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Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells
J. Steroid Biochem. Molec. Biol. Vol. 41, No. 3-8, pp. 621~31, 1992
0960-0760/92 $5.00+ 0.00 Copyright© 1992PergamonPresspie
Printed in Great Britain.All rightsreserved
DEMONSTRATION AND
OF
STEROID OF
STEROID
ACTION
IN
RAT
GLIAL
HORMONE PRIMARY
RECEPTORS CULTURES
CELLS
INGRIDJUNG-TESTAS,l* MICHELRENOIR,1 HELENEBUGNARD,I GEOFFREYL. GREENE2 and ETIENNE-EMILEBAULIEU1 ~Universit6 Paris XI and INSERM U33, Laboratoire Hormones, 94275 Bic&re, France and 2The University of Chicago, The Ben May Institute, Chicago, IL 60637, U.S.A. Summary--Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 antiPR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [3H]R5020, [3H]dexamethasone, [3H]OH-tamoxifen or [3H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4-6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presence of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found. During primary culture, the effects of progesterone (P) and estradioi (E2) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by Ev Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.
INTRODUCTION Steroid hormones are known to act in neural tissues to affect brain development and behavior. These actions are thought to be mediated by specific intracellular receptors since the brain contains receptors for all five classes of steroid hormones, progestin, estrogen, androgen, glucocorticoid and mineralocorticoid, and they resemble those found in non-neural target tissues (for review see Ref. [1]). In preliminary studies, our group has given evidence of the occurrence and accumulation of pregnenolone and dehydroepiendrosterone in the brain of several mammalian species even in Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.
*To whom correspondence should be addressed.
the absence of steroidogenic organs, suggesting an "in s i t u " mechanism unrelated to the peripheral endocrine glands [2, 3]. In further studies we have shown that newborn rat glial cells in primary culture can synthesize pregnenolone and progesterone [4-6] and recently we have identified the presence of progesterone, glucoeorticoid, estrogen and androgen receptors (PR, G R , ER and AR) in these glial cells [7]. Estradiol treatment of the cultures induced PR, but in contrast, levels of GR, ER and A R remained constant in the same cells. In the present work we have compared the estrogeninduction of PR in glial cultures established either from male or female offsprings, and the expression of PR was studied in separated oligodendrocytes and astrocytes with the monoclonal K C 146 antibody against PR[8] by immunohistochemical techniques. Furthermore, using gradient ultracentrifugations, the composition 621
622
INGRIDJUNO-TESTASet al.
of the heterooligomeric 8.5S structure of GR was studied with specific antibodies against GR, hsp90 and against the 59 kDa protein. Finally, we have shown direct effects of steroid hormones on glial cell multiplication, morphology and differentiation and we suggest that glial cells are targets for steroid hormones.
EXPERIMENTAL
Primary glial cell cultures Primary cultures of glial cells were established from newborn Sprague-Dawley rats (postnatal days 0-2) as described previously [5]. Briefly, cerebral hemispheres were mechanically dissociated in culture medium after removal of the meninges, the cell suspension was filtered through a 82/~m nylon mesh and cells were plated on poly-L-lysine coated plastic dishes in Dulbecco's Modified Eagle Medium (DME) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 3% calf serum (CS), insulin (4.8/~g/ml), glucose (1.8 mg/ml), penicillin (100 UI/ml) and streptomycin (100/~ g/ml). At complete cell density (days 7 to 9 of culture), cells were diluted 4 times (after trypsin-treatment) and kept growing for 2 to 4 weeks in the presence or not of hormones and/or antihormones, as will be specified for each experiment. Hormones and antihormones were added every 2 days, the media were changed twice a week. Three days before the experiments, steroid-free charcoal-treated CS was used. Cultures enriched in oligodendrocytes were obtained by plating cells at high density during the first subculture on days 7-9. At day 20, oligodendrocytes were then detached selectively from the dense culture by flushing medium several times with a pipet on the culture surface. The detached oligodendrocytes were replated on poly-L-lysine coated dishes. Pure astrocytes were obtained by replating the resting underlayer of astrocytes at very low density. Oligodendrocytes were identified by the measure of galactocerebrosid (Gal C) and myelin basic protein (MBP), astrocytes by the measure of glial fibrillary acidic protein (GFAP) using indirect immunofluorescence staining. The immunofluorescence procedure is described in Refs [4, 5 and 7]. Staining of Gal C was done using a monoclonal antibody kindly given by B. Ranscht [9]. Monoclonal anti-MBP was purchased from Boehringer-Mannheim, monoclonal anti-GFAP from Bio-Yeda (Flow Laboratories, France).
Receptor binding For receptor measurements, cells, after 3-4 weeks of culture, were harvested, centrifuged, and resuspended in 1 ml DME containing radiolabeled steroids from 2 to 10 nM, with or without 1 #M of the corresponding unlabeled steroid. For labeling of PR, 20 nM radioinert cortisol was added to avoid binding to GR. Cells were labeled for l h at 37°C and after centrifugation, washed three times with 3 ml icecold PBS. All subsequent steps were performed at 0°C. During each washing cycle, cells were incubated for 5 min in the cold, to allow for non-specifically bound radioactivity to diffuse out of the intact cells. After washing, the cell pellet was resuspended in 0.5 ml of PGW-buffer [10mM potassium phosphate, 10% glycerol (v/v), 20 mM tungstic acid, pH 7.8] and cells were homogenized and centrifuged at 10,000g for 20 min at 4°C, the supernatant was either submitted to linear 5-20% sucrose density gradients prepared as described previously [10], or the radioactivity was counted in a liquid scintillation counter to determine specific binding, which was expressed as the difference between total binding and non-specific binding (measured in the presence of unlabeled competitor). The nuclear pellet was washed once with 1 ml PGW plus 1% (v/v) Triton X-100 and twice with the same buffer without Triton. The final pellet was resuspended in 0.5 ml PGW buffer containing 0.4 M KCI and extracted for 1 h. Soluble nuclear proteins were obtained after centrifugation at 10,000g for 30min and analyzed either on linear 5-20% sucrose density gradients or by counting the specific radioactivity. Internal standards for density gradients were fungal glucose oxidase (GO, S20,w= 7.9) and peroxidase (PO, S20.w= 3.6). In case of GR, aliquots of the PGW-KCI 0.4 M extracts were incubated for 4h at 4°C with either 100 #1 of culture medium of BUGR2 [11] or ~ 100 pg of either a rabbit antibody against the conserved 17 aminoacids at the carboxyl terminal end of hsp90 (anti-hsp90-174), or a rabbit antibody against the 18 amino acids at the carboxyl terminal end of p59 (anti-p59-173)[12] prior to the ultracentrifugation analysis. It is to note that anti-hsp90-174 also recognizes hsp70, in addition to hsp90 [12]. Immunolocalization of PR The KC 146 monoclonal antibody to human PR is a mouse IgG that recognizes the rat PR
Steroid hormone receptors and steroid action
[8]. Its cross-reactivity with rat PR was tested on tissue sections of rat uterus before the experiments. Indirect immunofluorescence techniques were used for the localization of PR, as described previously [7]. Briefly, cells, cultured on glass coverslips, were washed, fixed in 4% paraformaldehyde and permeabilized with 1% Triton in PBS for 5 min. After washing, the KC 146 antibody (diluted 1:50) was applied for 30 min, followed, after washing, by a fluoresceincoupled goat anti-mouse IgG for another 30 rain. The coverslips were mounted in Moviol.
