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Vol. 15, No. 18, pp. 2367-2380, September 15, 2001
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch cedex, France
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ABSTRACT |
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 Top
 Abstract
 Introduction
Results
Discussion
Material and methods
References
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The glucocorticoid receptor (GR) and the tumor supressor p53 mediate different stress responses. We have studied the mechanism of their mutual inhibition in normal endothelial cells (HUVEC) in response to hypoxia, a physiological stress, and mitomycin C, which damages DNA. Dexamethasone (Dex) stimulates the degradation of endogenous GR and p53 by the proteasome pathway in HUVEC under hypoxia and mitomycin C treatments, and also in hepatoma cells (HepG2) under normoxia. Dex inhibits the functions of p53 (apoptosis, Bax, and p21WAF1/CIP1 expression) and GR (PEPCK and G-6-Pase expression). Endogenous p53 and GR form a ligand-dependent trimeric complex with Hdm2 in the cytoplasm. Disruption of the p53-HDM2 interaction prevents Dex-induced ubiquitylation of GR and p53. The ubiquitylation of GR requires p53, the interaction of p53 with Hdm2, and E3 ligase activity of Hdm2. These results provide a mechanistic basis for GR and p53 acting as opposing forces in the decision between cell death and survival.
[Key Words: Endothelial cells; hypoxia; Bax; p21WAF1/CIP1; PEPCK; G6Pase]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Material and methods
References
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The development of most tumors is associated with
loss of function of the tumor suppressor p53. Different physiological
stress, including DNA damage and hypoxia, activate p53. p53 is a
transcription factor that regulates genes involved in growth arrest and
apoptosis. p53 is down-regulated by its target gene product Mdm2 (human
homolog Hdm2). Mdm2 forms an autoregulatory loop with p53 by binding to its N-terminal domain, inhibiting its transcriptional activity and
increasing its degradation by the ubiquitin proteasome pathway (for
reviews, see Jimenez et al. 1999
; Lakin and Jackson 1999; Sionov and
Haupt 1999). Mdm2 is a RING finger-dependent ubiquitin protein ligase
for p53 and itself (Argentini et al. 2000; Fang et al. 2000; Honda and
Yasuda 2000). Mdm2 also inhibits p53 by nuclear export through a
mechanism involving either the nuclear export signal (NES) of Mdm2 (Tao
and Levine 1999) or the RING finger of Mdm2 and the NES of p53 (Boyd et
al. 2000; Geyer et al. 2000). The NES of p53 is masked in the
transcriptionally active heterodimer, but is exposed in the monomeric
form of p53 (Stommel et al. 1999). MdmX, a Mdm2 homolog that lacks a
NES, stabilizes p53 by retention in the nucleus (Jackson and Berberich
2000; Stad et al. 2000). Nuclear p53 levels can also be maintained by
ARF, which blocks nucleo-cytoplasmic shuttling of Mdm2 (Sherr and Weber 2000). Most studies of p53 involve DNA damaging drugs and radiation. Much less is known about the physiological stress, hypoxia. Under hypoxic conditions, p53 is stabilized by mitochondria through a
redox-dependent mechanism (Chandel et al. 2000) and by HIF-1
(An et
al. 1998), whereas p53 induces degradation of HIF-1 (Ravi et al.
2000). The response to hypoxia in vivo also involves the glucocorticoid
receptor (GR) (Bauer et al. 1999).
GR is a member of the steroid receptor superfamily that mediates
physiological processes controlled by glucocorticoids. In the unbound
state GR is located in the cytoplasm bound to chaperones. Upon ligand
binding the chaperones dissociate, exposing the NLS that enables GR to
enter the nucleus and regulate transcription (Cheung and Smith 2000).
GR activates transcription by binding to glucocorticoid response
elements (GREs), and represses by binding to negative and composite
elements (Kellendonk et al. 1999). GR mediates stress responses, and
animals that overexpress GR are more resistant to stress and endotoxic
shock (Reichardt et al. 2000).
Recently, evidence has been growing for cross talk between the p53 and
GR-mediated responses to stress. p53 physically interacts with and
represses the activities of GR (Sengupta et al. 2000b). Modulation of
glucocorticoid-induced apoptosis has been linked to p53 gene dosage in
mice (Mori et al. 1999). Glucocorticoids prevent p53-induced apoptosis
in immortalized human granulosa cells (Sasson et al. 2001). p53 and GR
regulate in opposite manners the type II hexokinase gene, whose
overexpression is associated with increased glucose catabolism in
cancer cells (Mathupala et al. 1997). Hypoxia leads to p53 activation
and decreased glucose metabolism (Riva et al. 1998). Because little is
known about the functional interactions between the signalling pathways
mediated by p53 and GR under physiological conditions, we have
investigated how p53 and GR regulate each other under hypoxia. We show
that they mutually inhibit each other's activity by cytoplasmic
sequestration in a ligand-dependent manner, which leads to increased
degradation through recruitment of the E3 ubiquitin ligase Hdm2 and the
proteasome pathway.
