the addition of the radiolabeled probe.
Western Blot
Proteins were obtained by direct
lysis of the cells in Laemmli's loading buffer containing 6
M urea and equal amounts were run on polyacrylamide gel
electrophoresis (Laemmli, 1970). After transfer onto a 0.45-µm
nitrocellulose membrane (Prolabo) according to standard procedures
(Towbin et al., 1979), immunodetection was carried out with an
affinity-purified rabbit antibody directed against Krox-24 (0.25
µg/ml), and a horseradish peroxidase-coupled anti-rabbit IgG
antibody and the Amersham chemiluminescence detection system.
Identification and Distribution of Cannabinoid Receptor
CB1 in Human Astrocytoma Cell Lines, Astrocytoma Tumors, and Rat
Astrocytes
The analysis by RT-PCR of the CB1 and CB2 expression
pattern revealed that human astrocytoma cell lines, as well as
astrocytoma tumors of different grades and rat normal astrocytes,
express CB1 but not CB2 mRNA, the latter being only expressed in tumor
cells originating from peripheral tissues, such as IM9 and HL60
(Fig. 2A). The PCR products blotted onto nylon membranes
hybridized specifically with the relevant oligonucleotide probes.
Comparative quantification, as measured by RT-PCR, indicated that not
only did astrocytoma cells (U373 MG, GL-15) express CB1 mRNA, its level
was similar to or even higher than that observed in the brain
(Fig. 2B). A high level of central cannabinoid receptors
on human U373 MG astrocytoma cells was confirmed by binding studies
using the cannabinoid receptor ligand [H]CP-55940
which binds to this receptor in a monophasic and saturable manner with
a K
value of 0.54 ± 0.07
nM (n = 2) and a B
value of 9.5(± 0.8)10
sites/cell (n = 2) (Fig. 3A). Displacement of
[
H]CP-55940 binding to U373 MG cell membranes by
various cannabinoid ligands showed a specificity pattern conforming to
that reported for central cannabinoid receptors in neural tissues with
CP-55940 (K
= 3.86 ± 1.28
nM) >
-THC (K
= 16.8 ± 12 nM) > WIN 55212-2
(K
= 97.6 ± 24 nM)
(Rinaldi-Carmona et al., 1994). In these experiments, the
cannabinoid antagonist SR 141716A displayed a K
of 6.6 ± 1 nM.
Figure 2:
Expression of CB1 mRNA in U373 MG cells.
A, RT-PCR analyses of CB1 and CB2 gene expression in tumor
cell lines, astrocytoma tumors and brain tissues. cDNAs were amplified
with primers specific for CB1 or CB2, and the products were analyzed
after 35 amplification cycles. Left, ethidium bromide staining
of PCR products separated on agarose gel. Right,
autoradiographs of the corresponding Southern blots hybridized with
internal CB1 or CB2 receptor probes conjugated to horseradish
peroxidase. B, comparison of the levels of CB1 mRNA in tumor
cell lines and brain tissue. The levels of CB1 mRNA in each sample were
determined by comparative RT-PCR as described under ``Materials
and Methods.'' The CB1 mRNA level was taken as 100% in human brain
tissue. The levels evaluated in U373 MG, GL-15, and IM9 were 174, 95,
and 6%, respectively. CB1 was not detectable in HL-60
cells.
Figure 3:
Expression of cannabinoid receptor in U373
MG cells. A, equilibrium binding of
[H]CP-55940 to U373 MG cells was measured with
the indicated concentrations of [
H]CP-55940 and
0.5
10
cells/assay. Specific binding (
) of
[
H]CP-55940 is the difference between the binding
in the absence (
) and the presence (
) of 1 µM
unlabeled CP-55940. Inset represents the Scatchard plot for
the specific [
H]CP-55940 binding component (B = specific bound fraction in pmol/milligram of protein;
F = free fraction in nanomolar). B,
cannabinoid-induced inhibition of forskolin-stimulated cAMP production
in U373 MG cells. U373 MG cells were treated with 5 µM
forskolin and various concentrations of CP-55940 (CP) or WIN 55212-2
(WIN) for 30 min. cAMP levels were determined as described in
under ``Materials and Methods.'' Data represent the average
of cAMP accumulation ± S.E. of two independent determinations
performed in triplicate. Left, dose-response curve of CP-55940
and WIN 55212.2 on cAMP accumulation. EC
was 0.8 ±
0.3 nM for CP-55940 and 32 ± 9 nM for WIN
55212.2. Treating cells with PTX (100 ng
ml
)
blocked all CP-55940 inhibition of cAMP synthesis. Right,
effect of the cannabinoid antagonist SR 141716A on the inhibition
induced by CP-55940 (1 nM); IC
was 41 ± 8
nM. The cAMP level in non-stimulated cells was 0.5 pmol of
cAMP/10
cells.
