Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: Dr. Donald DeFranco, Department of Pharmacology, University of Pittsburgh School of Medicine, Room E1352 BST, Pittsburgh, Pennsylvania 15261. E-mail: dod1{at}pitt.edu.
ABSTRACT
Steroid hormone receptors exert much of their effects on cellular physiology through regulating the rate of transcription from unique target genes. Much has been learned about the actions of steroid hormone receptors at regulated promoters through model in vitro studies, but it has always been a challenge to extrapolate these mechanistic insights to molecular events that occur in live cells. However, novel insights have recently been gained regarding the nature of receptor encounters with the transcriptional machinery from elegant experimental approaches that used advances gained in biochemical, molecular biological, cell biological, and biophysical disciplines. Although these is no doubt that steroid hormone receptors represent some of the most mobile proteins within the nucleus, they still maintain their ability to orchestrate a highly ordered recruitment of cofactors and coregulators at specific sites and remain accessible to alternative processing pathways that limit their action. As highlighted in this review, there may be interrelationships between seemingly distinct pathways of receptor trafficking and processing within the nucleus that impact receptor action at regulated promoters.
THE REQUIREMENT FOR nuclear localization of steroid hormone receptors to elicit the wide-ranging physiological effects of the steroid hormones was recognized in the earliest work on hormone action (1, 2). Although there is also an emerging interest in cytoplasmic targets of steroid receptors (3), many years of study have been devoted to probing steroid receptor function within the nucleus. In that regard, nuclear transport of the receptors is necessary for their direct action on target genes and is likely to serve as an important regulatory step in the steroid hormone signal transduction pathway. Furthermore, because steroid hormone receptors often exert rapid effects on gene transcription, specific mechanisms must exist to insure the timely and efficient localization of target sites by the receptors. In recent years, a number of exciting cell biological, genetic, and biochemical experiments have provided new insights into the complexities of steroid receptor trafficking within the nucleus. With the sophisticated tools and extensive reagents now available, the detailed molecular mechanisms of steroid hormone-regulated transcription revealed in model in vitro studies can now be elaborated in live cells.
STEROID RECEPTOR MOBILITY WITHIN THE NUCLEUS
Green fluorescence protein (GFP) chimeras have proven to be invaluable tools for cell biologists and allowed for the real-time visualization of protein movement and protein-protein interactions in live cells (4). For some steroid receptor proteins, GFP chimeras provided definitive proof of their hormone-dependent cytoplasmic to nuclear transport (5, 6, 7, 8, 9). Importantly, specific compartmentalization of steroid receptors within the nucleus could also be discerned in live cells (5, 6, 7, 8, 9, 10, 11). These studies confirmed earlier results obtained with fixed cells that implied that localization of bulk receptors within the nucleus was not random (12, 13). The nature and importance of steroid receptor foci visible within the nucleus at the light-microscope level has been controversial, but given the previous commentaries that have been written on this topic (e.g. see Ref. 14), this will not be discussed in detail here. However, it does seem likely that in addition to their concentration within regions of the nucleus associated with active transcription, receptors accumulate within visible foci that are transcriptionally inert (13) and likely to represent storage sites that transiently engage receptors destined for alternative processing fates.
Recently, the Hager and Mancini (15, 16) laboratories have used steroid receptor-GFP chimera to reveal the dynamic nature of steroid receptor movement within the nucleus. Both groups used fluorescence recovery after photobleaching and other techniques to provide real-time assessments of steroid receptor subnuclear trafficking. The Hager laboratory (15) took advantage of a cell line that contains a large array of integrated copies of the well-studied glucocorticoid-responsive promoter contained within the mouse mammary tumor virus long terminal repeat. Thus, they could visualize in real time and in live cells the movement of glucocorticoid receptors (GRs) within a specific nuclear site where receptors were actively engaged in transcriptional regulation (15). These elegant studies confirmed previous work from traditional biochemical experiments (17, 18) that implied that GR interactions with specific sequences within chromatin templates are dynamic. The model put forth by Hager and co-workers (15) proposes that GR occupies its target sites only transiently, relying on a "hit-and-run" mechanism to alter transcriptional efficiency from linked promoters. While bound, GR is likely to recruit essential coactivators and other cofactors to target genes (19), but continued occupancy by bound receptor may not be required for subsequent assembly of an active preinitiation complex and recruitment of RNA polymerase II.