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Results |
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Top
Abstract
Introduction
Results
Discussion
Material and methods
References
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Dexamethasone enhances the interaction of GR with the core domain and NLS of p53
We have reported previously that p53 and GR interact in vivo
(Sengupta et al. 2000b). We used in vitro GST pull-down assays to
determine which region of p53 interacts with GR (Fig. 1A,
left). The GST fusion proteins were
produced in Escherichia coli, purified on Glutathione
Sepharose beads, and equivalent amounts of protein (judged by Coomassie
blue staining, Fig. 1B), were used. Full-length p53 interacted with GR
weakly but specifically in the absence of ligand (Fig. 1A,C, lanes 3 and 6). This interaction was enhanced by the agonists Dexamethasone
(Dex) or Cortisol (Cort), and inhibited by the antagonist RU486 (Fig.
1C, lanes 2-6). These results show that the binding of GR to p53 is
enhanced by agonists in vitro. In the presence of ligand, GR interacted
with both the core domain [GST-p53(76-304)] and the NLS
[GST-p53(305-320)] of p53, and more strongly with a mutant
containing both regions [GST-p53(76-320)]. No interaction was
detected between GR and either the N-terminal [GST-p53(1-75)] or
C-terminal [GST-p53(321-362) and GST-p53(363-393)] domains. The
mutant containing both interaction domains [GST-p53(76-320)] interacted more strongly than the full-length protein, raising the
possibility that it lacks sequences that inhibit the interaction with
GR. The interactions between GR and deletion mutants containing the NLS
[GST-p53(76-320) and GST-p53(305-320)] were inhibited by a
peptide containing the NLS (amino acids 305-320, Pep, Fig. 1C, lanes
7-11), confirming that the GR-p53 NLS interaction is specific.
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GR interacts with the central region of p53, suggesting that it should
inhibit the Chimeric Tumor Supressor, CTS1, that harbors this domain of
p53 linked to heterologous activation and dimerization domains
(Conseiller et al. 1998) (Fig. 1D, top left). The transcriptional activity of CTS1 was measured in p53 null HSC-2 cells with the p53CON
reporter (Fig. 1D, top right). Vehicle alone (ethanol), Dex, and RU486
did not affect the basal activity of the reporter (Fig. 1D, lanes
1-3). Increasing amounts of CTS1 stimulated reporter activity in a
dose-dependent manner (Fig. 1D, lanes 4-6). CTS1 was inhibited around
threefold by endogenous GR activated with Dex, but was not affected by
RU486 (Fig. 1D, lanes 7-12). With a constant level of CTS1, exogenous
GR inhibited CTS1 activity to a small extent (Fig. 1D, lanes 13-16),
and the addition of Dex increased this inhibition (Fig. 1D, lanes
17-19). In contrast, GR, in the presence of RU486, had little effect
(Fig. 1D, lanes 20-22). Similar results were obtained in Saos-2 cells
(data not shown). These results indicate that the region of p53 that
interacts with GR in vitro is sufficient for efficient repression by GR in vivo.
Dex decreases p53 and GR levels and inhibits expression of their target genes in HepG2 cells
Cytoplasmic p53 in neuroblastoma cells is released from its complex
with GR and translocates to the nucleus following treatment with GR
antagonists (Sengupta et al. 2000b). We investigated whether cytoplasmic p53 in other tumor cell types, such as HepG2 (hepatoma) and SA1 (osteosarcoma) (Wasylyk et al. 1999; Lu et al. 2000), behaved
similarly. Under normal growth conditions, Dex increased the amount
of p53 and GR in the cytoplasm of both cell lines, although some
p53 always remained nuclear. In contrast, RU486 significantly
increased the amount of nuclear p53 (data not shown). Interestingly, in
HepG2 cells, Dex caused a drastic decrease in the protein levels of
both p53 and GR (Fig. 2A, lanes 1,2). This effect was blocked by the proteasome inhibitor LLnL (Fig. 2A, lanes
3,4), suggesting that the decrease is due to enhanced degradation. We
investigated whether Dex can inhibit the expression of genes downstream
from both p53 (Bax, Hdm2, and p21WAF1/CIP1) and GR (phospho
enol pyruvate carboxy kinase, PEPCK; Imai et al. 1990). Dex decreased
Bax and p21WAF1/CIP1 protein levels (Fig. 2B) and promoter
activity (reporter assays; data not shown). Dex did not affect either
the level of Hdm2 protein (Fig. 2B), or the activity of the promoter
(reporter assay, data not shown), showing that Hdm2 expression is less
sensitive than Bax or p21WAF1/CIP1 to changes in p53 levels.
