P53 and p73 differ in their ability to inhibit glucocorticoid receptor (GR) transcriptional activity
© Zhang et al; licensee BioMed Central Ltd. 2006
Received: 13 October 2006
Accepted: 06 December 2006
Published: 06 December 2006
p53 is a tumor suppressor and potent inhibitor of cell growth. P73 is highly similar to p53 at both the amino acid sequence and structural levels. Given their similarities, it is important to determine whether p53 and p73 function in similar or distinct pathways. There is abundant evidence for negative cross-talk between glucocorticoid receptor (GR) and p53. Neither physical nor functional interactions between GR and p73 have been reported. In this study, we examined the ability of p53 and p73 to interact with and inhibit GR transcriptional activity.
We show that both p53 and p73 can bind GR, and that p53 and p73-mediated transcriptional activity is inhibited by GR co-expression. Wild-type p53 efficiently inhibited GR transcriptional activity in cells expressing both proteins. Surprisingly, however, p73 was either unable to efficiently inhibit GR, or increased GR activity slightly. To examine the basis for this difference, a series of p53:p73 chimeric proteins were generated in which corresponding regions of either protein have been swapped. Replacing N- and C-terminal sequences in p53 with the corresponding sequences from p73 prevented it from inhibiting GR. In contrast, replacing p73 N- and C-terminal sequences with the corresponding sequences from p53 allowed it to efficiently inhibit GR. Differences in GR inhibition were not related to differences in transcriptional activity of the p53:p73 chimeras or their ability to bind GR.
Our results indicate that both N- and C-terminal regions of p53 and p73 contribute to their regulation of GR. The differential ability of p53 and p73 to inhibit GR is due, in part, to differences in their N-terminal and C-terminal sequences.
The p53 tumor suppressor pathway is inactivated in a majority of human cancers, either through mutation of the p53 gene or alterations of p53 regulators or p53-pathway proteins [1, 2]. Wild-type p53 is a transcription factor that binds the promoter regions of various target genes in a sequence-specific manner and activates their transcription. Some of these target genes are necessary for p53 to induce cell cycle arrest following stress, such as Waf1, which encodes the cyclin-dependent kinase inhibitor protein p21 . Other target genes are important for the apoptotic function of p53, including Bax, Fas/Apo1, PUMA, Noxa, and Apaf1 [4, 5]. Cancer-derived mutations in p53 block its sequence-specific DNA-binding capability, resulting in decreased expression of these growth-inhibitory target genes. This can lead to uncontrolled cell growth and eventual carcinogenesis.
P73 is a p53-related protein that shares a high degree of amino acid sequence identity with p53 and many of the same structural features [6, 7]. Given their similarities, it is important to determine whether p53 and p73 carry out similar or distinct functions, and whether they are regulated through similar or different mechanisms. Both p53 and p73 contain an N-terminal transactivation domain (TAD) and proline-rich domain (PRD), a central DNA-binding domain (DBD), and a C-terminal oligomerization domain (OD) [6, 7]. P53 and p73 differ considerably in their extreme C-terminal sequences. The p53 extreme C-terminus (residues 364–393) is a basic-charged region that contains a cluster of six lysine residues. These lysines are sites of post-translational modifications (acetylation, ubiquitination, neddylation, methylation) that can regulate p53 stability and transcriptional activity [8–12]. In contrast, p73 contains an extended C-terminus that can vary in length due to alternative splicing, and that has no sequence or structural homology with p53. P53 and p73 can carry out some redundant functions. For example, p73 can bind to and activate various p53 target genes, and can induce growth arrest or apoptosis when over-expressed [13, 14]. Further, levels of endogenous p73 protein increase in response to certain stresses, as does p53, and this p73 can induce apoptosis in p53-null cells [15, 16]. Despite these similarities, however, the consequence of p53 or p73 loss on development and cancer susceptibility is strikingly different. P53 loss-of-function mutations are found in over 50% or all human cancers, and p53-deficient mice develop multiple cancers and die at an early age [17–19]. This is consistent with p53s role as a bona fide tumor suppressor. In contrast, mutations in p73 are not commonly associated with cancer, and p73-deficient mice display neurological, pheromonal, and inflammatory defects without an apparent increased cancer incidence . These findings and others have suggested that p73 could play distinct roles in development that are not attributed to p53.
