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Biology of Reproduction 62, 1763-1771 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Transcriptional Interference Between Glucocorticoid Receptor and Estradiol Receptor Mediates the Inhibitory Effect of Cortisol on Fish Vitellogenesis1

C. Lethimoniera, G. Flouriota, Y. Valotairea, O. Kaha, and B. Ducouret2,a

a Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Endocrinologie Moléculaire des Poissons, INRA, Université de Rennes 1, 35042 Rennes cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In oviparous species, the synthesis of vitellogenin (Vg) takes place in the liver according to a strictly estrogen-dependent mechanism that first involves an up-regulation of the estrogen receptor (ER) by its own ligand. However, reports from the literature indicate that in trout stress or cortisol may cause a reduction of cytosolic E2-binding sites in the liver and a decrease in plasma Vg levels. To investigate the mechanisms underlying these effects, in vivo and in vitro experiments were designed in rainbow trout (Oncorhynchus mykiss). The results demonstrate that cortisol implanted into maturing females caused a marked decrease of rainbow trout ER (rtER) and rainbow trout Vg (rtVg) mRNA levels in the liver. In vitro experiments on hepatocyte aggregates also showed that dexamethasone (Dex) caused a strong decrease in the basal and E2-stimulated rtER mRNA and to a lesser extent rtVg mRNA. These effects were specific as no other hormones were able to mimic the inhibitory action of Dex. A study of rtER mRNA stability indicated that the effects of glucocorticoids are likely to take place at the transcriptional level. This was further indicated by transfection experiments in CHO-K1 cells, which showed that rainbow trout glucocorticoid receptor (rtGR) strongly inhibited the E2-stimulated transcriptional activity of the rtER promoter. Taken together, these results indicate that the rtGR exerts a transcriptional interference on the expression of the rtER that may explain some of the negative effects of stress or cortisol on vitellogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In all oviparous species, the liver plays a major role in the control of vitellogenin (Vg) production, a mechanism crucial for the subsequent development of ovarian vitellogenesis. However, there are different reports in the literature showing that in fish various stressful conditions have a negative effect on several reproductive parameters, such as plasma Vg, gonadal steroid, and gonadotropin levels [1]. The deleterious effects of stress on vitellogenesis are well documented in salmonids. For example, female brook trout exposed to acid stress have lower Vg levels [2], and in rainbow trout different stress conditions influence the quality of the gametes, the timing of ovulation, and the survival of the larvae [3, 4]. In fish, as in other vertebrates, these various types of stress are associated with elevated cortisol levels. Indeed, in male rainbow trout, plasma cortisol levels in fish subjected to an emersion stress were significantly higher than in the control fish [3]. The concentration of plasma cortisol changes with time after a handling stress in brown trout [5] or after a confinement stress in rainbow trout [6]. Moreover, there is a positive correlation between the elevation of plasma cortisol levels and the degree of handling stress in tilapia [7]. Cortisol implants that mimic the systemic cortisol concentrations observed during a stressful stimuli have also been shown to decrease gonadal weight and plasma E2 dramatically [8]. The observation that decreased production of Vg is detected under stress conditions can be partly explained by the fact that cortisol implants result in a decrease of the E2-binding capacity of liver cytosolic extracts [9]. Indeed, E2 is the key hormone responsible for the initiation and maintenance of Vg production, and the mechanisms underlying its action on the liver have been well established in the rainbow trout. Both in vivo [10] and in vitro [11], the first effect of E2 in the liver is to stimulate strongly the expression of its own receptor, and this positive autoregulation involves both transcriptional and post-transcriptional effects [12]. The molecular mechanisms underlying the transcriptional effects of E2 on the rainbow trout estrogen receptor (rtER) have been studied by transfection assays following the characterization of the rtER gene promoter [13]. It was found that a 0.2-kb (-248/-40) fragment of this promoter contains a virtually perfect estrogen-responsive element (ERE) and a consensus ERE half site [14, 15]. These two sequences are essential for the rtER autoregulation and are also implicated in a synergistic effect between rtER and the orphan receptor COUP-TF1 [15, 16].

