©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selectivity of Cell Cycle Regulation of Glucocorticoid Receptor Function (*)

(Received for publication, August 1, 1994; and in revised form, October 21, 1994)

Shu-chi Hsu Donald B. DeFranco (§)

From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The restricted expression of some genes to distinct stages of the cell cycle is often brought about through alterations in the activity and/or abundance of specific transcription factors. Many cells have been shown to be unresponsive to glucocorticoid hormone action during the G(2) phase of the mammalian cell cycle, suggesting that some activities of the glucocorticoid receptor (GR), a ligand-activated transcription factor, are subjected to cell cycle control. We show here that GR insensitivity in G(2) is selective, affecting receptor-mediated transactivation from a simple glucocorticoid response element, but not repression from a composite glucocorticoid response element. Since glucocorticoid-dependent down-regulation of GR protein levels is also unaffected in G(2), distinct activities of the receptor that participate in this homologous down-regulation must be operating as effectively in G(2)-synchronized cells as in asynchronous cells. Finally, the phosphorylation state of the GR is altered in G(2)-synchronized cells reflecting, in part, both site-specific phosphorylation and dephosphorylation events. These results suggest that, while GR may be a target for cell cycle regulated kinases and phosphatases, the resulting changes in receptor phosphorylation have an impact only on selected GR functions.


INTRODUCTION

Steroid hormones elicit complex responses within cells predominantly through the action of intracellular receptor proteins which are members of a large superfamily of nuclear hormone receptors (1) . Members of this superfamily of transcription factors have the capacity to regulate transcription either via their interactions with specific target sequences linked to hormonally responsive genes(2, 3) , or in the absence of direct DNA binding, by their interactions with other transcription factors(3, 4) . Given the fairly widespread occurrence of glucocorticoid receptors (GRs) (^1)within different tissues and cell types, multiple cellular factors must influence the receptor's activity imparting the complexity that often characterizes physiological responses to glucocorticoids(5) . In that regard, a number of transcription factors have been identified that direct cell type-specific (6, 7) and developmental stage-specific(8, 9) transcriptional activation activity of the GR.

In many cells which possess the complete machinery necessary to elicit a glucocorticoid response, GR activity can be blunted by the action of other signal transduction pathways. For example, in hepatoma cells glucocorticoid induction of the phosphoenolpyruvate carboxykinase gene can be inhibited by insulin(10, 11) . In addition, activation of protein kinase C by tumor-promoting phorbol esters inhibits glucocorticoid induction of the tyrosine aminotransferase (TAT) gene (12) , illustrating the diversity of intracellular signaling pathways that influence GR action. Since protein kinase C activation in other cases potentiates glucocorticoid induced transcription(13) , additional cell-specific factors must influence the nature of cross-talk between the glucocorticoid and protein kinase C signal transduction pathways. While steroid receptor function has been shown to be influenced by various independent signal transduction pathways which impact upon protein kinases and phosphatases(14, 15, 16, 17) , no detectable alterations in receptor phosphorylation have been observed under these conditions.

Many protein phosphorylation and dephosphorylation events play critical roles in regulating ordered progression through the eukaryotic cell cycle(18, 19) . In some cases, the targets of cell cycle-regulated kinases and phosphatases have been shown to be transcription factors whose activity can be altered in numerous ways by resultant changes in their phosphorylation state(20, 21, 22) . Tomkins and co-workers first noted an apparent glucocorticoid insensitivity during the G(2) phase of the cell cycle (23) which has more recently been shown to reflect, in part, inhibition of GR transactivation activity(24) . Given the various levels at which gene expression can be regulated by the GR(25, 26, 27) , the extent of receptor activities which may be subjected to cell cycle control remains unresolved.

We report here our examination of G(2) effects on GR transactivation and repression brought about through GR interactions with a promoter-linked simple and composite glucocorticoid response element (GRE), respectively. Likewise, we have extended our previous analysis (24) of cell cycle regulation of GR phosphorylation. Our results reveal that GR insensitivity during the G(2) phase of the cell cycle applies to selective GR functions, i.e. transactivation, but not repression. Furthermore, we show that complex changes in GR phosphorylation occur during G(2), which include alterations in both overall phosphorylation and site-specific phosphorylation and dephosphorylation.


