©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Cannabinoid Receptor CB1 Induces krox-24 Expression in Human Astrocytoma Cells (*)

Monsif Bouaboula , Bernard Bourrié , Murielle Rinaldi-Carmona , David Shire (2), Gérard Le Fur (1), Pierre Casellas (§)

From the (1) From Sanofi Recherche, 371 rue du Professeur Joseph Blayac, 34184 Montpellier, Sanofi Recherche, 32-34 rue Marbeuf, 75008 Paris, France, and (2) Sanofi Recherche, voie 1, BP 137, 31676 Labège Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Although the psychoactive and medicinal properties of marijuana have been well known for centuries, the mechanism of action of -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).

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.


MATERIALS AND METHODS

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.


RESULTS

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 ngml) 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 ngml PMA; lane8, 30 ngml 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 ngml pertussis toxin; lane6, 100 ngml pertussis toxin; lane7, 30 ngml PMA; lane8, 30 ngml PMA + 100 ngml 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/GProtein-dependent, cAMP-independent Transduction Pathway

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 or G family. The treatment did not affect a PMA activation, eliminating the possibility of a nonspecific effect of PTX (Fig. 5C).

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-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 ngml PMA; lane6, 30 ngml 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.


DISCUSSION

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.

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.

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.

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 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.

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 and C stimulation or ion channel modulation (data not shown).

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 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-67-10-62-90; Fax: 33-67-10-67-67.

The abbreviations used are: -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.


ACKNOWLEDGEMENTS

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.


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