In this study, we have tested the hypothesis that the rainbow trout glucocorticoid receptor (rtGR) [17] that mediates the genomic effects of cortisol interferes with the abovedescribed up-regulation of the rtER by its own ligand at the transcriptional level. The trout liver cells express both rtGR and rtER [11, 18, 19]. Moreover, as a member of the nuclear receptor family, the rtGR can modulate expression of specific target genes, either by binding to hormone-responsive elements [20] or by protein/protein interactions according to different models [21]. To test the possibility of cortisol actions on rtER and Vg expression in the liver of rainbow trout, we have examined the effects of cortisol or dexamethasone (Dex) on rtER and Vg mRNA levels in vivo and on hepatocyte aggregates in vitro.

The results indicate that cortisol or Dex consistently induce a strong decrease of rtER expression and, to a lesser extent, of Vg expression. This effect is likely to take place at a transcriptional level because Dex did not affect the rtER mRNA half-life in hepatocyte aggregates treated with actinomycin D. In addition, cotransfection of rtER and rtGR cDNAs with a reporter gene placed under the control of the rtER gene promoter confirmed the transcriptional inhibition of rtER gene expression mediated by ER/GR interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Animals were treated in agreement with the European Union regulation concerning the protection of experimental animals. Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Rainbow trout (Oncorhynchus mykiss) were supplied by the INRA fish farm (Le Drennec, France) and kept in the laboratory in a recirculating water system at 12–15°C under an artificial light regime mimicking the natural photoperiod (46° North). The animals were fed a trout diet ad libitum. For hepatocyte isolation, 1-yr-old males (1 kg) were used. For in vivo treatment, maturing females 16 mo old (Gonadal Somatic Index: 2.3–3.6%), weighing about 600 g were used. After 8 days of acclimatization in the laboratory, trout were divided into four experimental tanks (12 per tank). For implantation, the fish were anesthetized using 2-phenoxyethanol (1:3500; Sigma, St. Louis, MO). Cortisol (Sigma) was dissolved in molten cocoa butter at 42°C and injected intraperitoneally at a concentration of 60 mg/kg. Control fish (sham) received the same injection (1 ml/kg) with cocoa butter alone. The solidified cocoa butter pellet acts as a slow release implant over several days [22].

Sampling

Immediately after netting, all fish from one tank were rapidly anesthetized with phenoxyethanol (1:2000). The blood was collected from the caudal vessel using sodium citrate-treated syringes, diluted with 6 % sodium citrate, 9{per thousand} NaCl (50 µl/ml blood), and kept on ice until centrifugation. Fish were weighed and killed by severance of the spinal cord. Gonads were excised and weighed. Plasma was stored frozen and stored at -20°C prior to steroid radioimmunoassay (RIA). Samples of 100 mg of liver were taken and immediately frozen in liquid nitrogen until RNA extraction.

Radioimmunoassays

Plasma was extracted twice with five volumes of cyclohexane:ethyl acetate (50:50). The pooled organic extracts were evaporated under air flow and dissolved in ethanol. Before the assays were performed, ethanol was evaporated under air flow, and the dry residues were dissolved in phosphate buffer (0.01 M, pH 7.25) containing 0.1% gelatin. The steroids, 17ß-estradiol and cortisol were measured according to Fostier et al. [23] except that the bound steroids were precipitated with polyethylene glycol [24].

Hepatocyte Isolation and Culture

The liver was dissociated by collagenase A (Boehringer, Meylan, France) perfusion, as described by Seglen [25], and adapted to trout [26]. The cell suspension was filtered through a 70-mesh sieve, and the pellet was collected by centrifugation (50 x g for 5 min) at 18°C. The nonparenchymal and damaged cells were removed by centrifugation. The hepatocyte pellet was resuspended in a serum-free medium: Dulbecco's modified Eagle's medium:Ham's F-12 nutrient mixture (1:1 mixture, with L-glutamine and 15 mM HEPES, without phenol red), supplemented with 15 mM of TES (N-Tris [hydroxymethyl]methyl-2 amino-ethanesulfonic acid), 12 mM NaHCO3, 1% (v/v) antibiotics (penicillin, streptomycin, and amphotericin B; Sigma) and 2% (v/v) ultroser SF (Biosepra, Villeneuve la Garenne, France). The cells were plated in 60-mm untreated plastic petri dishes (Falcon; 1–2 x 107 cells/5 ml medium per dish). Aggregates were obtained by constant gyratory shaking at 55 rpm at 18°C (Novotron, INFORS AG, Massy, France). The culture medium was changed every 2 days. Steroid treatments were performed 8 days after plating: E2, Dex, or other steroids (0–1000 nM) were dissolved in ethanol and added to the culture medium (1:1000 v/v). An equal volume of ethanol was added to the controls. Peptidic hormones, salmon prolactin (PRL) or salmon growth hormone (GH), were added in aqueous solution. After hormonal treatment, cells were harvested, pelleted by centrifugation (at 50 x g for 5 min), and stored at -70°C until use. For mRNA half-life measurements, hepatocytes were pretreated for 48 h with 100 nM of estradiol and for 12 h either with or without Dex (1000 nM) before actinomycin D (0.4 µg/ml, Sigma) addition. Medium was renewed every 24 h, and steroid treatments were maintained after the actinomycin D addition. During the antibiotic treatment, cells were collected at different times until 0, 3, 6, 12, and 24 h.