CP-55940 Inhibits cAMP Production in Forskolin-stimulated
U373 MG Cells
Previous studies performed on cultured
neuroblastoma cells have demonstrated that cannabinoid drugs inhibit
cAMP production (Howlett et al., 1986; Howlett, 1987). The
ability of the cannabinoid receptor to elicit a similar functional
response was evaluated in U373 MG cells. When U373 MG cells were
stimulated with forskolin, cannabinoids led to a dramatic reduction of
cAMP in a dose-dependent manner. The EC for this
inhibition was in the nanomolar range in accordance with the
K
value for CP-55940. The higher potency
of CP-55940 compared with that of the agonist WIN 55212-2 to inhibit
adenylyl cyclase is consistent with their respective abilities to
displace [
H]CP-55940 binding
(Fig. 3B). The lack of a pharmacological antagonist for
cannabinoid receptors has hitherto prevented the discrimination between
specific and nonspecific effects (Felder et al., 1992). Using
the CB1 cannabinoid antagonist SR 141716A (Fig. 1), we could
establish that the effect on cAMP production is indeed
receptor-mediated (Fig. 3B). Treatment with pertussis
toxin (PTX), which is known to induce ADP-ribosylation of
G
/G
-type G proteins, thus preventing the
dissociation of their
and
/
subunits (Casey and Gilman,
1988), blocked this response as well, indicating that this mechanism
involves the GTP-binding protein G
/G
(Fig. 3B).
CP-55940 Induces krox-24, Krox-20, and jun-B but Not
c-fos Gene Expression in U373 MG Cells, an Effect That Is Specifically
Mediated by Cannabinoid Receptors
Immediate-early genes have
been described as acting as third messengers for astrocytes in that
they couple external stimuli to long term events related to cell
proliferation and differentiation (Hisanago et al., 1990). We
explored whether the activation of the cannabinoid receptor was coupled
to such gene expression in U373 MG cells. Because of the low amounts of
the corresponding mRNAs in these cells, we performed quantitative
RT-PCR using a competitive multispecific cRNA standard to analyze the
modulation of gene expression (Bouaboula et al., 1992; Legoux
et al., 1992). Among the different genes examined,
jun-B, Krox-20, and krox-24 genes were up-regulated
upon CP-55940 treatment, whereas c-fos mRNA was not affected
(Fig. 4). Northern blot analysis confirmed a reproducible 5-fold
increase in the krox-24 mRNA level. The response to CP-55940
was dose-dependent and detectable at concentrations as low as 0.1
nM (Fig. 5A). Agonist activation resulted in a
rapid but transient stimulation of krox-24 transcription,
which was maximal at 1 h and returned to near base line within 3 h
(data not shown). Because of their low expression levels,
jun-B and Krox-20 mRNAs were not detectable in the Northern
blot assay. Therefore, subsequent studies were focused on
krox-24. The cannabinoid antagonist SR 141716A, which had no
effect per se, completely prevented CP-55940-induced
krox-24 expression, whereas it had no effect when PMA was used
as krox-24 inducer (Fig. 5B).
Figure 4:
Effect of CP-55940 on immediate-early gene
expression in U373 MG cells. U373 MG cells were treated with or without
10 nM CP-55940 for 1 h at 37 °C, except for c-fos mRNA measurement where the incubation time was 30 min. Total RNA
were extracted from cells, and the respective levels of specific mRNAs
were determined by quantitative RT-PCR using a competitive
multispecific cRNA standard and primers specific for c-fos,
jun-B, Krox-20, krox-24, and
-microglobulin (
µ) as described
under ``Materials and Methods.'' mRNA content in cells
treated with CP-55940, arbitrarily taken as 100%, was compared to that
measured in cells not treated.
Figure 5:
krox-24 is specifically inducible
in U373 MG cells by the cannabinoid agonist CP-55940. A,
effect of the concentration of CP-55940 on krox-24 gene
expression in U373 MG cells. U373 MG cells were incubated for 1 h at 37
°C with or without various concentrations of CP-55940: lane1, untreated cells; lane2, 1000
nM CP-55940; lane3, 10 nM
CP-55940; lane4, 0.1 nM CP-55940. Total RNA
was fractionated on a 1.0% agarose gel under denaturing conditions, and
the Northern blot was hybridized with a P-labeled
krox-24 probe. Ethidium bromide staining of 18 and 28 S rRNAs
confirms that equal amounts of RNA were loaded (inset).
