Glucocorticoid Receptors in Hippocampal Neurons that Do Not Engage Proteasomes Escape from Hormone-Dependent Down-Regulation but Maintain Transactivation Activity

Xinjia Wang, Julie L. Pongrac and Donald B. DeFranco

Department of Pharmacology (X.W., J.L.P., D.B.D.) and Neuroscience (D.B.D.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Donald B. DeFranco, Department of Pharmacology, University of Pittsburgh School of Medicine, Room E1352 BST, Pittsburgh, Pennsylvania 15261. E-mail: dod1{at}pitt.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucocorticoid receptor (GR) protein is subjected to hormone-dependent down-regulation in most cells and tissues. This reduction in receptor levels that accompanies chronic hormone exposure serves to limit hormone responsiveness and operates at transcriptional, posttranscriptional, and posttranslational levels. The ability of glucocorticoid hormones to trigger GR down-regulation may be not universal, particularly in mature and developing neurons in which conflicting results regarding hormone control of GR protein have been reported. We find that endogenous GR is not down-regulated in the HT22 mouse hippocampal cell line and in primary hippocampal neurons derived from embryonic rats. Because GR has the capacity to be ubiquitylated in HT22 cells, receptor down-regulation must be limited by defects in either targeting of polyubiquitylated receptor to the proteasome or processing of the targeted receptor by the proteasome. Despite the lack of GR down-regulation in the HT22 cells, glucocorticoid-induced transcription from transiently transfected templates is attenuated upon prolonged hormone treatment. This termination of GR transactivation is not due to inefficient nuclear import or nuclear retention of the receptor. Furthermore, GR efficiently exports from HT22 cell nuclei in hormone-withdrawn cells, indicating that the receptor has access to both nuclear and cytoplasmic degradation pathways. Our results suggest that appropriate maturation of proteasomal degradative or targeting activities may be required, particularly in hippocampal neurons, for hormone-dependent down-regulation of GR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOCORTICOID HORMONES EXERT wide-ranging effects on cellular physiology and have an impact on nearly every organ system (1, 2). The secretion and synthesis of these hormones are subjected to exquisite regulation involving both pituitary and hypothalamic neuropeptides (3, 4). In addition, glucocorticoid responsiveness is also regulated at the cellular level by alterations in expression and accumulation of the glucocorticoid hormone receptor (GR) protein. The GR is a soluble intracellular receptor protein that is primarily responsible for cellular responses to glucocorticoids. GR is a member of a larger superfamily of nuclear receptors that share a highly conserved zinc finger DNA-binding motif (5).

Chronic glucocorticoid treatment typically leads to down-regulation of GR levels both in cell culture and in intact tissue (6). This homologous down-regulation of the receptor reflects both effects of glucocorticoids on GR gene transcription (7, 8) and protein turnover (9, 10). Because glucocorticoid responsiveness in cell culture (11, 12, 13) and in vivo (14) is related to the relative abundance of GR, homologous down-regulation of GR in response to chronic hormone treatment leads to a gradual loss of glucocorticoid-regulated transcription. This form of receptor desensitization is not limited to GR and is characteristic of other steroid receptors as well (15, 16).

Although most cells that have been examined exhibit homologous down-regulation of GR in vitro or in vivo, the impact of chronic hormone treatment on GR expression and accumulation in neurons is not fully resolved (17, 18, 19, 20, 21, 22). Furthermore, fetal exposure to glucocorticoids has been reported to have no effect on GR levels in whole embryos or embryonic liver (23). To assess the universality of GR down-regulation we have examined a hippocampal cell line and primary hippocampal neurons for their response to chronic glucocorticoid treatment. Our results suggest that hormone-dependent down-regulation of GR protein is not universal and may develop postnatally. This insensitivity of GR to down-regulation may reflect an inability of the receptor to effectively engage the proteasome degradation machinery. Interestingly, the lack of GR down-regulation does not lead to persistent activation of a glucocorticoid-responsive promoter, at least not with transiently transfected templates. Thus, multiple mechanisms may operate to limit glucocorticoid responsiveness, particularly under conditions of chronic hormone exposure.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Homologous Down-Regulation of GR in the GrH2 Hepatoma Cell Line
Figure 1AGo illustrates a profile of homologous GR down-regulation triggered by agonist that is characteristic of most cells examined in culture or in intact animals (6). The GrH2 cells used in this study were derived from HTC hepatoma cells and contain additional copies of the rat GR gene that were introduced by stable transfection (11). Despite the relatively high levels of GR expression in GrH2 cells (i.e. ~100,000 copies of GR/cell), receptor down-regulation is robust and leads to an approximate 80% loss of steady-state receptor levels within 48 h of hormone treatment. This extends the results of previous studies in our laboratory in which homologous down-regulation of GR in GrH2 cells was examined with shorter hormone exposure (24). As will be shown below, the reduction in GR levels that results from chronic hormone exposure is associated with decreased hormone-induced transcription.