Remarkably, the Mancini laboratory (16) found that the kinetics of bulk estrogen receptor (ER) movement within the nucleus was analogous to the rapid kinetics of GR exchange at a specific target site. We have confirmed that the movement of bulk GR within the nucleus of hormone-treated cells is also quite rapid (Rentsch, C., and D. DeFranco, unpublished observations). These results suggest that even receptors that are not intimately involved in transcriptional regulatory events at specific sites are nonetheless undergoing rapid movement within the nucleus. In fact, unliganded ER was found to exhibit the most rapid subnuclear movement, suggesting that there may be some retardation of receptor trafficking within the nucleus as activated receptors are scanning the genome in search of specific binding sites. ER bound to mixed antagonists are as mobile as agonist-bound ER, whereas the movement of receptors bound to pure antagonists is more retarded. Thus, the nuclear migration of antagonist-bound receptors may be limited by nonproductive interactions with some nuclear components (16). In this regard, it would be most informative when the dynamics of nuclear trafficking is followed for unliganded nuclear receptors, such as retinoic acid receptors or thyroid hormone receptors, that are associated with corepressors.
Another important contribution from the Mancini study (16) was the demonstrated role of ATP in receptor movement. The energy requirements for nuclear protein movement have not been generally agreed upon, as recent studies examining the nuclear trafficking of various GFP chimera did not show an effect of ATP depletion (20). Interestingly in the studies of Mancini and colleagues, ATP depletion led to a rapid and reversible inhibition of unliganded ER movement within the nucleus but did not affect the movement of agonist-bound ER (16). Perhaps the apparent energy requirement for unliganded ER movement is based upon their association with molecular chaperones. The binding and release of molecular chaperones to their substrate proteins is driven by a cycle of ATP hydrolysis and exchange (21). Disruption of this cycle upon ATP depletion may stabilize a nuclear ER/chaperone heteromeric complex and thereby limit its mobility. Ligand-bound ERs are not found in stable complexes with molecular chaperones (22) and may therefore not be subjected to limitations in nuclear migration upon ATP depletion. Furthermore, other proteins that do not show an ATP dependence for rapid nuclear trafficking (20) may likewise not assemble into stable heteromeric complexes with molecular chaperones. This hypothesis implicates molecular chaperones in nuclear migration of steroid receptors, an issue that has been addressed in previous reviews (23, 24). Alternatively, as discussed by Mancini and co-workers (16), unliganded ER in ATP-depleted cells might be immobilized by virtue of their stable association with the nuclear matrix. Previous biochemical studies have revealed an ATP requirement for GR release from the nuclear matrix (25).
RECRUITMENT OF COACTIVATORS TO SITES OF HORMONE-ACTIVATED TRANSCRIPTION
The regulation of promoter activity by nuclear receptors requires the highly orchestrated assembly of large multi-subunit complexes, which include components that either directly impact the basal transcription machinery (26) or remodel chromatin through specific modifications of core histones (19). Many cofactors (i.e. coactivators or corepressors) involved in nuclear receptor-regulated transcription have been identified and the biochemical basis for their impact on chromatin structure or the activity of basal transcription factors revealed (19). In fact, some of the first mechanistic studies of coactivator activity in model in vitro systems revealed the dynamic nature of coactivator action in nuclear receptor-mediated transcriptional activation. Using in vitro transcription reactions with purified ER and the p300 coactivator, Kraus and Kadonaga (27) showed that p300 acted to enhance the efficiency of transcriptional initiation from an estrogen-regulated template assembled into chromatin. The reassembly of active complexes during subsequent rounds of reinitiation did not require p300 in vitro. In contrast, agonist-bound ER enhanced both the efficiency of transcriptional initiation and reinitiation (27). In these model in vitro studies, it was not possible to assess the dynamic interaction of ER with its target site. However, given the recent work of the Hager and Mancini groups (Refs. 15 and 16 ; see above), it seems reasonable to assume that ER is not statically bound to chromatin templates in vitro and is likely to undergo rapid cycles of DNA binding and release. In fact, GR has been shown to be transiently associated with chromatin templates in vitro and requires ATP for chromatin release (28).