B, specificity of CP-55940 action on krox-24 gene
expression in U373 MG cells. RNA was fractionated on a 1.0% agarose gel
and the blot hybridized with the
P-labeled krox-24 probe. The blot was also hybridized with a
P-labeled
-microglobulin probe to provide an internal control
for the amount of RNA loaded in each lane. U373 MG cells were exposed
for 1 h at 37 °C to the following conditions: lanes1 and 2, untreated cells; lanes3 and
4, 30 nM CP-55940; lane5, 30
nM CP-55940 + 1 µM SR 141716A; lane6, 1 µM SR 141716A; lane7, 30 ng
ml
PMA; lane8, 30 ng
ml
PMA + 1
µM SR 141716A. C, effect of pertussis toxin and
cAMP on CP-55940-induced krox-24 transcription in U373 MG
cells. RNA was analyzed as in panelB. U373 MG cells
were incubated under the following conditions: lanes1 and 2, untreated cells; lanes3 and
4, 30 nM CP-55940; lane5, 30
nM CP-55940 + 100 ng
ml
pertussis
toxin; lane6, 100 ng
ml
pertussis toxin; lane7, 30
ng
ml
PMA; lane8, 30
ng
ml
PMA + 100 ng
ml
pertussis toxin; lane9, 30 nM
CP-55940 + 0.1 mM IBMX + 1 mM dibutyryl
cAMP; lane10, 0.1 mM IBMX + 1
mM dibutyryl cAMP.
CP-55940 Induces krox-24 mRNA in U373 MG Cells via a
G
To further investigate the effector pathway
responsible for krox-24 induction in U373 MG cells, we first
examined the effect of PTX. As shown in Fig. 5C, the
cannabinoid receptor-mediated expression of krox-24 was
entirely blocked by overnight pretreatment with 100 ng/ml PTX,
indicating that this effect was mediated by G protein(s) of the G/G
Protein-dependent, cAMP-independent
Transduction Pathway
or G
family. The treatment did not affect a PMA
activation, eliminating the possibility of a nonspecific effect of PTX
(Fig. 5C).
-mediated inhibition of adenylyl cyclase. If
so, augmenting cAMP levels should prevent krox-24 activation.
To explore this possibility, krox-24 gene expression was
analyzed in cells treated by a combination of CP-55940 and
hydrolysis-resistant cAMP analogues associated with phosphodiesterase
inhibitors to maintain high cellular levels of cAMP. As shown in
Fig. 5C, dibutyryl cAMP plus IBMX alone significantly
increased the level of krox-24 mRNA. In contradiction with the
above prediction, the combination of these treatments with CP-55940 did
not prevent cannabinoid stimulation but instead generated an enhanced
signal corresponding to an additive effect of the two stimuli, as might
be expected for agents acting on different transduction pathways.
Identical results were obtained by substituting 8-bromo-cAMP for
dibutyryl cAMP (data not shown). These results argue against the
possibility that krox-24 activation is secondary to a
G
-mediated fall in basal cAMP and suggest that CP-55940
acts independently of cAMP production.
CP-55940 Induces Krox-24 Protein and Krox-24 DNA Binding
Activity in U373 MG Cells
The observation of the enhancement of
krox-24 at the mRNA level prompted us to determine whether
such an effect could also be detected at the protein level. Cellular
extracts from U373 MG cells stimulated or not with CP-55940 were
subjected to Western blot analysis using anti-Krox-24 antibodies. The
results illustrated in Fig. 6A show that CP-55940
induced krox-24 protein and that the treatment with SR 141716A
completely prevented this induction.
Figure 6:
Induction by CP-55940 of Krox-24 binding
to its consensus sequence. A, Western blot analysis of Krox-24
accumulation in U373 MG cells (leftpanel),
CB1-transfected CHO cells (middlepanel), and
wild-type CHO cells (rightpanel) after 1.5 h of
CP-55940 treatment. B, nuclear proteins extracted from
10 U373 MG cells were incubated with a
P-labeled oligonucleotide probe containing the consensus
binding site for Krox-24 (upper panel) or for Oct-1 (lowerpanel). EMSA were processed as described under
``Materials and Methods.'' Lane1,
untreated U373 MG cells; lane2, cells treated with
30 nM CP-55940; lane3, 30 nM
CP-55940 + 1 µM SR 141716A; lane4,
1 µM SR 141716A; lane5, 30
ng
ml
PMA; lane6, 30
ng
ml
PMA + 1 µM SR 141716A.
