help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0585
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Conway-Campbell, B. L.
Right arrow Articles by Lightman, S. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Conway-Campbell, B. L.
Right arrow Articles by Lightman, S. L.
Endocrinology Vol. 148, No. 11 5470-5477
Copyright © 2007 by The Endocrine Society

Proteasome-Dependent Down-Regulation of Activated Nuclear Hippocampal Glucocorticoid Receptors Determines Dynamic Responses to Corticosterone

Becky L. Conway-Campbell, Mervyn A. McKenna, Crispin C. Wiles, Helen C. Atkinson, E. Ron de Kloet and Stafford L. Lightman

Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (B.L.C.-C., M.A.M., C.C.W., H.C.A., S.L.L.), Department of Medicine, University of Bristol, Bristol BS1 3NY, United Kingdom; and Department of Medical Pharmacology (E.R.d.K.), Leiden/Amsterdam Center for Drug Research, Leiden University Medical Center, 2300 RA Leiden, The Netherlands

Address all correspondence and requests for reprints to: Becky L. Conway-Campbell, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Dorothy Hodgkins Building, University of Bristol, Whitson Street, Bristol BS1 3NY, United Kingdom. E-mail: b.conway-campbell{at}bristol.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Timing is a critical factor in neuroendocrinology. Despite this, the temporal aspects of glucocorticoid signaling in the regulation of in vivo targets have been largely overlooked. Here, we present data showing that plasma glucocorticoid levels differ greatly from the constant signal predominantly used in cell culture experiments. Using an automated blood sampling system, we found that under basal conditions in nonstressed rats, corticosterone release occurs in discrete pulses of various amplitudes dependent on the circadian cycle. This basal pattern changes to a prolonged elevated nonpulsatile release in response to stressful stimuli. We have been able to recapitulate these different patterns of corticosterone presentation (short pulse vs. prolonged elevation) in adrenalectomized rats, and show that each pattern results in differential activation of hippocampal glucocorticoid and mineralocorticoid receptors. Finally, we provide evidence for a rapid proteasome-dependent clearance of activated glucocorticoid receptors, but not mineralocorticoid receptors, as a novel mechanism to allow dynamic interaction with rapidly changing physiological and environmental conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLUCOCORTICOID RECEPTOR (GR) and mineralocorticoid receptor (MR) are members of the nuclear receptor superfamily, one of the most abundant classes of transcriptional regulators in animals (1). These receptors function as ligand-activated transcription factors, providing a direct link between extracellular steroid levels and intranuclear transcriptional responses in target cells.

Glucocorticoids (GCs) are the endogenous ligand for both GR and MR, specifically corticosterone in the rat and cortisol in man, and aldosterone in the case of MR (2). GCs readily enter the brain and are known to regulate many aspects of neuronal function (3). Therefore, intracellular regulation of GC receptor activity is likely to play an important role in modulating the effects of GCs in the central nervous system. Specificity is determined in many GC target tissues by several well-characterized mechanisms, including tissue-specific protection from corticosterone binding due to expression of 11-β-hydroxysteroid dehydrogenase, which interconverts corticosterone and cortisol into inactive forms (4), and differential expression patterns of GR and MR in various regions (3). In the hippocampus, there are significant levels of active GCs, and abundant coexpression of both MR and GR (5). In this case, specificity is determined by the differing corticosterone binding affinities of GR and MR (6). The high-affinity receptor MR is activated at very low concentrations of GC, whereas the lower-affinity GR is only activated at high concentrations (7). Once ligand activated, both receptors can undergo active transport into the cell’s nucleus, where they bind to glucocorticoid responsive elements to initiate and regulate transcription of GC target genes (8). Thus, in normal physiological states, MR is activated and, hence, nuclear localized at very low concentrations of GC, whereas GR remains latent in the cytoplasm until GC concentrations increase above a threshold level.

Much of the work describing the activity of the GC receptors with respect to nuclear translocation and retention times has relied heavily on cell culture models using continuous treatment of GCs throughout the time course of the experiment. However, this is not representative of the physiology of GC secretion in vivo. In stark contrast to a constant rate of presentation to target cells, we have shown that circulating corticosterone exhibits highly fluctuating levels, following not only a diurnal pattern but also a striking ultradian rhythm characterized by discrete pulses at approximately hourly intervals (9, 10). This pattern changes to a sustained nonpulsatile release of GC after an episode of stress (9). Pulsatile secretion of many neuroendocrine hormones, including GH (11), LH (12), and GnRH (13), has been an essential element of their biological activity. However, the functional significance of pulsatile presentation of hormones that bind to nuclear receptors is unknown. Here, we test whether different patterns of corticosterone presentation have physiological sequelae in terms of GR and MR activity in the hippocampus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female (study 1) and male (studies 2–4) Sprague-Dawley rats (weight 200–250 g; age 9–11 wk) were obtained from Harlan (Bicester, Oxfordshire, UK) and maintained under standard housing conditions with a 14:10 light/dark cycle (lights on 05:15, lights off 19:15). Food and water (or saline when specified) were available ad libitum. All animal procedures were performed in accordance with the United Kingdom Home Office animal welfare regulations.

