Autoregulation of glucocorticoid receptor by cortisol in rainbow trout hepatocytes

Ramesh Sathiyaa and Mathilakath M. Vijayan

Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We used primary cultures of trout hepatocytes and a physiological dose of cortisol (100 ng/ml), mimicking stressed levels in salmonid fish, to address the impact of glucocorticoid stimulation on glucocorticoid receptor (GR) mRNA abundance and protein content. Cortisol significantly elevated GR mRNA content over a 24-h period; this increase was abolished by actinomycin D, suggesting transcriptional control of GR. However, cortisol significantly decreased GR protein content, leading us to hypothesize that lower GR protein content may be regulating GR mRNA abundance. Indeed, treatment of hepatocytes with MG-132, a proteasomal inhibitor shown to prevent GR degradation by cortisol, abolished cortisol-mediated GR mRNA upregulation. Also, geldanamycin, a heat shock protein 90-specific inhibitor, abolished the GR mRNA increase evident with cortisol but did not modify cortisol-induced increases in abundance of mRNA for phosphoenolpyruvate carboxykinase, a glucocorticoid-responsive gene, or hepatocyte glucose release. Together, our results suggest a negative feedback loop for GR gene regulation by cortisol in trout hepatocytes. The autoregulation of GR may be a crucial step in the physiological stress response process, especially in modulating energy-dependent processes that are glucocorticoid dependent, including gluconeogenesis.

hsp90; proteasome; geldanamycin; stress; gluconeogenesis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GLUCOCORTICOIDS ARE PRODUCED in response to stress and play a major role in the stress response process, especially in the metabolic adjustments to stress (2, 18). The mechanism(s) of action of glucocorticoids include the binding of ligand to a cytosolic glucocorticoid receptor (GR), translocation of ligand-GR protein heterocomplex to the nucleus, binding to specific DNA regions of the promoter, the glucocorticoid response elements, and initiating the transcription of glucocorticoid-responsive genes (2). Glucocorticoid binding to GR regulates transcriptional and translational activation and/or repression of several genes involved in cellular homeostasis, including GR in a cell type-specific manner (2, 7, 35). There are also a number of well-characterized metabolic responses associated with glucocorticoids, including activation of phosphoenolpyruvate carboxykinase (PEPCK) and the production of glucose via gluconeogenesis to fuel energy-dependent processes (2, 18).

Most studies on glucocorticoid impact on GR showed a reduction in GR mRNA by about half of that seen in untreated transformed cell lines (21, 38), except in immune cells where GR mRNA was upregulated (see Ref. 7); the GR protein changes normally mirrored GR mRNA content in these studies. The GR downregulation evident with glucocorticoids in most cell types may be due to glucocorticoid-mediated attenuation of gene transcription (21) and/or enhanced protein degradation via the proteasome (35). Because the majority of studies on GR regulation utilized transformed cell lines with reporter constructs to characterize the transcriptional and translational processes, the physiological relevance of such studies is less clear.

Using a physiologically relevant cell system, the trout hepatocytes in primary culture, we showed recently that the proteasomal pathway modulates glucocorticoid impact on glucocorticoid-responsive genes (5). Specifically, the heat shock protein (hsp) 70 response to heat shock was attenuated in cortisol-exposed hepatocytes, and this response correlated with lower GR content. Proteasomal inhibitors, MG-132 and lactacystin, prevented cortisol-mediated GR downregulation and also abolished the hsp70 response to cortisol, suggesting that the cellular GR concentration may be crucial for GR signaling (5). Indeed, in trout hepatocytes with lowered GR content, cortisol-induced glucose release was also completely abolished (4), confirming the impact of GR concentration on cellular response to glucocorticoids. Because the physiological responses to glucocorticoid stimulation depend on GR content, the autoregulation of GR may be a key process in the transcriptional signaling of glucocorticoid-responsive genes, but this has never been tested in a physiologically relevant model system.