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Figure 1. Differential Effects of Chronic Glucocorticoid Treatment on GR Levels in Distinct Cell Types

GrH2 cells (A), HT22 cells (B), or primary hippocampal neurons isolated from E16 rat embryos grown under serum-free conditions (C) were treated with 1 µM dexamethasone (Dex) for various times ranging from 4–96 h (A), 1–72 h (B), or 24–120 h following 4 d in culture (C). Total protein in whole-cell lysates was separated by SDS-PAGE and subjected to Western blot analysis to detect GR or actin. Inset in panel A shows Western blot result obtained after ECL detection, whereas the bar graph illustrates change in GR levels upon hormone treatment relative to actin. Blots shown in panels A and B are representative of five separate experiments, whereas that in panel C is representative of three separate experiments.

 
Lack of GR Down-Regulation in the HT22 Hippocampal Cell Line
Conflicting results have been generated regarding the effect of hormone on GR expression and accumulation in neurons (20, 21, 22). In most cases, GR function has not been assessed, making it difficult to evaluate the mechanisms responsible for potentially unique responses of GR to hormone treatment in neurons. The HT22 hippocampal cell line expresses functional GR, which can contribute to oxidative toxicity generated by glutathione depletion (25), and thus represents a useful model with which to evaluate the relationship between neuronal GR activity and degradation. As shown in Fig. 1BGo, chronic dexamethasone treatment of HT22 cells does not lead to down-regulation of GR levels. GR levels remained relatively unchanged even upon a 72-h treatment with 1 µM dexamethasone (Fig. 1BGo). An analogous treatment of GrH2 hepatoma cells led to an 80% reduction in GR levels (Fig. 1AGo). Although the dose of dexamethasone used (i.e. 1 µM) does not affect HT22 cell viability (Ref. 25 and data not shown), it does lead to GR-dependent activation of transcription (see below).

GR steady-state levels, as measured by Western blot analysis (e.g. Fig. 1Go), reflect the balance between receptor synthesis and degradation. Repeated attempts to directly measure GR turnover in HT22 cells using pulse-chase analysis were unsuccessful, most likely due to the relatively low level of GR expression in these cells. Therefore, to provide an alternative assessment of hormone effects on GR stability in HT22 cells, GR levels were examined at various times after the addition of the protein synthesis inhibitor, cycloheximide. As shown in Fig. 2Go, Western blot analysis of GR levels in cycloheximide-treated HT22 cells reveal that receptor stability is not significantly affected by dexamethasone. The relative half-life of GR in the presence or absence of dexamethasone is identical (i.e. ~12 h) when assessed in this manner (Fig. 2AGo). Furthermore, actin stability, determined after the reprobing of stripped blots processed initially for GR immunodetection, is identical in hormone-treated vs. untreated cells. Thus, the Western blot analysis of steady-state GR levels in HT22 cells (Fig. 1BGo) accurately depicts the lack of hormone effects on GR degradation.



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Figure 2. GR Stability Is Not Affected by Dexamethasone in HT22 Cells

HT22 cells were treated with 100 µg/ml cycloheximide (CHX) in the absence (-) or presence (+) of 1 µM dexamethasone. After the indicated incubation times, cells were harvested and equivalent aliquots of the whole-cell extract protein were subjected to SDS-PAGE and Western blotting using an anti-GR (top) or antiactin (bottom) antibody (panel A). Relative GR levels from blots shown are quantified in panel B. Blots shown in panel A are representative of three separate experiments.

 
Lack of GR Down-Regulation in Embryonic Hippocampal Neurons
Previous studies of GR down-regulation in neurons have mainly used in vivo models in which precise hormone exposure of individual neurons is difficult to assess (20, 21, 22). We therefore examined the effects of dexamethasone in nonproliferating primary hippocampal neurons isolated from E17 rat embryos that were grown under serum-free conditions. As shown in Fig. 1CGo, GR levels were not altered by dexamethasone in primary hippocampal neurons, even when examined after 5 d of hormone treatment. Thus, steady-state levels of endogenous GR in both proliferating and nonproliferating neurons of hippocampal origin are not affected by chronic exposure to pharmacological doses of glucocorticoid.