Coactivator recruitment to hormone-regulated templates has also been assessed at specific target sites through the use of chromatin immunoprecipitation assays. In addition to providing one of the first demonstrations of site-specific histone hyperacetylation at hormonally responsive promoters, Chen et al. (29) showed that the binding of coactivators to endogenous estrogen-responsive promoters is transient. The release of promoter-bound coactivators, despite the persistence of ER binding, leads to an attenuation of histone hyperacetylation and hormone-induced transcription (29). For the activator of thyroid hormone and retinoic acid receptors (ACTR) coactivator, release from promoter-bound ER may be brought about via its acetylation by p300 (29). It is curious that the chromatin immunoprecipitation assay used in this study could reveal dynamic interactions of coactivators but not of the ER with a hormone-responsive promoter. A differential stability of ER vs. the steroid hormone receptor coactivator-1 and CBP (cAMP response element binding protein-binding protein) coactivators has also been observed in vivo on a high-copy array of ER binding sites (30). However, these results are not consistent with the results of Hagers group (15) showing rapid exchange of GR from a high-copy array of active glucocorticoid-responsive promoters in vivo. It is possible that the precise makeup of high-copy arrays used to visualize receptor binding to target sites in live cells, i.e. tandem repeats of hormone-responsive promoter (15) vs. tandem arrays of lac operator sites (28), influences receptor exchange.
Recent chromatin immunoprecipitation experiments from the Brown lab (31), while confirming the dynamic association of coactivators with hormonally responsive promoters in vivo, also distinguished "early" and "late" events in hormone-regulated transcription. Specifically, agonist-bound ER, and recruited coactivators amplified in breast cancer-1 (AIB1)/ACTR and peroxisome proliferator activator receptor-binding protein (PBP) show a biphasic association with chromatin at an estrogen-responsive promoter during chronic hormone exposure (31). ER and coactivators recruited to the promoter within 1530 min of hormone treatment are released after an additional 60-min exposure to hormone and then reassembled on to the hormone-responsive promoter for an additional 3045 min (31). Thus ER, AIB1/ACTR, p300/CBP-associated factor, CBP, and PBP appear to continually cycle onto a regulated promoter during a continuous hormone exposure. Interestingly, p300 is recruited to the promoter during the first phase of ER binding (i.e. within the initial 60 min of hormone treatment), but does not reassemble onto this same template with the recycled ER, AIB1/ACTR, and PBP (31). This result is consistent with those reported by Kraus and Kadonaga (27), which showed a requirement for p300 during initiation but not reinitiation of ER-dependent transcription from chromatin templates in vitro.
Although it may be inappropriate to compare the kinetics of nuclear receptor "occupancy" at target sites as revealed with GFP chimera in live cells vs. chromatin immunoprecipitation assays performed with nuclear extracts, the seemingly disparate findings obtained with these two approaches may in fact be compatible. Thus, the results of Hager and colleagues (15) imply that during the first 60 min of hormone treatment, where chromatin immunoprecipitation assays show stable receptor association with hormonally responsive templates, the receptor is in fact undergoing rapid exchange with kinetics that appear to be indistinguishable from their exchange within bulk chromatin. Although this rapid (i.e. within 510 sec) exchange of receptors at active sites of transcription may not be revealed when isolated chromatin is analyzed (29, 31), the in vitro assays do reveal a distinct cycling of hormone-bound receptors and coactivator proteins that occurs over a 2-h period of hormone exposure. Such an ordered exchange of coactivators may be elicited by rapidly cycling receptors that encounter biochemically distinct targets during different kinetic phases of a hormone-induced transcriptional response. The conditions used for chromatin immunoprecipitation assays may differentially stabilize or trap rapidly cycling receptors on unique templates that form in vivo during hormone-induced transcription. In any event, distinct patterns of coactivator cycling, which is prompted by hormone-bound receptor association with specific binding sites, might be necessary to insure the appropriate assembly of components required for efficient initiation and reinitiation of hormone-induced transcription.