Lane7, control with free probe. Lane8, mobility shift competition assay with a nuclear
extract from CP-55940-induced U373 MG and
P-labeled probe
in the presence of a 30-fold excess of unlabeled probe (Krox-24 and
Oct-1 in upper and lowerpanels,
respectively) for nonspecific DNA-protein complex. Lane9, the binding reaction was performed after a 60-min
preincubation of nuclear extracts prepared from 30 nM
CP-55940-treated U373 MG cells with 0.5 µg of Krox-24 rabbit
antiserum.
Krox-24 is a nuclear
phosphoprotein containing three zinc finger motifs of the
Cys-His
subclass, which binds to a specific
GC-rich consensus DNA sequence (CGCCCCCGC) in a zinc-dependent manner
(Lemaire et al., 1990). We quantified the Krox-24 binding
activity by EMSA on nuclear extracts from U373 MG cells using a labeled
oligonucleotide containing the consensus Krox-24 binding site. As shown
in Fig. 6B, a significant increase in the intensity
ofa retarded band could be detected. This band reflects the
binding of specific factors, since its formation is prevented by the
presence of an excess of unlabeled Krox-24 oligonucleotide. The complex
could also be supershifted by incubation with a Krox-24 antibody,
consistent with the binding of the antibody to the complex, thereby
inducing a decrease in its mobility. SR 141716A, as expected, had no
effect on PMA activation and blocked CP-55940 induction. As a control
for specific Krox-24 binding, the binding activity of the ubiquitous
Oct-1 sequence was not affected by CP-55940 or PMA treatments.
Cannabinoid Stimulation Induces krox-24 through the
Cannabinoid Receptor Subtype CB1
Although only the cannabinoid
receptor subtype CB1 was found to be expressed in astrocytoma cells,
the possibility cannot be excluded that a cannabinoid agonist might act
through different unknown receptor subtypes, which might account for
the different types of responses displayed by U373 MG cells upon
CP-55940 activation. To investigate this, we transfected CHO cells with
human CB1 cDNA and generated a stable transfectant exhibiting specific
[H]CP-55940 binding. Scatchard analysis revealed
that the dissociation constant (K
) and
the maximal binding (B
) were 0.65 ± 0.065
nM and 2.36 ± 0.16 pmol/mg of protein, respectively.
The results obtained from these cells showed that not only was adenylyl
cyclase inhibited (data not shown) but also that Krox-24 expression was
activated by cannabinoid stimulation. CP-55940 treatment of CHO-CB1
cells induced Krox-24 protein as assessed by Western blot analysis
(Fig. 6A). This induction was inhibited by SR 141716A
(Fig. 6A) or PTX treatment (data not shown). In
wild-type CHO cells, PMA, but not CP-55940, could induce Krox-24
expression (Fig. 6A). This suggests that the CB1
activation leads to both cAMP and Krox-24 modulation. These findings
also strongly support the proposal that the effects described above for
U373 MG cells treated with CP-55940 are very likely to be mediated by
the CB1 receptor.
Expression of CB1 Receptor in Human Astrocytoma Cell
Lines
By both mRNA and radioligand binding studies, we here
provide evidence for the expression of CB1 receptors by human
astrocytoma tumors of different grades, as well as astrocytoma cell
lines and normal rat astrocytes. The alternative receptor for
cannabinoids of the peripheral type (CB2) is not expressed in these
cells. Quantitative mRNA analysis revealed that astrocytoma cells
exhibited unexpectedly high levels of CB1. These results were confirmed
by radioligand binding studies on the astrocytic U373 MG cells, which
displayed binding characteristics similar to those previously described
in neuronal tissues. Collectively, these data are consistent with the
expression of the CB1 receptor in astrocytes. This conclusion is in
agreement with the recent observation that astrocytes can bind
[H]anandamide (Di Marzo et al., 1994).
The high level of expression observed here, along with the absence or
low expression in the white matter described previously (Mailleux and
Vanderhaeghen, 1992; Jansen et al., 1992), predicts that CB1
receptor could be differentially expressed in some subpopulations of
glial cells that remain to be identified.