With this in mind, we tested the hypothesis that cellular GR protein content regulates GR transcriptional machinery in primary cultures of trout hepatocytes. Because hsp90, a ubiquitous molecular chaperone, is essential to maintain GR in a ligand-binding conformation (11, 19), we also investigated the role of hsp90 in the regulation of GR and glucocorticoid-responsive genes. To this end, we used geldanamycin (GA), an hsp90-selective inhibitor that disrupts the function of hsp90 substrate proteins including GR (9, 23, 25, 37), as a tool to identify specific roles of hsp90 on GR regulation. Induction of PEPCK, a key gluconeogenic enzyme induced by glucocorticoids (12), and the capacity for glucose release by hepatocytes were used as indicators of glucocorticoid-responsive gene activation and physiological responses. Our results suggest negative feedback regulation of GR mRNA abundance by GR protein content in trout hepatocytes treated with cortisol. We implicate the proteasome and hsp90 in the autoregulation of GR by glucocorticoids in trout hepatocytes.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Primary Cell Culture

Rainbow trout (Oncorhynchus mykiss) were obtained from Rainbow Springs Trout Farm (Thamesford, ON, Canada) and kept at the University of Waterloo Aquatic Facility at 12 ± 1°C on a 12:12-h light/dark cycle and fed once daily to satiety (3 pints of sinking food; Martin Mills, Elmira, ON, Canada). The fish were acclimated for at least 2 wk before the experiments. Hepatocytes were isolated using collagenase perfusion according to established protocols (22) and plated in six-well Primaria plates (GIBCO) in L15 medium (Sigma, St. Louis, MO) at a density of 0.75 × 106 cells/ml (1.5 × 106 cells/well). The hepatocytes were maintained at 13°C for either 24 or 48 h (only the GA study) before the start of the experiments. The experimental protocol consisted of replacing the media with either fresh media (control) or fresh media containing MG-132 (50 µM; Calbiochem, San Diego, CA), actinomycin D (0.5 mM; Sigma), RU-486 (1,000 ng/ml), GA (1,000 ng/ml; Sigma), or hydrocortisone (100 ng/ml; Sigma) and sampling the media and cells at 24 h, except for the time-course study (sampling at 1, 4, 8, and 24 h after cortisol treatment). In combined treatments (inhibitors and cortisol), cells were incubated with the inhibitors first (for 2.5 h) and then cortisol was added to the media. Cortisol, RU-486, and GA were dissolved in ethanol, whereas MG-132 was dissolved in DMSO, and the final concentration of either ethanol or DMSO in the incubation medium did not exceed 0.01%. The control cells received the same amount of ethanol and DMSO as the treatment groups. Individual experimental details including sampling time are included in the figure legends. The sampling consisted of collecting medium and washing cells with diethyl pyrocarbonate-treated ice-cold rinsing medium (50 mM Tris, pH 7.4). The cells were centrifuged (13,000 g for 1 min), supernatants were removed, and the cell pellets were flash frozen in liquid nitrogen and stored at -70°C.

Glucose Release and PEPCK Activity

Media glucose was determined colorimetrically by using a commercially available kit (Trinder method; Sigma). Hepatocyte PEPCK activity was determined spectrophotometrically (at 340 nm) on a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA) according to an established protocol (32).

RNA Isolation and Northern Blot Analysis

RNA isolation was performed using RNeasy mini kits (Qiagen), and Northern blot analysis of hsp90 mRNA was performed as described previously (22). Briefly, 5 µg of total RNA was separated on a 1% agarose-formaldehyde gel (2.5 h at 90 V), transferred, using a capillary method, to a positively charged nylon membrane (Roche Diagnostics), and the RNA was fixed to the nylon membrane by UV cross-linking. The blot was stained with reversible Northern blot stain (Sigma) to confirm the transfer of RNA. Hsp90 primers were designed from a conserved region of Chinook salmon (GenBank accession no. U89945) and zebrafish hsp90 cDNA sequences (AF068773) to give a 500-bp fragment by PCR amplification for use as a probe. The primers used for making the probe were: forward, 5'-GAG GAG ATC TCC ATG GTC-3'; reverse, 5'-TTG TTG TCC TTG GTC ATG GCT CTC-3'.

Plasmid DNA containing the hsp90 probe was linearized using the EcoRI restriction enzyme (MBI Fermentas) and radiolabeled (32P) by using a random prime labeling system (Amersham Pharmacia). The unincorporated nucleotides were cleaned using a QIAquick gel extraction kit (Qiagen). The membrane was prehybridized (Rapid-hyb buffer; Amersham Pharmacia) for 1.5 h at 65°C followed by hybridization (Rapid-hyb buffer) for 3 h at 65°C with a probe concentration of 2 ng/ml (with ~109 counts/min). After hybridization, the membrane was washed for 20 min with 2× SSC and 0.1% SDS at room temperature, followed by two 15-min washes with 0.5× SSC and 0.1% SDS at 65°C. The membrane was visualized by autoradiography using a Storm phosphorimager (Molecular Dynamics).