Dex decreased PEPCK protein levels (Fig. 2B). These results show that
Dex down-regulates both p53 and GR protein levels and the expression of
their downstream targets in HepG2 cells.
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Dex activation of endogenous GR inhibits p53 induction by hypoxia in normal endothelial cells (HUVEC)
HepG2 is a tumor cell line with undefined genetic alterations. Hence, we investigated in normal human cells (HUVEC) the response to hypoxia, a natural inducer of p53. Normally growing HUVEC express very little or no p53 protein (Fig. 3A, lane 1). p53 was strongly induced after 12 h of hypoxia, and increased further after 18 h (Fig. 3A, lanes 3,5). Dex inhibited the induction of p53 up to 36 h (Fig. 3A, lanes 2-5; data not shown). The proteasome inhibitor, LLnL, decreased Dex-dependent inhibition of p53 induction, without substantially affecting the level of p53 in the absence of ligand (Fig. 3A, lanes 6-9). p53 RNA levels detected by Northern blotting were not affected by Dex (Fig. 3B), showing that the inhibitory effect of Dex on p53 is not due to changes in transcription or RNA stability. In conclusion, Dex decreases p53 stability by enhanced proteasomal degradation.
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The products of the p53 downstream genes Bax and p21WAF1/CIP1
were induced by hypoxia (Fig. 3C, lanes 1,3,5,7).
p21WAF1/CIP1 has also been shown to be induced by hypoxia in
normal fibroblasts but not in several transformed cell lines (Ashcroft
et al. 2000; Koumenis et al. 2001). Bax and p21WAF1/CIP1
induction was inhibited by Dex (Fig. 3C, lanes 2,4,6). Under normoxia,
in which p53 levels are very low, Dex had no effect, indicating that
Dex inhibition of p53 downstream genes is p53 dependent (Fig. 3C, lanes
8-11). HIF-1 stabilizes p53 in response to hypoxia (An et al.
1998), which raised the possibility that it, too, is affected by Dex.
However, HIF-1 induction by hypoxia in HUVEC was not significantly
affected by Dex (Fig. 3D). The apparently weak induction of HIF-1 is
consistent with the low level of HIF-1 RNA in endothelial cells
(Tian et al. 1997). Dex did, however, inhibit Hdm2 induction until 12 h
under hypoxia (Fig. 3E, lanes 2,3). In the presence of LLnL the
difference in the level of induction was no longer observed, indicating
that the difference is due to Dex-induced proteasomal degradation (Fig. 3E, lanes 6,7). After 18 and up to 36 h the levels of Hdm2 were similar, irrespective of the presence of Dex and LLnL (Fig. 3E, lanes
4,5,8,9; data not shown). Hypoxia induces apoptosis of HUVEC in a
p53-dependent manner (Stempien-Otero et al. 1999). We, therefore, investigated whether Dex affected apoptosis by following PARP cleavage
and DNA fragmentation. PARP cleavage, an early marker of apoptosis, was
detected at 18 h and increased up to 36 h (Fig. 3F, lanes 1-5). Dex
inhibited PARP cleavage up to 36 h under hypoxia (Fig. 3F, lanes 6-8).
Similar results were obtained using DNA fragmentation (data not shown).
These results show that the activation of endogenous GR with Dex in
HUVEC, as well as HepG2, dramatically decreases p53 downstream functions.
Dex-dependent GR degradation by a proteasome pathway and inhibition of its target genes under hypoxia
Hypoxia has been shown to affect GR functions and levels of
expression in several systems (Jenq et al. 1996; Bauer et al. 1999).
Therefore, we investigated whether hypoxia affects GR in HUVEC. Hypoxia
alone caused a reproducible increase in GR protein levels (Fig. 4A,
lanes 1,3,7), which was down-regulated by
Dex (Fig. 4A, lanes 2,3,6,7). This down-regulation was blocked by the
proteasome inhibitor LLnL (Fig. 4A, lanes 4,5,8,9). As Dex had no
effect on GR at the RNA level under hypoxia (data not shown), we
conclude that GR is decreased at the protein level by a
proteasome-mediated process in response to Dex under hypoxic
conditions, similar to p53 (see above).