Cannabinoid Receptor Stimulation Induces krox-24 Gene
Expression in U373 MG Cells
It is well established that one
pathway of the signal transduction from CB1 is mediated by the
PTX-sensitive inhibition of adenylyl cyclase in neuroblastoma cells
(Howlett, 1985; Matsuda et al., 1990). We show here that U373
MG cells also share this functional property. Interestingly, we noted
that whereas the cannabinoid-induced inhibition of adenylyl cyclase
stimulated by forskolin in neuroblastoma cell lines was limited to
30-40% (Howlett and Fleming, 1984), more than 95% inhibition
could be attained in U373 MG cells. Moreover, in experiments using a
10-fold higher concentration of forskolin (50 µM),
inducing a 10-fold higher cAMP production, treatment with 10
nM of the cannabinoid agonist CP-55940 still inhibited cAMP by
more than 90% (data not shown). This indicates that the cannabinoid
receptor is coupled, after activation, to most of the cellular adenylyl
cyclases in U373 MG cells, making this cell line a very useful model to
study the functional aspect of this receptor. It is noteworthy that the
adenylyl cyclase activity of C6 glioma cell membranes was not affected
by cannabimimetic agents (Howlett et al., 1986), supporting
the above notion that cannabinoid receptor expression is restricted to
a subpopulation of glial cells.
Specificity of CP-55940 Action
The possibility
that cannabinoids might produce their effects merely by perturbating
membranes due to their lipophilic properties (Marx, 1990) has often
been raised (Tahir and Zimmerman, 1991; Watzl et al., 1991;
Felder et al., 1992). SR 141716A, which is a potent and
selective antagonist for the cannabinoid receptor CB1 subtype, was used
to establish the specificity of CP-55940 action in U373 MG cells. SR
141716A is a very selective ligand for CB1 since it has a 1000-fold
higher affinity for CB1 than for the CB2 receptor (Rinaldi-Carmona
et al., 1994). Using this molecule, our results unequivocally
demonstrate that the effects observed on both cAMP production and IEG
induction are cannabinoid receptor-mediated. We further documented that
CP-55940 acts specifically on the receptor by extending the
demonstration to CB1-expressing CHO cells, whereas wild-type CHO cells
remained unaffected.
Cannabinoid Stimulation Induces Multiple Signal
Transduction Pathways in U373 MG Cells through the Cannabinoid Receptor
Subtype CB1
We further showed that krox-24 induction,
although blocked by PTX treatment, was not affected by treatment with
hydrolysis-resistant cAMP analogues. This implies that the transduction
pathway between the cannabinoid receptor and krox-24 involves
a heterodimeric G protein of the G or G
subfamily and is not secondary to the inhibition of adenylyl
cyclase. Therefore, we conclude that CP-55940 inhibits cAMP production
and induces krox-24 expression through simultaneous but
independent pathways.
values, both pathways being
completely blocked by treatment with PTX or SR 141716A. These results
strongly suggest that the cannabinoid receptor CB1 independently
promotes both the inhibition of cAMP production and IEG induction via a
PTX-sensitive G protein in U373 MG cells.
and C stimulation or ion channel
modulation (data not shown).
and krox-24. The release of
subunits through the activation of G
-coupled receptors is
generally responsible for positive signal induction (Clapham and Neer,
1993). An interesting possibility, which remains to be explored, would
be that
subunit dimers rather than
subunits have a direct role in G
-krox-24 coupling through tyrosine kinase.
Concluding Remarks
The psychoactive effects of
cannabinoids on mood, memory, movements, and pain have been attributed
to neuronal signaling (Hollister, 1986). Our data presented here
suggest that the potential cannabinoid action in brain could be
extended to subpopulations of glial cells via the CB1 receptor. We show
that activation of this receptor results in multiple cellular responses
including both adenylyl cyclase inhibition and expression of the IEG
krox-24, Krox-20, and jun-B. The absence of c-fos modulation by CP-55940 indicates a very selective response to
cannabinoid agonist. The rapid changes in IEG expression, which are
believed to be involved in long term biological events, support a
regulatory role for CB1 in astrocyte functions in vivo.
Several physiological roles have been allotted to astrocytes: metabolic
and trophic support of neurons, guidance for neuronal migration, and
axonal elongation during brain development, immunological functions as
antigen-presenting cells, cellular reactivity to brain injuries, and
the recently described direct signaling from astrocytes to neurons
(Nedergaard, 1994). Further studies are needed to elucidate the
potential contribution of the CB1 receptor in these functions.
-THC,
-tetrahydrocannabinol; IEG, immediate-early gene; PMA,
phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; PTX,
pertussis toxin; CHO, Chinese hamster ovary; PCR, polymerase chain
reaction; RT, reverse transcriptase; bp, base pair(s); IBMX,
isobutylmethylxanthine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.