MATERIALS AND METHODS

Cell Lines and Cell Synchronization

GrH2 cells, a rat HTC hepatoma cell line which expresses elevated levels of GR protein (28) , were maintained in Dulbecco's modified essential medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (FBS; Life Technologies, Inc.). Cells were synchronized in G(0) by serum starvation (72 h in 0.01% FBS), then following stimulation with 10% FBS, arrested in early S by incubating for 16 h with 1.5 mM hydroxyurea (Sigma). For G(2) synchrony(29) , cells released from S phase arrest were cultured for 8-14 h in Dulbecco's modified essential medium with 5% FBS and 1.5 µg/ml Hoechst 33342 (Calbiochem). Cell cycle analysis was carried out by flow cytometry performed at the Flow Cytometry Center of the Pittsburgh Cancer Institute(24) .

Chloramphenicol Acetyl Transferase (CAT) Assay

Whole cell extracts were prepared and assayed for CAT activity as described previously(14) . Significance of the data was determined using a two-tailed Student's t test.

Western Blot Analysis

GRs were immunoprecipitated from whole cell lysates (30) using the BuGR2 monoclonal antibody (31) and separated by electrophoresis on 7.5% polyacrylamide-SDS gels. The quantitative recovery of GR from cell lysates was confirmed by a second round of immunoprecipitations which typically did not yield detectable levels of GR. Western blots were performed(32) , and GRs were visualized using the enhanced chemiluminescence technique (Amersham Corp.). Relative levels of GR were measured by densitometric analysis of autoradiographs.

S- and P-Labeling and Tryptic Peptide Mapping of GRs

Cells (1 times 100-mm diameter culture dish) were incubated with 70 µCi of [S]methionine and 3 mCi of [P]orthophosphate for 7 h. Immunoprecipitated GRs were gel-purified (30) and extracted with 30% hydrogen peroxide. S and P incorporated into GR was quantified using a Beckman LS 1701 liquid scintillation system. Tryptic digests and two-dimensional mapping of P-labeled GR peptides were performed as described previously(30) . The relative phosphorylation within individual peptides was quantified using an Ambis Radioanalytic Imager. To compare the relative phosphorylation within individual peptides between different thin layer plates, a reference peptide was chosen whose phosphorylation, in multiple trials, was unchanged by G(2) synchronization.


RESULTS

GR Function at a Simple, but Not a Composite GRE, Is Inhibited in G(2)-synchronized GrH2 Cells

The impairment of glucocorticoid-induced gene expression during the G(2) phase of the cell cycle has been shown to reflect, in part, an inhibition of GR-mediated transactivation(24) . The precise arrangement (33) and occupancy (34) of distinct transcription factors at glucocorticoid-responsive promoters can influence the ability of the GR bound to linked GREs to activate transcription. We therefore set out to determine whether the inhibition of GR transactivation during G(2) is due to a disruption of complex interactions between the receptor and transcription factors obligatory for hormone-induced transcription. To eliminate the contribution to glucocorticoid induced transcription of other transcription factors such as OTF-1(35) , whose activity may be cell cycle-regulated(20) , we analyzed the function of a simple GRE linked to a minimal promoter possessing only a TATA-box element directing basal transcription. The TAT3CAT reporter plasmid, which possesses three tandem copies of a simple GRE derived from the TAT gene linked to a minimal promoter from the Drosophila alcohol dehydrogenase gene(36) , was stably transfected into GrH2 rat hepatoma cells(28) , and glucocorticoid induction of CAT activity was measured in asynchronous and G(2)-synchronized cells.