Glucocorticoid receptor (GR) is a nuclear receptor and ligand-dependent transcription factor (reviewed in [21, 22]). GR belongs to the superfamily of steroid nuclear receptors that also includes estrogen receptor (ER), androgen receptor (AR), and progesterone receptor (PR). In the absence of ligand, GR is inactive and resides in the cytoplasm in complex with chaperones such as Hsp90. Ligand-binding promotes dissociation of GR from cytoplasmic complexes and its translocation to the nucleus, where it can then activate transcription of its target genes. The effect of activating GR appears to be cell-type specific. GR activation has been reported to promote survival and inhibit apoptosis in mammary epithelial cells, breast cancer cells, neuroblastoma cells, and other cell types [23–28]. In contrast, GR activation in thymocytes triggers apoptosis (reviewed in ). There is compelling evidence for cross-talk between GR and p53. Binding between GR and p53 has been demonstrated both in vitro and in vivo, and their interaction can lead to the mutual inhibition of both proteins [27, 28]. The GR ligand dexamethasone has been reported to enhance binding between GR and p53 and, under certain conditions, to cause their cytoplasmic sequestration and degradation . Complexes of p53 and GR may also contain MDM2, a p53-responsive protein that can act as an E3 ubiquitin ligase to promote degradation of both p53 and GR . These findings have suggested that GR could enhance survival by sequestering p53 in the cytoplasm and, conversely, that p53 might enhance cell death by sequestering GR in the cytoplasm and blocking its survival function. To date, neither physical nor functional interactions between GR and p73 have been described.
In the current study, we examined whether p73 could bind GR and participate in negative cross-talk with GR, similar to p53. We found that both p53 and p73 could bind GR, and that p53 and p73-mediated transcriptional activity was inhibited by GR co-expression. Wild-type p53 efficiently inhibited GR transcriptional activity in cells expressing both proteins. Surprisingly, however, p73 was either unable to effectively inhibit GR, or increased GR activity slightly. To examine the basis for this difference, we generated a series of p53:p73 chimeric proteins in which corresponding regions of either protein have been swapped. These studies revealed that N- and C-terminal regions of p53 and p73 contribute to their regulation of GR. Fusion of p53 N- and C-terminal sequences to p73 allowed p73 to inhibit GR and, conversely, fusion of p73 N- and C-terminal sequences to p53 prevented p53 from inhibiting GR. We conclude that the differential ability of p53 and p73 to inhibit GR is due, in part, to differences in their N-terminal and C-terminal sequences.
Both p53 and p73 can bind GR
We next asked whether binding between GR and either p53 or p73 could be detected. For this, Saos-2 cells were transfected with expression DNAs (1 μg each) encoding GR and HA-tagged p53 or p73β. Transfected cells were then either untreated or treated with Dex for 17 hrs, followed by incubation for an additional 6 hrs in the presence or absence of MG132 to block their potential degradation. Cell lysates were immunoprecipitated with a GR antibody, followed by immuno-blotting with an HA antibody to detect co-immunoprecipitating p53 or p73β. As shown in Fig. 1C, both p53 and p73β co-immunoprecipitated with GR. Interestingly, binding between transfected GR and p53 or p73β was most evident in cells treated with MG132, suggesting the ability to detect GR complexes with p53 or p73 may be limited by their proteasomal degradation. p53 appeared to bind GR to a slightly greater extent than did p73β.
GR inhibits p53 and p73 transcriptional activity
P53, but not p73, can efficiently inhibit GR transcriptional activity
P73 fails to inhibit GR when MDM2 is over-expressed
Chimeric p53:p73 proteins have altered abilities to inhibit GR
Binding between p53 and GR has been demonstrated in vivo and in vitro, and p53 and GR can inhibit each others transcriptional activity in transfected cells [27, 28]. Moreover, activation of endogenous GR by Dex treatment inhibited p53 induced cell cycle arrest in stressed cells, and activation of endogenous p53 by DNA damage inhibited GR transcriptional activity [27, 28]. These findings and others support the existence of negative cross-talk between the p53 and GR pathways. The GR-binding site in p53 has been mapped to the p53 DNA-binding domain (DBD) , and Dex treatement was reported to both enhance p53-GR binding and promote their proteasomal degradation to some extent. P53 and p73 are structurally similar and share high amino acid sequence identity in their DBD and other domains. However, to date, neither physical nor functional interactions between p73 and GR have been reported. In some but not all experiments, GR and p53 levels decreased upon Dex treatment in cells co-expressing both proteins, and this was partially blocked by proteasome inhibition. GR and p73 levels were also decreased in Dex-treated cells in some experiments (Fig. 1B). This suggests Dex treatment may promote proteasomal degradation of GR, p53, and p73. Our co-immunoprecipitation studies (Fig. 1C) indicate that p73 can also bind GR, similar to p53. Interestingly, binding between GR and p53 or p73β was most evident in cells treated with the proteasome inhibitor MG132. This suggests the ability to detect GR complexes with p53 or p73 may be limited by their proteasomal degradation. Alternatively, MG132 may stabilize one or more factors that enhance the interaction between GR and either p53 or p73.