RESULTS
Progestin receptor induction by estradiol (E2) in mixed glial cells Primary cell cultures prepared from neonatal rat forebrains consist of a bedlayer of astrocytes overlaid by process-bearing oligodendrocytes. Both cell types were identified by morphology and by immunostaining with antibodies against GFAP for astrocytes and against Gal C and MBP for oligodendrocytes [4, 5, 7]. After 3 weeks of culture, when cells are still actively proliferating, the presence of PR was measured by whole cell assays after incubating cells with [3H]Organon 2058 at different concentrations between 0.3 to 10nM, in the presence of 10 nM unlabeled cortisol to avoid binding to
GR. Parallel incubations were performed in the presence of excess unlabeled progesterone (P) to determine non-specific binding. Specific binding was expressed as bound [3H]Organon per mg of protein in cytosol and nuclear fractions. The treatment of the cells with E2 (50 nM) from day 10 onward of primary culture considerably increased the number of PR binding sites in both cytosol and nuclear fractions, and corresponded to ~2.700 and 10.000 binding sites per cell, respectively, in untreated and E~-treated cells (Fig. 1). When the antiestrogen tamoxifen was added to the cultures together with E2, the estrogen induction of PR was completely inhibited, whereas tamoxifen alone did not influence PR levels [7].
PR induction by E2 in glial cells prepared from male or female offsprings Primary cultures from newborn male or female pups were prepared and cells treated or not with E2 in order to examine if sex differences were seen in PR concentration and in its induction by the estrogen. Cells were labeled with two different ligand concentrations (2.0 and 5.0 nM [3H]Organon) and PR was measured by whole cell assays. As shown in Fig. 2, both male and female cultures possess specific PR binding sites PR I-1 []
d
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623
~
Control *E 2
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9
x
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Q. '*0
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/I
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5.0 7.5 10.0 nM [3H]-Org 2058
Fig. 1. Measure of PR in estrogen-treated or untreated glial cells. Cells were treated (or not) from days 10 to 25 of primary culture with 50nM E2. Control cells received vehicle only. On day 26, cells were recovered, incubated for ! h at 37°C with increasing concentrations of [3H]Org 2058 in presence of 20 nM radioinert cortisol, and with or without 1 # M radioinert P. Specific P R binding was determined in cytosol and nuclear fractions as described in Experimental. Nuclear fractions: A - - & + E2; • - - • control, cytosol: /x - - A + E2; O - - O control.
2.0 nM
5.0 nM
p"]-Org 2058 Fig. 2. Measure of PR in cultures from male or female offsprings. Primary cultures were established from male or female pups at day 1 of birth. Cells were treated with 50 nM E 2 (or not) and P R specific binding was measured after incubating cells with [3H]Org 2058 at a concentration of 2.0 or 5.0 nM, in presence of 20 nM radioinert cortisol, as described in Fig. I. Specific PR binding sites were determined. [] control cultures; l cells treated with E 2.
624
]NGRID JUNG-TESTAS et al.
Nuclear
Cytosol [3H]-Dex
[3H]-Org 2058 PR
Fractions
[3H]-Org 2058
GR
[3H]-Dex
PR
GR
60
[] 50
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[]
Control
Control
[] +E2
[] FE2
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20 10
10
30
90
08
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Ligand Concentration (nM) Fig. 3. Measure of PR and G R in estrogen-treated or untreated glial cells. Cells were treated with 50 nM E: (or not) as described in Fig. 1. PR and G R were determined after incubating cells for 1 h at 37°C with increasing concentrations of [3H]Org 2058 in presence of 20 nM radioinert cortisol, or with increasing concentrations o f [3H]dexamethasone, both in presence or absence of excess unlabeled competitor. Specific binding was determined in cytosol and nuclear fractions. [] cytosol control; m cytosol + E2; [] nuclear fraction, control; [] nuclear fraction + E 2.
and little difference of uninduced PR was seen at the two ligand concentrations. However, the estrogen-inducibility of PR was substantially increased in female cultures.
Among the four classes of receptors, PR, GR, ER and AR, only PR is inducible by E2 Glial cells in primary culture contain, in addition to PR, also GR, ER and AR binding
Cytosol
Nuclear
Fractions
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[3.)_. 1881
[3H]- Org 2 0 5 8
[3HI- OH-Tam
[3H1- R 1 8 8 1
PR
ER
AR
PR
ER
AR
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3O
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J~l Control
B+E2
~I+E 2
x /
x
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r
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~
I 7.O
:
rl
20
"
18o
Concentration (nM)
2.o 17.o
N_ ] 2,0 17.o
-
2.o l e o
2.o 17.~
Concentration (nM)
Fig. 4. Measure of PR, ER and A R in estrogen-treated or untreated glial cells. Cells were treated with 50 aM E 2 (or not) as described in Fig. I. For receptor determinations, cells were incubated with 2 concentrations of [3H]Org 2058 for PR, or of [3H]OH-tamoxifen for ER, or of [3HlR1881 for AR, during l h at 37°C. Excess unlabeled competitor was added in parallel incubations. Specific binding was determined in cytosol and nuclear fractions. [] cytosol control; [] cytosol + E~; [] nuclear fraction, control; [] nuclear fraction + E 2.
Steroid hormone receptors and steroid action
sites, as we have shown previously [7]. Therefore it was of interest to measure in parallel the different receptors in cells treated (or not) with E 2. We first compared the amount of PR and GR in the same primary culture, and as shown in Fig. 3, only PR binding sites in cytosol and nuclear fractions increased significantly in estrogen-treated cells, whereas the amount of GR binding sites remained unchanged. We then compared in another primary culture the amount of PR with those of ER and AR and again, only PR binding sites were increased by the estrogen treatment (Fig. 4). ER binding sites had decreased in E2 treated cultures. This can be explained by a number of ER binding sites still occupied by endogenous E2, despite the culture in steroid-free medium 2 to 3 days before the experiments.
Analysis of PR and GR by gradient ultracentrifugations As mixed glial cells proliferate actively until 4 to 5 weeks after primary culture, sufficient material could be obtained to analyze PR and GR on sucrose density gradients. In the presence of agonist, ([3H]Organon and [3H]dexamethasone, respectively), both receptors displayed a
625
9S form in the cytosol and a 4-6S form in nuclear 0.4 M KC1, tungstate ions containing fractions. In contrast, when the receptors were labeled with the antagonist [3H]RU486, a nonactivated 8.5S receptor complex was found in the nuclear fractions for both PR and GR [7]. The composition of the GR-8.5S complex was further analyzed after incubation with specific antibodies against the GR itself (BUGR2 [1 l]) and against non-hormone binding proteins known to associate with steroid hormone receptors in their non-activated, non-DNA binding form[13]. In whole cell extracts (containing both cytosoluble and nuclear GR), GR displayed a heterogenous structure in the presence of 0.4 M KCI and tungstate ions and migrated as 8.5 and 7.0S forms, respectively. Both peaks represented the receptor since BUGR2 shifted them to a 10S form [Fig. 5(a)]. However, the 7.0S form does not contain hsp90 nor hsp70 since it remained in a 7.0S form after incubation with the 174 anti-hsp antibody [Fig. 5(b)]. As described for other steroid hormone receptors, a 59 kDa protein is also present, at least partly, in the 8.5S complex since the anti p59 antibody (173) partially displaced the GR 8.5S forms [Fig. 5(c)].
15
~10
==
10
20
10 Fraction
20 Number
10
20
Fig. 5. Analysis of G R by gradient ultracentrifugations. Cells were exposed to [3H]RU486 for l h at 37°C, harvested and treated as described in Experimental. Aliquots (200/~l) of whole cells extracts in PGW-KC1 0.4 M were centrifuged prior ( O - - O , panels a, b and c) or after incubation with 50/~ l of BUGR 2 anti-GR monoclonal antibody ( A - - A , panel a) or after incubation with 100/~g of anti-hsp90 antibody 174 ( A - - A , panel b) or after incubation with 120/~g of anti-p59 antibody 173 ( k - - A , panel c). In panel b, BUGR 2 (50/~l) and anti-hsp90 antibody 174 (100/~g) were added simultaneously to the incubation medium (ll--r-l). Centrifugation was performed as described in Experimental with GO = glucose oxydase 7.9S, and PO = peroxidase 3.6S as internal standards.