As shown in Fig. 1A, with a cell synchronization paradigm that uses Hoescht 33342(29) , we were able to generate a population of GrH2 cells >80% enriched in the G(2) phase of the cell cycle. This synchronization was readily reversible (Fig. 1B) as a population of cells 45% enriched in G(1) was obtained following a 6-h withdrawal from medium containing Hoescht 33342. As shown in Fig. 2A, transcription from a stably integrated TAT3CAT reporter was effectively induced by dexamethasone in asynchronous GrH2 cells, but not in G(2)-synchronized cells. Transcriptional activity of the Rous sarcoma virus promoter, which is not hormonally responsive(37) , was not significantly affected by G(2) synchronization of GrH2 cells (Fig. 2C). Importantly, the inhibition of GR transactivation from the TAT3CAT reporter in G(2)-synchronized cells demonstrates that G(2) effects on GR transactivation activity apply even when expression from the GRE-linked promoter is driven by basal components of the transcriptional machinery.


Figure 1: FACS analysis of GrH2 cells. Asynchronously growing GrH2 cell cultures (A) were subjected to G(2) synchrony (B) utilizing Hoechst 33342 as described under ``Materials and Methods.'' Reversibility of the G(2) arrest is revealed by the FACS profiles obtained following removal of Hoechst 33342 and culturing cells for an additional 6 (C) or 10 (D) h.




Figure 2: GR function at a simple, but not composite GRE, is inhibited in G(2)-synchronized GrH2 cells. Asynchronous (asyn) and G(2)-synchronized GrH2 cell lines stably transfected with TAT3 (A), plfG3 (B), or Rous sarcoma virus long terminal repeat (C) CAT reporter plasmid were either untreated(-) or treated (+) with 0.1 µM dexamethasone (dex) for 6 h. CAT assays were performed using equivalent amounts of protein in crude cell-free lysates. Average CAT activity (n = 3) is expressed relative to that obtained in untreated asynchronous cells. The 30 and 50% dexamethasone repression of plfG3 CAT activity observed in asynchronous and G(2)-synchronized cells, respectively, was statistically significant (p < 0.005) as determined using a two-tailed Student's t test.



GRs are bifunctional transcription factors which can act, in certain contexts, as transcriptional repressors as well as activators(36, 38, 39, 40) . While various mechanisms account for GR-mediated transcriptional repression(4) , a composite GRE (cGRE) located within the rat proliferin gene can direct the GR to activate or repress transcription from a linked promoter depending upon the composition of AP-1 transcription factors (41) that co-occupy the cGRE(36) . To compare the activity of the proliferin cGRE with a simple GRE in G(2)-synchronized cells, GrH2 cells were stably transfected with a reporter (plfG3) (36) possessing the proliferin cGRE linked to the identical Drosophila alcohol dehydrogenase minimal promoter that was used to monitor the activity of a simple GRE. As shown in Fig. 2B, plfG3 promoter activity was repressed upon dexamethasone treatment of both asynchronous and G(2)-synchronized GrH2 cells. The extent of dexamethasone repression of plfG3 promoter activity was similar in asynchronous and G(2)-synchronized GrH2 cells, despite the fact that basal activity of the plfG3 promoter was reduced in G(2)-synchronized cells (Fig. 2B). Thus GRs are not completely disabled in G(2)-synchronized cells, and although they are severely compromised in their ability to activate transcription, they still retain the capacity to repress transcription.

GR Protein Levels Are Similarly Down-regulated in Response to Dexamethasone Treatment of Asynchronous and G(2)-synchronized GrH2 Cells

GRs act at multiple levels to control the ultimate production of specific gene products. For example, in many cells, GR protein levels are down-regulated in response to glucocorticoid treatment(27, 42, 43) . This homologous down-regulation in rat HTC hepatoma cells results from glucocorticoid effects on GR protein stability and perhaps GR transcription(27) . To reveal whether this multilevel regulation of GR expression is sensitive to G(2) effects, we used Western blot analysis to quantify GR protein levels. As shown in Fig. 3, a 12-h dexamethasone treatment of either asynchronous or G(2)-synchronized GrH2 cells led to an analogous down-regulation of GR protein levels. It was impractical to extend the hormone treatment much beyond 12 h in G(2)-synchronized cells given the decreased ability of GrH2 cells to recover from G(2) synchronization upon prolonged treatment with Hoescht 33342 (not shown). Nevertheless, the extent of GR down-regulation apparent following a 12-h dexamethasone treatment is identical (approximately 60% in two separate experiments) in both asynchronous and G(2)-synchronized GrH2 cells.