DEGRADATION OF NUCLEAR RECEPTORS BY THE UBIQUITIN-PROTEASOME PATHWAY
Proteasomes are multi-subunit complexes that serve as the major soluble protein degradative machinery within eukaryotic cells (32). The targeting of proteins to proteasomes requires their covalent modification with multiple residues of the 76-amino acid ubiquitin protein (32). Although passage through the inner core of the proteasome leads to the degradation of target proteins into small peptides, intact ubiquitin moieties are liberated after target protein proteolysis and released for subsequent reutilization. In addition to serving as the major degradative pathway to eliminate damaged and denatured proteins, proteasomes operate to degrade proteins with both short and long half-lives (32). GR (33, 34, 35), like other nuclear receptors (36, 37, 38), is degraded via the ubiquitin-proteasome pathway. The efficiency of GR (33) and ER (38) degradation by the proteasome is enhanced upon chronic hormone treatment and leads to down-regulation of receptor levels.
NUCLEAR RECEPTOR DEGRADATION AND NUCLEAR EXPORT
The efficiency of proteasome-mediated degradation of nucleocytoplasmic shuttling proteins has been linked in some cases with their rate of nuclear export (39, 40). For example, proteasome-mediated degradation of the cyclin-dependent kinase inhibitor protein p27Kip1 is stimulated when its nuclear export rate is increased via its interaction with the Jun activation domain-binding protein-1 coactivator protein (41). The murine double minute (MDM2) RING-finger protein serves an analogous role to enhance the nuclear export and proteasome-mediated degradation of the p53 tumor suppressor protein (42, 43). 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 sequence-GR is enhanced (44).
Although degradation of nuclear receptors may be coupled to their nuclear export, recent results with p53 and GR suggest that nuclear export and degradation of shuttling proteins are not always inextricably linked. For example, enhancement of p53 nuclear export upon expression of the chromosome maintenance region-1/exportin nuclear export factor did not lead to its increased degradation (45). Furthermore, proteasome-mediated degradation of a p53 mutant with reduced capacity for nuclear export was still increased upon overexpression of MDM2 (45). These results provide convincing evidence for the existence of active proteasomes within both the cytoplasm and nucleus and suggest that proteasome-dependent degradation of shuttling proteins may not be limited to a distinct subcellular compartment. The human ortholog of MDM2 (HDM2) may also be involved in GR ubiquitylation and degradation, perhaps acting in concert with p53 (46). HDM2 and p53 have been implicated in altered GR trafficking in hypoxic cells, but the impact of these proteins on the nucleocytoplasmic shuttling of the receptor under conditions of normoxia has not been fully resolved (46). The nuclear export pathway used by GR is clearly distinct from that of p53, as the receptor utilizes the calcium-binding protein calreticulin (47), and not chromosome maintenance region-1/exportin, as an essential nuclear export factor. The relationship between calreticulin and MDM2-stimulated ubiquitylation and subsequent degradation of GR has not been examined. Other proteins, such as the heat shock protein 90 (hsp90)-binding cochaperone carboxyl terminus of heat shock cognate protein-70-interacting protein (34), also influence GR ubiquitylation and degradation but have not been assessed for effects on GR trafficking.
ROLE OF PROTEASOMES IN NUCLEAR RECEPTOR TRANSACTIVATION
Hormone-dependent down-regulation of steroid hormone receptors, while limiting the duration of hormone responsiveness, also affects the efficiency of receptor transactivation (48). The relationship between steroid receptor abundance and efficiency of hormone- induced transcriptional responses was also revealed in model transfection studies (49) and recently shown to be relevant in vivo (50). It was therefore surprising when progesterone receptor and thyroid hormone receptor transactivation was found to be inhibited in transiently transfected cells treated with an inhibitor of proteasome function (51). In this case, elevated receptor levels caused by proteasome inhibition reduced, rather than potentiated, receptor transactivation. Curiously, proteasome inhibition does not reduce GR transactivation (51), but in fact leads to enhanced GR transactivation from both transiently transfected (35) and stably integrated templates (DeRoo, B., D. B. DeFranco, and T. Archer, unpublished). Nonetheless, a link between transcriptional activation and proteasome-mediated degradation had been revealed in previous model studies with chimeric transcriptional activators of differing potencies. Specifically, the rate of activator degradation was found to directly correlate with its transactivation potency (52, 53). One hypothesis that has emerged from these studies proposes a role for dynamic, proteasome-mediated turnover of transcriptional activators for efficient transcription (51, 53). This hypothesis is consistent with the observed effects of proteasome inhibition on ER mobility within the nucleus and implies that proteasomes may be involved in the bulk movement of transactivators within the nucleus (16). Proteasome inhibition likewise affects GR mobility within the nucleus, but these effects are not always correlated with effects on receptor transactivation (DeRoo, B., T. Archer, and D. B. DeFranco, unpublished data).
In addition to GR, other cases have been reported in which nuclear receptor transactivation is differentially responsive to receptor degradation. For example, an uncoupling of transactivation and degradation has been observed with specific mutants of the retinoid X receptor (54). In addition, a progesterone receptor 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 (55). 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.
In addition to potential indirect effects on transcription activation through regulation of receptor turnover and trafficking, specific proteasomal subunits may play a more direct role in regulating transcription. For example, the 19S regulatory subunit of the proteasomes functions in transcriptional elongation of RNA polymerase II, as measured in model in vitro transcription reactions (56). Genetic and biochemical studies imply that the 19S proteasome subunit-associated SUG1 ATPase (57) participates in transcriptional elongation by RNA polymerase II in vitro supplies the activity necessary for proteasome effects on transcription independent of its affects on protein degradation (56). Interestingly, SUG1 was identified as a nuclear receptor-interacting protein in yeast two-hybrid screens (58, 59). However, SUG1 has been found to impact nuclear receptor degradation and has not been directly shown to participate in nuclear receptor-activated transcription, particularly at the level of elongation (60). It is possible that SUG1, or other proteasome components, exert dual functions during RNA polymerase II transcription, facilitating global transcription elongation while at the same time limiting the efficiency of selected transcription initiation events through the recruitment of specific transactivators (e.g. nuclear receptors) for proteasome-mediated degradation.
In summary, as a result of recent advances in nuclear receptor action at chromatin templates and live cell imaging, a more complete understanding of the dynamic interplay between nuclear receptors and the multitude of cofactors and coregulators at transcriptionally active templates is beginning to emerge. In fact, unique regulatory features may be imparted onto specific hormonally responsive loci by variations in the order of assembly or turnover of individual components of the transcriptional machinery. The ubiquitin-proteasome system has emerged as a potential regulator of nuclear receptor transactivation acting through effects on receptor trafficking and turnover (Fig. 1). Furthermore, many posttranslational modifications (e.g. acetylation, methylation, phosphorylation, ubiquitylation) and novel proteins (i.e. specific components of chromatin remodeling machines, molecular chaperones, proteasomal subunits) may have a role in maintaining efficient exchange and turnover of receptors and associated factors at active sites of transcription. Perhaps what is the most exciting aspect of the current state of receptor research is that we may now be able to elucidate these complex mechanisms in real time and in live cells.
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FOOTNOTES
Abbreviations: ACTR, Activator of thyroid hormone and retinoic acid receptors; AIB1, amplified in breast cancer-1; CBP, cAMP response element binding protein-binding protein; ER, estrogen receptor; GFP, green fluorescence protein; GR, glucocorticoid receptor; hsp90, heat shock protein 90; MDM2, murine double minute; PBP, peroxisome proliferator activator receptor-binding protein.
Received for publication January 18, 2002. Accepted for publication March 1, 2002.
REFERENCES