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We next investigated whether hypoxia affects Dex induction of the GR
downstream genes PEPCK (Imai et al. 1990) and Glucose-6-Phosphatase (G-6-Pase) (Argaud et al. 1996). Whereas Dex induced RNA expression from both of these genes under normoxia (Fig. 4B, lanes 1,2), hypoxia
blocked this induction (Fig. 4B, lanes 3,4). Similarly, hypoxia blocked
PEPCK protein induction (data not shown). These results concur with
studies in rat hepatocytes, where in vivo hypoxic exposure inhibits
gluconeogenesis at the PEPCK level due to decreased PEPCK gene
transcription (Pison et al. 1995).
p53, GR, and Hdm2 colocalize in the cytoplasm in the presence of ligand
Our experiments show that p53 and GR are degraded by proteasomes in HepG2 under normal growth conditions and in HUVEC under hypoxia. Because subcellular localization affects p53 degradation, we studied the localization of p53 and GR in HUVEC cells (Fig. 5). As expected, under normal conditions p53 was not detectable (Fig. 5A), because of its very low level of expression (see above). GR was located in the cytoplasm in the absence of ligand, but accumulated in the nucleus in the presence of Dex. Under hypoxic conditions, in the absence of Dex, p53 was detectable and was localized to both the nucleus and the cytoplasm in the majority of cells (65%), and exclusively in the nucleus in about 25%, whereas GR remained in the cytoplasm (Fig. 5B). In the presence of Dex, p53 was completely cytoplasmic in around 60% of the cells, and was colocalized extensively with GR. The immunofluorecence of both GR and p53 was, however, considerably lower, as expected from their degradation (see above). To confirm these data, LLnL was used to stabilize the proteins and increase the signal. Typical cells are shown in Figure 5C-E, and the quantification in Figure 5F. The presence of LLnL did not change the results overall, but the effects were more pronounced. p53 was detected in the nucleus in almost 90% of the cells in the absence of Dex. However, in the presence of ligand, p53 and GR colocalized extensively in the cytoplasm (Fig. 5C).
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Hdm2 is involved in p53 degradation and export from the nucleus, raising the possibility that it may also be implicated in GR turnover and localization. As expected, Hdm2 was found to be predominantly nuclear under hypoxia and in the absence of ligand. Interestingly, Hdm2 was cytoplasmic and extensively colocalized with GR in the presence of ligand (Fig. 5D). Similarly, Hdm2 was found to colocalize with p53 in the nucleus in the absence of ligand and in the cytoplasm in the presence of Dex (Fig. 5E).
The ligand-dependent trimeric complex between p53, GR, and Hdm2 enhances proteasomal degradation
The colocalization of p53, GR, and Hdm2 in the cytoplasm led us to investigate whether they form a trimeric complex. Coimmunoprecipitation assays were performed on extracts from HUVEC that had been exposed to hypoxia in the presence of LLnL and in the presence or absence of Dex. Equivalent amounts of p53 were immunoprecipitated with the anti-p53 antibody, irrespective of the presence of ligand, as expected from the inhibition of p53 degradation by LLnL (Fig. 6A, c). The p53-antibody immunoprecipitate contained both GR and Hdm2 in significantly higher amounts in the presence of Dex than in its absence (Fig. 6A, a,b, lanes 3,4). In the reciprocal experiment, the GR-antibody immunoprecipitates contained equivalent amounts of GR irrespective of Dex treatment (Fig. 6A, f). They also contained both p53 and Hdm2 in significantly higher amounts in the presence of Dex (Fig. 6A, d,e, lanes 7,8). Similar results were obtained in HepG2 cells, where p53, GR and Hdm2 formed a trimeric complex more efficiently following Dex treatment of the cells (Fig. 6B).
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GR has been postulated to be degraded by the ubiquitin-proteasome
pathway (Whitesell and Cook 1996; Segnitz and Gehring 1997), but there
was no direct evidence. Our results do indicate, however, that during
cross talk between the GR and p53 pathways, GR is degraded
concurrently with p53 by a pathway involving the E3 ubiquitin ligase
activity of MDM2. To test this hypothesis HUVEC cells were transfected
with HA-tagged ubiquitin (HA-Ub) in the presence or absence of IP3, a
small peptide that specifically disrupts the p53-Hdm2 interaction
(Wasylyk et al. 1999). Twenty-four hours post-transfection, the cells
were placed under hypoxic conditions in the presence of the proteasome
inhibitor, LLnL. Western blotting of HA-immunoprecipitates with GR
antibodies showed that GR was poly-ubiquitinated, but only when the
cells were incubated in the presence of Dex (Fig. 6C, a, lanes 1,2).
IP3 expression inhibited GR poly-ubiquitylation, showing that it
requires the p53-Hdm2 interaction (Fig. 6C, a, lanes 1,3). The
poly-ubiquitinated forms are specific, because they were not detected
when HA-Ub was not transfected (Fig. 6C, a, lanes 5,6). Interestingly,
polyubiquitylation of p53 is also enhanced in the presence of Dex (Fig.
6C, b, lanes 1,2). Polyubiquitylation of p53 was inhibited by the
expression of IP3, as expected from the disruption of the p53-Hdm2
interaction (Fig. 6C, b, lanes 1,3). These results show that Dex
stimulates GR and p53 polyubiquitylation through a pathway involving
Hdm2 and its interaction with p53.