Surgery
Rats (n = 6 per time point per group) were anesthetized with im injection of Hypnorm (Vetapharma Ltd., Leeds, UK) (fentanyl citrate 0.252 mg/kg and fluanisone 8 mg/kg) after ip injection of diazepam (4 mg/kg), then subjected to one of four surgical procedures: 1) jugular vein cannulation for the automated blood sampling; 2) bilateral adrenalectomy (adx) for the ip corticosterone administration study; 3) bilateral adx and jugular cannulation for the iv corticosterone administration study; and 4) bilateral adx, jugular cannulation, and intracerebroventricular (icv) cannulation into the right lateral ventricle for the proteasome inhibitor study. Coordinates 0.40 dorsal/ventral, –0.15 lateral, –1.0 anterior/posterior relative to Bregma were used for cannula placement. After surgery the rats were allowed to recover for 5 d, with adx rats receiving corticosterone replacement (50 µg/ml) in 0.1% ethanol saline for drinking.

Automated blood sampling
Frequent blood sampling at 5-min intervals in nonstressed, free running rats in their home cage was achieved as previously described (9, 10).

Experimental design
Study 1: intraperitoneal corticosterone administration.
The rats were allowed to recover with corticosterone replacement (50 µg/ml) in saline for drinking for 5 d after surgery. Corticosterone was withdrawn 24 h before the experiment. On the day of the experiment, each rat was given a 750-µg ip bolus of corticosterone (equivalent to ~3 mg/kg) in the form of a preformed water-soluble complex of corticosterone and 2-hydroxypropyl-β-cyclodextrin (C-174; Sigma-Aldrich, St. Louis, MO) (14). This route of delivery provides a prolonged corticosterone profile in the blood similar to that of an acute stress response (15). Animals were killed at times 0, 30, 60, and 120 min.

Study 2: intravenous corticosterone administration.
After recovery for 5 d as described previously, rats were given two bolus iv injections of corticosterone and 2-hydroxypropyl-β-cyclodextrin, each at a dose of 100 µg corticosterone/rat to mimic the plasma profile of two endogenous basal pulses. The iv injections were administered at time 0 and 120 min, and animals were killed at 0, 10, 15, 30, 60, 120, 130, 135, 150, 180, and 240 min.

Study 3: proteasome inhibitor study.
After recovery for 5 d as described previously, rats were infused via the icv cannula with either 2 µl of 100 µM MG132 in 5% dimethyl sulfoxide (DMSO)/saline or 5% DMSO/saline vehicle alone. After 16 h, the rats were given a bolus iv injection of 100 µg corticosterone. Animals were killed at times 0, 15, and 120 min. Cannula placement into right lateral ventricle was verified postmortem using infusion of 2 µl cresyl violet stain (result not shown).

Brain dissections
After decapitation, the brains were rapidly removed, then sliced in 3-mm coronal sections using a rat brain matrix (Harvard Apparatus, Holliston, MA) on ice at all times. The hippocampus was dissected from the slices and rapidly frozen in liquid N2, then stored at –80 C until nuclear, and cytoplasmic extracts were prepared. Blood was collected from the trunk into heparinized tubes and the plasma obtained by centrifugation, then stored at –20 C until measurement of corticosterone by RIA.

Nuclear and cytoplasmic fractionation
Nuclear and cytoplasmic fractions were prepared according to the method of Vallone et al. (16). All procedures were performed at 4 C and on ice. Each hippocampus was homogenized in 1 ml/100 mg tissue in S1 buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA (pH 8), supplemented with 0.5 mM dithiothreitol, 0.2 mM Na orthovanadate, 2 mM NaF, and Complete Protease Inhibitor (Roche Diagnostics Ltd., Burgess Hill, UK)] using a Dounce homogenizer (Jencons, Leeds, UK). Nuclear proteins were extracted in 1.2 pellet volume of S2 buffer [10 mM HEPES (pH 7.9), 400 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA (pH 8), and 5% glycerol, supplemented with 0.5 mM dithiothreitol, 0.2 mM Na orthovanadate, 2 mM NaF, and Complete Protease Inhibitor] and stored at –80 C. Protein concentrations were determined by bicinchoninic acid assay (BCA) (Pierce, Rockford, IL). The integrity of cytoplasmic and nuclear fractions was verified by Western blot with antibodies specific to either the nuclear (anti-Histone H1 sc8030; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or cytoplasmic fraction (anti-RAS 05-516; Upstate Biotechnology, Lake Placid, NY).

Western blot
Western blot was performed according to the original method of Laemmli (17). Aliquots of each sample (20 µg) were run on an 8–15% polyacrylamide gel, and transferred to polyvinylidene fluoride membrane (PVDF) (Amersham Biosciences, Uppsala, Sweden). The membranes were probed with either anti-GR antibody M20 sc-1004 (Santa Cruz Biotechnology) or anti-MR antibody mAb 1D5, which has been fully characterized previously (18) (kindly supplied by Dr. Gomez-Sanchez (University of Mississippi Medical Center, Jackson, MS) at 1:1000 dilution, then with either antirabbit IgG-HRP (NA934V; Amersham Biosciences) for GR or antimouse IgG-HRP (NA931V; Amersham Biosciences) for MR, both at 1:10,000 dilution. The signal was detected using enhanced chemiluminescence (ECLPlus) reagent and enhanced chemiluminescence hyperfilm (Amersham Biosciences).

The upper portion of each membrane (above 50 kDa) was probed for either GR or MR, and the lower portion of each membrane (<50 kDa) was probed for Histone H1 as a loading control and Ras for cytoplasmic contamination.

Corticosterone RIA
Total corticosterone concentration in whole blood for the automated sampling study in intact rats, or in plasma for the studies in corticosterone replaced adx rats, was quantified by RIA using a specific corticosterone antibody (kindly supplied by G. Makara, Institute of Experimental Medicine, Budapest, Hungary), as previously described (9, 10).