Figure 3: GR protein levels are similarly down-regulated in response to dexamethasone (dex) treatment of asynchronous and G(2)-synchronized GrH2 cells. Asynchronous and G(2)-synchronized GrH2 cells were treated with 0.1 µM dex for the lengths of time indicated (in hours). GRs were immunoprecipitated from the same amount of total protein in whole cell extracts, electrophoresed on 7.5% polyacrylamide-SDS gels, and transferred to nitrocellulose. GR protein was visualized as described under ``Materials and Methods.'' The migration of protein molecular mass standards (in kilodaltons) is indicated. The intensely stained rapidly migrating band represents the heavy chain of the BuGR2 antibody which was used to immunoprecipitate the GR and is detected upon development of the Western blot.



Alterations in GR Phosphorylation and Dephosphorylation in G(2)-synchronized GrH2 Cells

G(2) synchronization of mouse L cell fibroblasts was previously shown to lead to minor changes in GR phosphorylation as detected by two-dimensional tryptic mapping(24) . Tryptic mapping of rat GR phosphopeptides using the identical method (30) has generated more well resolved patterns than mouse GR maps(24) . Thus, GR phosphorylation was analyzed in rat GrH2 cells, not only to confirm our previous studies in mouse fibroblasts(24) , but also to potentially uncover any novel alterations in GR phosphorylation that were not well resolved in mouse GR tryptic maps. Initially, we examined overall GR phosphorylation and observed two striking differences between asynchronous and G(2)-synchronized GrH2 cells. As shown in Table 1, basal phosphorylation of GRs was increased in G(2)-synchronized cells while no further increase in GR phosphorylation was observed upon dexamethasone treatment. In asynchronous GrH2 cells, dexamethasone treatment led to a 1.9-fold increase in overall GR phosphorylation (Table 1).



Glucocorticoid-induced hyperphosphorylation of the GR has been observed in mouse (44, 45) and rat cells (30) and reflects increased overall phosphorylation (Table 1) (44) as well as hyperphosphorylation at specific sites(30) . As seen in Fig. 4(compare Panels A and B), dexamethasone treatment of GrH2 cells led to prominent hyperphosphorylation of a few specific peptides (peptides g and h), in agreement with our observations in other cultured cell lines(14, 15, 30) , and prominent dephosphorylation of another peptide (peptide c). Other minor variations in spot intensity were not reproducible in GrH2 cells or in different cell lines that we have previously examined(14, 15, 30) . Despite the fact that overall GR phosphorylation was not increased upon dexamethasone treatment of G(2)-synchronized cells (Table 1), the identical prominent hyperphosphorylation of GR peptides g and h was observed in G(2)-synchronized GrH2 cells (Fig. 4, compare Panels C and D). In fact, the only striking difference in GR phosphorylation between asynchronous and G(2)-synchronized cells was observed on peptide d. In the absence of dexamethasone treatment, peptide d was hyperphosphorylated 2-fold in G(2)-synchronized cells relative to asynchronous cells (Fig. 4, compare Panels A and C). The closely migrating peptide c was shown to be phosphorylated to the same extent in asynchronous and G(2)-synchronized cells using identical means for quantifying phosphorylation within individual peptides (see ``Materials and Methods''). It seems unlikely that this hyperphosphorylation accounts entirely for increased overall phosphorylation of GR observed in G(2)-synchronized cells (Table 1), as we have previously determined that this peptide accounts for only approximately 10% of total GR phosphorylation in rat cell lines (not shown). In addition to this change in constitutive phosphorylation of peptide d, dexamethasone treatment of G(2)-synchronized cells led to a dephosphorylation of this peptide (Fig. 4, compare Panels C to D). Using a peptide whose level of phosphorylation was not significantly affected by dexamethasone as a reference, the relative extent of peptide d phosphorylation was shown to be reduced by 60% upon dexamethasone treatment of G(2)-synchronized cells. A less dramatic dephosphorylation of peptide d was noted upon dexamethasone treatment of asynchronous cells (Fig. 4, compare Panels A to B). Thus, the lack of overall hormone-induced hyperphosphorylation of GRs in G(2)-synchronized cells is misleading as analysis of GR phosphorylation at specific sites revealed both hormone-induced phosphorylation and dephosphorylation during G(2).