The E3 ligase activity of Hdm2 is responsible for ligand-dependent degradation of GR
To study GR degradation in transfection assays, in which exogenous
proteins are expressed in cells, we used HCT116 isogenic cell lines in
which p53 is either wild type or disrupted by homologous recombination
(Bunz et al. 1998). The isogenic cell lines were transfected with
recombinants that express HA-Ub and IP3. Posttransfection, the cells
were subjected to hypoxia in the presence of Dex and LLnL. HA-antibody
immunoprecipitates contained lower mobility HA-tagged polyubiquitylated
forms of GR (Fig. 6D, a, lane 1). IP3 expression inhibited
ubiquitylation of GR (Fig. 6D, a, lane 2). Ubiquitylated GR was not
detected in cells lacking p53, whether or not IP3 was expressed (Fig.
6D, a, lanes 3,4). These results indicate that GR ubiquitylation
requires p53 and its interaction with Hdm2. In cells that were not
treated with LLnL, IP3 expression increased the levels of both GR and
p53 (Fig. 6D, a, lanes 5,6), as expected from decreased degradation of
both proteins by a common mechanism involving Hdm2. Irrespective of the
presence or absence of IP3, GR levels were higher in the isogenic
p53
/ cells (Fig. 6D, a, lanes 7,8), as expected from the
lack of p53 that is required to recruit Hdm2.
To confirm that p53-Hdm2 interactions are required for GR degradation,
we used p53(22,23), which cannot interact with Hdm2 (Lin et al. 1994).
Expression of wild-type p53, Hdm2, and exogenous GR in HCT116
p53/ cells in the presence of Dex resulted in a
significant decrease in GR levels (Fig. 6E, lanes 1-4). In
contrast, the GR levels remained unchanged when the p53(22,23)
mutant was expressed (Fig. 6E, lane 5). Interestingly, similar to GR,
the levels of p53 and Hdm2 were also higher with p53(22,23) compared to
p53 wild type (Fig. 6E, lanes 5,6). These results suggest that
Dex-dependent ternary complex formation induces degradation of all
three proteins.
To determine whether the E3 ligase activity of Hdm2 is responsible for
GR ubiquitylation and degradation, HCT116 p53/ cells were
transfected with GR, p53, and the Mdm2 mutants 
R and C462A, which
abolish the E3 ligase activity of Mdm2 (Argentini et al. 2000). Mdm2
promoted the degradation of both p53 and GR, whereas the mutants
protected both proteins from degradation (Fig. 6F). These results show
that Mdm2 E3 ligase activity is required for GR degradation.
Dex-dependent ternary complexes involving p53, GR, and Hdm2 form during mitomycin C treatment
To determine whether DNA damage could stimulate Dex-dependent
degradation of GR, we treated HUVEC cells with mitomycin C (Fritsche et
al. 1993). Similar to hypoxia, mitomycin C stabilized p53 and GR in
HUVEC cells (Fig. 7A, lanes 1,3). Dex
reduced induction of both GR and p53 (Fig. 7A, lanes 2,3), and
inhibited a downstream function of p53, apoptosis (data not shown).
Inhibition of proteasome-mediated degradation with LLnL stabilized both
proteins (Fig. 7A, lanes 4,5). These results show that both p53 and GR
undergo enhanced degradation in the presence of Dex during mitomycin C
treatment.
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We determined whether the cellular localization of p53 and GR are the same in mitomycin C as in hypoxia-treated cells. HUVEC cells were treated with mitomycin C in the presence of LLnL, and the cells examined by indirect immunofluorecence. Typical cells are shown in Figure 7B-D and the quantitation in Figure 7E. In the absence of Dex, mitomycin C treatment resulted in nuclear accumulation of p53, whereas GR remained cytoplasmic. However, in the presence of Dex, p53 as well as GR were cytoplasmic and colocalized extensively (Fig. 7B). Similar Dex-dependent colocalization was observed when cells were examined for GR and Hdm2 (Fig. 7C) or for p53 and Hdm2 (Fig. 7D). These results show that p53, GR, and Hdm2 are colocalized in the presence of Dex during mitomycin C treatment, and suggest that they could form a triple complex. Indeed, GR antibody immunoprecipitates from mitomycin C-treated HUVEC cells grown in the presence of LLnL were found to contain both Hdm2 and p53, to a much larger extent in the presence than in the absence of Dex (Fig. 7F, cf. lanes 3 and 4).
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Material and methods
References
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Glucocorticoids and their receptors play important roles in both
apoptotic cell death and cellular proliferation. An agonist of the
glucocorticoid receptor, Dex, is known to induce apoptosis in T cells,
B lymphocytes, and multiple myoloma cells (Martin and Green 1995).
However, it also represses apoptosis and increases proliferation in
cancer cell lines (Chang et al. 1997), neutrophils (Daffern et al.
1999), retinal pigment cells (He et al. 1994), fibroblasts (Li et al.
1998), rat hepatoma (Yamamoto et al. 1998), and serum-depleted T
lymphocytes that express Bcl2 (Huang and Cidlowski 1999). The divergent
functions of glucocorticoids and GR in different contexts can be
attributed to interactions with different pro- and antiproliferation
factors. For example, GR interacts with and affects the functions of
the proapoptotic protein DAP3 (Hulkko et al. 2000). Here we show that
GR and the tumor supressor p53 regulate, by novel mechanisms, each
other's functions in normal cells in response to a physiological stress.
We have demonstrated that Dex induces mutual down-regulation of p53 and
GR-dependent downstream target genes, both under hypoxia in HUVEC and
under normoxia in HepG2 cells. HUVEC are normal human cells with
typical GR-dependent functions. Under normoxic conditions, in which p53
levels are very low, HUVEC display a ligand-dependent translocation of
the receptor to the nucleus and transactivation of GR downstream genes
such as PEPCK and G-6-Pase. However, under hypoxia, which induces a
large increase in p53 levels, GR functions are seriously impaired. p53
induction may not be required in HepG2 because its levels are
constitutively high. Down-regulation of GR, which occurs within 3 h in
HepG2, takes about 12-18 h in HUVEC, probably due to the time required
to increase p53 levels. The constitutively high level of p53 in HepG2
could account for the reported insensitivity of HepG2 to Dex induction
of GR activity (Xing and Quinn 1993).
We have shown, in vitro and in vivo, that GR and p53 interact weakly in the absence of ligand and more strongly in the presence of Dex. Interestingly, GR interacts with the NLS of p53, and apparently only in the presence of ligand. This interaction could result in ligand-dependent masking of the NLS, sequestration in the cytoplasm, and in consequence, inhibition of transactivation. However, we cannot exclude that the complex accelerates nuclear export of both proteins.
We have found that GR and p53 inhibit each other's activities by
cytoplasmic sequestration in response to hypoxia and DNA damage in a
ligand-dependent manner. Hypoxia and DNA damage can be expected to have
similar effects. Hypoxia is a potent inducer of Nitric Oxide Synthase
in rats and endothelial cells of bovine origin (Arnet et al. 1996; Gess
et al. 1997). Nitric oxide oxy-radicals damage DNA (Xu et al. 2000),
and up-regulate p53 (Forrester et al. 1996). We have previously shown
that neuroblastoma cells represent a pathological situation in which
p53 is inhibited by cytoplasmic sequestration (Sengupta et al. 2000b).
There is growing evidence that p53 sequestration in the cytoplasm may
be a common mechanism of p53 inactivation. For example, both Mot-2
(Wadhwa et al. 1999) and hepatitis B virus X protein (Elmore et al.
1997) have been shown to physically interact with p53 and repress its
transcriptional activity by sequestration in the cytoplasm. Estradiol
inactivates p53 by intracellular redistribution (Molinari et al. 2000).
The results from experiments with in vitro translated proteins and overexpression in cells suggest that p53 down-regulates ER-responsive genes by interfering with the binding of ER to ERE (Liu et al. 1999).
Moreover, ER protects p53 from Hdm2-mediated degradation through the
formation of a triple complex (Liu et al. 2000). Cytoplasmic sequestration and functional repression of p53 in mammary epithelium can be reversed with a hormone cocktail (Kuperwasser et al. 2000). However, in none of these studies was the subcellular localization of
the complex and the relevance of the interactions to physiological conditions addressed.
We have established that Dex stimulates the formation of a triple
complex that contains Hdm2 in addition to p53 and GR. This triple
complex apparently forms by GR and Hdm2 binding to different domains of
p53. Hdm2 binds to the N-terminal transactivation domain, whereas GR
binds to the DNA binding domain and the NLS. Interestingly, Hdm2
recruitment is associated with increased ubiquitylation and proteasome-mediated degradation of p53 and GR. In contrast, ER apparently inhibits Hdm2-mediated degradation of p53 (Liu et al. 2000).
Dex induces cytoplasmic accumulation of Hdm2, triple complex formation,
and degradation, suggesting that degradation is mediated by cytoplasmic
proteasomes. Hdm2-mediated degradation of p53 is generally thought to
occur in the cytoplasm. However, a recent study suggests that it may
also occur in the nucleus (Yu et al. 2000). In neuroblastoma cells both
p53-Hdm2 (Zaika et al. 1999) and p53-GR (Sengupta et al. 2000b)
complexes are found in the cytoplasm, suggesting that the cytoplasmic
degradation function of Hdm2 is defective in these cells. Thus, lack of
degradation as well as cytoplasmic sequestration distinguish
neuroblastoma from normal cells.
We have discovered that GR is a new substrate for Hdm2, thereby
extending the range of targets for this E3 ubiquitin ligase. Mdm2
mediates degradation of the cell fate regulator Numb (Juven-Gershon et
al. 1998) and E2F (Loughran and La Thangue 2000) in a p53-independent manner. In contrast, it targets GR (this study) and HIF-1 (Ravi et
al. 2000) in a p53-dependent manner. It is conceivable, therefore, that
p53-mediated recruitment of Mdm2 is an important mechanism for the
degradation of proteins that interact with p53 under different physiological conditions.
It is interesting to consider why p53 and GR cross talk under physiological conditions. Both p53 and GR mediate stress responses, but with intrinsic differences in their actions. GR, when complexed to glucocorticoids, is involved in fight-or-flight responses, and maintains homeostasis during internal or environmental changes. Hence, GR is generally involved in a survival response. On the other hand, p53, which has been described as the "guardian of the genome," responds to extensive genomic and other stresses by inducing cell death. p53 can be considered to be a death response. Hence, p53 and GR can be considered to be protagonists in the question of life or death, and should, therefore, have antagonistic effects on each other's functions. Hypoxia and the subsequent DNA damage represent such a situation where p53 and GR have opposite effects. The death response via p53 is held in check by ligand-activated GR, which induces p53 degradation. GR itself is also restrained by increased degradation induced by p53. Mutual negative cross talk between these factors thereby functions as a balance between the cellular responses to conflicting signals.
In summary, we have demonstrated for the first time that p53 and GR functionally cross talk under physiological conditions. We have also identified p53 and GR downstream genes that are down-regulated during this process. The repression of each other's functions is enhanced by ligand. The core domain and NLS of p53 bind to GR in a ligand-enhanced manner, resulting in the cytoplasmic relocalization of both proteins. The p53-GR complex in the cytoplasm also contains the E3 ubiquitin ligase Hdm2, which mediates degradation of both p53 and GR. Although GR degradation by the ubiquitin proteasome machinery has been postulated, our studies provide the first direct evidence for it in a physiological situation.
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Material and methods |
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Abstract
Introduction
Results
Discussion
Material and methods
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Recombinants
GST constructs GST-p53(305-321) was generated by ligating oligonucleotides into the BamHI and EcoRi sites of pGEX-2T (Pharmacia Biotech). The other p53 mutants were generated by PCR and cloning into the same sites of pGEX-2T.
Reporters, expression vectors, and cDNA probes
p53CON (Funk
et al. 1992); CTS1 (Conseiller et al. 1998); pcDNA3GR (Sengupta et al.
2000b); CMV-LacZ, IGBMC core facility; pBC-IP3.2 (Wasylyk et al.
1999); HA-Ub (Treier et al. 1994); pBS-
-actin (IGBMC core
facility); pBS-glucose-6-phosphate (Lange et al. 1994); pBS-PEPCK
(Bartels et al. 1989); pRCp53(WT) and pRCp53(22,23) (Lin et al. 1994);
pxMDM2, pxR, pxC462A (Argentini et al. 2000); pEGGFP-C1 (Clonetech).
Cells
HSC-2 (Momose et al. 1989); HUVEC, ATCC (CRL-1730); HepG2, ECACC
(Ref. 85011430); HCT116 p53(WT) and HCT116 p53(
/) (Bunz et al.
1998).
Protein expression, purification, and GST interactions
GST proteins were induced with 1 mM IPTG in E. coli DH5
grown at 25°C, and extracted by sonication in STE buffer (10 mM
Tris-HCl at pH 8.0; 150 mM NaCl; 1 mM EDTA supplemented with 5 mM DTT, 1mM PMSF, 2.5 µg/mL proteinase inhibitors and 1.5% Sarkosyl). The
sonicate was adjusted to 2% Triton X-100, agitated for 30 min at
4°C, centrifuged, and the crude extract stored in aliquots at
70°C. Glutathione-S-Sepharose beads were equilibrated in GST buffer
(50 mM Tris-HCl at pH 7.5, 100 mM KCl, 10 mM MgCl2, 5% glycerol, 0.5% NP-40 supplemented with 0.3 mM DTT, 1 mM PMSF and 2.5 µg/mL proteinase inhibitors), incubated with the crude extracts at
4°C for 90 min, and washed. Bound proteins were analyzed by SDS-PAGE
and Coomassie staining. GR was produced by in vitro translation with
pcDNA3GR and the TNT Quick coupled transcription/translation system
(Promega) for 90 min at 30°C. Equivalent amounts of bound protein
were incubated with 5 µL of in vitro translated GR for 1 h at 4°C
in the presence of vehicle (ethanol), Dex, Cortisol, or RU486 (Sigma).
The bound proteins were washed and analyzed by SDS-PAGE and fluorography.