Analysis of results
The Western blot bands were quantified by densitometry using an Epson perfection scanner (Epson Europe, Amsterdam, The Netherlands) in transmission mode and the associated Quantity One software (Bio-Rad Laboratories, Hercules, CA). Data were analyzed as adjusted volume OD for the GR (or MR) bands, as well as the Histone H1 loading control bands. Each data point was then normalized relative to the Histone H1 present in that sample (presumed to remain constant per µg of total protein). Each Western blot contained a complete time course, such that each time point after corticosterone injection could be analyzed as fold inductions of basal values (at time zero). Only samples run at the same time were compared, after which, fold inductions from all data sets were pooled to give the final summary graphs depicting means ± SEM.

Statistical analyses were performed using one-way ANOVA with the Tukey post test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ultradian pattern of corticosterone
Figure 1Go shows a typical profile of whole blood corticosterone throughout the active phase in a representative nonstressed free-running female rat in its home cage. The circulating corticosterone concentrations can be seen to exhibit discrete peaks with low nadir levels, characteristic of the ultradian pattern of corticosterone secretion from the adrenals in basal nonstressed conditions (19). This pattern of corticosterone presentation contrasts greatly with the pattern initiated by a stress response. After a restraint stress, corticosterone levels increase, reaching a maximum at 30 min, maintain elevated values at 60 min, then decrease to basal values at 120 min (15, 20). During this time there is no evidence of superimposed pulsatility in circulating corticosterone levels (20). This stress-initiated pattern of corticosterone release in intact animals has previously been described to be closely recapitulated by ip injection of corticosterone into adx rats (15).


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 1. An individual profile of circulating corticosterone in whole blood sampled throughout the active phase of the circadian cycle in a female Sprague-Dawley rat. An individual profile from an adult female rat showing the ultradian rhythm of corticosterone detected in whole blood sampled at 5-min intervals commencing 1 h before the onset of the dark phase (active phase) of the circadian cycle and continuing through to the light phase (inactive phase). This data set is a representative profile of one of eight rats sampled in this experiment. Mean data are not shown due to a lack of pulse synchronicity between animals.

 
Response to ip corticosterone
Intraperitoneal corticosterone injection caused prolonged elevated plasma corticosterone levels, as depicted in Fig. 2AGo. At 30 min after injection, corticosterone concentrations reached 416 ± 115 ng/ml. The levels remained high at 177 ± 35 ng/ml at 60 min (lower but not significantly different from the 30-min levels; P = 0.1313), then decreased significantly to 34 ± 9 ng/ml by 120 min (P = 0.0203). In response to this sustained corticosterone signal, nuclear translocation and retention of both GR and MR in the hippocampus were prolonged, as shown in Fig. 2BGo, with summary data from six animals per time point shown in Figs. 2CGo (GR) and 2D (MR). We detected a 21.1 ± 5.8-fold induction of nuclear translocation of GR and a 21.7 ± 1.3-fold induction of nuclear translocation of MR at 30 min after the ip injection of corticosterone. The nuclear levels were still high at 60 min after ip injection, with a 14.6 ± 2.6-fold induction for GR and 24.0 ± 3.4-fold induction for MR. By 120 min after ip injection, there was a significant decline in both GR and MR nuclear levels. However, both receptors were still retained in the nucleus above baseline levels at 6.0 ± 1.7-fold for GR and 10.8 ± 2.2-fold for MR. There was no significant difference between GR and MR in terms of their initial maximum nuclear translocation (nonsignificant; P = 0.8738) or their nuclear retention levels at 120 min (nonsignificant; P = 0.9739).


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 2. Intraperitoneal injection of corticosterone results in prolonged elevation of circulating corticosterone, and prolonged nuclear retention of hippocampal GR and MR. A, Plasma corticosterone levels were undetectable in the adx rats before corticosterone injection (time 0 min) and increased to a maximal value 30 min after injection, remaining elevated at 60 min and reducing to near baseline levels at 120 min. B, On the left, nuclear translocation of both hippocampal GR (upper panel) and MR (middle panel) was induced in parallel with the increase in circulating corticosterone at 30 min after injection. Nuclear levels remained elevated at 60 min, decreasing toward baseline levels by 120 min. Histone H1 (lower panel) indicates loading control for each sample. On the right, cytoplasmic levels of both hippocampal GR (upper panel) and MR (middle panel) remain high throughout the time course. No significant quantitative depletion can be detected (P = 0.5911). Ras (lower panel) indicates loading control for each sample. C, Summary graph of fold inductions of nuclear translocation of GR for six per time point. D, Summary graph of fold inductions of nuclear translocation of MR for six per time point. There is no significant difference between GR and MR for either the initial induction of nuclear translocation (nonsignificant; P = 0.8738) or nuclear retention levels at 120 min (nonsignificant; P = 0.9739).

 
No significant depletion of cytoplasmic levels of either GR or MR was observed at any time point after ip injection of corticosterone, as can clearly be seen in Fig. 2BGo (nonsignificant in one-way ANOVA, P = 0.5911). Of course, it is well established that both GR and MR are actively transported into the nucleus from the cytoplasm after ligand binding. In cell culture models, depletion of cytoplasmic GR has been demonstrated upon application of supraphysiological levels of both dexamethasone and corticosterone. However, in vivo, both our study and others (15) have been unable to demonstrate a significant depletion of cytoplasmic GR. This may be due to the technical difficulty in measuring small changes in highly abundant cytoplasmic protein levels using Western blot.