GR can inhibit the transcriptional activity of p53. To test whether GR can also inhibit the transcriptional activity of p73, we monitored the effect of GR co-expression on p53 and p73-dependent activation of different luciferase reporter genes. GR inhibited transactivation of all three reporter genes by p53 or p73 to comparable extents. Thus, p53 and p73 can bind GR, and the transcriptional activity of both proteins is inhibited by GR. We also monitored the effect of p53 or p73 co-expression on the transcriptional activity of GR. In all experiments, p53 caused a pronounced inhibition of GR activity. Surprisingly, however, p73 was either unable to efficiently inhibit GR, or activated GR slightly (Fig. 3). Thus, p53 and p73 can bind GR, but only p53 and not p73 can efficiently inhibit GR activity. One interesting possibility based on these results is that the mechanism by which GR inhibits p53 and p73 may be different from the mechanism by which p53 or p73 inhibits GR. Binding to p53 or p73 may be sufficient for GR to inhibit p53 and p73 activity. However, binding alone may not be sufficient for p53 or p73 to inhibit GR, since p73 can bind GR just as p53 can, but is unable to efficiently inhibit GR activity.
Wasylyk and colleagues reported one way in which p53 can inhibit GR is through its activation of MDM2 . According to this model, p53 activates MDM2 gene expression, and increased levels of MDM2 protein then promote the degradation of both p53 and GR. We observed that p53 could still efficiently inhibit GR activity either when endogenous MDM2 levels were decreased via siRNA, or in MDM2/p53 double-knockout cells. While MDM2 may contribute to GR inhibition by p53, our results indicate p53 can also inhibit GR through an MDM2-independent mechanism. P73 induced lower levels of MDM2 than did p53. However, p73 remained unable to inhibit GR activity when expressed with increasing amounts of MDM2, and MDM2 alone also had no effect on GR activity. These results indicate the different abilities of p53 and p73 to inhibit GR results from something other than differences in MDM2 levels.
We considered two possibilities might explain the different abilities of p53 and p73 to inhibit GR. First, p53 may contain one or more unique sequence elements that are absent from p73, and that allow p53 to inhibit GR. Second, p73 may contain one or more unique sequence elements that are missing from p53, and that prevent p73 from inhibiting GR. To investigate these possibilities, a series of p53:p73 chimeric proteins were generated in which corresponding regions of either protein have been swapped (Figs. 5A and 8A). The high degree of structural homology between p53 and p73 should allow one to switch the various domains of each protein without disrupting protein conformation. This approach was used previously by Yuan and colleagues to identify specific sequence elements in p53 that confer its degradation by MDM2 . Our studies revealed that replacing C-terminal domains (oligo and C-term) in p53 with the corresponding domains of p73 had little effect, or only slightly diminished, the ability of p53 to inhibit GR. Conversely, deleting the unique C-terminus of p73, or replacing C-terminal domains (oligo and C-term) in p73 with the corresponding domains of p53 allowed p73 to inhibit GR to a large extent. Replacing the N-terminal domains (TAD and PRD) in p53 with the corresponding domains of p73 diminished the ability of p53 to inhibit GR, whereas replacing the N-terminal domains in p73 with the corresponding domains of p53 allowed p73 to inhibit GR to some extent. These results suggested that the different abilities of p53 and p73 to inhibit GR can be explained in part, but not fully, by individual differences in their N- and C-terminal sequences. Confirmation of this came from analysis of p53:p73 chimeric proteins in which their N- and C-terminal sequences were swapped simultaneously (Fig. 11). Replacement of p53s N- and C-terminal sequences with the N- and C-terminal sequences of p73 at the same time completely blocked its ability to inhibit GR, while replacement of p73s N- and C-terminal sequences with the N- and C-terminal sequences of p53 at the same time allowed it to efficiently inhibit GR. Importantly, the different ability of these chimeric proteins to inhibit GR was not due to differences in their transcriptional activity or ability to bind GR. The results suggest that p53 N- and C-terminal sequences contribute to its inhibition of GR, since these sequences cannot be replaced by the corresponding sequences of p73. In contrast, p73 N- and C-terminal sequences appear to prevent GR inhibition, since deletion of C-terminal sequences from p73 restored some ability to inhibit GR, and replacing p53s N- and C-terminal sequences with those of p73 blocked p53 from inhibiting GR. This raises the question of how the N- and C-terminal sequences of p53 or p73 might participate in GR inhibition. A recent study suggested that p53 could repress transcription of the IGFBP-1 gene through recruitment of a histone deacetylase (HDACs) . Interestingly, this recruitment of HDAC required both N-terminal and C-terminal sequences in p53. Perhaps p53 can bind GR on promoter DNA and repress its activity through N- and C-terminal sequences and HDAC recruitment. p73 might lack the ability to recruit HDAC protein(s) or only recruit HDACs inefficiently, accounting for its inability to inhibit GR.
p53 and p73 transcriptional activity is inhibited by glucocorticoid receptor (GR) co-expression. However, whereas wild-type p53 efficiently inhibits GR transcriptional activity, p73 is either unable to efficiently inhibit GR, or increases GR activity slightly. These differences are not due to differences in the ability of p53 and p73 to either bind GR, or activate MDM2 gene expression. Analysis of multiple p53:p73 chimeric proteins revealed that the differential ability of p53 and p73 to inhibit GR is due to differences in their N-terminal and C-terminal sequences.