Figure 4: Site specific alterations in GR phosphorylation and dephosphorylation in G(2)-synchronized GrH2 cells. P-Labeled GR protein was isolated from asynchronous and G(2)-synchronized GrH2 cells treated or untreated with 0.1 µM dexamethasone for 1 h and subjected to two-dimensional tryptic mapping on thin layer cellulose plates. A, B, C, and D show autoradiographs of corresponding tryptic maps from untreated asynchronous (A), dexamethasone-treated asynchronous (B), untreated G(2)-synchronized (C), and dexamethasone-treated G(2)-synchronized (D) samples. Phosphopeptides g and h (A) become hyperphosphorylated upon dexamethasone treatment of asynchronous (B) and G(2)-synchronized (D) cells (highlighted by filled horizontal arrows in B and D). Phosphopeptide c (A) is relatively dephosphorylated upon dexamethasone treatment of asynchronous (B) and G(2)-synchronized (D) cells (unfilled, vertical arrows in B and D). Phosphopeptide d (A) is hyperphosphorylated in G(2)-synchronized cells relative to asynchronous cells (filled, vertical arrowhead in C) but becomes dephosphorylated upon dexamethasone treatment of G(2)-synchronized cells (unfilled, vertical arrowhead in D). Analogous results were obtained in another independent tryptic mapping experiment.




DISCUSSION

There are a limited number of genes whose expression is restricted to specific stages of the cell cycle(46) . This feature applies both to genes whose products serve an important regulatory role in cell cycle progression, such as G(1)(47) and G(2)(48) cyclins, and to others which encode proteins more intimately involved in the mechanics of the chromosomal replication (49) condensation(50) , and segregation(51) . Alterations in the abundance and/or activity of the transcription factors that are required for efficient expression of cell cycle regulated genes often accounts for their restricted expression(46) . Our previous work(24) , and that reported herein, establish that GR transactivation activity is severely impaired in G(2)-synchronized cells, even from a promoter that utilizes only basal components of transcription machinery. It therefore seems unlikely that G(2)-specific alterations in the activity of transcription factors required for efficient glucocorticoid induction (35) are solely responsible for the refractoriness of endogenous promoters to GR transactivation. Since GR in crude nuclear extracts prepared from G(2)-synchronized GrH2 cells can bind DNA efficiently in vitro, (^2)we likewise do not believe that the failure to detect glucocorticoid induction is due to inhibition of GR binding to target GREs.

A novel finding presented in this report is that not all nuclear functions of GR are disrupted as GR-mediated repression directed by a cGRE is apparently unaffected in G(2)-synchronized cells. The mechanism of repression in this case involves the co-occupancy at the cGRE of GR with a specific cohort of AP-1 family members(36) , further supporting the notion that GR DNA binding activity is not grossly affected by G(2) synchronization. The expression of this transcriptional modulatory function of the GR argues against the possibility that receptors are sequestered during G(2) within a subnuclear compartment (52) that restricts their ability to productively interact with active transcription factors (i.e. AP-1), or that chromatin structural changes in G(2) render all target sites inaccessible to the GR. Since the proliferin cGRE can also direct GR to activate transcription from linked promoters (36) in the appropriate context, it would be interesting to examine whether GR transactivation from that element is likely impaired during G(2).