Quantitative Real-Time PCR

First-strand cDNA synthesis. The first-strand cDNA was synthesized from 1 µg of total RNA using a cDNA synthesis kit (MBI) according to the manufacturer's instructions. A relative standard curve for each gene of interest was constructed using cDNA synthesized by this method. An appropriate volume of cDNA for each gene was determined during quantitative real-time PCR (Q-PCR) optimization (iCycler; Bio-Rad).

Primers. The following primers were designed by using rainbow trout PEPCK, GR, and beta -actin cDNA sequences (GenBank accession nos. AF246149, Z54210, and AF157514) to amplify a ~100-bp product for Q-PCR quantitation: rainbow trout PEPCK, forward primer 5'-TGC TGA GTA CAA AGG CAA GG-3' and reverse primer 5'-GAA CCA GTT GAC GTG GAA GA-3'; rainbow trout GR, forward primer 5'-AGA AGC CTG TTT TTG GCC TGT A-3' and reverse primer 5'-AGA TGA GCT CGA CAT CCC TGA T-3'; and rainbow trout beta -actin, forward primer 5'-AGA GCT ACG AGC TGC CTG AC-3' and reverse primer 5'-GCA AGA CTC CAT ACC GAG GA-3'.

Relative standard curve. Relative standard curves for target genes (GR and PEPCK) and a housekeeping gene (beta -actin) were constructed by using cDNA (assumed concentration 500 pg/µl) ranging from 10 to 3,000 pg per PCR. Platinum Quantitative PCR Supermix-UDG (Invitrogen) was used for Q-PCR, and the samples were treated according to the manufacturer's instructions. Forward and reverse primers (0.2 µM) and 1:100,000 SYBR green I nucleic acid gel stain (Roche) was used in each PCR reaction. Samples and standards were run in triplicate on 96-well PCR plates (Bio-Rad) according to the manufacturer's instructions.

Quantification of samples. An optimized volume of cDNA was used for the amplification of each gene. The reaction components were exactly the same as the previous section, and for every single test sample, a Q-PCR for both the target (PEPCK and GR) and the housekeeping gene (beta -actin) was performed. The following PCR cycles were used for gene amplification: 95°C for 3 min, 40 cycles at 95°C for 20 s, 49°C for 20 s, and 72°C for 20 s, followed by a 4°C hold.

Data analysis for quantification of gene expression. Calculation of the threshold cycle values for every sample was performed using iCycler iQ real-time detection software (Bio-Rad). An Excel chart (xy scatter plot) was used to construct a standard curve with log input amount and threshold cycle values. The input amount for each sample was calculated for target gene and actin using the appropriate standard curve. The amount of target gene was divided by the amount of actin to determine the normalized amount of the target gene. The normalized amount of target gene (a relative unit) was then standardized by using an internal calibrator (control samples of each experiment). The mRNA content with treatments was expressed as percentage of control (internal calibrator).

Preparation of Trout-Specific GR Antibodies

Cloning and expression of rainbow trout GR in Escherichia coli. Full-length rainbow trout GR cDNA was cloned by RT-PCR technique using the gene-specific primers (forward, 5'-GGT GGT GCA TGC ATG GAT CCA GGT GGA CTG AAA C-3'; reverse, 5'-GGT GGT CCC GGG TAA GGC ATT GTG TCA TGG-3') designed from a published rainbow trout GR sequence (10). After confirmation of the full-length GR insert by sequencing, the GR cDNA was directionally subcloned into a pQE-30 (QIAexpress kit; Qiagen). Cloning, transformation, screening of transformed colonies, expression, and purification of the NH2-terminal poly his-tagged GR were performed according to the manufacturer's instructions (Qiagen). The recombinant GR was purified under denaturing conditions using nickel-nitrilotriacetic acid metal affinity chromatography matrix (supplied by the manufacturer) according to established protocols (28). The protein expression level was estimated to be ~125 µg/100 ml culture.

Antibody generation. Antibodies for the purified recombinant rainbow trout GR were generated in rabbits according to established protocols (6) at the animal care facility, Department of Biology, and approved by the University of Waterloo animal care committee. The specificity of our polyclonal antibody for trout GR was confirmed by probing parallel gels with another trout-specific GR polyclonal antibody (30), which was a generous gift from Dr. Ducouret (Université de Rennes).