GR Is Not Engaged with the Proteasome in HT22 Cells or Primary Embryonic Hippocampal Neurons
GR, like other steroid receptors (26, 27), is a substrate for the proteasome degradation machinery (28, 29). One criterion that has been used to reveal proteasome involvement in protein turnover takes advantage of specific proteasomal inhibitors such as MG132 (27). As shown in Fig. 3AGo, a 16-h treatment of GrH2 cells with MG132 reduces the extent of hormone-induced GR down-regulation. This result is consistent with those obtained in COS-1 cells where hormone-induced down-regulation of transfected mouse GR was also diminished by MG132 (30). In contrast, GR levels are not affected by MG132 in dexamethasone-treated or untreated HT22 cells (Fig. 3BGo) and primary hippocampal neurons (Fig. 3CGo). As one measure of the effectiveness of MG132 in HT22 cells and hippocampal neurons, we examined expression levels of the 70-kDa heat shock protein, hsp70. Hsp70 can be induced in some cells by MG132 treatment (31, 32). As shown in Fig. 4Go, A and B, an MG132 treatment of HT22 cells and primary hippocampal neurons, which does not affect GR levels, leads to a robust induction of hsp70.



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Figure 3. GR Levels Are Not Elevated upon MG132 Treatment of HT22 Cells or Primary Hippocampal Neurons

GrH2 cells (A), HT22 cells (B), or primary hippocampal neurons (C) were treated with 1 µM, 15 µM, or 5 µM MG132, respectively, both in the absence (-) and presence of 1 µM dexamethasone (Dex) for 16 h. Aliquots of the whole-cell extract protein were subjected to SDS-PAGE and Western blotting using an anti-GR antibody. Blot shown is representative of three separate experiments.

 


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Figure 4. Hsp-70 Induction upon MG132 Treatment of HT22 Cells and Primary Hippocampal Neurons

HT22 cells (A) or primary hippocampal neurons (B) were treated with 15 µM or 5 µM MG132, respectively, both in the absence (-) and presence of 1 µM dexamethasone (Dex) for 16 h. Total protein in whole-cell lysates was separated by SDS-PAGE and subjected to Western blot analysis to detect hsp70. This blot shown is representative of three separate experiments.

 
MG132 Treatment Enhances GR Transactivation in HT22 Cells
Unlike other steroid receptors the transactivation activity of which is impaired by proteasome inhibition (27), GR transactivation from either transiently transfected or stably integrated templates is enhanced by MG132 treatment (30, 33). In these cases, the enhancement of GR transactivation by proteasome inhibition is accompanied by an increase in receptor levels (30, 33). Because GR levels are not affected by MG132 treatment in HT22 cells, we examined whether proteasome inhibition affected hormone induction of a transiently transfected glucocorticoid-responsive promoter [i.e. derived from the mouse mammary tumor virus (MMTV) long terminal repeat]. As shown in Fig. 5Go, a 16-h MG132 treatment leads to enhanced glucocorticoid induction of MMTV transcription, consistent with previously published results (30, 33). However, in HT22 cells, increased GR transactivation brought about by MG132 treatment is not associated with elevated receptor levels (Fig. 3BGo).



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Figure 5. MG132 Treatment Enhances GR Transactivation in HT22 Cells

HT22 cells were transfected with an MMTV-CAT reporter plasmid and, where indicated, treated with 1 µM dexamethasone and/or 2 µM MG132 for 16 h. Whole-cell extracts were prepared and equivalent amounts of total extract protein were assayed for CAT activity. Background values for CAT assays (i.e. ~50 cpm) were typically 2.5% of that obtained in extracts from MMTV-CAT-transfected cell extracts not treated with hormone (i.e. ~2000 cpm). Effects of MG132 on dexamethasone induction are statistically significant (P < 0.005) as assessed by Student’s t test. Data shown (averages of three determinations per time point ± SE) are representative of three independent experiments.