Dot-Blot Hybridization

Total RNA was prepared from either 100 mg of liver or from cultured hepatocytes using Trizol reagent (Gibco BRL, Germany). Total RNA samples (5 µg) were spotted onto a nylon Biodyne A membrane (Pall, St. Germain en Laye, France), using a BioRad dot-blot apparatus, as described by Cheley and Anderson [27]. The membrane was prehybridized (50% formamide, 5x standard saline citrate [SSC], 5x Denhardt's solution, 5 mM NaH2PO4 pH 6.5, 0.1 mg/ml calf thymus DNA, and 0.1% SDS) at 42°C for 6 h, and hybridized under stringent conditions (50% formamide, 5x SSC, 1x Denhart's solution, 20 mM NaH2PO4 pH 6.5, 0.05 mg/ml calf thymus DNA, 0.1% SDS, and 2 x 106 cpm/ml of a radiolabeled probe). Rainbow trout Vg (rtVg) and actin cDNA were radiolabeled by random priming as previously described [28]. The estradiol receptor (rtER) single-strand probe was labeled using Dynabeads M-280 (Dynal, Oslo, Norway). After 16 h of hybridization, blots were washed four times with 2x SSC, 0.1% SDS for 5 min at room temperature, then three times for 15 min at 50°C with 0.1x SSC, 0.1% SDS. The washed blots were autoradiographed at -70°C. Radioactivity was quantified by instantimager (Packard) or densitometry.

Transient Transfections

The CHO-K1 cells were grown in 24-well plates at 37°C in phenol red-free Dulbecco's modified Eagle's medium-F12 (Sigma) containing 10% fetal calf serum (BioWhittaker). One hour before transfection, the medium was replaced with 10% charcoal-treated serum medium. Cells were transfected by using a calcium phosphate DNA precipitation method [29] with 875 ng of Basic luciferase reporter vector or 875 ng of 2.0 Basic (containing 2 kb of the rtER promoter) or 250 ng of ERE TK luciferase or 175 ng of MMTV luciferase. In each case, three expression vectors were transfected together: CMVrtER (50 ng), CMVrtGR (50 ng), and CMV-COUP-TF1 (25 ng). Each dish received 1 µg of DNA, using Bluescript vector for complementation. After 18 h of incubation at 37°C with 2% CO2, cells were washed once with PBS, and 1 µM of the steroids was added. Cells were harvested 36 h later for luciferase and protein assays. Luciferase activities were normalized to total cellular protein.

Reporter vectors Basic is the pGL2 Basic vector with the luciferase reporter gene from Promega (Madison, WI). 2.0 Basic is the pGL2, containing the 2.0-kb rtER promoter sequence (-2078/+28) with its own starting site prepared by Lazennec et al. [15]. ERE-TK-luc is a generous gift from Dr. P. Webb (EMBL, Heidelberg). MMTV-luc is the pCF 31 luciferase that was constructed by Dr. F. Gouilloux (Kremlin Bicètre, Paris).

Expression vectors The CMV-rtER (containing the strong cytomegalovirus promoter from pCMV5) was provided by Dr. B. Katzenellenbogen (University of Illinois, Urbana). The CMV-rtGR was also derived from pCMV5 [17], and CMV-COUP-TF1 was derived from pcDNA3 from Invitrogen and contains the human COUP-TF1.

Statistics

Statistical analysis was performed using Statview 4.02 software. Comparisons were made using analysis of variance and Fisher test. Any P values less than 0.05 were considered as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cortisol Implants Inhibit Liver rtER and rtVg Expression In Vivo

This experiment was performed in female fish at the beginning of vitellogenesis (GSI around 2.5%). Cortisol implants caused a 100% increase in plasma cortisol concentration 5 days after implantation, but levels returned to control values by Day 15 (Fig. 1b). Although E2 levels significantly increased between 5 and 15 days after implantation, cortisol implants had no significant effect on plasma E2 levels either 5 days or 15 days after implantation (Fig. 1a). The gonadal weight was not affected by the implants (data not shown).



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  		FIG. 1. Effect of cortisol implants on plasma estradiol (a) and cortisol (b) levels in maturing female trout: maturing female rainbow trout (600 g) were implanted with cocoa butter (sham) or cocoa butter containing cortisol (60 mg/kg). At Day 5 or 15 postimplantation, fish were rapidly anesthetized, and blood was taken for steroid measurements. Values represent the mean (±SE) of 12 individual fish. *Significantly different from the corresponding sham group, P < 0.05. ##Significantly different from sham at Day 5, P < 0.01

After total RNA extraction, rtER and rtVg were analyzed by dot-blot hybridization with specific DNA probes. The rtER mRNA levels in control fish were constant over time but decreased by 80% 5 days after implantation and by 50% after 15 days (Fig. 2a). The rtVg mRNA levels were also constant in control fish between 5 and 15 days. Cortisol treatment caused 73% decrease in the rtVg mRNA levels 5 days after implantation, but by Day 15 rtVg mRNA levels were not significantly different between sham and treated fish (Fig. 2b).



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  		FIG. 2. Effect of cortisol implants on rtER (a) and rtVg (b) liver mRNA levels in maturing females rainbow trout (see legend to Fig. 1 for details). Total RNA was measured by dot blot using rtER, rtVg, and actin probes 5 or 15 days after implantation. Actin was used as an internal standard for quantification. Values represent the mean (±SE) of 12 individual fish. **Significantly different from the corresponding sham group, P < 0.01. *Significantly different from the corresponding sham group, P < 0.05

Dexamethasone Inhibits rtER and rtVg Expression in Aggregated Hepatocyte Cultures

To investigate whether the inhibitory effect of cortisol observed in vivo on the rtER and rtVg mRNA levels is a direct cortisol effect, a series of experiments was performed in vitro on hepatocyte aggregates. These culture conditions allow the maintenance of differentiated hepatocytes, which have shown that E2 stimulates rtER and rtVg expression [11]. To avoid steroid contamination, ultroser-SF (steroid free) was used. In a preliminary experiment, hepatocyte aggregates were maintained in culture for 8 days and then different concentrations of E2 (0–1000 nM) were added together with 1000 nM of Dex. After 24 h, cells were harvested and the rtER and rtVg mRNA were measured by dot blots. Under these conditions, the basal levels of rtER and rtVg mRNA were detectable (Fig. 3 a,b), and a 24-h treatment with E2 increased rtER and rtVg mRNA in a dose-dependent manner. At the highest dose of E2 (1000 nM) both mRNA levels were increased by about 500%. The addition of Dex strongly inhibited the basal rtER mRNA levels (by about 80%) and also the E2-stimulated mRNA levels (between 80% for 1 nM of E2 and 35% for the 1000 nM dose, Fig. 3a). With respect to rtVg, the basal mRNA levels were unaffected by Dex, and a significant decrease was only detected for the highest dose of E2 (about 25% for the 100 and 1000 nM doses, Fig. 3b).



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  		FIG. 3. Effects of Dex on rtER (a) and rtVg (b) mRNA levels in E2-stimulated cultured hepatocytes. Hepatocytes from male trout liver were isolated by collagenase perfusion. Aggregated hepatocytes were treated 8 days after plating, with increasing concentrations of estradiol (E2, 0–1000 nM) or by a combination of the same E2 concentrations and 1000 nM of Dex. After 24 h of treatment, the cells were harvested and total RNA was prepared and hybridized with rtER, rtVg, and actin DNA probes using dot-blot technique. Actin was used as an internal standard for quantification. Values are expressed as the mean ± SE from three separate culture dishes. #Significant differences between E2-stimulated cells and unstimulated control; #P < 0.05 and ##P < 0.01. *Significant differences between Dex-treated and Dex-untreated cells, *P < 0.05 and **P < 0.01

To determine the Dex concentrations that cause a decrease in rtER and rtVg mRNA levels in aggregated hepatocytes, hepatocytes were incubated either with or without 100 nM of E2 and an increasing amount of Dex (Fig. 4 a,b). The selected E2 concentration is within the physiological range in maturing female trout. In this experiment, the basal levels of rtER and rtVg were detectable and stimulation with 100 nM of E2 causing a 240% and 370% increase in rtER and rtVg mRNA levels, respectively. In the case of rtER mRNA, Dex significantly decreased both basal and E2-stimulated levels in a dose-dependent manner (Fig. 4a). The maximum inhibition was achieved with the highest dose of Dex on both basal (80%) and E2-stimulated (70%) levels (Fig. 4a). For the rtVg messenger RNA, Dex also significantly reduced the basal and stimulated levels according to the Dex concentration (Fig. 4b), but the inhibition was lower than in the case of the rtER mRNA.



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  		FIG. 4. Effects of increasing doses of Dex on basal and E2-stimulated levels of rtER (a) and rtVg (b) mRNA. Aggregated hepatocytes were incubated either with or without 100 nM E2 and exposed to increasing concentrations of Dex (0–1000 nM). After 24 h, rtER, rtVg, and actin mRNA were measured by dot-blot hybridization. Values are expressed as the mean ± SE from three separate culture dishes. *Significant effect of Dex versus controls without Dex (0 nM). *P < 0.05 and **P < 0.01

In hepatocyte aggregates, maximal rtVg mRNA stimulation is achieved after a 48-h incubation with E2. Consequently, the effect of Dex was also tested on E2-prestimulated hepatocyte aggregates. Hepatocytes were pretreated for 24 h with different E2 concentrations. The medium was then changed and 1000 nM of Dex together with renewed E2 were added. The basal rtER mRNA level was detectable and was significantly increased by E2 in a dose-dependent manner (by 700% for the highest dose, Fig. 5). As observed previously, Dex also strongly inhibited both the basal and E2-stimulated rtER mRNA levels (Fig. 5). Under, these conditions there was a dramatic increase in rtVg mRNA levels that was not significantly affected by Dex (data not shown).



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  		FIG. 5. Effect of Dex on rtER mRNA levels in E2-prestimulated hepatocytes. Aggregated hepatocytes were cultured for 8 days. Culture dishes were incubated with or without different concentrations of E2 (0–1000 nM). After 24 h, the culture medium was renewed with the same E2 concentrations, either with or without 1000 nM Dex. After another 24 h, rtER and actin mRNA were monitored by dot-blot hybridization. Values are expressed as the mean ± SE from three separate culture dishes. #Significant differences between E2-stimulated cells and unstimulated controls; #P < 0.05. *Significant effect between Dex-treated and Dex-untreated cells; *P < 0.05

The Inhibitory Effect of Dex on rtER mRNA Is Specific

In order to ensure that the effects of Dex were specific and not due to interference with other hormones potentially acting on the liver, the effects of various hormones were tested. Among the different hormones tested, only Dex and cortisol were able to inhibit basal (Fig. 6a) and E2-stimulated (Fig. 6b) rtER mRNA levels. Other hormones, such as 17{alpha},20ß-dihydroxyprogesterone (a maturational steroid in teleosts), testosterone, T3, salmon prolactin, and salmon growth hormone, had no effect.



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  		FIG. 6. Specificity of glucocorticoid effects on rtER mRNA basal (a) and E2-stimulated (b) levels. Aggregated hepatocytes were stimulated (b) or not (a) by E2 (10 nM) together with different hormones: Dex, cortisol, testosterone (T), 17{alpha}-20ß-dihydroxy 4-pregnen 3-one (17,20P), triiodo-L-thyronine (T3) at 1 µM, salmon PRL (50 ng/ml), and salmon GH (100 ng/ml). After 24 h of treatment, rtER and actin mRNA were measured by dot blot. Values are the mean ± SE of four different experiments. *Significant differences between hormone-treated cells and untreated cells (a) or between combined treatment by E2/hormones and E2 alone (b). *P < 0.05; **P < 0.01