Response to pulses of iv corticosterone
Each iv injection into the peripheral circulation via the jugular cannula caused a pulse of corticosterone (720 ± 81 and 831 ± 97 ng/ml, respectively) into the circulation at 1 min after injection as measured by RIA and depicted in Fig. 3AGo. Although somewhat higher in amplitude than an endogenous pulse, each corticosterone spike was rapidly cleared from the circulation in a manner recapitulating the clearance rate of corticosterone in a basal pulse. The time frame of corticosterone clearance in both cases is consistent with the predicted half-life of corticosterone in blood of less than 10 min (9).


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 3. Intravenous injection of corticosterone results in brief elevation of circulating corticosterone and differential nuclear retention patterns of hippocampal GR and MR. A, Plasma corticosterone levels were undetectable in the adx rats before corticosterone injection (time 0 min) and increased to a maximal value 1 min after injection, then cleared according to the approximate 10-min half-life of corticosterone in blood. A second injection at 120 min exhibited a similar profile to the first. B, Nuclear translocation of both hippocampal GR (upper panel) and MR (middle panel) was rapidly induced after the increase in circulating corticosterone. Both GR and MR were detected in the nucleus as early as 10 min after each pulse. Nuclear retention time for GR was extremely brief, with clearance evident at 30 min after injection and returning to near basal levels by 60 min. Nuclear retention time for MR was longer in duration, with levels remaining high up to 60 min. C, Summary graph of fold inductions of nuclear translocation for the entire time course for GR. D, Summary graph of fold inductions of nuclear translocation for the entire time course for MR. GR and MR both exhibit similar rapid kinetics in the initial induction of nuclear translocation in response to each pulse of corticosterone. The clearance rate for GR is significantly more rapid than for MR. Nuclear GR levels decline to baseline by 60 min after injection, but MR still remains maximally elevated at 60 min after injection. Nuclear GR levels at 60 min are not significantly different from basal levels (P = 0.1331). Nuclear MR levels are significantly different from basal levels (P = 0.0207) and not significantly different from maximal levels (P = 0.9249).

 
Extremely rapid nuclear translocation of both GR and MR was observed within 15 min of each iv injection, as can clearly be seen in Fig. 3BGo. The summary graphs in Figs. 3CGo (GR) and 3D (MR) show a 19.0 ± 2.1-fold nuclear translocation of GR and 17.9 ± 4.0-fold nuclear translocation of MR in the hippocampus at 10 min. Nuclear levels of both receptors reach a maximum at 15 min, with a 24.7 ± 2.4-fold induction for GR and 21.3 ± 3.5-fold induction for MR.

The retention time of GR in the nucleus was significantly shorter in duration than that observed after the ip injection. The depletion of nuclear GR after each pulse was evident by 30 min, decreasing to 35.2% of maximal levels and 8.7 ± 2.0-fold above baseline, and returning to near basal levels at only 13.4% of maximum and 3.3 ± 1.2-fold above baseline by 60 min (nonsignificant difference at 60 min compared with basal levels; P = 0.1331). The rapid clearance kinetics of nuclear GR observed here seems consistent with the hypothesis that nuclear GR levels oscillate during basal pulsatility in a manner that reflects the peaks and troughs of the pulses.

MR displayed a much slower nuclear depletion with high nuclear levels of MR throughout the time course, maintaining elevated levels of 18.1 ± 4.6-fold induction above baseline at 60 min (nonsignificant difference at 60 min compared with maximal levels, P = 0.9249; significant difference at 60 min compared with basal levels, P = 0.0207). Nuclear depletion of MR only occurred at 120 min after the pulse, decreasing to 3.3 ± 0.9-fold above baseline levels, at which time peripheral corticosterone levels were undetectable by RIA. Because endogenous corticosterone pulses exhibit a periodicity of approximately 60 min (9, 10), the physiological relevance of the 60-min time point is most pertinent to MR bioactivity. It can be concluded that MR would remain maximally activated throughout basal pulsatility.

As was the case with ip corticosterone treatment, no significant depletion of cytoplasmic levels for either GR or MR could be detected at any time point after iv injection of corticosterone (nonsignificant in one way ANOVA, P = 0.9973) (data not shown). During basal pulsatility throughout the active phase of the circadian cycle, the presence of an abundant cytoplasmic pool of responsive receptors may be necessary for continued cellular responsiveness to discrete hourly pulses of corticosterone secretion.

The 26S proteasome regulates the rapid turnover of activated GR in the nucleus
To investigate the mechanism by which activated nuclear GR is down-regulated, we next used the specific irreversible 26S proteasome inhibitor MG132. MG132 is a substrate for the multidrug resistance efflux transporter p glycoprotein at the blood-brain barrier, therefore, we could not introduce the inhibitor peripherally. We infused the drug directly into the lateral ventricle at a dose that has been reported to inhibit protein degradation in several cell types, including neurons derived from rat brain (21), as well as progesterone and estrogen receptors (ERs) in vivo in the rat brain (22).

Corticosterone was again delivered via iv injection, as indicated in Fig. 4AGo. This dose of corticosterone (772 ± 67 ng/ml at 1 min) resulted in the expected rapid induction then down-regulation of hippocampal nuclear GR in the animals treated with vehicle alone (5% DMSO in saline), as shown in Fig. 4CGo. In the animals treated with the MG132 inhibitor, the rapid induction of GR nuclear translocation was still evident. However, the clearance from the nucleus was markedly diminished, and nuclear GR levels remained elevated 120 min after the iv bolus injection of corticosterone. Densitometric analysis revealed levels of GR detected in the nucleus to be 9.0 ± 2.6-fold higher than baseline in the presence of MG132, compared with 0.9 ± 0.1-fold in the absence of MG132 (significant difference; P = 0.0199), as shown in the summary data in Fig. 4BGo.