 
GR Is Ubiquitylated in HT22 Cells Chronically Exposed to Dexamethasone
The targeting of protein substrates to the proteasome requires their modification with ubiquitin (34, 35). To assess whether the limitation in GR degradation in HT22 cells results from its reduced targeting to the proteasome, we examined GR ubiquitylation in HT22 cells transiently transfected with a Myc-His6-tagged ubiquitin expression plasmid. The tagged form of ubiquitin facilitates detection of ubiquitylated proteins, which have proven difficult to visualize with available ubiquitin antibodies (36). Thus, GR recovered by immunoprecipitation from HT22 whole-cell extracts was subjected to Western blot analysis with an anti-Myc antibody to detect ubiquitylated forms of the receptor. As shown in Fig. 6Go, ubiquitylated GR could be detected in anti-GR antibody immunoprecipitates from HT22 cells transfected with Myc-His6-tagged ubiquitin and treated with MG132. Ubiquitylated GR was not detected above background levels in the absence of MG132, which is likely to be required to stabilize ubiquitylated GR. Importantly, ubiquitylated GR was recovered both in untreated and hormone-treated HT22 cells exposed to MG132 (Fig. 6Go). Although it is difficult to precisely quantify the extent of GR ubiquitylation in this type of experiment, control Western blots using an anti-GR antibody showed that GR recovery in immunoprecipitates was similar in hormone-treated vs. untreated cells. Thus, GRs in hormone-treated HT22 cells have the capacity to be ubiquitylated, suggesting that the defect in hormone-dependent receptor down-regulation in these cells occurs at the level of proteasome targeting or activity.



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Figure 6. GR Is Ubiquitylated in HT22 Cells Exposed to Dexamethasone

Where indicated, HT22 cells were transfected with a myc-his6-tagged ubiquitin (Ub) expression plasmid. Transfected cells were allowed to recover for 8 h and then treated, where indicated, with 5 µM MG132 and/or 1 µM dexamethasone for 18 h. Whole-cell lysates were immunoprecipitated with an anti-GR antibody. Immunoprecipitated protein was subjected to SDS-PAGE and Western blotting using an anti-GR (upper panel) or anti-myc (lower panel) antibody. High molecular mass ubiquitin-GR conjugates are noted in MG132-treated cells. Analogous results were obtained in three separate experiments.

 
GR Transactivation Is Attenuated upon Chronic Hormone Stimulation, Even in the Absence of Receptor Down-Regulation
Both in vitro and in vivo studies have shown a correlation between glucocorticoid responsiveness and GR levels (11, 13, 14). We therefore performed kinetic analysis of GR transactivation from transiently transfected templates in both GrH2 and HT22 cells, which differ with respect to glucocorticoid-dependent GR down-regulation (e.g. see Fig. 1Go). As shown in Fig. 7Go, dexamethasone induction of a glucocorticoid response element-driven luciferase reporter reached a maximum within 24 h of hormone exposure in both GrH2 and HT22 cells. Interestingly, even though GR levels are not reduced upon chronic exposure of HT22 cells to dexamethasone (Fig. 1BGo), GR-mediated transactivation does not persist but is attenuated to control, untreated levels within 72 h of hormone treatment (Fig. 7Go). In fact, the loss of GR transactivation from the transiently transfected luciferase reporter is similar in dexamethasone-treated GrH2 and HT22 cells, despite the markedly different effects of this hormone treatment on GR levels.



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Figure 7. GR Transactivation from Transiently Transfected Templates Is Attenuated upon Chronic Hormone Stimulation in Both GrH2 and HT22 Cells

HT22 and GrH2 cells were transfected with an MMTV-luciferase reporter plasmid. Whole-cell extracts were prepared and assayed for luciferase activity, which was normalized to total protein content per sample. The data shown (averages of six determinations per time point) are representative of three independent experiments.

 
GR Nuclear Import Is Not Attenuated by Chronic Dexamethasone Treatment
We had previously shown that inefficient nuclear retention or recycling of GR leads to desensitization of glucocorticoid-induced transcription in v-mos transformed cells (37). An analogous mechanism could be responsible for the loss of glucocorticoid responsiveness in HT22 cells chronically exposed to dexamethasone. To aid in our examination of GR trafficking in HT22 cells, we analyzed transiently transfected GR-green fluorescent protein (GR-GFP) chimera. Transiently expressed GR-GFP was not down-regulated upon dexamethasone exposure of HT22 cells (data not shown). As shown in Fig. 8Go, GR-GFP efficiently imports into the nucleus of HT22 cells within 15 min of dexamethasone treatment. GR-GFP is also efficiently retained within the nucleus of HT22 cells exposed to dexamethasone for 24 h. Thus, the desensitization of glucocorticoid responsiveness in HT22 cells chronically exposed to glucocorticoid is not due to inefficient nuclear retention of GR.



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Figure 8. GR Is Retained within the Nucleus of HT22 Cells Chronically Exposed to Dexamethasone

HT22 cells transfected with a GR-GFP chimera were grown in DMEM containing charcoal-stripped serum for 16 h before hormone treatment was initiated. Cells were then either untreated (A) or treated with 1 µM dexamethasone for 5 min (B), 15 min (C), or 24 h (D). GR-GFP fluorescent images are shown in representative cells.