Cell culture and transfection
Cells were maintained in DMEM plus 10% serum and antibiotics at
37°C with 5% CO2. HUVEC medium was supplemented with ECGF (50 µg/mL), Heparin (50 µg/mL), and Glutamine (1 mM). The HCT116 cell lines were grown in McKoy's 5A medium plus 10% serum,
antibiotics, and 2 mM glutamine. HSC-2 were transfected by calcium
phosphate precipitation (Chen and Okayama 1987) with 4 µg of plasmid
in 36-mm plates (six-well cluster, Costar 3516). After 16 h the cells were washed with medium and incubated in full medium with ligands or
vehicle (ethanol). To express HA-Ub and IP3, low-passage HUVEC (<15)
was transfected with Superfect (QIAGEN) according to the manufacturer's instructions. After 3 h with Superfect the cells were
incubated in normal medium for 24 h. HCT116 cells were transfected with
Lipfectamine 2000 (Life Technologies) in the presence of serum for 6 h,
washed, and incubated in normal medium for 12 h before hypoxic
treatment. Post-transfection is defined as after the removal of the
facilitator. Hypoxia experiments were carried out in anaerobic chambers
(Anaerocult, Merck, Cat. no. 1.13829) at 37°C according to the
manufacturer's instructions; 10 µM mitomycin C (Sigma) was used.
Transcriptional activity
Cell lysates were analyzed for luciferase and -galactosidase
activities as described previously (Sengupta et al. 2000a).
Western blots
Equal quantities of protein, estimated by the Bio-Rad assay on RIPA
buffer lysates, were fractionated by SDS-PAGE, transferred to
nitrocellulose membranes (Schleicher and Schuell), and Western blotted.
Primary and secondary antibodies: Bax, N20 (Santa Cruz Biotechnology);
p21WAF1/CIP1, 1WA-IC581 (Sengupta et al. 2000a); TBP, 3G3
(Brou et al. 1993); p53, DO-1 (Vojtesek et al. 1992); p53, PAb 421 (Wade-Evans and Jenkins 1985); p53, #588, raised against a GST-human
p53(320-393) (C. Wasylyk and the IGBMC core facility); GR, E-20 and
P-20 (Santa Cruz Biotechnology); PARP (Kaufmann et al. 1993); HA, 12CA5
(Boerhinger); PEPCK (Zimmer and Magnuson 1990); Hdm2, 2A10 (Chen et al.
1993); HIF-1, C-19 (Santa Cruz Biotechnology); peroxidase-coupled
anti-rabbit or anti-mouse antibodies (Jackson Laboratories); p53, 1801 (Ab-2) (Oncogene Research Products); GFP (Clontech). The blots were
revealed by chemiluminescence (Pierce Super Signal).
Northern blots
Forty micrograms of total RNA, extracted with Trizol (GIBCO BRL), was electrophoresed on 6% formaldehyde-1% agarose gels, transferred to Hybond N+ membranes, hybridized with random-primed probes at 55°C (for PEPCK and G-6-Pase) or 65°C (for p53, GR, and actin), washed, and autoradiographed.
Immunofluorescence
Cells were fixed with acetone:methanol (1:1), blocked with 10%
goat serum, incubated with primary antibodies for 2 h, secondary antibodies linked to either Cy3 or FITC (Moll et al. 1996) and Hoechst
(to stain nuclei), then visualized by confocal microscopy (Leica TCS 4D).
Immunoprecipitations
Cells were lysed on ice in NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl at pH 8.0) and the cleared supernatants used for immunoprecipitation. The antigen-antibody complex was allowed to form on ice for 1 h, then immobilized on protein G beads, washed three times, boiled in Laemmli buffer, and fractionated by SDS-PAGE.
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Acknowledgments |
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We thank Julia Young for critical reading of the manuscript; D. Bohmann, E. Conseiller, M.R. el-Maghrabi, T. Kietzmann, and J. Shay for the gifts of recombinants; N. Tsuchida for HSC-2 cells; D. Granner, G. de Murcia, and L. Tora for the gift of antibodies; the IGBMC core facilities for help and support; and BioAvenir (Aventis, Rhone-Poulenc), the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Ligue Nationale Française contre le Cancer (Equipe labellisée), the Ligue Régionale (Haut-Rhin) contre le Cancer, and the Ligue Régionale (Bas-Rhin) contre le Cancer for financial assistance.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received March 5, 2001; revised version accepted July 26, 2001.
1 Present address: Laboratory of Human Carcinogenesis, Building 37, Room 2C23, 37 Convent Drive, National Cancer Institute, NIH Bethesda, MD 20892, USA.
2 Corresponding author.
E-MAIL boh{at}igbmc.u-strasbg.fr; FAX 33-3-88-65-32-01.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.202201.
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Abstract
Introduction
Results
Discussion
Material and methods
References
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.
Nature
392:
405-408[CrossRef][Medline].