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 4. Inhibition of the proteasome reverses the rapid down-regulation of hippocampal activated nuclear GR. A, Plasma corticosterone levels were undetectable in the adx rats before corticosterone injection (time 0 min) and increased to a maximal value 1 min after injection, then cleared according to the approximate 10-min half-life of corticosterone in blood. B, In control rats subjected to icv injection of the vehicle, nuclear translocation of hippocampal GR is induced by the iv injection of corticosterone then cleared as expected. In rats treated with MG132 via icv injection, nuclear translocation of hippocampal GR is induced maximally at 15 min after iv injection of corticosterone, but the clearance is significantly affected. At 120 min after corticosterone injection, nuclear levels remain high in the MG132-treated rats (***, significant difference compared with the vehicle icv-treated rats at the 120-min post corticosterone injection; P = 0.0199). C, Results from the nuclear fractions are shown on the left-hand side panel. There is an induction of nuclear GR levels at 15 min, followed by clearance at 120 min in icv vehicle control rats. In contrast, the induction of nuclear GR levels at 15 min was not followed by clearance at 120 min in icv MG132-treated rats. Hippocampi were dissected into left (L) and right (R) hemispheres. No significant difference was found between the left and right hemisphere groups, therefore, the data were pooled together for the analysis shown in B. There was no nuclear retention of GR at 120-min post-corticosterone injection in the pituitaries taken from the MG132-treated rats used for the hippocampal study. Histone H1 (lower panel) indicates loading control for each sample. Results from the cytoplasmic fractions are shown on the right-hand side panel. Cytoplasmic levels of GR from the left and right hemispheres of hippocampi from both vehicle-treated and MG132-treated rats remain high throughout the time course. No significant quantitative change in cytoplasmic GR levels can be detected (P = 0.6031). Ras (lower panel) indicates loading control for each sample.

 
The effect appeared to be centrally localized because the MG132 inhibitor did not affect clearance of nuclear GR in pituitaries taken from the same animals as shown in Fig. 4CGo. This is a salient additional point that verifies that the higher levels of nuclear GR retention at 120 min after the pulse are due to the local action of the inhibitor rather than a possible incomplete adx in individual animals. The RIA also supports complete adx because corticosterone was undetectable in the plasma from these animals.

Interestingly, the effect of proteasomal inhibition is specific to nuclear GR. There was no increase in the levels of cytoplasmic GR after MG132 treatment compared with vehicle control, as can clearly be seen in Fig. 4CGo (P = 0.6031). Together, these data strongly indicate that proteasome-mediated protein degradation regulates the turnover of activated nuclear GR.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoids are secreted in a pulsatile manner that alters over the sleep-wake cycle (10), and responds to inflammation (23, 24), early life stress (25), and levels of sex hormones (26, 27, 28). We have developed an iv injection model that provides a much better representation of endogenous basal corticosterone pulsatility in the temporal domain than has been achieved before, and this has allowed us to demonstrate that the kinetics of nuclear clearance of GR in the hippocampus is much more rapid than previously thought. This is in striking contrast to the prolonged nuclear retention of GR after a stress response (15). The significantly slower kinetics for MR nuclear depletion is consistent with its remaining maximally activated throughout the endogenous cycles of pulsatile corticosterone presentation. Nuclear retention of MR was found to exceed the 1-h pulse interval with the current dose of 100 µg iv, as well as previously with tracer doses of 3H-corticosterone (29). In contrast, the fluctuating pattern of nuclear residence of GR implies a more rapid and transient response to these phasic changes in corticosterone concentration.

These data have led us to propose a novel model of GC receptor regulation that may be important in determining dynamic interactions with pulsatile corticosterone presentation, as depicted in the schematic model in Fig. 5Go. Here, nuclear GR is being constantly and rapidly cleared via a proteasome-dependent mechanism, thus ensuring a continuous turnover of activated GR in the face of phasic GC presentation.


Figure 5
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 5. Model of pulsatile corticosterone regulation of GRs in the hippocampus. Schematic depiction of circulating corticosterone pulsatility, showing how the peak and trough of each pulse differentially affect GR and MR. During the peak of each pulse, both GR and MR are maximally activated and localized to the nuclear compartment, where they are known to bind to glucocorticoid responsive elements in the promoters of GC target genes in the genome. Then, during the trough of each pulse, nuclear GR is specifically down-regulated by a proteasome-dependent mechanism, whereas MR is retained in the nucleus. This mechanism allows nuclear GR to be constantly and rapidly cleared, ensuring dynamic interaction with fluctuating GC levels.

 
The results from our study alone cannot definitively distinguish whether proteasomal clearance of GR occurs within the nucleus, or whether GR is shuttled out of the nucleus to the cytoplasm for degradation. It is clear that GR is not exported via the classical Exportin 1 (CRM1)-mediated nuclear export pathway (30), but a calreticulin-mediated nuclear export mechanism has been described (31). We detected prolonged elevation of nuclear GR levels with no increase in cytoplasmic GR levels after treatment with MG132, which might argue against nuclear export of GR as a mechanism for its rapid clearance. Although we cannot exclude the possibility that inhibiting the proteasome may have caused an indirect effect on GR nuclear export, there is considerable evidence for direct action of an intranuclear proteasome. Various subunits of the proteasome complex have been localized within the nucleus (32), and the nuclear proteasome has been shown to be involved in specific degradation of nuclear receptors and key signaling molecules, including ERs (ER{alpha} and ERβ), androgen receptor, progesterone receptor, and the signal transducer and activator of transcription protein (STAT 5) (33, 34, 35, 36).