 
GR Is Capable of Exporting from Nuclei in HT22 Cells after Hormone Withdrawal
Recent results from our laboratory have suggested that the rate of GR degradation is linked to its rate of nuclear export (38). To examine whether this relationship operates in HT22 cells, we examined the export of GR-GFP in hormone-withdrawn, transiently transfected cells. As shown in Fig. 9Go, GR-GFP that imports into nuclei of HT22 cells treated with 1 µM corticosterone is returned to the cytoplasm after a 24-h withdrawal of hormone. Thus, the insensitivity of GR to homologous down-regulation is not due to defects in receptor nucleocytoplasmic shuttling.



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Figure 9. GR exports from Nuclei of HT22 Cells upon Withdrawal of Glucocorticoid Hormone

HT22 cells transfected with a GR-GFP chimera were grown in DMEM containing charcoal-stripped serum for 16 h before hormone treatment was initiated. Cells were then treated with 1 µM corticosterone for 1 h and then switched to hormone-free medium for 0 min (A), 30 min (B), or 24 h (C). GR-GFP fluorescent images are shown in representative cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GRs are expressed in the developing central nervous system (39, 40, 41), particularly the hippocampus (42, 43). Within embryonic neurons, GRs are functional and participate in the development of specific neuroendocrine and neurotransmitter systems (44, 45). Although fetal exposure to maternal glucocorticoids is limited by placental glucocorticoid metabolism (46), significant amounts of hormone must gain access to the brain to activate GRs. However, excessive fetal glucocorticoid exposure, which can either result from prenatal stress (47, 48) or antenatal administration of metabolically stable synthetic glucocorticoids such as dexamethasone, may have detrimental effects on brain development (49, 50). In most adult tissues, chronic glucocorticoid treatment leads to down-regulation of GR protein and desensitization of glucocorticoid responsiveness (6). However, fetal tissues, particularly the brain, may lack supportive homeostatic systems to limit glucocorticoid action under conditions of hormone excess. Our results suggest that this may indeed be the case, as we show that GR in embryonic rat hippocampal neurons, and a mouse hippocampal cell line, is not subjected to hormone-dependent down-regulation. This result confirms results obtained in whole rat embryos in which glucocorticoid treatment was found not to affect GR protein and mRNA expression (23).

The insensitivity of GR to hormone-dependent down-regulation is not limited to embryonic hippocampal neurons, as chronic glucocorticoid exposure did not lead to GR down-regulation in primary embryonic rat cortical neurons (data not shown) or embryonic liver (23). Thus, GR-processing pathways that contribute to hormone-dependent down-regulation may not be fully operational in late-term embryos. In established cell lines, GRs, like other nuclear receptors (26, 51, 52, 53), are degraded via the ubiquitin-proteasome pathway (28, 29). However, because proteasome inhibition does not stabilize GR in hormone-treated HT22 cells or embryonic hippocampal neurons, we hypothesize that the receptor may not be effectively engaged with the proteasomes under these conditions. Proteins must be polyubiquitylated for their recruitment to the proteasome (54). GR can be ubiquitylated in HT22 cells, both in the presence and absence of hormone, indicating that the lack of GR down-regulation is not due to a deficit in the activity of an ubiquitin ligase used by the receptor. In transfected COS-7 cells, the C terminus of Hsc70-interacting protein cochaperone participates in GR ubiquitylation and degradation (29). It is therefore possible that GR down-regulation in hippocampal neurons may be limited by the expression of C terminus of Hsc70-interacting protein, an analogous cochaperone, or other ancillary proteins that prepare substrates for proteasome recognition (54). Ubiquitylated p53 protein has been found to accumulate in cells expressing a mutant Mdm2 protein but not be targeted for proteasome degradation (55). Thus, protein ubiquitylation can be uncoupled from degradation.

Recent Affymetrix oligonucleotide microarray analysis revealed distinct expression profiles of various proteasome subunits, ubiquitin-conjugating enzymes, and molecular chaperones in developing mouse hippocampus (56). Developmental regulation of proteasome subunit mRNAs has also been reported in rat midbrain (57). In addition to transcriptional regulation of proteasome expression, alternative splicing of specific proteasome subunits has been found to generate distinct proteasome forms during development (58). These studies illustrate that the ubiquitin-proteasome machinery is not static. Through changes in its composition and function, the ubiquitin-proteasome system may differentially regulate the expression levels and turnover of specific substrates at distinct periods of development. Differential expression or function of specific components of the ubiquitin-proteasome pathway seems to be particularly important in the brain. For example, mutations in the E6-AP ubiquitin-protein ligase are associated with Angelman’s syndrome, a human disease that is characterized by severe mental retardation and motor dysfunction (59, 60). Increased abundance of one E6-AP substrate, the p53 oncoprotein, results from an E6-AP deficiency and could underlie deficits in contextual learning and long-term potentiation that occur in mouse models of Angelman’s syndrome (61). Although E6-AP has been found to interact with the progesterone receptor, its impact on nuclear receptor function, including GR, appears to be more related to its transcriptional coactivator activity (62).

Hormone-dependent down-regulation of steroid hormone receptors, while limiting the duration of hormone responsiveness, also affects the efficiency of receptor transactivation. Thus, estrogen receptor {alpha}, progesterone receptor, and thyroid hormone receptor transactivation in transiently transfected cells is reduced when proteasome-mediated degradation of the receptors is inhibited (27). In fact, model studies with chimeric transcriptional activators of differing potencies established a link between transcriptional activation and proteasome-mediated degradation. Specifically, the rate of activator degradation was found to directly correlate with transactivation potency (62, 63). This seems at odds with previous results where GR transactivation was enhanced accompanying proteasome inhibition (30, 33). In these cases, it has been hypothesized that the extent of GR transactivation was simply a reflection of receptor levels, which were elevated upon proteasome inhibition. In HT22 cells, MG132 enhances GR transactivation, consistent with previous results with this receptor (30, 33), but does not lead to elevations in GR levels. Thus, the extent of nuclear receptor transactivation may not be strictly related to receptor levels and can be differentially responsive to the activity state of proteasomes. An uncoupling of transactivation and degradation has recently been observed with specific mutants of the retinoid X receptor (65). Furthermore, a PR mutant that does not undergo hormone-dependent degradation maintains some degree of hormone response in transfected HeLa cells, even though its ability to respond to the MAPK pathway is completely abrogated (66). Thus, the link between proteasome-mediated degradation and transactivation may be gene and receptor specific and responsive to a unique subset of signal transduction pathways that affect nuclear receptor activity.

The efficiency of proteasome-mediated degradation of nucleocytoplasmic shuttling proteins has been linked in some cases with their rate of nuclear export (67, 68). For example, proteasome-mediated degradation of the cyclin-dependent kinase inhibitor protein p27Kip1 is stimulated when its nuclear export is enhanced via its interaction with the Jab1 coactivator protein (69). The HDM2 RING-finger protein serves an analogous role to enhance the nuclear export and proteasome degradation of the p53 tumor suppressor protein (70, 71). When the rate of GR nuclear export is stimulated through linkage of a potent nuclear export signal sequence to its amino terminus, hormone-dependent down-regulation of the chimeric nuclear export signal-GR is enhanced (38). This result implies that degradation of nuclear receptors may likewise be linked to their nuclear export. However, inefficient down-regulation of GR in HT22 cells is not linked to an inhibition of its nuclear export as assessed under conditions of hormone withdrawal. Thus, GRs that export from the nucleus are not strictly directed to proteasomes. It is possible that some interactions linking GR nuclear export with proteasome targeting are intact in the HT22 cells but rendered ineffective by a deficit in a downstream step in the ubiquitin-proteasome pathway. The HT22 cells will provide a useful tool in future studies directed toward unraveling the relationship between nuclear receptor trafficking and degradation.

What is the impact of sustained GR expression in hormone-treated hippocampal neurons? Because down-regulation of GR limits glucocorticoid responsiveness in transfected cells (12), hormone-dependent transactivation might be maintained in cells that do not have the capacity to down-regulate the receptor when chronically exposed to hormone. This is in fact not the case, as we found a densensitization of GR transactivation, from transiently transfected templates, in HT22 cells treated with glucocorticoid for extended periods of time. The kinetic profile of GR transactivation assessed with luciferase assays exhibited a similar pattern of desensitization in cells (i.e. HT22 and GrH2) that differed with respect to the response of GR to chronic hormone. Thus, GR responsiveness, at least from transiently transfected templates, can be limited in cells that do not possess the capacity to down-regulate receptor levels in response to chronic hormone stimulation.