HA-tagged and Flag-tagged p53 wt DNAs were described previously . HA-tagged human p73β DNA was from Frank Mckeon (Harvard Medical School). Flag-tagged p73β was generated by PCR amplifying p73β sequences from HA p73β DNA and cloning them into Bam HI and Xba I sites downstream of the Flag epitope. p53(73βaa1–54), and p73β(53aa1–45) DNAs were gifts from Zhimin Yuan (Harvard School of Public Health. All other DNAs encoding human p53:p73β chimeric proteins were generated by two-step PCR method, as described previously . All chimeric DNAs were confirmed by DNA sequencing. Oligonucleotide sequences used in cloning are available on request. DNA encoding glucocorticoid receptor (GR) and GR-responsive luciferase reporter gene (GR-luc) was from E.A. Thompson (University of Texas Medical Branch, Galveston, TX). GR-luc contains two copies of a GR-responsive element from the rat angiotensin gene upstream of minimal promoter. PG13-luc DNA was from Bert Vogelstein (Johns Hopkins). P21-luc and MDM2-luc DNA were from Moshe Oren (Israel). DNA encoding siRNA against MDM2 was described  and was from Ruiwen Zhang (University of Alabama-Birmingham).
Cell culture and transfections
Saos-2 cells are a p53-null human osteosarcoma cell line. MDM2/p53 double knockout (DKO) mouse embryo fibroblasts were from Rudy Alarcon (Stanford University). Cell lines were maintained at 37°C in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (1% penicillin and streptomycin). Transfections in Saos-2 or MDM2/p53 DKO cells were done using Fugene-6 transfection reagent (Roche) according to the manufacturers protocol when cells were approximately 60% confluent. Total DNA in each transfection was equalized by addition of empty plasmid. Dexamethasone (Dex) was purchased from Sigma and was dissolved in ethanol at a concentration of 1 mM. Dex was added to cells at a final concentration of 100 nM to activate GR, and cells harvested 20–24 hrs later. Where indicated, the proteasome inhibitor MG132 (Boston Biochem) was added to a final concentration of 30 μM and the cells incubated for an additional 6 hrs before harvesting.
Immunoprecipitations and Immunoblotting
To harvest cell lysates for immunoblotting, cells were rinsed with 2 ml PBS and then scraped into 500 μl lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40, PMSF, leupeptin) and transferred to microfuge tubes. The cells were then incubated on ice for 30 min with occasional vortexing, and spun at 4°C, 14,000 rpm for 15 min to remove cellular debris. For co-immunoprecipitations, lysates were immunoprecipitated overnight with 200 ng anti-GR polyclonal antibody (clone E-20, Santa Cruz Biotechnology). GR immunoprecipitates were isolated on protein A agarose beads as described previously . For immunoblotting, protein lysates or GR immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes for immunoblotting. Antibodies used in immunoblotting included anti-HA monoclonal antibody (HA.11, Covance), anti-Flag monoclonal antibody Ab-5 (Sigma-Aldrich), anti-GR polyclonal antibodies (E-20 and P-20, Santa Cruz Biotechnology), anti-MDM2 monoclonal antibody SMP-14 (Santa Cruz Biotechnology).
To monitor p53 and p73 transcriptional activity, Saos-2 cells were transfected with 100 ng of each luciferase reporter DNA (pG13-luc, p21-luc, MDM2-luc) and 50 ng DNA encoding Flag p53 wt or Flag p73β wt. In some cases, GR DNA was included in the transfection (100, 250, 500, or 1000 ng) and cells were treated with Dex (100 nM) for 16–20 hrs before harvesting. To monitor GR transcriptional activity, cells were transfected overnight with 200 ng GR-luc DNA and 100 ng DNA encoding GR. Cells were then treated with Dex (100 nM) for 20–24 hrs before harvesting. In some cases, p53 wt or p73 wt, or the various chimeric proteins were included in the transfection. PRL-TK DNA (5 ng) was included in each transfection as a normalization control. Luciferase activity was monitored using the dual-luciferase reporter assay kit (Promega Corp.), according to the manufacturers protocol. P53 and p73 protein expression levels were compared in these luciferase experiments by densitometric scanning of films using Image-J software. This allowed comparison of GR-luc activity under conditions of comparable p53 and p73 protein expression.
This work was supported by grant 1RO1 CA108843-02 (to C.G.M) from the National Institutes of Health.
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