Western Blot Analysis

Total protein (50 µg) was separated on an 8% SDS-PAGE set at 200 V for 40 min using 1 × TGS (250 mM Tris, 1.92 M glycine, 1% SDS) and transferred onto a nitrocellulose membrane (Bio-Rad) using a Trans-blot SD semidry electrophoretic transfer cell (Bio-Rad). A 5% solution of nonfat dry milk in 1× TTBS (2 mM Tris, 30 mM NaCl, 0.01% Tween 20, pH 7.5) was used as a blocking agent (1 h at room temperature) and for diluting antibodies. The blots were incubated with primary antibodies for 1 h at room temperature [For hsp90, rat anti-human hsp90 monoclonal antibody (1:1,000), Stressgen; For GR, rabbit anti-recombinant trout GR polyclonal antibody (1:1,000) followed by a 1-h incubation with alkaline phosphatase-conjugated secondary antibody (Bio-Rad or Stressgen)]. The membranes were washed after incubation in either primary [2 × 15 min washes in TTBS (20 mM Tris, 300 mM NaCl, pH 7.5, 0.1% Tween 20)] or secondary antibodies [2 × 15 min in TTBS followed by 2 × 15 min in TBS (20 mM Tris, 300 mM NaCl, pH 7.5)]. The protein bands were detected using 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium color substrate (Bio-Rad). The protein bands were quantified using Chemi imager (Alpha Innotech).

Data Analysis

Percentages were arcsine transformed or log transformed for homogeneity of variance, and the means were compared using either Student's paired t-test or one-way analysis of variance (see Fig. 1A). However, nontransformed values are shown in the figures. Values of P <=  0.05 were considered significant.


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Fig. 1.   Impact of cortisol on glucocorticoid receptor (GR). A: temporal profile of cortisol effects on GR mRNA abundance in primary cultures of rainbow trout hepatocytes. GR mRNA was quantified using real-time PCR. Cells were treated with cortisol (100 ng/ml), and samples were taken at 0, 1, 4, 8, and 24 h after cortisol treatment. Bars represent %change from control (0 ng/ml cortisol) and are shown as means ± SE (n = 4, except 4 h, where n = 2). * Significantly different (P < 0.05). B: effect of cortisol (100 ng/ml) on GR protein expression at 24 h in trout hepatocytes. Bars represent means ± SE (n = 4). * Significantly different from control (paired Student's t-test; P <=  0.05). A representative GR Western blot is shown at top, and the membranes were probed with polyclonal antibodies raised against recombinant trout GR (see METHODS for details). C: effect of cortisol, actinomycin D (0.5 mM), and MG-132 (50 µM) on GR mRNA abundance in primary cultures of trout hepatocytes. GR mRNA was quantified using quantitative real-time PCR (Q-PCR). Bars represent %change from control (0 ng/ml cortisol) and are shown as means ± SE (n = 5). * Significantly different from control (paired Student's t-test; P <=  0.05).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cortisol Impact on GR Regulation

After exposure to a concentration of cortisol simulating stressed salmonid fish (20), GR mRNA of trout hepatocytes was significantly elevated at 24 h after treatment (Fig. 1, A and C). In contrast, GR protein content was significantly lower following cortisol treatment than in the control group (Fig. 1B). The significant inhibition of cortisol-induced GR mRNA abundance in trout hepatocytes with actinomycin D (Fig. 1C), a transcriptional inhibitor, suggests that the cortisol impact on GR was regulated at the transcriptional level.

MG-132, a proteasomal inhibitor that prevents GR protein degradation (8), significantly decreased GR mRNA compared with the control trout hepatocytes (Fig. 1C). Also, MG-132 completely abolished cortisol-mediated GR mRNA abundance in trout hepatocytes, suggesting the possibility that GR protein content may regulate GR mRNA abundance (Fig. 1C).

GA impact on GR regulation

GA, an hsp90 inhibitor, significantly increased hsp90 mRNA abundance in trout hepatocytes either in the presence or absence of cortisol (Fig. 2A). This response corresponded with a similar increase in hsp90 protein expression with GA (Fig. 2B). Inhibition of hsp90 binding with GA did not significantly affect GR mRNA (Fig. 3A). Cortisol significantly increased GR mRNA abundance even in this study (Fig. 3A). However, the magnitude of cortisol-induced GR mRNA abundance was lower than in the previous experiments (Fig. 1, A and C) and is due to the temporal differences in hormone addition (cells exposed to hormones only after 24 h of plating in the GA study as opposed to immediately on plating in the other experiments). Despite the lower GR mRNA response, GA completely abolished this cortisol-mediated GR mRNA abundance in trout hepatocytes, suggesting a role for hsp90 in the transcriptional regulation of GR (Fig. 3A). GA also decreased GR protein levels in trout hepatocytes but independently of cortisol treatment (Fig. 3B).