Lee and Archer (72) have shown that the dephosphorylation of histone H1, which results from chronic dexamethasone treatment of a mouse fibroblast cell line, leads to a specific reduction in GR transactivation from a stably integrated MMTV promoter. GR transactivation from a transiently transfected MMTV promoter was not affected by hormone-dependent histone H1 dephosphorylation (72), implying that this mechanism may not apply to the desensitization of MMTV transactivation that we observe in transiently transfected HT22 cells. Nonetheless, if histone H1 dephosphorylation is enhanced by chronic glucocorticoid treatment in embryonic hippocampal neurons, some subset of endogenous glucocorticoid-responsive genes could be affected. Since enhanced histone H1 dephosphorylation does not exert global effects on GR-responsive genes (72), distinct mechanisms could operate to limit glucocorticoid responsiveness of specific genes in distinct cells, including hippocampal neurons. The lack of GR down-regulation in hippocampal neurons eliminates one widely used mechanism to limit GR action under conditions of chronic hormone exposure. Minimal (i.e. 20–60%) forced overexpression of GR in transgenic mice leads to a number of effects on the hypothalamus-pituitary-adrenal axis and stress responses (14), illustrating the physiological importance of maintaining homeostasis of GR expression.

Antenatal glucocorticoid treatment is widely used clinically in attempts to increase survival of premature infants (73). Despite the benefits to pulmonary function of premature infants from antenatal glucocorticoid treatment, some studies report detrimental effects on cognitive function in children or juvenile animals that had been exposed to chronic dexamethasone in utero (74). If GR down-regulation is not fully developed, chronic activation of receptor may alter the transcription rate of genes the precisely coordinated expression of which may be critical for the appropriate neuronal development. Using HT22 cells and primary embryonic hippocampal neurons as models, we may be able to delve more deeply into the functioning of GR in this critical phase of brain development and understand the mechanisms responsible for detrimental effects of fetal glucocorticoid exposure.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells
The rat GrH2 hepatoma cell line (Ref. 11 ; gift of K. R. Yamamoto, University of California at San Francisco, San Francisco, CA) and the mouse HT22 hippocampal cell line (Ref. 75 ; gift of D. Schubert, Salk Institute, La Jolla, CA) were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Atlantic Biological, Norcross, GA) unless otherwise indicated. For hormone treatment, dexamethasone (Sigma, St. Louis, MO) or corticosterone (Sigma) was added to a final concentration of 1 µM from 1 mM stock solutions prepared in ethanol. Where indicated, cells were also cultured in DMEM containing charcoal-stripped FBS. For hormone withdrawal, cells were pretreated with 1 µM corticosterone for 2 h, washed three times, and then maintained in hormone-free medium (DMEM containing 10% charcoal-stripped FBS) for 5 min to 24 h. For proteasome inhibition studies, cells were treated for 18 h with MG132 (Biosciences, Inc., Darmstadt, Germany), which was prepared as a 10 mM stock solution in dimethylsulfoxide. Control cultures were treated with an equal volume of dimethylsulfoxide alone. For measures of GR stability, HT22 cells were treated for 1 h with 100 µg/ml of cycloheximide (Sigma) before initiating treatment with 1 µM dexamethasone.

Primary Hippocampal Cultures
Cultures enriched for hippocampal neurons were prepared from embryonic d 17 rat embryos using a previously described procedure (76). Basically, cells from dissected hippocampi were dispersed by mechanical dissociation using fire-polished Pasteur pipettes. Cells were then plated into culture wells containing 100 µg/ml poly-D-lysine (Sigma) in Neurobasal medium (Life Technologies, Inc.) and a DMEM/F12 nutrient supplement (DMEM/F12, 1:1) containing 10% FBS. Cultures were enriched for neurons by removing the plating medium at 45 min and replacing it with phenol red- and serum-free Neurobasal/DMEM/F12 (3:1:1) medium supplemented with L-glucose (6 g/liter), insulin (10 µg/ml), progesterone (20 nM), putrescine (62 µM), transferrin (20 µg/ml), sodium selenite (30 nM), and penicillin/streptomycin (1 U/ml: 1 µg/ml). Neurons were maintained in 37 C and 5% CO2.

Western Blot Analysis
Whole-cell extracts were prepared from GrH2 and HT22 cells using conditions described previously (77). After clarification of lysates by centrifugation, identical amounts of total lysate protein (i.e. 5 µg) were loaded onto sodium dodecyl sulfate-7.5% polyacrylamide gels and subjected to electrophoresis. Separated proteins were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) and subjected to Western blot analysis to detect GR or actin. The BuGR2 anti-GR monoclonal antibody (78) was used to detect GR, whereas a commercially available antibody C-2 monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to detect actin. Immunoreactive bands were visualized on Western blots using an Enhanced Chemiluminescence (ECL) Kit (NEN Life Science Products, Boston, MA). In some cases, ECL signals were quantified using an EPSON PERFECTION scanner and Molecular Dynamics, Inc. (Sunnyvale, CA) image analysis system.