Interestingly, proteasome-dependent degradation has been found to be an integral part of the transcriptional function of some nuclear receptors, notably ER{alpha} (37). How proteasome-dependent degradation affects the transcriptional outcome of GR appears controversial and cell type specific (38, 39). In neuronal cultures (38) and other cell culture models (39, 40, 41), MG132 inhibition of the proteasome has been shown to enhance transcription. So a simplistic answer may be that GR transcriptional activity is switched off by specific degradation of the activated protein. In parallel with the concept that the rapid turnover of ER{alpha} may allow continued responsiveness to ever-changing levels of estradiol (37), a similar mechanism could facilitate the ability of GR to respond in a sensitive and dynamic manner to rapidly changing levels of GC.

Proteasome-dependent degradation of GR has occurred in several cell types (38, 39, 40, 41, 42, 43) and may be either a generalized or a cell-specific regulatory mechanism in GC target tissues. We do know that cell-specific relative expression of components of the ubiquitin proteasome pathway, in particular carboxy terminus of heat shock protein 70-interacting protein (CHIP) E3 ligase (42) and murine double min 2 (mdm2) (43), confers specificity for regulation of GR levels in varying conditions in vitro, and we now have evidence that these mechanisms may have a functional significance in vivo.

Why MR is not cleared so rapidly from the nucleus is another interesting question arising from our study. MR has been described as a target for proteasomal degradation (44). Most likely, the differences in in vivo clearance rates of nuclear GR and MR may simply reflect their different ligand binding affinities and kinetics (45). First, MR is maximally activated at very low levels of corticosterone, far below the threshold required to activate GR. Once activated, MR remains bound for a significantly longer time (t1/2 = 45 min), whereas GR dissociates from corticosterone quite rapidly (t1/2 = 5 min). Therefore, it is possible that the longer duration of ligand binding may stabilize MR and protect it from degradation. Alternatively, low levels of corticosterone throughout the pulse trough may still be able to cause continuous new maximal activation of MR, keeping nuclear MR levels high.

Functionally, rapidly changing levels of GR relative to constant levels of MR in the nucleus of target cells would be expected to have significant consequences (46), especially on gene regulation (47). If distinct subsets of GC target genes are regulated differentially by MR or GR homodimers, or MR/GR heterodimers (47), then the rapidly changing levels of nuclear GR relative to MR may be one of the critical determinants regulating specificity of the GC response. The hippocampus is an obvious candidate target for such precise regulatory control, with its abundant expression of MR and GR (45), and its ability to fine-tune its neural synaptic output and, therefore, memory functions (48, 49, 50) in response to differing circulating levels of glucocorticoids.

In conclusion, our study provides the first evidence for a novel mechanism whereby receptor-mediated responses can differentiate between distinct patterns of glucocorticoid secretion.


   Acknowledgments
 
We thank Susan A. Wood, R. Louise Harrison, and Emma Castrique for their excellent technical assistance.


   Footnotes
 
This work was supported by a Biotechnology and Biological Sciences Research Council grant (to S.L.L.). E.R.d.K. is supported by the Royal Netherlands Academy of Arts and Sciences.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 9, 2007

Abbreviations: adx, Adrenalectomy; DMSO, dimethyl sulfoxide; ER, estrogen receptor; GC, glucocorticoid; GR, GC receptor; icv, intracerebroventricular; MR, mineralocorticoid receptor.

Received May 3, 2007.