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Fig. 2.   Impact of geldanamycin (GA) on HSP90. A: representative Northern blot showing the effect of GA (1,000 ng/ml) on HSP90 mRNA abundance in primary cultures of trout hepatocytes, and the band intensities are shown as %control (0 ng/ml cortisol) on the histogram. Bars represent means ± SE (n = 3). * Significantly different from control (paired Student's t-test; P <=  0.05). B: representative Western blot showing the impact of GA on hsp90 protein in primary cultures of trout hepatocytes. Bars (means + SE; n = 5) represent band intensities expressed as %control (0 ng/ml cortisol). * Significantly different from control (paired Student's t-test; P <=  0.05).



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Fig. 3.   Impact of GA on GR. A: effect of cortisol and GA on GR mRNA abundance in primary cultures of trout hepatocytes. GR mRNA was quantified using Q-PCR. Bars represent %change from control (0 ng/ml cortisol) and are shown as means ± SE (n = 3). * Significantly different from control (paired Student's t-test; P <=  0.05). B: representative Western blot showing the impact of GA on GR protein in primary cultures of trout hepatocytes. Bars (means ± SE; n = 5) represent band intensities expressed as %control (0 ng/ml cortisol and GA). * Significantly different from control (paired Student's t-test; P <=  0.05).

Effect of GA on PEPCK mRNA Abundance and Enzyme Activity

To address the notion that the hsp90 impact on GR mRNA may be specific to GR transcriptional machinery and not due to its lack of binding to GR protein, we examined changes to a glucocorticoid-responsive gene, PEPCK, with GA either in the presence or absence of cortisol. As expected, cortisol significantly elevated PEPCK mRNA abundance compared with the control group in trout hepatocytes (Fig. 4A). GA showed no significant effect on PEPCK mRNA levels, and neither did it modify the cortisol-induced PEPCK mRNA abundance (Fig. 4A).


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Fig. 4.   Impact of GA on gluconeogenesis. A: effect of cortisol (100 ng/ml) and GA (1,000 ng/ml) on phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance (A), PEPCK activity (B), and media glucose release (C) in primary cultures of trout hepatocytes. PEPCK mRNA was quantified by using Q-PCR; RU-486 was added at a concentration of 1,000 ng/ml (C). Bars represent %control (0 ng/ml cortisol) and are shown as means ± SE (n = 3). * Significantly different from control (paired Student's t-test; P <=  0.05).

The PEPCK activity was significantly lower at 24 h after cortisol treatment in trout hepatocytes (Fig. 4B). There was no significant effect of GA on PEPCK activity compared with the control group, and GA also did not modify the cortisol-mediated lowering of PEPCK activity (Fig. 4B).

Effect of GA on Glucose Release

As expected, cortisol exposure significantly increased media glucose levels in primary cultures of trout hepatocytes. RU-486, a GR antagonist, abolished this glucose elevation, clearly implying a GR-mediated glucose response to glucocorticoid stimulation (Fig. 4C). GA neither had any significant effect on media glucose levels nor modified cortisol-induced glucose release in trout hepatocytes (Fig. 4C).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GR Autoregulation in Trout Hepatocytes

Our study for the first time shows a mismatch between GR mRNA abundance and protein content in response to glucocorticoid stimulation in trout hepatocytes (Fig. 1, A and B). This is contrary to most other studies, in which changes in GR mRNA correlated positively with GR protein content (7, 31). The reason for the differing response between our results and other studies, mostly mammalian models, are not clear, but may be related to species/cell type differences in glucocorticoid signaling as well as cellular responsiveness. Also, the majority of these studies utilized transformed cell lines with reporter constructs, and, although these studies are important for understanding the mechanisms of GR regulation, the physiological relevance of such studies have not been fully characterized.

The utility of primary cultures of trout hepatocyte as a model system for glucocorticoid responsiveness is well established (5). Also, the concentration of cortisol used in the present study mimics plasma cortisol levels seen in stressed salmonid fish (3), clearly suggesting a glucocorticoid-mediated upregulation of GR mRNA during stress in these fish. Moreover, the in vitro results concur with a recent in vivo study showing a similar mismatch between higher GR mRNA and lower protein content in the liver of rainbow trout chronically stimulated by cortisol (33), confirming the physiological relevance of our cell system.

The downregulation of GR protein seen with chronic cortisol stimulation in the present study (Fig. 1B) concurs with most other studies (2, 18); however, that is not the case with GR mRNA response. The higher GR mRNA abundance at 24 h with cortisol in the present study likely reflects differences in either GR transcription and/or mRNA stability. The lower GR protein content despite higher GR mRNA levels points to posttranscriptional modifications affecting mRNA stability. However, the inhibition of cortisol-induced GR mRNA abundance at 24 h with actinomycin D (inhibitor of transcription; Fig. 1C) clearly implies a glucocorticoid-mediated upregulation of the GR transcriptional machinery in trout hepatocytes. In bony fishes, only one GR has been cloned and sequenced in rainbow trout, and Northern blot analysis showed only a single mRNA species of 7.5 kb (10). Although the GR promoter region is not characterized in fish, mammalian studies have clearly shown the presence of multiple promoters regulated by several transcription factors, including GR (see Ref. 38 for a review). This flexibility in the promoter sequence may, therefore, allow for a multitude of signaling molecules coordinating GR gene expression either positively or negatively in a cell-specific manner but remains to be characterized in fish.

The ubiquitin-proteasomal pathway is thought to play a key role in this GR-signaling process (35). In trout, a recent study showed that the proteasomal inhibitors, lactacystin and MG-132, abolished cortisol-induced GR protein downregulation in heat-shocked trout hepatocytes (5). This lack of GR degradation coincided with higher hsp70 response to cortisol treatment, implying that the proteasome may modulate cortisol signaling by altering the GR protein content. The upregulation of the ubiquitin-proteasomal pathway by glucocorticoids (1, 20, 29) may be a mechanism favoring this GR response. Although GR protein changes may be involved in the proteasome-mediated impact on GR signaling, a direct impact of the proteasome on transcriptional regulation of GR and/or GR-responsive genes cannot be excluded. This is especially true given the recent studies showing that the proteasome is directly involved in the regulation of GR (35) and androgen receptor signaling (16).

Because the higher GR protein content, resulting from proteasomal inhibition, enhanced GR-mediated transcriptional activation in COS-1 cells (35) and hsp70 response in trout hepatocytes (5), we hypothesized that the proteasome also plays a role in the autoregulation of GR. Indeed, addition of MG-132 abolished cortisol-mediated GR downregulation in trout hepatocytes (5) and also abolished cortisol-induced GR mRNA abundance in trout hepatocytes (Fig. 1C). Together, our results argue for negative feedback regulation of GR gene expression by GR protein levels in trout hepatocytes. Although the mechanism(s) involved is not known, it appears likely that the proteasome is involved in this transcriptional regulation of GR (Fig. 1C). We propose that this glucocorticoid-mediated autoregulation of GR may be a key cellular stress response allowing for the regulation of glucocorticoid-responsive genes to maintain cellular homeostasis during stress in fish.

HSP90 is Involved in GR Regulation

GA, a selective hsp90-binding agent (25), was used to characterize the role of hsp90 on GR regulation in trout hepatocytes. The significantly higher hsp90 mRNA with GA in the present study (Fig. 2A) and the concomitant higher hsp90 protein content are in agreement with studies showing elevations in hsp90 transcription and translation with GA (14, 24). This hsp90 response to GA exposure appears to be an indirect effect mediated by accumulation of incorrectly folded proteins (15) and/or activation of heat shock factor-I (14). Because hsp90 is an essential chaperone protein involved in cell signaling, it appears likely that the inhibition of hsp90 action by GA binding activates a negative feedback loop resulting in the upregulation of hsp90 transcriptional and translational machineries (25). Despite the higher hsp90, the GA-mediated inhibition of hsp90 binding to signaling molecules, including protein kinases and GR, causes proteasomal degradation of these proteins (8, 17, 23, 27, 36). The lower GR protein content in the present study with GA, independent of cortisol, supports the role of hsp90 as an important chaperone involved in the steroid receptor stability.

In our study, GA had no significant impact on GR mRNA abundance in the absence of cortisol but abolished cortisol-induced GR mRNA abundance, suggesting a role for HSP90 on GR autoregulation in trout hepatocytes (Fig. 3A). Because GA affects GR stability (23), it seems logical that the lower GR response with cortisol may be due to a lower GR capacity. However, that does not appear to be the case, especially because GA did not have any impact on glucocorticoid-mediated elevation of PEPCK mRNA, a key glucocorticoid-responsive gene, suggesting that the effect of hsp90 on GR mRNA abundance is independent of GR capacity.

Previous studies alluded to a direct interaction between the ratio of free hsp90 to GR levels on transcriptional repression of glucocorticoid-responsive genes (13). Since GA increases GR breakdown, one would expect to see an increase in GR mRNA especially due to a negative feedback regulation (see GR autoregulation in trout hepatocytes). However, the abolishment of glucocorticoid-induced GR mRNA response with GA supports the argument that the impact of hsp90 may be direct and independent of GR protein regulation. The higher free hsp90 pool with GA, because of lack of binding to other protein molecules, coupled with lower GR in the presence of cortisol results in a higher hsp90-to-GR ratio and is perhaps responsible for the negative regulation of glucocorticoid response elements as proposed for mammalian cells (13). Together, these results implicate a specific role for hsp90 not only in the GR protein stability but also in the transcriptional regulation of GR in trout hepatocytes.

It is interesting to note that, despite the decreased GR protein levels, cortisol resulted in higher glucose release and higher PEPCK mRNA abundance in trout hepatocytes (Fig. 4), confirming the gluconeogenic role of cortisol in fish (18). The absence of cortisol-induced glucose release with RU-486 (Fig. 4C) clearly confirms GR as a mediator of the gluconeogenic response in trout hepatocytes (34). A key step in the gluconeogenic pathway involves the activation of PEPCK both at the transcriptional and translational levels (12). Our study is the first to show a direct impact of cortisol on PEPCK mRNA abundance in fish, whereas studies have already noted higher hepatic PEPCK activity with glucocorticoids in vivo (see Ref. 18 for a review). The glucocorticoid response unit for the PEPCK gene has been well characterized in mammals, involving a battery of coactivators and corepressors (12, 26), but little is known in fish. The reason for the lower PEPCK activity, despite higher PEPCK mRNA, is not clear and may be due to increased turnover of the protein. Mammalian studies reported that PEPCK has a half-life of 6-8 h, and even though factors regulating PEPCK turnover have not been identified (12), it is tempting to speculate that cortisol may enhance PEPCK turnover in trout hepatocytes. Also, the fact that medium glucose was higher with cortisol, despite lower PEPCK activity, argues for an increased turnover of the protein. However, despite lower GR protein content with cortisol, the higher abundance of PEPCK mRNA and glucose release with cortisol suggests activation of the GR signaling pathway before glucocorticoid-mediated GR breakdown. Consequently, proteasome-mediated GR downregulation may be one mechanism preventing excessive activation of glucocorticoid-responsive genes and proteins in response to chronic glucocorticoid stimulation. In support of this notion, a recent study (4) showed a lack of glucocorticoid-induced glucose release in trout hepatocytes with artificially lowered GR protein content.

In conclusion, our study shows, for the first time, a negative feedback regulation of GR transcriptional machinery by using a physiologically relevant cell system. In trout hepatocytes, ligand-dependent GR activation of the proteasome is involved in this GR autoregulation and includes the degradation of GR, although the precise mechanisms are yet unclear. We have identified that the molecular chaperone hsp90 is also specifically involved in this glucocorticoid-induced transcriptional regulation of GR. The autoregulation of GR may be a crucial step in the stress response process and may be a mechanism to increase tissue responsiveness to glucocorticoid stimulation, thereby offsetting the physiological consequences of GR protein downregulation evident with hypercortisolism.


    ACKNOWLEDGEMENTS

We thank Dr. B. Ducouret for the trout GR antibody and Dr. A. N. Boone for assistance with the study.


    FOOTNOTES

This study was supported by the Natural Sciences and Engineering Research Council, Canada, operating grant to M. M. Vijayan.

Address for reprint requests and other correspondence: M. M. Vijayan, Dept. of Biology, Univ. of Waterloo, Waterloo, ON N2L 3G1, Canada (E-mail: mvijayan{at}sciborg.uwaterloo.ca).

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

First published February 12, 2003;10.1152/ajpcell.00448.2002

Received 28 September 2002; accepted in final form 3 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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