Immunoprecipitation of Ubiquitylated GR
HT22 cells grown in 100 mm-diameter tissue culture plates were transfected with 9 µg of a myc-tagged ubiquitin expression plasmid (79) using Lipofectamine (Life Technologies, Inc.) under conditions specified by the supplier. At 16 h post transfection, the medium was replaced with DMEM containing 10% FBS, and cells were allowed to recover for 8 h before treatment with MG132 was initiated. Cells were collected and pelleted for 2 min at 14,000 x g at 4 C and then lysed in 50 µl of lysis buffer (50 mM Tris-Cl, pH 8.8; 5 mM EDTA; 2.0% sodium dodecyl sulfate; 10 mM dithiothreitol; 100 µM sodium vanadate; 10 mM sodium fluoride; and protease inhibitors). After lysis, the extract was diluted with 950 µl of dilution buffer (20 mM Tris-Cl, pH 8.8; 150 mM NaCl; 2 mM EDTA; 100 µM sodium vanadate; 10 mM sodium fluoride; and protease inhibitors) and DNA was sheared by passing the extract through a 22-gauge needle five times. The extract was centrifuged for 10 min at 14,000 x g at 4 C, and the resulting supernatant was incubated for 1 h at 4 C with 20 µl of protein A/G-agarose (sc-2003; Santa Cruz Biotechnology, Inc.). After this incubation, the agarose beads were pelleted, and the supernatants added to 5 µl of BuGr2 anti-GR antibody prebound to 15 µl of protein A/G-agarose. After an overnight incubation at 4 C, the immunoprecipitation reaction was washed three times for 10 min each at room temperature with 1 ml of high-salt buffer (20 mM Tris-HCl, pH 8.8; 500 mM NaCl; 2 mM EDTA; 0.2 mM dithiothreitol; 100 µM sodium vanadate; 10 mM sodium fluoride; and protease inhibitors). Bound protein was eluted with 40 µl of 1x sodium dodecyl sulfate sample buffer.

Assays of Luciferase or Chloramphenicol Acetyl Transferase (CAT) Activity in Transiently Transfected Cells
For experiments with luciferase reporter plasmids, GrH2 or HT22 cells were transfected with 0.1 µg of MMTV-luciferase (gift of K. R. Yamamoto) using lipofectamine as described above. Cells were allowed to recover for 6–24 h following transfection before treatment with 1 µM dexamethasone was initiated. Whole-cell extracts, prepared using the Luciferase Assay Buffer (Promega Corp., Madison, WI) were assayed for luciferase activity (33) at various times after treatment with dexamethasone. For experiments with CAT reporter plasmids, HT22 cells was transfected with 0.5 µg MMTV-CAT (gift of T. Archer) using lipofectamine and allowed to recover for 16 h. Where indicated, cells were then treated with 2 µM MG132 for 1 h before a 16-h treatment with 1 µM dexamethasone was initiated. Whole-cell extracts were prepared by sonication in 0.25 M Tris-HCl (pH 7.8), and CAT activity in equivalent amounts of total protein was measured (80) after a pretreatment of extracts for 10 min at 60 C.

Localization of Transiently Transfected GR-GFP
GrH2 or HT22 cells grown on coverslips were transfected with 2 µg of an expression vector for a GR-GFP chimera (Ref. 81 ; gift of I. Macara, University of Virginia, Charlottesville, VA) using the lipofectamine transfection reagent as described above. Hormone treatments and withdrawal were performed as described above and detailed in the figure legends. GR-GFP was visualized in either live cells or paraformaldehyde-fixed cells (82) using a E800 fluorescence microscope (Nikon, Melville, NY).


    ACKNOWLEDGMENTS
 
We thank Drs. T. Archer, R. Kopito, I. Macara, D. Schubert, and K. Yamamoto for supplying DNA or cells and Dr. M. Stallcup for his critical reading of the manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant CA-43037 (to D.B.D.).

Abbreviations: CAT, Chloramphenicol acetyltransferase; ECL, enhanced chemiluminescence; FBS, fetal bovine serum; GFP, green fluorescent protein; GR, glucocorticoid receptor; MMTV, mouse mammary tumor virus.

Received for publication October 22, 2001. Accepted for publication May 14, 2002.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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