626
INGRID JUNG-TESTAS et al.
Table 1. Indirect immunofluorescent staining of PR in oligodendrocytes and astrocytes prepared from male or female newborn rats
Rat
Glial
cells
PR-positive cells Oligodendrocytes
Astrocytes
~' CX ~CX 3'+E2
+ + + + + +
none + (+ -)
~+E 2
+ + + +
+
lmmunofluorescence staining of PR was done with the monoclonal anti-PR antibody KC 146, as described in Experimental. CX: control cultures; +E:: 50nM E2 was present from days 10 to 25 of culture. ( + - ): very few positive cells; + : some positive cells; + +: many positive cells; + + +: ~70% positive cells; + + + + : almost all cells were PR-positive.
{7"
2,0 +E 2 +RU
16 - -
In previous experiments with the anti-PR monoclonal antibody KC 146, we were able to visualize PR in glial cells by the biotin-avidinimmunoperoxidase procedure and by indirect immunofluorescence staining [7]. By both techniques it was clearly seen that PR staining was preferentially localized in oligodendrocytes. In order to get more information about PR distribution in glial cells and as double-labeling was not possible (all available antibodies were raised in mice), we prepared pure cultures of oligodendrocytes or astrocytes from either male or female newborn pups. The male and female cells were treated or not with E2 (50 nM) from days 10 to 25 of primary culture, and oligodendrocytes were separated from astrocytes on day 20. Indirect immunofluorescence staining of PR was done on day 25 with the KC 146 monoclonal antibody [8]. As seen on Table 1, PR was almost exclusively localized in oligodendrocytes. The number of PR-positive cells in control cultures was low, however, in female cultures, the percentage of PR-positive oligodendrocytes was higher. After E2-treatment, the number of PR-positive cells increased substantially in both types of culture, and again, the percentage of PR-positive oligodendrocytes and the staining intensity for PR was higher in female cultures. In astrocytes, only few cells expressed PR and most of them were seen in female cultures. However, the percentage of astrocytes that were PR-positive was similar in control and E2treated cultures. The staining intensity for PR was lower in astrocytes than in oligodendrocytes.
Steroid hormone effects on glial ceils in culture Cell growth. The effect of E2, P and of the antiprogestin RU486 was tested on glial cell multiplication. After 6 days of primary culture,
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Immunohistochemical localization of PR in pure cultures of oligodendrocytes or astrocytes
s~" ~
1.0
Proa + RU 486
z :.'~=
+ Prog
/ ..,.~' .,'"
o,5
I 6
I 10 Days of
I I 15 20 primary culture
Fig. 6. Growth curves of glial cells in primary culture. Primary culture were established at day I (day of birth) as described in Experimental. At day 6, cells were subcultured and replated in poly-L-lysine treated 6 0 m m Petri-dishes in the absence of hormone. 24 h later, when cells were attached, hormone-containing medium was given. Media were changed every 2 days. At the indicated days of culture, triplicate dishes were counted with a hemocytometer. • --• control v a l u e s + E 2 (100nM), A - - A + P (100riM), r-q--Fq+RU486 (200nM), A--A+P (100 nM) and + RU486 (200 nM).
cells were replated at low density in 60 mm Petri dishes and, after attachment, hormones and, or, antihormones were added to the culture medium and cells were counted after different days of hormone treatment. As shown in Fig. 6, E2 (100nM) increased, whereas P (100nM) decreased cell multiplication. This decrease was inhibited when the antiprogestin RU486 (200 nM) was added together with P. RU486 alone slightly stimulated cell growth at high cell density. Cell morphology. During the cell growth experiments we also observed changes in cell morphology due to the presence of hormones in the culture medium. For a better observation, cells were plated at high and low density from the beginning of the culture (day of birth) in presence or absence of P, E2, RU486 or P + RU486 (1/zM of each). After several days of culture, both types of glial cells went through
Steroid hormone receptors and steroid action
627
Primary culture of rat glial cells (3 weeks old)
+ progesterone
no hormone
01igodendrocytes (low density)
+ progesterone
no hormone
Fig. 7. Phase contrast microscopy of glial cells, 3 weeks after primary culture. At day 14, cells were plated at high or low density and cultured during the last week (or not) in the presence of P (1 #M), ( x 200).
a clear morphological change. Oligodendrocytes developed processes which appeared longer, straighter and more numerous in the presence of P (Fig. 7). E 2, RU486 and P + R U 4 8 6 had similar effects. Astrocytes also developed long fine fibers in the presence of the hormones and the anti-hormone as was observed by immunofluorescence staining of GFAP. The morphologic changes of astrocytes could be seen from day 3 of culture (Fig. 8). Cell differentiation. The expression of MBP, one of the markers of differentiation of oligo-
dendrocytes[14] and of GFAP, the major intermediate filament protein of differentiated astrocytes[15] was measured in glial cells treated or not with P or E2 (100nM) from day 1 of primary culture. In oligodendrocytes, the progestin treatment considerably increased the expression of MBP as was measured by immunostaining. This increase was more evident in early cultures ranging between day 6 and day 14. In older cultures, MBP-positive cells are very abundant making the evaluation of hormone effects more difficult (Table 2). E2-treatment
628
INGRID JUNG-TESTAS et al.
i
+P
Control
!
i Fig. 8. Immunofluorescence staining of GFAP in astrocytes. Cells were cultured on glass coverslips in the presence (or not) of P (1 pM) fro day 1 onwards after primary culture, lmmunofluorescence staining was done 3 days (3d, upper part, x 800) or 10 days (10d, lower part, x 400) after culture, using a monoclonal anti-GFAP antibody, followed by a second antimouse FITC-conjugated goat antibody.
also increased MBP-expression, but to a smaller extent. Astrocytes express G F A P from the beginning of primary culture. The number of GFAP-positive astrocytes was about three times increased in P- or E2-treated cultures. This increase was visible from days 3 to 10,
after that time the percentage of GFAPpositive cells was too high for precise evaluation of hormone effects. The immunofluorescence intensity of MBP and of G F A P was measured with a Leitz Photoautomat (Table 2).
Steroid h o r m o n e receptors a n d steroid action Table 2. Immunofluorescence intensity of MBP and GFAP in oligodendrocytes and astrocytes MBP (oligodendrocytes)
GFAP (astrocytes)
Days of culture
Control
+P
Control
+P
2 3 6 10 14
--14.0 7.2 4.3
--9.0 3.5 2.1
25.2 20.0 5.4 4.1 3.0
15.1 12.1 1.9 3.8 3.1
Glial cells were plated at day 1 on glass cover slips and cultured in the presence or absence of P (1/aM). At the indicated days of culture, MBP or GFAP was measured by indirect immunofluorescence staining with monoclonal anti-MBP or anti-GFAP antibodies. The fluorescence intensity of 20 to 30 cells of different fields was measured by spot metering with a Leitz sensor. The sensor signal, converted to an electrical signal, was expressed as seconds which are necessary for the automatic exposure. The average numbers are indicated.
DISCUSSION
Based on our earlier findings that newborn rat glial cells after 3 weeks of primary culture are able to synthetize progesterone [4-6], we have demonstrated in recent studies the presence of estrogen-inducible PR in these cultures. Moreover, we also identified during these studies specific GR, ER and AR binding sites in the same cells. E 2 treatment of the cultures increased significantly the amount of PR, whereas levels of GR, ER and AR binding sites remained constant in parallel experiments. As sex differences in PR and ER levels in certain regions of the rat brain have been reported by Rainbow et aL [16], and significant differences in PR induction by E2 treatment of male and female rats[17], we wondered if levels of PR and estrogen-induction of PR by E2 were different in cultures prepared from male or female newborn pups. The results of the present study demonstrate that the amount of uninduced PR was similar in male and female cultures, but sex differences in PR levels became apparent after E2 treatment of the cells. Indeed, the estrogeninducibility of PR was substantially increased in female cultures. These results were obtained after binding studies in mixed glial cultures. In further experiments, PR expression was measured in purified oligodendrocytes and purified astrocytes, prepared from either male or female offsprings and treated or not with E2. PR expression, as measured by indirect immunofluorescence staining, was almost exclusively localized in oligodendrocytes and the number of PR-positive cells and the staining intensity of the uninduced and E2-induced receptor was higher in female oligodendrocytes. In astrocytes, PR immunoreactivity was either undetectable or weak and restricted to
629
female cultures. Treatment of astrocytes with E2 did not increase the staining pattern in female astrocyte cultures. Therefore we can conclude that PR is mainly present in oligodendrocytes and it appears that female cultures contain higher levels of estrogen-inducible PR. Different groups have shown the presence of GR in glial cell cultures by binding studies and immunofluorescence microscopy [1, 18-20]. In our recent studies on steroid hormone receptors in rat glial cells, we have analyzed GR and PR on sucrose density gradients and found that both receptors, when labeled with the corresponding agonists, displayed a 9S form in the cytosol and a 4-6S form in the nuclear high salt, tungstate ions containing fractions. In contrast, when the receptors were labeled with the antagonist RU486, which has high affinity for both receptors [21, 22] a non-activated 8.5S complex was found in the nuclear fractions for GR and PR [7, 23, 24]. In the present study we further analyzed the non-activated GR with specific antibodies against GR itself (BUGR2, [11]) and against non-hormone binding proteins. As described for other steroid hormone receptors [25], hsp90 and p59 are also present in the non-activated GR [26]. The partial shift of 8.5S GR (Fig. 5) after incubation with the 173 anti-p59 antibody, may be due to heterogeneity of the GR population, only a portion still containing the p59-hsp90 complex [13] while the other part lacks p59 which had dissociated during KC1 extraction [24]. The use of the antiglucocorticosteroid RU486, known to stabilize either non-activated GR [23] or PR [27], combined with the presence of tungstate ions during nuclear receptor extractions, was helpful to elucidate this heterooligomeric structure of GR in glial cells. However, it is not possible to assess, at present, if hsp70 is also present in this system as observed in the heterooligomeric GR, overexpressed in chinese hamster ovary cells [26]. For the study of steroid hormone effects, we first measured glial cell proliferation in the presence or absence of P or Ez and we found that E2 stimulated cell growth, whereas P had inhibitory effect. This inhibition was abolished by the antagonist RU486. Even if these effects were rather modest, similar results were obtained in many different primary cultures. In contrast, the hormone effects on cell morphology were much stronger, especially those induced by P, as can be seen on Figs 7 and 8. Both oligodendrocytes and astrocytes developed long, fine
630
|NGRID JUNG-TESTASet al.
processes in the presence of P and surprisingly RU486 had no antagonistic effect under these conditions. From in vitro studies it is known that glucocorticoids may regulate the expression of MBP in oligodendrocytes [28], as well as astrocytic expression of GFAP [29, 30]. We now report that P and to a lesser extent E2 induce MBP expression in oligodendrocytes and these effects were most dramatic in early cultures, between day 6 and 10. GFAP expression in astrocytes was also considerably increased by P and E 2 during the first days of primary culture. In conclusion, evidence was presented that rat glial cells in primary culture contain 4 classes of steroid hormone receptors, PR, GR, ER and AR. Only PR is estrogen-inducible and this is a first report on sex differences in PR-induction by E: under in vitro conditions. Steroid hormones, as P or E2, elicit a variety of effects on glial cell development in primary culture and therefore this cell system will be a promising model for future investigation of hormonal control during central nervous system development.
8.
9.
10.
11. 12. 13.
14.
15. Acknowledgements--We thank C. Legris and F. Boussac for her efficient assistance in preparing the manuscript, J. C. Lambert and P. Beaufils for preparing the figures and B. Ranseht, R. W. Harrison and W. Hendry for the generous gifts of antibodies. This work was supported by INSERM and the Ligue Franqaise contre le Cancer.
16. 17.
REFERENCES
I. McEwen B. S., Biegon A., Davis P. G., Krey L. C., Luine V. N., McGinnis Y., Paden C. M., Parsons B. and Rainbow T. C.: Steroid hormones: humoral signals which alter brain cell properties and functions. Recent Prog. Horm. Res. 38 (1982) 41-83. 2. Robel P., Bourreau E., Corprchot C., Dang D. C., Halberg F., Clarke C., Haug M., Schlegel M. L., Synguelakis M., Vourch C. and Baulieu E. E.: Neurosteroids: 3fl-hydroxy-A5-derivatives in rat and monkey brain. J. Steroid Biochem. 27 (1987) 649-655. 3. Baulieu E. E., Robel P., Vatier O., Haug M., Le Goaseogne C. and Bourreau E.: Neurosteroids: proegnenolone and dehydroepiandrosterone in the brain. In Receptor-Receptor Interactions (Edited by K. Fuxe and L. F. Agnati). Macmillan Press, Basingstoke Vol. 48 (1987) pp. 89-104. 4. Jung-Testas I., Hu Z. Y., Baulieu E. E. and Robel P.: Neurosteroids: biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells. Endocrinology 125 (1989) 2083-2091. 5. Jung-Testas I., Hu Z. Y., Baulieu E. E. and Robel P.: Steroid synthesis in rat brain cell cultures. J. Steroid Biochem. 34 (1989) 511-519. 6. Hu Z. Y., Jung-Testas I., Robel P. and Baulieu E. E.: Neurosteroids: steroidogenesis in primary cultures of rat giial cells after release of aminoglutethimide blockade. Biochem. Biophys. Res. Commun. 161 (1989) 917-922. 7. Jung-Testas I., Renoir J. M., Gasc J. M. and Baulieu E. E.: Estrogen-inducible progesterone receptor in
18.
19.
20.
21. 22.
23.
24.
primary cultures of rat glial cells. Exp. Cell Res. 193 (1991) 12-19. Greene G. L., Harris K., Bova R., Kinders R., Moore B. and Nolan C.: Preparation and characterization of monoclonal antibodies to human progesterone receptor. Molec. Endocr. 2 (1988) 714-726. Ranscht B., Clapshaw P. A., Price J., Noble M. and Seifert W.: Development of oligodendrocytes and Schwann cells studies with a monoclonal antibody against galactocerebroside. Proc. Natn. Acad. Sci. U.S.A. 79 (1982) 2709-2713. Renoir J. M., Yang C. R., Formstecher P., Lustenberger P., Wolfson A., Redeuilh G., Mester J., Richard-Foy H. and Baulieu E. E.: Chick oviduct progesterone receptor: purification of a molybdatestabilized form and preliminary characterization. Eur. J. Biochem. 127 (1982) 71-79. Gametchu B. and Harrison R. W.: Characterization of a monoclonal antibody to the rat liver glucocorticoid receptor. Endocrinology 114 (1984) 274-279. Radanyi C., Renoir J. M. and Baulieu E. E.: Production and characterization of antibodies against synthetic peptides from hsp90 and p59. In preparation. Renoir J. M., Radanyi C., Faber L. E. and Baulieu E. E.: The non-DNA binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59 kDa protein. J. Biol. Chem. 265 (1990) 10740-10745. Dubois-Dalcq M., Behar T., Hudson L. and Lazzarini R. A.: Emergence of three myelin proteins in oligodendrocytes cultured without neurons. J. Cell Biol. 102 (1986) 384-392. Bignami A., Eng L. F., Dahl D. and Uyeda C. T.: Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43 (1972) 429-435. Rainbow T. C., Parsons B. and McEwen B. S.: Sex differences in rat brain oestrogen and progestin receptors. Nature 300 (1982) 648-649. Coirini H. and McEwen B. S.: Progestin receptor induction and sexual behavior by estradiol treatment in male and female rats. J. Neuroendocr. 2 (1990) 467-472. McGinnis J. F. and DeVellis J.: Cell surface modulation of gene expression in brain cells by down regulation of glucocorticoid receptors. Proc. Natn. Acad. Sci. U.S.A. 18 (1981) 1288-1292. Gustafsson J. A., Carlstedt-Duke J., Poellinger L., Okret S., Wikstr6m A. C., Br6nneg~ird M., Gillner M., Dong Y., Fuxe K., Cintra A., H/irfstrand A. and Agnati L.: Biochemistry, molecular biology, and physiology of the glucocorticoid receptor. Endocrine Rev. 8 (1987) 185-234. Vielkind U., Walencewicz A., Levine J. M. and Churchill-Bohn M.: Type II glucocorticoid receptors are expressed in oligodendrocytes and astrocytes. J. Neurosci. Res. 27 (1990) 360-373. Baulieu E. E.: Contragestion and other clinical applications of RU486, an antiprogesterone at the receptor. Science 245 (1989) 1351-1357. Jung-Testas I. and Baulieu E. E.: Inhibition of glucocorticoid action in cultured L-929 mouse fibroblasts by RU486, a new anti-giucocorticosteroid of high affinity for the glucocorticosteroid receptor. Exp. Cell Res. 147 (1983) 177-182. Groyer A., Schweizer-Groyer G., Cadepond F., Mariller M. and Baulieu E. E.: Antiglucocorticosteroid effects suggest why steroid hormone is required for receptors to bind DNA in vivo but not in vitro. Nature 328 (1987) 624-626. Renoir J. M., Radanyi C., Jung-Testas I., Faber L. E. and Baulieu E. E.: The non-activated progesterone receptor is a nuclear heterooligomer. J. Biol. Chem. 265 (1990) 14402-14406.
Steroid hormone receptors and steroid action 25. Joab I., Radanyi C., Renoir J. M., Buchou T., Catelli M. G., Binart N., Mester J. and Baulieu E. E.: Immunological evidence for a common non hormone-binding component in "non-transformed" chick oviduct receptors of four steroid hormones. Nature 308 0984) 850-853. 26. Sanchez E. R., Hirst M., Scherrer L. C., Tang H. Y., Welsh M. J., Harmon J. M., Simons S. S., Ringold G. M. and Pratt W. B.: Hormone-free mouse glucocorticoid receptors overexpressed in chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90. J. Biol. Chem. 265 (1990) 20123-20130. 27. Renoir J. M., Radanyi C. and Baulieu E. E.: The antiprogesterone RU486 stabilizes the heterooligomefic, non-DNA-binding, 8S form of the rabbit uterus cytosol progesterone receptor. Steroids 53 (1989) 1-20.
631
28. Kumar S., Cole R., Chiapeili F. and DeVellis J.: Differential regulation of oligodendrocyte markers by glucocorticoids: post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase. Proc. Natn. Aead. Sei. U.S.A. 86 (1989) 6807-6811. 29. Kumar S. and DeVellis J.: Glucocorticoid-mediated functions in glial cells. In Glial Cell Receptors (Edited by H. K. Kimelberg). Raven Press, New York (1988) pp. 243-264. 30. O'Callaghan J. P., Brinton R. E. and McEwen B. S.: Glucocorticoids regulate the concentration of glial fibrillary acidic protein throughout the brain. Brain Res. 494 (1989) 159-161.
0960-0760/92 $5.00+ 0.00 Copyright© 1992PergamonPresspie
Printed in Great Britain.All rightsreserved
DEMONSTRATION AND
OF
STEROID OF
STEROID
ACTION
IN
RAT
GLIAL
HORMONE PRIMARY
RECEPTORS CULTURES
CELLS
INGRIDJUNG-TESTAS,l* MICHELRENOIR,1 HELENEBUGNARD,I GEOFFREYL. GREENE2 and ETIENNE-EMILEBAULIEU1 ~Universit6 Paris XI and INSERM U33, Laboratoire Hormones, 94275 Bic&re, France and 2The University of Chicago, The Ben May Institute, Chicago, IL 60637, U.S.A. Summary--Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 antiPR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [3H]R5020, [3H]dexamethasone, [3H]OH-tamoxifen or [3H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4-6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presence of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found. During primary culture, the effects of progesterone (P) and estradioi (E2) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by Ev Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.
INTRODUCTION Steroid hormones are known to act in neural tissues to affect brain development and behavior. These actions are thought to be mediated by specific intracellular receptors since the brain contains receptors for all five classes of steroid hormones, progestin, estrogen, androgen, glucocorticoid and mineralocorticoid, and they resemble those found in non-neural target tissues (for review see Ref. [1]). In preliminary studies, our group has given evidence of the occurrence and accumulation of pregnenolone and dehydroepiendrosterone in the brain of several mammalian species even in Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.
*To whom correspondence should be addressed.
the absence of steroidogenic organs, suggesting an "in s i t u " mechanism unrelated to the peripheral endocrine glands [2, 3]. In further studies we have shown that newborn rat glial cells in primary culture can synthesize pregnenolone and progesterone [4-6] and recently we have identified the presence of progesterone, glucoeorticoid, estrogen and androgen receptors (PR, G R , ER and AR) in these glial cells [7]. Estradiol treatment of the cultures induced PR, but in contrast, levels of GR, ER and A R remained constant in the same cells. In the present work we have compared the estrogeninduction of PR in glial cultures established either from male or female offsprings, and the expression of PR was studied in separated oligodendrocytes and astrocytes with the monoclonal K C 146 antibody against PR[8] by immunohistochemical techniques. Furthermore, using gradient ultracentrifugations, the composition 621
622
INGRIDJUNO-TESTASet al.
of the heterooligomeric 8.5S structure of GR was studied with specific antibodies against GR, hsp90 and against the 59 kDa protein. Finally, we have shown direct effects of steroid hormones on glial cell multiplication, morphology and differentiation and we suggest that glial cells are targets for steroid hormones.
EXPERIMENTAL
Primary glial cell cultures Primary cultures of glial cells were established from newborn Sprague-Dawley rats (postnatal days 0-2) as described previously [5]. Briefly, cerebral hemispheres were mechanically dissociated in culture medium after removal of the meninges, the cell suspension was filtered through a 82/~m nylon mesh and cells were plated on poly-L-lysine coated plastic dishes in Dulbecco's Modified Eagle Medium (DME) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 3% calf serum (CS), insulin (4.8/~g/ml), glucose (1.8 mg/ml), penicillin (100 UI/ml) and streptomycin (100/~ g/ml). At complete cell density (days 7 to 9 of culture), cells were diluted 4 times (after trypsin-treatment) and kept growing for 2 to 4 weeks in the presence or not of hormones and/or antihormones, as will be specified for each experiment. Hormones and antihormones were added every 2 days, the media were changed twice a week. Three days before the experiments, steroid-free charcoal-treated CS was used. Cultures enriched in oligodendrocytes were obtained by plating cells at high density during the first subculture on days 7-9. At day 20, oligodendrocytes were then detached selectively from the dense culture by flushing medium several times with a pipet on the culture surface. The detached oligodendrocytes were replated on poly-L-lysine coated dishes. Pure astrocytes were obtained by replating the resting underlayer of astrocytes at very low density. Oligodendrocytes were identified by the measure of galactocerebrosid (Gal C) and myelin basic protein (MBP), astrocytes by the measure of glial fibrillary acidic protein (GFAP) using indirect immunofluorescence staining. The immunofluorescence procedure is described in Refs [4, 5 and 7]. Staining of Gal C was done using a monoclonal antibody kindly given by B. Ranscht [9]. Monoclonal anti-MBP was purchased from Boehringer-Mannheim, monoclonal anti-GFAP from Bio-Yeda (Flow Laboratories, France).
Receptor binding For receptor measurements, cells, after 3-4 weeks of culture, were harvested, centrifuged, and resuspended in 1 ml DME containing radiolabeled steroids from 2 to 10 nM, with or without 1 #M of the corresponding unlabeled steroid. For labeling of PR, 20 nM radioinert cortisol was added to avoid binding to GR. Cells were labeled for l h at 37°C and after centrifugation, washed three times with 3 ml icecold PBS. All subsequent steps were performed at 0°C. During each washing cycle, cells were incubated for 5 min in the cold, to allow for non-specifically bound radioactivity to diffuse out of the intact cells. After washing, the cell pellet was resuspended in 0.5 ml of PGW-buffer [10mM potassium phosphate, 10% glycerol (v/v), 20 mM tungstic acid, pH 7.8] and cells were homogenized and centrifuged at 10,000g for 20 min at 4°C, the supernatant was either submitted to linear 5-20% sucrose density gradients prepared as described previously [10], or the radioactivity was counted in a liquid scintillation counter to determine specific binding, which was expressed as the difference between total binding and non-specific binding (measured in the presence of unlabeled competitor). The nuclear pellet was washed once with 1 ml PGW plus 1% (v/v) Triton X-100 and twice with the same buffer without Triton. The final pellet was resuspended in 0.5 ml PGW buffer containing 0.4 M KCI and extracted for 1 h. Soluble nuclear proteins were obtained after centrifugation at 10,000g for 30min and analyzed either on linear 5-20% sucrose density gradients or by counting the specific radioactivity. Internal standards for density gradients were fungal glucose oxidase (GO, S20,w= 7.9) and peroxidase (PO, S20.w= 3.6). In case of GR, aliquots of the PGW-KCI 0.4 M extracts were incubated for 4h at 4°C with either 100 #1 of culture medium of BUGR2 [11] or ~ 100 pg of either a rabbit antibody against the conserved 17 aminoacids at the carboxyl terminal end of hsp90 (anti-hsp90-174), or a rabbit antibody against the 18 amino acids at the carboxyl terminal end of p59 (anti-p59-173)[12] prior to the ultracentrifugation analysis. It is to note that anti-hsp90-174 also recognizes hsp70, in addition to hsp90 [12]. Immunolocalization of PR The KC 146 monoclonal antibody to human PR is a mouse IgG that recognizes the rat PR
Steroid hormone receptors and steroid action
[8]. Its cross-reactivity with rat PR was tested on tissue sections of rat uterus before the experiments. Indirect immunofluorescence techniques were used for the localization of PR, as described previously [7]. Briefly, cells, cultured on glass coverslips, were washed, fixed in 4% paraformaldehyde and permeabilized with 1% Triton in PBS for 5 min. After washing, the KC 146 antibody (diluted 1:50) was applied for 30 min, followed, after washing, by a fluoresceincoupled goat anti-mouse IgG for another 30 rain. The coverslips were mounted in Moviol.
RESULTS
Progestin receptor induction by estradiol (E2) in mixed glial cells Primary cell cultures prepared from neonatal rat forebrains consist of a bedlayer of astrocytes overlaid by process-bearing oligodendrocytes. Both cell types were identified by morphology and by immunostaining with antibodies against GFAP for astrocytes and against Gal C and MBP for oligodendrocytes [4, 5, 7]. After 3 weeks of culture, when cells are still actively proliferating, the presence of PR was measured by whole cell assays after incubating cells with [3H]Organon 2058 at different concentrations between 0.3 to 10nM, in the presence of 10 nM unlabeled cortisol to avoid binding to
GR. Parallel incubations were performed in the presence of excess unlabeled progesterone (P) to determine non-specific binding. Specific binding was expressed as bound [3H]Organon per mg of protein in cytosol and nuclear fractions. The treatment of the cells with E2 (50 nM) from day 10 onward of primary culture considerably increased the number of PR binding sites in both cytosol and nuclear fractions, and corresponded to ~2.700 and 10.000 binding sites per cell, respectively, in untreated and E~-treated cells (Fig. 1). When the antiestrogen tamoxifen was added to the cultures together with E2, the estrogen induction of PR was completely inhibited, whereas tamoxifen alone did not influence PR levels [7].
PR induction by E2 in glial cells prepared from male or female offsprings Primary cultures from newborn male or female pups were prepared and cells treated or not with E2 in order to examine if sex differences were seen in PR concentration and in its induction by the estrogen. Cells were labeled with two different ligand concentrations (2.0 and 5.0 nM [3H]Organon) and PR was measured by whole cell assays. As shown in Fig. 2, both male and female cultures possess specific PR binding sites PR I-1 []
d
"E2
/ //
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I
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623
~
Control *E 2
d
9
x
o.-" E
Q. '*0
I,/, /
"~ I01-I ,'
2.0
/I
,~" Cy,osol
5.0 7.5 10.0 nM [3H]-Org 2058
Fig. 1. Measure of PR in estrogen-treated or untreated glial cells. Cells were treated (or not) from days 10 to 25 of primary culture with 50nM E2. Control cells received vehicle only. On day 26, cells were recovered, incubated for ! h at 37°C with increasing concentrations of [3H]Org 2058 in presence of 20 nM radioinert cortisol, and with or without 1 # M radioinert P. Specific P R binding was determined in cytosol and nuclear fractions as described in Experimental. Nuclear fractions: A - - & + E2; • - - • control, cytosol: /x - - A + E2; O - - O control.
2.0 nM
5.0 nM
p"]-Org 2058 Fig. 2. Measure of PR in cultures from male or female offsprings. Primary cultures were established from male or female pups at day 1 of birth. Cells were treated with 50 nM E 2 (or not) and P R specific binding was measured after incubating cells with [3H]Org 2058 at a concentration of 2.0 or 5.0 nM, in presence of 20 nM radioinert cortisol, as described in Fig. I. Specific PR binding sites were determined. [] control cultures; l cells treated with E 2.
624
]NGRID JUNG-TESTAS et al.
Nuclear
Cytosol [3H]-Dex
[3H]-Org 2058 PR
Fractions
[3H]-Org 2058
GR
[3H]-Dex
PR
GR
60
[] 50
x o~
[]
Control
Control
[] +E2
[] FE2
4O
E
230
Y,
20 10
10
30
90
08
22
10
60
30
90
02
22
60
Ligand Concentration (nM) Fig. 3. Measure of PR and G R in estrogen-treated or untreated glial cells. Cells were treated with 50 nM E: (or not) as described in Fig. 1. PR and G R were determined after incubating cells for 1 h at 37°C with increasing concentrations of [3H]Org 2058 in presence of 20 nM radioinert cortisol, or with increasing concentrations o f [3H]dexamethasone, both in presence or absence of excess unlabeled competitor. Specific binding was determined in cytosol and nuclear fractions. [] cytosol control; m cytosol + E2; [] nuclear fraction, control; [] nuclear fraction + E 2.
and little difference of uninduced PR was seen at the two ligand concentrations. However, the estrogen-inducibility of PR was substantially increased in female cultures.
Among the four classes of receptors, PR, GR, ER and AR, only PR is inducible by E2 Glial cells in primary culture contain, in addition to PR, also GR, ER and AR binding
Cytosol
Nuclear
Fractions
[~.]-o,o 2os8
PH1-OH-r.m
[3.)_. 1881
[3H]- Org 2 0 5 8
[3HI- OH-Tam
[3H1- R 1 8 8 1
PR
ER
AR
PR
ER
AR
[]
3O
Control
J~l Control
B+E2
~I+E 2
x /
x
E I(3 E
"o
r
2.O
~
I 7.O
:
rl
20
"
18o
Concentration (nM)
2.o 17.o
N_ ] 2,0 17.o
-
2.o l e o
2.o 17.~
Concentration (nM)
Fig. 4. Measure of PR, ER and A R in estrogen-treated or untreated glial cells. Cells were treated with 50 aM E 2 (or not) as described in Fig. I. For receptor determinations, cells were incubated with 2 concentrations of [3H]Org 2058 for PR, or of [3H]OH-tamoxifen for ER, or of [3HlR1881 for AR, during l h at 37°C. Excess unlabeled competitor was added in parallel incubations. Specific binding was determined in cytosol and nuclear fractions. [] cytosol control; [] cytosol + E~; [] nuclear fraction, control; [] nuclear fraction + E 2.
Steroid hormone receptors and steroid action
sites, as we have shown previously [7]. Therefore it was of interest to measure in parallel the different receptors in cells treated (or not) with E 2. We first compared the amount of PR and GR in the same primary culture, and as shown in Fig. 3, only PR binding sites in cytosol and nuclear fractions increased significantly in estrogen-treated cells, whereas the amount of GR binding sites remained unchanged. We then compared in another primary culture the amount of PR with those of ER and AR and again, only PR binding sites were increased by the estrogen treatment (Fig. 4). ER binding sites had decreased in E2 treated cultures. This can be explained by a number of ER binding sites still occupied by endogenous E2, despite the culture in steroid-free medium 2 to 3 days before the experiments.
Analysis of PR and GR by gradient ultracentrifugations As mixed glial cells proliferate actively until 4 to 5 weeks after primary culture, sufficient material could be obtained to analyze PR and GR on sucrose density gradients. In the presence of agonist, ([3H]Organon and [3H]dexamethasone, respectively), both receptors displayed a
625
9S form in the cytosol and a 4-6S form in nuclear 0.4 M KC1, tungstate ions containing fractions. In contrast, when the receptors were labeled with the antagonist [3H]RU486, a nonactivated 8.5S receptor complex was found in the nuclear fractions for both PR and GR [7]. The composition of the GR-8.5S complex was further analyzed after incubation with specific antibodies against the GR itself (BUGR2 [1 l]) and against non-hormone binding proteins known to associate with steroid hormone receptors in their non-activated, non-DNA binding form[13]. In whole cell extracts (containing both cytosoluble and nuclear GR), GR displayed a heterogenous structure in the presence of 0.4 M KCI and tungstate ions and migrated as 8.5 and 7.0S forms, respectively. Both peaks represented the receptor since BUGR2 shifted them to a 10S form [Fig. 5(a)]. However, the 7.0S form does not contain hsp90 nor hsp70 since it remained in a 7.0S form after incubation with the 174 anti-hsp antibody [Fig. 5(b)]. As described for other steroid hormone receptors, a 59 kDa protein is also present, at least partly, in the 8.5S complex since the anti p59 antibody (173) partially displaced the GR 8.5S forms [Fig. 5(c)].
15
~10
==
10
20
10 Fraction
20 Number
10
20
Fig. 5. Analysis of G R by gradient ultracentrifugations. Cells were exposed to [3H]RU486 for l h at 37°C, harvested and treated as described in Experimental. Aliquots (200/~l) of whole cells extracts in PGW-KC1 0.4 M were centrifuged prior ( O - - O , panels a, b and c) or after incubation with 50/~ l of BUGR 2 anti-GR monoclonal antibody ( A - - A , panel a) or after incubation with 100/~g of anti-hsp90 antibody 174 ( A - - A , panel b) or after incubation with 120/~g of anti-p59 antibody 173 ( k - - A , panel c). In panel b, BUGR 2 (50/~l) and anti-hsp90 antibody 174 (100/~g) were added simultaneously to the incubation medium (ll--r-l). Centrifugation was performed as described in Experimental with GO = glucose oxydase 7.9S, and PO = peroxidase 3.6S as internal standards.
626
INGRID JUNG-TESTAS et al.
Table 1. Indirect immunofluorescent staining of PR in oligodendrocytes and astrocytes prepared from male or female newborn rats
Rat
Glial
cells
PR-positive cells Oligodendrocytes
Astrocytes
~' CX ~CX 3'+E2
+ + + + + +
none + (+ -)
~+E 2
+ + + +
+
lmmunofluorescence staining of PR was done with the monoclonal anti-PR antibody KC 146, as described in Experimental. CX: control cultures; +E:: 50nM E2 was present from days 10 to 25 of culture. ( + - ): very few positive cells; + : some positive cells; + +: many positive cells; + + +: ~70% positive cells; + + + + : almost all cells were PR-positive.
{7"
2,0 +E 2 +RU
16 - -
In previous experiments with the anti-PR monoclonal antibody KC 146, we were able to visualize PR in glial cells by the biotin-avidinimmunoperoxidase procedure and by indirect immunofluorescence staining [7]. By both techniques it was clearly seen that PR staining was preferentially localized in oligodendrocytes. In order to get more information about PR distribution in glial cells and as double-labeling was not possible (all available antibodies were raised in mice), we prepared pure cultures of oligodendrocytes or astrocytes from either male or female newborn pups. The male and female cells were treated or not with E2 (50 nM) from days 10 to 25 of primary culture, and oligodendrocytes were separated from astrocytes on day 20. Indirect immunofluorescence staining of PR was done on day 25 with the KC 146 monoclonal antibody [8]. As seen on Table 1, PR was almost exclusively localized in oligodendrocytes. The number of PR-positive cells in control cultures was low, however, in female cultures, the percentage of PR-positive oligodendrocytes was higher. After E2-treatment, the number of PR-positive cells increased substantially in both types of culture, and again, the percentage of PR-positive oligodendrocytes and the staining intensity for PR was higher in female cultures. In astrocytes, only few cells expressed PR and most of them were seen in female cultures. However, the percentage of astrocytes that were PR-positive was similar in control and E2treated cultures. The staining intensity for PR was lower in astrocytes than in oligodendrocytes.
Steroid hormone effects on glial ceils in culture Cell growth. The effect of E2, P and of the antiprogestin RU486 was tested on glial cell multiplication. After 6 days of primary culture,
~
,
£k/l, . ~:. ..."'r" /_/
_/_Z,,,j/ ....f"
x 1,5
•
::"
I I ..:.
"6
Immunohistochemical localization of PR in pure cultures of oligodendrocytes or astrocytes
s~" ~
1.0
Proa + RU 486
z :.'~=
+ Prog
/ ..,.~' .,'"
o,5
I 6
I 10 Days of
I I 15 20 primary culture
Fig. 6. Growth curves of glial cells in primary culture. Primary culture were established at day I (day of birth) as described in Experimental. At day 6, cells were subcultured and replated in poly-L-lysine treated 6 0 m m Petri-dishes in the absence of hormone. 24 h later, when cells were attached, hormone-containing medium was given. Media were changed every 2 days. At the indicated days of culture, triplicate dishes were counted with a hemocytometer. • --• control v a l u e s + E 2 (100nM), A - - A + P (100riM), r-q--Fq+RU486 (200nM), A--A+P (100 nM) and + RU486 (200 nM).
cells were replated at low density in 60 mm Petri dishes and, after attachment, hormones and, or, antihormones were added to the culture medium and cells were counted after different days of hormone treatment. As shown in Fig. 6, E2 (100nM) increased, whereas P (100nM) decreased cell multiplication. This decrease was inhibited when the antiprogestin RU486 (200 nM) was added together with P. RU486 alone slightly stimulated cell growth at high cell density. Cell morphology. During the cell growth experiments we also observed changes in cell morphology due to the presence of hormones in the culture medium. For a better observation, cells were plated at high and low density from the beginning of the culture (day of birth) in presence or absence of P, E2, RU486 or P + RU486 (1/zM of each). After several days of culture, both types of glial cells went through
Steroid hormone receptors and steroid action
627
Primary culture of rat glial cells (3 weeks old)
+ progesterone
no hormone
01igodendrocytes (low density)
+ progesterone
no hormone
Fig. 7. Phase contrast microscopy of glial cells, 3 weeks after primary culture. At day 14, cells were plated at high or low density and cultured during the last week (or not) in the presence of P (1 #M), ( x 200).
a clear morphological change. Oligodendrocytes developed processes which appeared longer, straighter and more numerous in the presence of P (Fig. 7). E 2, RU486 and P + R U 4 8 6 had similar effects. Astrocytes also developed long fine fibers in the presence of the hormones and the anti-hormone as was observed by immunofluorescence staining of GFAP. The morphologic changes of astrocytes could be seen from day 3 of culture (Fig. 8). Cell differentiation. The expression of MBP, one of the markers of differentiation of oligo-
dendrocytes[14] and of GFAP, the major intermediate filament protein of differentiated astrocytes[15] was measured in glial cells treated or not with P or E2 (100nM) from day 1 of primary culture. In oligodendrocytes, the progestin treatment considerably increased the expression of MBP as was measured by immunostaining. This increase was more evident in early cultures ranging between day 6 and day 14. In older cultures, MBP-positive cells are very abundant making the evaluation of hormone effects more difficult (Table 2). E2-treatment
628
INGRID JUNG-TESTAS et al.
i
+P
Control
!
i Fig. 8. Immunofluorescence staining of GFAP in astrocytes. Cells were cultured on glass coverslips in the presence (or not) of P (1 pM) fro day 1 onwards after primary culture, lmmunofluorescence staining was done 3 days (3d, upper part, x 800) or 10 days (10d, lower part, x 400) after culture, using a monoclonal anti-GFAP antibody, followed by a second antimouse FITC-conjugated goat antibody.
also increased MBP-expression, but to a smaller extent. Astrocytes express G F A P from the beginning of primary culture. The number of GFAP-positive astrocytes was about three times increased in P- or E2-treated cultures. This increase was visible from days 3 to 10,
after that time the percentage of GFAPpositive cells was too high for precise evaluation of hormone effects. The immunofluorescence intensity of MBP and of G F A P was measured with a Leitz Photoautomat (Table 2).
Steroid h o r m o n e receptors a n d steroid action Table 2. Immunofluorescence intensity of MBP and GFAP in oligodendrocytes and astrocytes MBP (oligodendrocytes)
GFAP (astrocytes)
Days of culture
Control
+P
Control
+P
2 3 6 10 14
--14.0 7.2 4.3
--9.0 3.5 2.1
25.2 20.0 5.4 4.1 3.0
15.1 12.1 1.9 3.8 3.1
Glial cells were plated at day 1 on glass cover slips and cultured in the presence or absence of P (1/aM). At the indicated days of culture, MBP or GFAP was measured by indirect immunofluorescence staining with monoclonal anti-MBP or anti-GFAP antibodies. The fluorescence intensity of 20 to 30 cells of different fields was measured by spot metering with a Leitz sensor. The sensor signal, converted to an electrical signal, was expressed as seconds which are necessary for the automatic exposure. The average numbers are indicated.
DISCUSSION
Based on our earlier findings that newborn rat glial cells after 3 weeks of primary culture are able to synthetize progesterone [4-6], we have demonstrated in recent studies the presence of estrogen-inducible PR in these cultures. Moreover, we also identified during these studies specific GR, ER and AR binding sites in the same cells. E 2 treatment of the cultures increased significantly the amount of PR, whereas levels of GR, ER and AR binding sites remained constant in parallel experiments. As sex differences in PR and ER levels in certain regions of the rat brain have been reported by Rainbow et aL [16], and significant differences in PR induction by E2 treatment of male and female rats[17], we wondered if levels of PR and estrogen-induction of PR by E2 were different in cultures prepared from male or female newborn pups. The results of the present study demonstrate that the amount of uninduced PR was similar in male and female cultures, but sex differences in PR levels became apparent after E2 treatment of the cells. Indeed, the estrogeninducibility of PR was substantially increased in female cultures. These results were obtained after binding studies in mixed glial cultures. In further experiments, PR expression was measured in purified oligodendrocytes and purified astrocytes, prepared from either male or female offsprings and treated or not with E2. PR expression, as measured by indirect immunofluorescence staining, was almost exclusively localized in oligodendrocytes and the number of PR-positive cells and the staining intensity of the uninduced and E2-induced receptor was higher in female oligodendrocytes. In astrocytes, PR immunoreactivity was either undetectable or weak and restricted to
629
female cultures. Treatment of astrocytes with E2 did not increase the staining pattern in female astrocyte cultures. Therefore we can conclude that PR is mainly present in oligodendrocytes and it appears that female cultures contain higher levels of estrogen-inducible PR. Different groups have shown the presence of GR in glial cell cultures by binding studies and immunofluorescence microscopy [1, 18-20]. In our recent studies on steroid hormone receptors in rat glial cells, we have analyzed GR and PR on sucrose density gradients and found that both receptors, when labeled with the corresponding agonists, displayed a 9S form in the cytosol and a 4-6S form in the nuclear high salt, tungstate ions containing fractions. In contrast, when the receptors were labeled with the antagonist RU486, which has high affinity for both receptors [21, 22] a non-activated 8.5S complex was found in the nuclear fractions for GR and PR [7, 23, 24]. In the present study we further analyzed the non-activated GR with specific antibodies against GR itself (BUGR2, [11]) and against non-hormone binding proteins. As described for other steroid hormone receptors [25], hsp90 and p59 are also present in the non-activated GR [26]. The partial shift of 8.5S GR (Fig. 5) after incubation with the 173 anti-p59 antibody, may be due to heterogeneity of the GR population, only a portion still containing the p59-hsp90 complex [13] while the other part lacks p59 which had dissociated during KC1 extraction [24]. The use of the antiglucocorticosteroid RU486, known to stabilize either non-activated GR [23] or PR [27], combined with the presence of tungstate ions during nuclear receptor extractions, was helpful to elucidate this heterooligomeric structure of GR in glial cells. However, it is not possible to assess, at present, if hsp70 is also present in this system as observed in the heterooligomeric GR, overexpressed in chinese hamster ovary cells [26]. For the study of steroid hormone effects, we first measured glial cell proliferation in the presence or absence of P or Ez and we found that E2 stimulated cell growth, whereas P had inhibitory effect. This inhibition was abolished by the antagonist RU486. Even if these effects were rather modest, similar results were obtained in many different primary cultures. In contrast, the hormone effects on cell morphology were much stronger, especially those induced by P, as can be seen on Figs 7 and 8. Both oligodendrocytes and astrocytes developed long, fine
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|NGRID JUNG-TESTASet al.
processes in the presence of P and surprisingly RU486 had no antagonistic effect under these conditions. From in vitro studies it is known that glucocorticoids may regulate the expression of MBP in oligodendrocytes [28], as well as astrocytic expression of GFAP [29, 30]. We now report that P and to a lesser extent E2 induce MBP expression in oligodendrocytes and these effects were most dramatic in early cultures, between day 6 and 10. GFAP expression in astrocytes was also considerably increased by P and E 2 during the first days of primary culture. In conclusion, evidence was presented that rat glial cells in primary culture contain 4 classes of steroid hormone receptors, PR, GR, ER and AR. Only PR is estrogen-inducible and this is a first report on sex differences in PR-induction by E: under in vitro conditions. Steroid hormones, as P or E2, elicit a variety of effects on glial cell development in primary culture and therefore this cell system will be a promising model for future investigation of hormonal control during central nervous system development.
8.
9.
10.
11. 12. 13.
14.
15. Acknowledgements--We thank C. Legris and F. Boussac for her efficient assistance in preparing the manuscript, J. C. Lambert and P. Beaufils for preparing the figures and B. Ranseht, R. W. Harrison and W. Hendry for the generous gifts of antibodies. This work was supported by INSERM and the Ligue Franqaise contre le Cancer.
16. 17.
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