Dex Does Not Affect the Stability of the rtER mRNA

Transcriptional and/or post-transcriptional regulations may be implicated in the mechanisms mediating the effects of Dex on rtER mRNA levels. Consequently, the potential post-transcriptional contribution was investigated by measurement of the rtER mRNA half-life. To this end, the transcriptional activity was blocked by addition of actinomycin D to cultured cells and the decrease of specific rtER mRNA levels was evaluated over time. Because the low basal levels of rtER mRNA at time zero would not allow accurate monitoring of values expected to decrease, the effect of Dex on the half-life of rtER mRNA was tested on E2-stimulated hepatocytes, in order to achieve high levels of rtER mRNA at the beginning of the experiment. In both control and Dex-treated cells, the rtER mRNA levels (Fig. 7) decreased over time and, after 24 h, reached a level near 20% that at the start. At each sampling time, the rtER mRNA levels were not significantly different between Dex-treated and control cells. Consequently, the half-life of rtER mRNA in E2-stimulated control cells (12 h) was not significantly different from that of rtER mRNA in Dex-treated cells (15 h). In the same conditions, the half-life of actin mRNA was longer (24 h) and not modified by Dex treatment (data not shown).



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  		FIG. 7. Stability of rtER mRNA in E2-stimulated cultured hepatocytes after Dex treatment. Aggregated hepatocytes were treated with 100 nM E2 for 36 h. They were then exposed to a combination of E2 (100 nM) either with or without dexamethasone (1000 nM) for 12 h. Actinomycin D (0.4 µg/ml) was added and cells were harvested at 0, 3, 6, 12, and 24 h for mRNA measurement. Values are expressed as the % of rtER mRNA levels at time 0. Each value represents the mean of three culture dishes. No significant difference was observed between Dex-treated and untreated cells

Effect of rtGR on the Transcriptional Activity of rtER Gene Promoter

The previous experiment suggests that Dex does not influence the rtER mRNA stability but inhibits rtER mRNA accumulation by transcriptional repression. This potential transcriptional effect was tested by means of transfection studies. Indeed, as cortisol receptor mRNA and protein are detected in the liver of trout [17, 18], the transcriptional effect of Dex may be explained by the negative effect of rtGR on the transcriptional activity of the rtER gene. This hypothesis was tested using cotransfection experiments in CHO-K1 cell line, with the luciferase reporter gene of the pGL2 basic vector controlled by the rtER (2 kb) complete promoter region (2.0 basic) and the rtGR and rtER expression vectors. In these experiments, a COUP-TF expression vector was also added, as this transcription factor was shown to positively cooperate with rtER for this promoter induction and also as COUP-TF is also present in fish liver cells [15]. With the control pGL2 basic vector, the level of expression was low, and no significant effect of E2 or Dex was observed (Fig. 8). For the rtER promoter reporter construct (referred to as 2.0 Basic), E2 increased the luciferase expression by 340%. Dex addition strongly inhibited the E2-stimulated luciferase expression but had no effect on the basal level (Fig. 8). Under the same conditions, the MMTV luciferase reporter gene was strongly stimulated by Dex, demonstrating the transactivation capacity of the expressed rtGR protein. On this reporter gene, E2 had no significant effect on either the basal or the Dex-stimulated luciferase expression. Using another luciferase reporter gene, controlled by an ERE-TK promoter (ERE-TK-luc), a classical induction by E2 was observed. After Dex treatment, the basal level was not changed, and the E2-stimulated level was not decreased as in the case of the 2.0 Basic construct. In contrast, Dex in combination with E2 induced a slight increase in luciferase activity (Fig. 8). These results indicate that the inhibition observed by the rtGR on the 2.0 Basic reporter gene expression is specific for this promoter, as no Dex effect was seen on the Basic reporter nor on another ERE-dependent promoter, such as the ERE-TK-luc.



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  		FIG. 8. Effects of rtGR on the transcriptional activity of the rtER gene promoter in transfection experiments in the CHO-K1 cell line. Cells were cotransfected by the calcium phosphate precipitation method [29] with 875 ng of 2.0 Basic luciferase reporter gene, and three expression vectors: 50 ng CMV-rtGR, 50 ng CMV-rtER, and 25 ng of CMV-COUP-TF1. Controls were transfected with the Basic reporter gene, which gives the basal activity of the pGL2-Basic, or with the ERE-TK-luciferase vector (250 ng) sensitive to ER stimulation, or with the MMTV-luciferase (175 ng) sensitive to GR stimulation. NT is the negative control (untransfected cell). The cells were washed with PBS 18 h after transfection and treated with steroids (1000 nM), E2, and/or Dex. Cells were harvested 36 h after steroid treatment for luciferase and protein assays. Luciferase activities were normalized to total cellular protein and each value represents the mean of six culture dishes


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results reported here demonstrate that cortisol, or its analog Dex, can inhibit the liver expression of two genes crucial in fish reproduction, the ER and Vg genes. Our in vivo investigations on previtellogenic female trout indicate that the inhibitory effects of glucocorticoids on the expression of these two genes occur at the mRNA level, suggesting a transcriptional regulation. However, the long-term effects of cortisol may be explained either by a direct action of cortisol on the liver or by indirect mechanisms implicating other hormones, themselves regulated by cortisol. It is well known that ER and Vg are under estrogenic regulation [10, 12], and a decrease in plasma E2 and gonadotropin (GTHs) levels were reported after cortisol implantation in maturing female brown trout [8], which could explain the decrease of plasma Vg. In tilapia, cortisol implants also decreased E2 levels and ovary size [30]. However, under our experimental conditions, cortisol implants did not affect plasma E2 levels, excluding a possible indirect implication of E2 in the inhibitory effects of cortisol on rtER and rtVg expression. Nevertheless, these results cannot exclude indirect effects of other hormones such as GH or thyroid hormone (T3) on the regulation of Vg or E2. Such effects have been reported in other species such as the frog Rana esculata [31], and Xenopus [32], and rat [33]. However, our results seem to rule out the possibility of indirect effects mediated by other steroids or metabolic hormones as our data show that none of the hormones tested in culture were able to mimic the effects of Dex.

To demonstrate a direct glucocorticoid action on liver cells, we used cultured hepatocytes under conditions required for the formation of cell aggregates [11]. Under these culture conditions, we consistently observed a strong up-regulation of rtER and rtVg mRNA by E2, with a higher sensitivity of rtER induction in comparison to rtVg, in agreement with Flouriot et al. [11, 12]. Whatever the experimental conditions of E2 stimulation, we always observed a strong inhibition by Dex of the rtER mRNA stimulated levels and a moderate decrease of rtVg mRNA levels. These results obtained in cultured hepatocytes after short-term treatment are comparable to those obtained previously after long-term in vivo treatment of previtellogenic females and demonstrate the direct effect of glucocorticoids on the rtER and rtVg mRNA in E2-stimulated conditions. On the other hand, basal levels of rtER and rtVg mRNA slightly fluctuated between the different cultures as each culture was obtained from a single animal. Hence, in the basal or stimulated conditions, Dex always presented a stronger inhibitory effect on rtER than on rtVg mRNA levels. As with the rtER gene, the rtVg gene is also up-regulated by E2-activated ER, but these two genes present a completely different pattern of stimulation with a lower sensitivity of the rtVg gene and a delay in the time course of stimulation compared to rtER [12]. It was also demonstrated that the Vg gene transcriptional response is directly proportional to the amount of synthetized ER, in contrast with the rtER gene [34]. Therefore, it is not surprising that a decrease in rtER mRNA levels after Dex treatment does not induce the same range of decrease in rtVg mRNA.