Accepted for publication August 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
  2. Funder JW 1996 Mineralocorticoid receptors in the central nervous system. J Steroid Biochem Mol Biol 56:179–183[CrossRef][Medline]
  3. de Kloet ER, Reul JM, Sutanto W 1990 Corticosteroids and the brain. J Steroid Biochem Mol Biol 37:387–394[CrossRef][Medline]
  4. Seckl JR 1997 11beta-Hydroxysteroid dehydrogenase in the brain: a novel regulator of glucocorticoid action? Front Neuroendocrinol 18:49–99[CrossRef][Medline]
  5. Nishi M, Kawata M 2006 Brain corticosteroid receptor dynamics and trafficking: implications from live cell imaging. Neuroscientist 12:119–133[Abstract/Free Full Text]
  6. De Kloet ER 2004 Hormones and the stressed brain. Ann NY Acad Sci 1018:1–15[CrossRef][Medline]
  7. Reul JM, van den Bosch FR, de Kloet ER 1987 Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J Endocrinol 115:459–467[Abstract/Free Full Text]
  8. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465–474[CrossRef][Medline]
  9. Windle RJ, Wood SA, Shanks N, Lightman SL, Ingram CD 1998 Ultradian rhythm of basal corticosterone release in the female rat: dynamic interaction with the response to acute stress. Endocrinology 139:443–450[Abstract/Free Full Text]
  10. Atkinson HC, Wood SA, Kershaw YM, Bate E, Lightman SL 2006 Diurnal variation in the responsiveness of the hypothalamic-pituitary-adrenal axis of the male rat to noise stress. J Neuroendocrinol 18:526–533[CrossRef][Medline]
  11. Waxman DJ, Ram PA, Pampori NA, Shapiro BH 1995 Growth hormone regulation of male-specific rat liver P450s 2A2 and 3A2: induction by intermittent growth hormone pulses in male but not female rats rendered growth hormone deficient by neonatal monosodium glutamate. Mol Pharmacol 48:790–797[Abstract]
  12. Papavasiliou SS, Zmeili S, Khoury S, Landefeld TD, Chin WW, Marshall JC 1986 Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone {alpha} and β subunits in male rats. Proc Natl Acad Sci USA 83:4026–4029[Abstract/Free Full Text]
  13. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631–633[Abstract/Free Full Text]
  14. Rekharsky MV, Inoue Y 1998 Complexation thermodynamics of cyclodextrins. Chem Rev 98:1875–1918[CrossRef][Medline]
  15. Kitchener P, Di Blasi F, Borrelli E, Piazza PV 2004 Differences between brain structures in nuclear translocation and DNA binding of the glucocorticoid receptor during stress and the circadian cycle. Eur J Neurosci 19:1837–1846[CrossRef][Medline]
  16. Vallone D, Battista S, Pierantoni GM, Fedele M, Casalino L, Santoro M, Viglietto G, Fusco A, Verde P 1997 Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product. EMBO J 16:5310–5321[CrossRef][Medline]
  17. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  18. Gomez-Sanchez CE, de Rodriguez AF, Romero DG, Estess J, Warden MP, Gomez-Sanchez MT, Gomez-Sanchez EP 2006 Development of a panel of monoclonal antibodies against the mineralocorticoid receptor. Endocrinology 147:1343–1348[Abstract/Free Full Text]
  19. Jasper MS, Engeland WC 1991 Synchronous ultradian rhythms in adrenocortical secretion detected by microdialysis in awake rats. Am J Physiol 261(5 Pt 2):R1257–R1268
  20. Ma XM, Lightman SL 1998 The arginine vasopressin and corticotrophin-releasing hormone gene transcription responses to varied frequencies of repeated stress in rats. J Physiol 510(Pt 2):605–614
  21. Sawada H, Kohno R, Kihara T, Izumi Y, Sakka N, Ibi M, Nakanishi M, Nakamizo T, Yamakawa K, Shibasaki H, Yamamoto N, Akaike A, Inden M, Kitamura Y, Taniguchi T, Shimohama S 2004 Proteasome mediates dopaminergic neuronal degeneration, and its inhibition causes alpha-synuclein inclusions. J Biol Chem 279:10710–10719[Abstract/Free Full Text]
  22. Villamar-Cruz O, Manjarrez-Marmolejo J, Alvarado R, Camacho-Arroyo I 2006 Regulation of the content of progesterone and estrogen receptors, and their cofactors SRC-1 and SMRT by the 26S proteasome in the rat brain during the estrous cycle. Brain Res Bull 69:276–281[CrossRef][Medline]
  23. Windle RJ, Wood SA, Kershaw YM, Lightman SL, Ingram CD, Harbuz MS 2001 Increased corticosterone pulse frequency during adjuvant-induced arthritis and its relationship to alterations in stress responsiveness. J Neuroendocrinol 13:905–911[CrossRef][Medline]
  24. Harbuz MS, Windle RJ, Jessop DS, Renshaw D, Ingram CD, Lightman SL 1999 Differential effects of psychological and immunological challenge on the hypothalamo-pituitary-adrenal axis function in adjuvant-induced arthritis. Ann NY Acad Sci 876:43–52[CrossRef][Medline]
  25. Shanks N, Windle RJ, Perks PA, Harbuz MS, Jessop DS, Ingram CD, Lightman SL 2000 Early-life exposure to endotoxin alters hypothalamic-pituitary-adrenal function and predisposition to inflammation. Proc Natl Acad Sci USA 97:5645–5650[Abstract/Free Full Text]
  26. Seale JV, Wood SA, Atkinson HC, Bate E, Lightman SL, Ingram CD, Jessop DS, Harbuz MS 2004 Gonadectomy reverses the sexually diergic patterns of circadian and stress-induced hypothalamic-pituitary-adrenal axis activity in male and female rats. J Neuroendocrinol 16:516–524[CrossRef][Medline]
  27. Seale JV, Wood SA, Atkinson HC, Harbuz MS, Lightman SL 2004 Gonadal steroid replacement reverses gonadectomy-induced changes in the corticosterone pulse profile and stress-induced hypothalamic-pituitary-adrenal axis activity of male and female rats. J Neuroendocrinol 16:989–998[CrossRef][Medline]
  28. Seale JV, Wood SA, Atkinson HC, Harbuz MS, Lightman SL 2005 Postnatal masculinization alters the HPA axis phenotype in the adult female rat. J Physiol 563(Pt 1):265–274