In G(2)-synchronized cells, quantitative effects on GR phosphorylation and dephosphorylation were observed. Hormone treatment of both asynchronous and G(2)-synchronized cells led to dephosphorylation of a single peptide (peptide c), while more extensive dephosphorylation of a different peptide (peptide d) was observed in hormone treated G(2)-synchronized cells. Since peptide d is hyperphosphorylated in G(2)-synchronized cells not treated with hormone, the phosphorylation state of GR in hormone treated G(2) cells appears remarkably similar to that obtained from asynchronous cells. Thus, basal phosphorylation of the GR, and not its phosphorylation state in hormone treated cells, is what distinguishes the receptor in asynchronous versus G(2)-synchronized GrH2 cells. If increased basal phosphorylation of GR is responsible for the inhibition of its transactivation activity, perhaps the GR, analogous to other transcription factors such as c-jun(53) , possesses sites that can exert an inhibitory effect on its activity when phosphorylated. The inability of hormone-induced dephosphorylation of GR in G(2) to overcome this inhibition could be explained if this dephosphorylation occurs subsequent to receptor interactions with the transcriptional machinery.

The amino acid sequences surrounding most mouse GR phosphorylation sites (54) implies that they are likely to be the targets of proline-directed protein kinases which could include members of both the cyclin-directed and mitogen-activated protein (MAP) kinase families (55) . Obviously, any members of this family that are activated during G(2)(56) could account for the site-specific hyperphosphorylation of GR in cells not treated with hormone. Regardless of the identity such GR kinase(s), the resultant hyperphosphorylation of GR does not affect all its nuclear functions, and appears to be associated only with inhibition of transactivation. Both PP-1 and PP-2A have been shown to dephosphorylate specific GR sites in vitro and are implicated in the in vivo dephosphorylation of GR(30) . In fact, the hormone-induced dephosphorylation of GR occurs at sites that are major in vitro substrates for PP-1 and PP-2A. Although we have not detected differences in PP-1 and PP-2A activity in crude cell extracts prepared from asynchronous and G(2)-synchronized cells,^2 specific phosphatase targeting subunits (57) could be involved in regulating their activity on GR during G(2). Since hormone-induced dephosphorylation in G(2) restores a normal GR phosphorylation pattern but not transactivation, protein phosphatases may act at a step in the nuclear processing of GR that follows receptor interactions with the transcriptional machinery. This notion is supported by the fact that a population of GR that is relatively dephosphorylated has been found to be confined to a distinct subnuclear compartment, defined biochemically(58) .

The diversity of endogenous and synthetic glucocorticoid-responsive genes whose induction is inhibited in G(2)(23, 24, 59) (this report) implicates the receptor as a likely target of G(2)-specific regulators. While GR interactions with components of the transcriptional machinery that participate in transactivation may be sensitive to some G(2)-specific biochemical alteration in the receptor, interactions of the receptor with distinct transcription factors that bring about transcriptional repression must be transparent to these biochemical changes. Our results point out the potential influence of both kinases and phosphatases in regulating GR activity, and by demonstrating the specificity in affected GR functions, illustrate the diversity of effects that intracellular signaling pathways could exert on steroid hormone responsiveness.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA43037 (to D. B. D.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 412-624-4259; Fax: 412-624-4759; DOD1{at}vms.cis.pitt.edu.

(^1)
The abbreviations used are GR, glucocorticoid receptor; GRE, glucocorticoid response element; cGRE, composite glucocorticoid response element; TAT, tyrosine aminotransferase; FBS, fetal bovine serum; CAT, chloramphenicol acetyl transferase; FACS, fluorescence-activated cell sorting; MAP, mitogen-activated protein.

(^2)
S.-C. Hsu and D. B. DeFranco, unpublished observations.


ACKNOWLEDGEMENTS

We thank Keith Yamamoto for the gifts of DNA (TAT3CAT and plfG3CAT) and GrH2 cells. In addition, Michael Garabedian, Marija Krstic, Allan Munck, and Keith Yamamoto are thanked for sharing unpublished data and for providing useful suggestions throughout the course of these studies. Finally, Uma Chandran, Todd Evans, Marija Krstic, Anu Madan, and Catharine Smith are thanked for their critical reading of the manuscript.


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