Because rtER mRNAs were strongly and consistently modified by Dex treatment, we decided to investigate the mechanisms of the down-regulation of rtER gene expression that can involve either a transcriptional suppression or a post-transcriptional regulation. For example, glucocorticoids can repress c-jun transcription by interferences with others transcriptional factors [35]. However, a combination of two mechanisms is involved in the repression of the cyclooxygenase-2 gene (COX-2). In fact, GR partially transrepressed the COX-2 gene, but there is also a major role for a post-transcriptional mechanism caused by the loss of the COX-2 messenger RNA poly A tail [36], which is also of major importance. To investigate the mechanisms involved, we first analyzed the half-life of rtER mRNA in the presence of Dex, using actinomycin D in vitro. In E2-stimulated hepatocytes, the mRNA half-life was evaluated to 12 h, in agreement with Flouriot et al. [12], and after Dex addition the half-life was not significantly different from the controls. As Dex does not affect the stability of rtER mRNA, under stimulated conditions we can conclude that post-transcriptional pathways are not involved in the present case. Regulation at the transcriptional level was confirmed by the use of cotransfection assays in a heterologous system, the CHO cell line. This cell line is devoid of ER, has very few GR, and can be efficiently transfected by the rainbow trout expression vectors, rtER and rtGR, together with a reporter vector controlled by the complete rtER promoter fragment previously isolated and extensively studied in this cell system [14, 15, 37]. In these transient cotransfection assays, the two expression vectors are functional for transactivation, i.e., Dex activates a classical GRE-dependent reporter vector (MMTV-luc) and E2 activates a classical ERE-dependent reporter vector (ERE-TK-luc). Moreover, on the rtER 2.0 Basic vector, in which the E2 induction is similar to that reported in previous studies [15], Dex partially blocks the E2 transcriptional induction, demonstrating the inhibitory function of rtGR on the E2-stimulated transcriptional activity. Under the same experimental conditions, the promoters of the ERE-dependent reporter gene (ERE-TK-luc) and of the Basic reporter gene are not sensitive to Dex inhibition. This demonstrates the specificity of the rtER promoter response to Dex and excludes a nonspecific inhibitory effect of rtGR on the general transcriptional machinery. The transcriptional interference between rtGR and rtER on the rtER gene regulation is also well correlated with the physiological responses observed in vivo after cortisol implants and explains the direct effect of Dex on rtER mRNA levels in cultured hepatocytes.

What Is the Molecular Mechanism for the ER/GR Interference?

Interactions between GR and other transcription factors (TFs) have been extensively investigated to explain the inhibitory effects of GR on the transactivation of different genes [21, 38]. For example, in the proliferin model, the binding of GR to DNA has been shown to inhibit AP1 binding and thus AP1 transcriptional activity [39]. In the case of collagenase, AP1 activity is repressed by direct protein/protein interaction between the Jun-Fos heterodimer and GR, with no DNA binding of GR [40, 41]. Such direct protein/protein interactions between GR and ER have never been reported and are unlikely to explain GR/ER transcriptional interference. An alternative explanation could involve interactions between GR and other TFs implicated in the stimulation of ER-activated transcription. Some of the present data could support this hypothesis. Firstly, some TFs have been reported to cooperate positively with ER for gene transactivation, for example, SF1, SP1, and AP1 in the cases of salmon gonadotropin IIß [42], heat shock protein of breast cancer cells [43], and ovalbumin [44], respectively. In addition, GR has been shown to interfere with ER at the AP1 site of the collagenase promoter [45]. In the case of the rtER, the positive autoregulation of the gene by E2 is enhanced by different TFs depending on the cellular or promoter context. In the CHO cell line, COUP-TF1 cooperates with rtER in enhancing autoregulation [15]. In the MCF7 cell line, ER cooperates with cell-specific transcription factors to enhance the rtER promoter autoregulation on an rtER-SV-luciferase reporter gene [37]. These TFs could be potential targets for GR. Interestingly, the basal activity of the rtER promoter (2.0 Basic) is not decreased by Dex treatment, contrary to the basal levels of rtER mRNA of cultured hepatocytes. This result may be explained by the lack of specific TF or specific coactivator in CHO cells necessary for the basal expression of rtER gene in liver cells. One hypothesis could be that the specific constitutive transcriptional activity of the rtER, as already described [16], is responsible for the maintenance of the rtER basal level in the liver cell and implicates interactions with specific coactivators that are missing in CHO cells.

In conclusion, our results indicate that opposite influences of E2 and Dex can be mediated at the transcriptional level by ER/GR interferences and could explain the physiological responses such as inhibition of ER and Vg gene expression by stress or cortisol in trout hepatocytes. This ER/GR cross-talk could also interfere in other physiological responses, when ER and GR are colocalized in the same cells, which is the case with some brain cells [46] (C. Teitsma and B. Ducouret, unpublished results), osteoblast-like cells [47], and breast tumor cell lines [48].


   ACKNOWLEDGMENTS
 
We are grateful to Nic and Sarah Bury for manuscript revision.


   FOOTNOTES
 
First decision: 6 December 1999.

1 This work was supported by the Ministère de l'Education Nationale et de la Recherche, the CNRS, the INRA, and the Fondation Langlois. Back

2 Correspondence: B. Ducouret, Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Campus de Beaulieu, Bat 13, 35042 Rennes cedex, France. FAX: 33 2 99 67 94; bernadette.ducouret{at}univ-rennes1.fr Back

Accepted: January 18, 2000.

Received: November 8, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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