  29. De Kloet R, Wallach G, McEwen BS 1975 Differences in corticosterone and dexamethasone binding to rat brain and pituitary. Endocrinology 96:598–609[Abstract/Free Full Text]
  30. Liu J, DeFranco DB 2000 Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin 1/CRM1-directed nuclear export pathway. Mol Endocrinol 14:40–51[Abstract/Free Full Text]
  31. Holaska JM, Black BE, Love DC, Hanover JA, Leszyk J, Paschal BM 2001 Calreticulin Is a receptor for nuclear export. J Cell Biol 152:127–140[Abstract/Free Full Text]
  32. Gonzalez F, Delahodde A, Kodadek T, Johnston SA 2002 Recruitment of a 19S proteasome subcomplex to an activated promoter. Science 296:548–550[Abstract/Free Full Text]
  33. Chen Y, Dai X, Haas AL, Wen R, Wang D 2006 Proteasome-dependent down-regulation of activated Stat5A in the nucleus. Blood 108:566–574[Abstract/Free Full Text]
  34. Tateishi Y, Sonoo R, Sekiya Y, Sunahara N, Kawano M, Wayama M, Hirota R, Kawabe Y, Murayama A, Kato S, Kimura K, Yanagisawa J 2006 Turning off estrogen receptor beta-mediated transcription requires estrogen-dependent receptor proteolysis. Mol Cell Biol 26:7966–7976[Abstract/Free Full Text]
  35. Lange CA, Shen T, Horwitz KB 2000 Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome. Proc Natl Acad Sci USA 97:1032–1037[Abstract/Free Full Text]
  36. Conaway RC, Brower CS, Conaway JW 2002 Emerging roles of ubiquitin in transcription regulation. Science 296:1254–1258[Abstract/Free Full Text]
  37. Reid G, Hubner MR, Metivier R, Brand H, Denger S, Manu D, Beaudouin J, Ellenberg J, Gannon F 2003 Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling. Mol Cell 11:695–707[CrossRef][Medline]
  38. Wang X, Pongrac JL, DeFranco DB 2002 Glucocorticoid receptors in hippocampal neurons that do not engage proteasomes escape from hormone-dependent down-regulation but maintain transactivation activity. Mol Endocrinol 16:1987–1998[Abstract/Free Full Text]
  39. Deroo BJ, Rentsch C, Sampath S, Young J, DeFranco DB, Archer TK 2002 Proteasomal inhibition enhances glucocorticoid receptor transactivation and alters its subnuclear trafficking. Mol Cell Biol 22:4113–4123[Abstract/Free Full Text]
  40. Wallace AD, Cidlowski JA 2001 Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. J Biol Chem 276:42714–42721[Abstract/Free Full Text]
  41. Garside H, Waters C, Berry A, Rice L, Ardley HC, White A, Robinson PA, Ray D 2006 UbcH7 interacts with the glucocorticoid receptor and mediates receptor autoregulation. J Endocrinol 190:621–629[Abstract/Free Full Text]
  42. Wang X, DeFranco DB 2005 Alternative effects of the ubiquitin-proteasome pathway on glucocorticoid receptor down-regulation and transactivation are mediated by CHIP, an E3 ligase. Mol Endocrinol 19:1474–1482[Abstract/Free Full Text]
  43. Kinyamu HK, Archer TK 2003 Estrogen receptor-dependent proteasomal degradation of the glucocorticoid receptor is coupled to an increase in mdm2 protein expression. Mol Cell Biol 23:5867–5881[Abstract/Free Full Text]
  44. Yokota K, Shibata H, Kobayashi S, Suda N, Murai A, Kurihara I, Saito I, Saruta T 2004 Proteasome-mediated mineralocorticoid receptor degradation attenuates transcriptional response to aldosterone. Endocr Res 30:611–616[CrossRef][Medline]
  45. Reul JM, de Kloet ER 1985 Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117:2505–2511[Abstract/Free Full Text]
  46. De Kloet ER, Vreugdenhil E, Oitzl MS, Joels M 1998 Brain corticosteroid receptor balance in health and disease. Endocr Rev 19:269–301[Abstract/Free Full Text]
  47. Meijer OC 2002 Coregulator proteins and corticosteroid action in the brain. J Neuroendocrinol 14:499–505[CrossRef][Medline]
  48. Joels M, de Kloet ER 1991 Effect of corticosteroid hormones on electrical activity in rat hippocampus. J Steroid Biochem Mol Biol 40:83–86[CrossRef][Medline]
  49. Joels M, de Kloet ER 1993 Corticosteroid actions on amino acid-mediated transmission in rat CA1 hippocampal cells. J Neurosci 13:4082–4090[Abstract]
  50. Joels M, Pu Z, Wiegert O, Oitzl MS, Krugers HJ 2006 Learning under stress: how does it work? Trends Cogn Sci 10:152–158[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
D. M. Keenan, S. Alexander, C. Irvine, and J. D. Veldhuis
Quantifying Nonlinear Interactions within the Hypothalamo-Pituitary-Adrenal Axis in the Conscious Horse
Endocrinology, April 1, 2009; 150(4): 1941 - 1951.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
T. Dickmeis
Glucocorticoids and the circadian clock
J. Endocrinol., January 1, 2009; 200(1): 3 - 22.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
F. Hu, E. J. Crespi, and R. J. Denver
Programming Neuroendocrine Stress Axis Activity by Exposure to Glucocorticoids during Postembryonic Development of the Frog, Xenopus laevis
Endocrinology, November 1, 2008; 149(11): 5470 - 5481.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
E. R. de Kloet and R. A. Sarabdjitsingh
Everything Has Rhythm: Focus on Glucocorticoid Pulsatility
Endocrinology, July 1, 2008; 149(7): 3241 - 3243.
[Full Text] [PDF]

Home page
 EndocrinologyHome page
S. K. Droste, L. de Groote, H. C. Atkinson, S. L. Lightman, J. M. H. M. Reul, and A. C. E. Linthorst
Corticosterone Levels in the Brain Show a Distinct Ultradian Rhythm but a Delayed Response to Forced Swim Stress
Endocrinology, July 1, 2008; 149(7): 3244 - 3253.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Conway-Campbell, B. L.
Right arrow Articles by Lightman, S. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Conway-Campbell, B. L.
Right arrow Articles by Lightman, S. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals