Repression of Glucocorticoid Receptor Transactivation and DNA Binding of a Glucocorticoid Response Element within the Serum/Glucocorticoid-Inducible Protein Kinase (sgk) Gene Promoter by the p53 Tumor Suppressor Protein
Anita C. Maiyar,
Phan T. Phu,
Arthur J. Huang1 and
Gary L. Firestone
Department of Molecular and Cell Biology and The Cancer
Research Laboratory University of California at Berkeley
Berkeley, California 94720
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ABSTRACT
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sgk is a novel member of the
serine/threonine protein kinase family that is transcriptionally
regulated by serum and glucocorticoids in Rat2 fibroblasts and in
mammary epithelial cells. 5'-Deletion analysis of the sgk
promoter, using a series of sgk-CAT (chloramphenicol
acetyltransferase) chimeric reporter gene plasmids, defined a
glucocorticoid-responsive region that contains a glucocorticoid
response element (sgkGRE) between -1000 and -975 bp. The
sgkGRE is specifically bound by glucocorticoid receptors
and is sufficient to confer glucocorticoid responsiveness to a
heterologous promoter in several cell lines. Strikingly, cotransfection
of either the murine or human wild type p53, but not a mutant p53,
repressed the dexamethasone-stimulated transactivation of reporter
plasmids containing either the sgkGRE or a consensus GRE.
Gel shift analysis revealed that in vitro synthesized p53
prevented binding of the glucocorticoid receptor both to the
sgkGRE as well as to a consensus GRE. The p53-mediated
repression of dexamethasone-induced sgkGRE activity
required both the DNA binding and transactivation functions of the p53
protein. Activation of endogenous p53, by exposure to UV light,
repressed the glucocorticoid receptor transactivation of a consensus
GRE-CAT reporter plasmid in transfected cells. Conversely, activated
glucocorticoid receptors suppressed the transactivation function of
p53, while transrepression by p53 was largely unaffected. The presented
data demonstrate that sgk is a primary
glucocorticoid-responsive protein kinase gene that implicates a new
pathway of cross-talk between steroid receptor signaling and cellular
phosphorylation cascades. In addition, our study provides the first
evidence of mutual interference of transactivation functions of p53 and
the glucocorticoid receptor, possibly through their direct interaction.
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INTRODUCTION
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Glucocorticoid hormones govern a wide spectrum of cellular
functions and physiological processes in animal tissues by interacting
with their cognate intracellular receptors (1, 2, 3, 4, 5). These steroid
receptor complexes act as ligand-activated transcriptional stimulators
or repressors of primary response genes either by their specific
binding to glucocorticoid-responsive elements (GRE), within the
promoter of steroid-sensitive genes (3, 6, 7), and/or by direct
protein-protein interactions with other transcription factors (6, 8, 9, 10, 11). It has been proposed that pleiotropic responses of
steroids occur through cellular cascades in which changes in the
expression and activity of a network of early-response regulatory
molecules control a subsequent series of events. From this viewpoint,
transcriptional modulations in the activities of primary
steroid-responsive gene products provide mechanisms for potential
cross-talk with signal transduction pathways that functionally
complement or compete with steroid-dependent processes. For example,
phosphorylation-dephosphorylation networks are commonly used by a
variety of extracellular regulators, such as growth and differentiation
factors, to rapidly and reversibly transduce signals from the
extracellular environment to the cytoplasm and nucleus (12). There are
many potential mechanisms by which the regulation of protein kinase and
phosphatase signaling can be coordinated with steroid-responsive
pathways at the cellular level. One direct, but not well characterized,
mechanism of regulation is steroid-dependent changes in transcription
of components of phosphorylation-dephosphorylation pathways. Consistent
with this concept, we previously reported the cloning of a novel
serine/threonine protein kinase gene, sgk, that is
transcriptionally stimulated by glucocorticoid hormones and by serum in
both Rat2 fibroblasts and in rat mammary tumor epithelial cells (13, 14), which implicates the existence of a direct interplay between
glucocorticoid receptor (GR)-mediated and phosphorylation-dependent
cell signaling.
The sgk gene encodes a 49-kDa putative protein kinase, which
exhibits strong homology (4555% amino acid identity) with the
catalytic domains of the protein kinase C family, cAMP-dependent
protein kinase, rac protein kinase, and ribosomal protein S6 kinase
(13). Nuclear run-on and Northern blot analysis demonstrated that the
induction of sgk transcripts by glucocorticoids and serum in
both fibroblasts and epithelial cells are rapid transcriptional
responses that do not require de novo protein synthesis (13, 14). These studies strongly indicate that sgk may be a
primary glucocorticoid-responsive gene with its transcription regulated
by a cis-acting GRE located within the promoter of
sgk. Interestingly, in Rat2 fibroblasts, serum induces a
rapid and transient increase in sgk transcription,
reminiscent of the well characterized immediate early class of genes
expressed during the transit of mitogen-stimulated cells through the G1
phase of cell cycle (14, 15). Apart from the serum and glucocorticoid
up-regulation of sgk transcription, expression of
sgk is strongly modulated by a combination of androgen,
testosterone, and FSH in rat ovarian granulosa cells (16). Moreover,
using differential display, sgk was isolated from a rat
brain tissue after central nervous system injury, implicating a role
for sgk in axonal regeneration upon central nervous system
injury and development of specific groups of neurons in postnatal brain
(17). Thus, diverse sets of cellular signals generated by steroid and
peptide hormones, as well as serum-derived growth factors, appear to
transcriptionally regulate the expression of sgk in a
tissue-specific manner.
sgk is the second described member of a newly emerging
subfamily of serine/threonine protein kinases that are predominantly
regulated at the transcriptional level (18, 19, 20, 21), with sgk,
snk, and fnk the only known mitogen-inducible
immediate early response protein kinase genes (13, 19, 21). However,
relatively little is known about the transcriptional control of this
potentially important subfamily of protein kinases. To elucidate the
molecular basis of the transcriptional regulation of sgk by
trans-acting regulatory factors, 4.0 kb of the
sgk promoter region upstream of the transcriptional start
site was cloned from a rat genomic library. Consistent with the nuclear
run-on results, showing that dexamethasone strongly stimulates
sgk gene transcription, sequence analysis revealed the
existence of a putative GRE approximately 1.0 kb upstream of
sgk promoter (13). In addition, the sgk promoter
contains multiple Sp-1 sites, TATA sequence at -35 bp relative to the
transcriptional start site as well as several putative binding sites
for transcription factors implicated in cell proliferation, development
and differentiation such as the AP-1 transcriptional complex, GATA,
hunchback, and Kruppel proteins, CCAAT enhancer
binding protein (C/EBP), and Ets-2 factors (our unpublished data). A
feature unique to this kinase promoter is the presence of putative p53
DNA recognition sequences dispersed throughout the 5'-flanking region
of sgk promoter. We have recently shown that four of the p53
binding sites are specifically recognized by the p53 protein and that
at least one of these sites within the sgk promoter (between
-1380 and -1345 bp) can confer p53 transactivation to a heterologous
promoter in mammary epithelial cells (22). Our observations are
consistent with the p53 protein being a transcriptional regulator
implicated in a wide range of cellular processes including cell
proliferation, DNA replication, and apoptosis (23, 24, 25, 26, 27). The structure
of this phospho-nucleoprotein contains features typical of
transcription factors including DNA binding and transactivation domains
(28, 29, 30), and several studies have documented the ability of p53 to
either activate or repress a variety of cellular target genes (23, 24, 25, 26).
Moreover, in addition to its sequence-specific DNA-binding properties,
p53 also selectively interacts with several cellular proteins that
include transcription factors, such as TATA-binding factor (TBF),
CCAAT-binding factor (CBF), specificity protein 1 (Sp1), and the
thyroid hormone receptor, as well as viral proteins via specific
domains (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41).
The availability of particular sets of transcription factors has been
shown to dramatically modulate the transcriptional effects of steroid
receptors on specific target genes in a tissue-specific manner (6, 9, 10, 11). The regulation of sgk gene expression by
glucocorticoids and several other extracellular signals and the
existence of many putative regulatory elements in the sgk
promoter implicate important combinatorial actions of
trans-acting transcription factors in mediating
sgk gene expression. Given the existence of functional p53
DNA-binding sites and a near-consensus GRE in the sgk
promoter and the known involvement of protein kinases and p53 in
complex cellular functions, an important issue was to examine whether
GRs and p53 coordinately regulate sgk promoter activity. In
this study, we show that the sgk promoter is a direct target
of GRs through a functional GRE sequence, and that in Rat2 fibroblasts,
cotransfection of p53 inhibits the glucocorticoid-stimulated
transactivation of the sgk promoter. The molecular basis for
this inhibitory effect is a reciprocal repression of transactivation
activities of p53 and the GR. Our results provide evidence for
functional interactions between the GR and the p53 protein that
implicates a potential coupling between these two classes of
transcriptional regulators.
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RESULTS
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The sgk Promoter Contains a Functional GRE that Confers
Dexamethasone Responsiveness to a Heterologous Promoter
In both Rat2 fibroblasts and Con8 mammary epithelial cells,
dexamethasone stimulated transcription of the sgk
serine/threonine protein kinase gene without an apparent time lag and
in the absence of de novo protein synthesis, suggesting that
sgk is a primary target gene of GRs (13, 14). To
functionally determine which region within the sgk promoter
is responsible for its glucocorticoid-mediated transcriptional
activation, -4.0 kb of sgk 5'-flanking region was cloned
from a rat genomic library (13), and a series of 5'-progressive
deletions starting at -4000 bp and terminating at +51 were generated
by controlled exonuclease III digestions. Rat2 cells were transiently
transfected with sgk-CAT reporter constructs, which were
generated by linking the various sgk promoter fragments to
the CAT reporter gene, transfected cells treated with or without 1
µM dexamethasone for 24 h and cell lysates assayed
for CAT activity. As shown in Fig. 1
, dexamethasone
stimulated transcriptional activity of the sgk-CAT
constructs containing the four largest sgk promoter regions
(deletions ending at -4000, -2321, -1428, and -1148 bp). Maximum
stimulation (4- to 5-fold) of sgk-CAT activity by
dexamethasone was observed with both the -1428sgk-CAT and
-1148sgk-CAT constructs. Glucocorticoid inducibility of the
sgk promoter was completely lost in the deletions at and
beyond -901 bp, implicating sequences between -1148 and -901 bp of
the sgk promoter as the key glucocorticoid-responsive
region. The variation in basal level of CAT activity between the
deletion constructs was likely due to the selective elimination of
functional transcription factor-binding sites.

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Figure 1. Identification of a Dexamethasone-Responsive Region
within the sgk Promoter
A series of sgk-CAT reporter plasmids containing the
indicated 5'-deletions of sgk promoter fused to the
bacterial CAT reporter gene were transfected by electroporation into
Rat2 fibroblasts. Cells received 10 µg of reporter plasmid and 20
µg of the promoterless CAT vector, pBLCAT3, and then were incubated
for 24 h with (+) or without (-) 1 µM
dexamethasone. CAT activity was assayed by quantification of the
conversion of [3H]acetylCoA into
[3H]acetylchloramphenicol by the two phase fluor
diffusion assay described in Materials and Methods and
normalized to protein levels to determine the CAT-specific activity.
Experiments were performed in triplicate, and the reported values
represent the means and SD values derived from at least
three separate transfections.
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Sequence analysis of 4.0 kb of the sgk promoter revealed the
presence of one putative GRE between -1000 and -975 within the -1148
to -901 glucocorticoid-responsive region functionally defined by the
deletion studies. The sgkGRE bears strong homology with the
consensus GRE sequence (Fig. 2
, upper panel)
and contains the base pairs critical for direct contact with the GR (6, 42). To functionally test whether the sgkGRE can confer
dexamethasone responsiveness to a heterologous promoter, a 25-bp
oligonucleotide representing the regions between -1000 to -975 bp of
the sgk promoter was linked immediately upstream of the
herpes simplex virus thymidine kinase minimal promoter sequences (-105
to +51) driving the bacterial CAT gene (sgkGREtk-CAT). Rat2
cells were transiently transfected with either sgkGREtk-CAT,
tk-CAT containing only the -105 to +51 minimal promoter sequences,
GRE-CAT containing six copies of a functional GRE encoded by the mouse
mammary tumor virus (MMTV) gene, or the constitutively expressed Rous
sarcoma virus (RSV)-CAT reporter gene. CAT reporter gene activity was
monitored in cell extracts isolated from dexamethasone-treated and
untreated cells. As shown in Fig. 2
(lower panel),
dexamethasone induced CAT expression driven by the
sgkGREtk-CAT chimeric reporter plasmid by 6-fold compared
with Rat2 fibroblasts not treated with dexamethasone. Reporter gene
activity in cells transfected with the minimal promoter thymidine
kinase (tk)-CAT alone was low and unaffected by dexamethasone
treatment. As expected, the positive control GRE-CAT reporter plasmid
was strongly induced (30-fold) by dexamethasone, whereas the
constitutive RSV-CAT reporter construct displayed high basal activity
that was unresponsive to dexamethasone. These results establish that
the putative DNA element located between -1000 and -975 bp within the
sgk promoter is a functional GRE capable of rendering
hormone responsiveness to a heterologous promoter. The hormone
sensitivity of the sgkGREtk-CAT reporter construct was also
observed in several other glucocorticoid responsive epithelial cell
lines (data not shown).

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Figure 2. The sgk GRE Confers Dexamethasone
Inducibility to a Heterologous Gene Promoter
The upper panel shows the sequence of
sgkGRE present between -1000 and -975 within
sgk promoter in comparison to the consensus GRE. An
oligonucleotide corresponding to the sgkGRE was fused
upstream of the tk-CAT reporter gene driven by the tk minimal promoter
to form sgkGREtk-CAT. Lower panel, Rat2
fibroblasts were transfected either with 10 µg each of
sgkGREtk-CAT, tk-CAT containing only the tk minimal
promoter, GRE-CAT containing six copies of the MMTV GRE, or a
constitutively active RSV-CAT reporter plasmid. Transfected cells were
treated with 1 µM dexamethasone for 24 h and assayed
for CAT activity as described in Materials and Methods.
CAT specific activity was determined as the amount of
[3H]acetylchloramphenicol formed per µg of protein. The
data represent the means and SD obtained from three
separate transfections each carried out in triplicate.
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Gel Shift Analysis of GR Binding to the sgkGRE
Gel shift analysis of 32P-labeled oligonucleotides
corresponding to the sgkGRE and the consensus GRE
demonstrated that GR can specifically recognize the sgkGRE.
The radiolabeled oligonucleotides were incubated with cytoplasmic
extracts prepared from Rat2 fibroblasts, which were transfected with a
GR expression plasmid, and electrophoretically fractionated by native
polyacrylamide gel electrophoresis. As shown in Fig. 3
, a single protein/DNA complex was detected with the
sgkGRE-labeled probe (No competitor). Addition of excess
unlabeled oligomer corresponding to the sgkGRE abolished the
complex formation, while the same amount of unlabeled consensus GRE
(Con GRE) completely impaired formation of specific protein-DNA
complexes. This competition was specific for GRE oligonucleotides
because the addition of excess unlabeled DNA fragments containing an
unrelated sequence was unable to compete for protein binding
(Non-specific DNA). Importantly, the addition of BuGR-2 antibodies
specific for the GR caused a supershift of the protein-DNA complex
(BuGR-2) demonstrating the presence of GRs in this protein/DNA complex.
A parallel set of gel shift reactions carried out with the
[32P]oligonucleotide corresponding to the Con GRE
revealed formation of specific protein/DNA complexes that was competed
off with excess unlabeled Con GRE as well as with excess unlabeled
sgkGRE but not with an unrelated sequence (Non-specific
DNA). Binding reactions containing anti-GR antibodies caused supershift
of this specifically retarded band (BuGR-2), establishing the
specificity of binding. Our results show that the GR is able to
specifically bind to the sgkGRE with nearly the same
efficiency as the Con GRE.

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Figure 3. Gel Shift Analysis of GR Binding to the
sgk GRE and a Consensus GRE
Cytoplasmic extracts (10 µg), prepared from Rat2 fibroblasts
transfected with a GR expression plasmid, were incubated either with
32P-end-labeled double-stranded oligonucleotide DNA probes
containing the GRE sequences between -1000 to -975 of the
sgk promoter shown in Fig. 2 (sgk GRE
probe) or the consensus GRE sequence shown in Fig. 2 (Consensus GRE
probe). One set of reactions for each radiolabeled probe did not
contain any cytoplasmic extracts (No extract lanes). The reaction
mixtures contained no unlabeled competitor DNA (No competitor) or
100-fold molar excess of double-stranded oligonucleotides corresponding
to GRE in the sgk promoter (sgk GRE), the
consensus GRE (Con GRE), or an unrelated oligonucleotide (Non-specific
DNA). One binding reaction was preincubated for 1 h with anti-GR
antibodies (BuGR-2). The protein-DNA complexes formed were resolved by
4% native polyacrylamide gel electrophoresis and visualized by
autoradiography. Arrows indicate the position of
protein-DNA complexes and free probe.
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Wild Type p53 Protein Represses the Dexamethasone-Induced
sgk Promoter Activity in Rat2 Fibroblasts
The presence of specific transcription factors and the promoter
context of a given GRE have been shown to profoundly influence the
direction and magnitude of glucocorticoid regulation of target genes
(6, 9, 11). The magnitude and kinetics of glucocorticoid-regulated
sgk gene expression differ among several cell types (13),
suggesting that nuclear factors along with GRs may have combinatorial
or antagonistic effects on sgk transcription. Consistent
with this possibility, four DNA recognition sites for the p53 protein,
located at -1380/-1345, -1155/-1125, -285/-255, and -235/-205,
are all contained within the glucocorticoid-responsive
-1428sgk promoter fragment, and one of these sites, at
-1380/-1345, can independently mediate p53 responsiveness in mammary
epithelial cells (22). To functionally test whether p53 modulates
glucocorticoid responsiveness of the sgk promoter, Rat2
fibroblasts were transfected with the -1428sgk-CAT reporter
plasmid alone or along with expression plasmids encoding either the
murine wild type p53 or a mutant p53 that is defective in DNA binding.
Transfected cells were treated with or without 1 µM
dexamethasone for 24 h,and cell extracts were assayed for CAT
activity. As shown in Fig. 4
(upper panel),
cotransfection of an expression plasmid encoding wild type p53
abolished the 4-fold dexamethasone-induced promoter activity of the
-1428sgk-CAT reporter plasmid. Transfection of wild type
p53 had only a minor inhibitory effect on basal promoter activity. In
contrast to the effects of wild type p53, dexamethasone-inducible
activity of -1428sgk-CAT was only partially attenuated by
cotransfection of the murine mutant p53 gene (Fig. 4
, upper
panel). The wild type p53-dependent inhibition of
dexamethasone-induced -1428sgk-CAT activity occurred in the
presence of a 3-fold molar excess of mutant p53 (data not shown).

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Figure 4. Repression of Dexamethasone-Induced
sgk Promoter and AGP Promoter Activities by Wild Type
p53
Rat2 fibroblasts were transfected with 10 µg of the
-1428sgk-CAT reporter plasmid (upper
panel) or AGP3xGRE-CAT (lower panel) either
alone or along with 10 µg of expression plasmids encoding either wild
type p53 (wt p53), or mutant p53 (mt p53). Transfected cells were
treated with (+) or without (-) 1 µM dexamethasone for
24 h, and CAT activity was determined and normalized to protein
levels as described in Fig. 1 . The results represent the mean and
SD of three independent transfections performed in
triplicate.
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To evaluate whether the repressive effects of p53 on GR transactivation
are unique to the sgk promoter or are representative of a
more general biological regulation of glucocorticoid responsiveness,
the effects of wild type and mutant p53 were examined on the
transcriptional activity of the glucocorticoid-responsive
-1-acid
glycoprotein (AGP) gene promoter (43). Cells were cotransfected with
the AGP-CAT reporter plasmid, which contains a small fragment of the
AGP promoter and includes three GREs, but no p53-binding sites, along
with the expression vectors for either the wild type or mutant p53
gene. As also shown in Fig. 4
(lower panel), the 5-fold
induction of the AGP promoter by dexamethasone was strongly inhibited
in cells transfected with wild type p53, while the basal activity of
the AGP-CAT reporter was unaffected. The dexamethasone-dependent
activation of the AGP-CAT construct was mildly inhibited in the
presence of mutant p53. Taken together, these results demonstrate that
p53 is capable of inhibiting the GR transactivation of two distinct
glucocorticoid-responsive genes, implicating potential functional
interactions between p53 and GR activities.
The direction and magnitude of transcriptional activity observed with
certain gene promoters can differ depending on the amount of
transfected p53 (44). To test the dose-dependent effects of wild type
p53 on glucocorticoid-responsive sgk promoter activity, Rat2
fibroblasts were cotransfected with -1428sgk-CAT along with
various amounts of expression plasmids for either the wild type or
mutant p53 protein, and CAT activity was monitored in cells treated
with or without dexamethasone for 24 h. As shown in Fig. 5
, cotransfection of increasing amounts of wild type
p53-encoding plasmid evoked only a dose-dependent inhibition of the
dexamethasone-induced -1428sgk-CAT activity without any
activation effects in Rat2 fibroblasts. In contrast, coexpression of
mutant p53 had little effect on the dexamethasone-induced
sgk promoter activity except at the highest tested amounts
of transfected plasmid.

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Figure 5. Dose-Dependent Inhibition of Dexamethasone-Induced
-1428sgk-CAT Reporter Plasmid Activity by Wild Type p53
Rat2 fibroblasts were transfected with 10 µg of
-1428sgk-CAT reporter plasmid and the indicated amounts
of either wild type or mutant p53 expression plasmids. Cells were
cultured in the presence or absence of 1 µM dexamethasone
for 24 h, and CAT specific activity was determined as described in
Fig. 1 . Reporter gene activity is expressed as fold induction by
dexamethasone. The data are expressed as the means and SD
obtained from three separate transfections performed in triplicate.
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Wild Type p53 Represses Dexamethasone-Stimulated Activity of the
sgkGRE in the Absence of a p53 DNA-Binding Site
One explanation for the p53-mediated inhibition of
dexamethasone-induced -1428sgk-CAT activity is that p53 may
directly or indirectly interfere with receptor-stimulated
transcriptional events converging on the GRE located at -1000 bp
within the sgk promoter. To directly test whether p53 can
disrupt the GR activation of the sgkGRE, in the absence of a
p53-binding site, Rat2 fibroblasts were transfected with CAT reporter
plasmids driven either by the minimal tk promoter containing the
sgkGRE (sgkGREtk-CAT) or without this GRE
sequence (tk-CAT). Cells were transfected with the reporter plasmids
alone or along with expression plasmids for either the murine wild type
p53 or mutant p53. Extracts isolated from dexamethasone-treated or
untreated cells were assayed for CAT activity. As shown in Fig. 6
, the 6-fold dexamethasone-stimulated activation of
sgkGREtk-CAT was strongly repressed by cotransfection of
wild type p53, whereas, this reporter plasmid was
glucocorticoid-inducible in the presence of the mutant p53. Both the
wild type and mutant p53 caused a minor reduction in the basal activity
of sgkGREtk-CAT, which was also observed in cells
transfected with the glucocorticoid nonresponsive tk-CAT reporter
plasmid. These results show that the murine wild type p53 can
functionally interfere with the ability of the GR to transactivate a
simple GRE present within the sgk promoter.

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Figure 6. p53-Mediated Inhibition of Dexamethasone-Inducible
sgkGRE Activity
Rat2 fibroblasts were transfected with 10 µg of the
sgkGREtk-CAT or tk-CAT reporter plasmids, as described
in Fig. 2 , either alone or cotransfected with 10 µg of either the
murine wild type p53 (wt p53) or the murine mutant p53 (mt p53).
Transfected cells were treated with (+) or without (-) 1
µM dexamethasone for 24 h, and CAT activity was
determined and normalized to protein levels as described in Fig. 1 . The
results represent the mean and SD of three independent
transfections performed in triplicate.
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Inhibition of Dexamethasone-Induced sgkGRE Activity
Requires the DNA Binding and N-Terminal Domains of the p53 Protein
Cytomegalovirus (CMV)-driven expression plasmids for the human
wild type p53 and two of its mutant counterparts were used to determine
whether either the DNA binding and/or the N-terminal transactivation
domains of the p53 protein play a role in the repression of
dexamethasone-induced sgkGRE activity. Rat2 fibroblasts were
transfected with sgkGREtk-CAT or with the minimal promoter
tk-CAT lacking the sgkGRE sequence either alone or in
combination with expression vectors encoding either the human wild type
p53 (CMV-p53wt), a human mutant p53
(CMV-p53179) that is incapable of binding DNA (45), or a
human mutant p53 (CMV-p53
43) that lacks the N-terminal
43 amino acids, rendering it incapable of its transactivation function
(45). Transfected cells were treated with or without dexamethasone for
24 h and asssayed for CAT activity. Cotransfection of the human
wild type p53 expression plasmid strongly repressed the
dexamethasone-induced activity of sgkGREtk-CAT (Fig. 7
) without any detectable effects on the basal reporter
gene activity. In contrast, the dexamethasone-mediated induction of the
sgkGREtk-CAT construct was completely retained in cells
cotransfected with either the CMV-p53179 DNA- binding
mutant or the transactivation deletion mutant, CMV-p53
43
(Fig. 7
). Cotransfection of the wild type or both mutant p53 expression
plasmids had a negligible effect on the tk-CAT reporter gene activity
both in the presence or absence of dexamethasone (Fig. 7
, right
panel). These data demonstrate that the wild type p53-mediated
repression of dexamethasone-induced sgkGRE activity requires
both the DNA binding and transactivation functions of the p53
protein.

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Figure 7. Requirement of p53 DNA Binding and Transactivation
Domains for the Inhibition of sgkGRE Activity
Rat2 fibroblasts were transiently transfected with 10 µg of the
sgkGREtk-CAT reporter plasmid or tk-CAT minimal promoter
construct either alone or cotransfected with 10 µg of expression
plasmids for the human wild type p53 (p53wt), a mutant human p53 with a
point mutation in the DNA-binding region (p53179), or a
truncated p53 protein that lacks the transactivation region
(p53 43). Cells were treated with (+) or without (-) 1
µM dexamethasone for 24 h and assayed for CAT
activity as described in Fig. 1 . The CAT activity is expressed relative
to protein content and the values shown are the means and
SD from three different transfections done in
triplicate.
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Repression of Dexamethasone-Induced Consensus GRE Activity by Wild
Type p53
Although the sgkGRE sequence is highly homologous to
the consensus GRE (Fig. 2
, upper panel), the actual sequence
differs by 2 bp. Therefore, we examined whether the transcriptional
inhibition by p53 is unique to the GRE within the sgk
promoter or is a more general phenomenon that modulates transcriptional
activity of other functional GREs. Several glucocorticoid- responsive
cell lines were cotransfected with the consensus GRE-CAT reporter
plasmid alone or in combination with p53 expression plasmids encoding
either the murine wild type or mutant p53 protein. CAT reporter
activity was monitored in cells treated with or without dexamethasone
for 24 h. As shown in Table 1
, coexpression of wild
type p53, but not mutant p53, strongly repressed the 80-fold
stimulation of GRE-CAT activity by dexamethasone in nontumorigenic
NMuMG mammary epithelial cells and the 30- to 35-fold stimulated CAT
activities observed in transformed Con8Hd6 mammary epithelial cells,
BDS1 hepatoma cells, and Rat2 fibroblasts, respectively. Neither the
wild type nor the mutant p53 had any appreciable effect on basal
activity of the GRE-CAT reporter plasmid in the different cell lines
tested. As expected for a receptor-dependent response, the p53-mediated
inhibition of GR transactivation was notably absent in hepatoma cells
that lack the GR, and upon rescue of these cells with cotransfection of
GR expression plasmid, transfected p53 was able to inhibit the
GR-induced transactivation of a GRE-CAT reporter plasmid (data not
shown). Thus, the functional consequences of coexpression of wild type
p53 in different cell types is to blunt glucocorticoid responsiveness
by preventing GR transactivation of its corresponding GRE.
Wild Type p53 Inhibits DNA Binding of the GR either to the
sgkGRE or to a Consensus GRE
One explanation for the p53-mediated repression of GR
transactivation of a simple GRE is an inhibition of GR DNA-binding
activity. To test this possibility, the effect of p53 on GR DNA binding
in gel shift assays was examined using in vitro synthesized
p53 protein from rabbit reticulocyte lysates in the presence of
unlabeled amino acids. Cytoplasmic extracts (10 µg) prepared from
GR-transfected cells were incubated with either the
32P-labeled sgkGRE or the consensus GRE
oligomers in the absence or presence of in vitro translated
p53, and the protein-DNA complexes were resolved by native
polyacrylamide gel electrophoresis. Preincubation of extracts with
either 5 µl or 15 µl of in vitro synthesized p53 (wt
p53, 5 µl and 15 µl lanes) but not with unprogrammed lysate (Un.
lysate) resulted in a profound inhibition of binding of GR either to
the sgkGRE (Fig. 8
, upper panel)
or to the consensus GRE (middle panel)-labeled probes as
compared with samples with extract alone (Extract). Moreover, the
specific GR-retarded protein-DNA complexes formed with the
sgkGRE and the consensus GRE probes were retained in
p53-containing reaction mixtures preincubated with anti-p53 antibodies
(wt p53+PAb421) or in which the in vitro translated p53 was
heat inactivated for 15 min at 90 C before incubation with the extracts
(wt p53, heat treated). The specificity of GR binding to both DNA
probes was confirmed by preincubation of extracts with anti-GR
antibodies (BuGR-2), which caused the disappearence of the protein-DNA
complex in samples containing GR antibodies (BuGR-2) but not in
extracts preincubated with preimmune sera (Preimmune). The observed
protein-DNA complexes were shown to be specific by competition with
100-fold excess of unlabeled self-oligonucleotides but not with an
unrelated DNA sequence (data not shown). As expected, no binding was
observed when in vitro synthesized p53 alone (no extract) or
unprogrammed lysate alone (no extract) was present in the binding
reaction without any added extracts.

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Figure 8. Wild Type p53 Inhibits Binding of the GR to the
sgkGRE and the Consensus GRE in Gel Shift Assays
Cytoplasmic extracts (10 µg) prepared from NMuMG mammary cells
cotransfected with a GR expression plasmid were preincubated for 1
h on ice either with rabbit reticulocyte lysates containing in
vitro synthesized unlabeled wild type p53 (wt p53) or
unprogrammed lysate followed by the addition of radiolabeled
sgkGRE (upper panel) or consensus GRE
(middle panel) probes and incubated for 30 min on ice.
The protein-DNA complexes were resolved on low ionic strength native
4% polyacrylamide gels. Extracts were incubated either with anti-GR
antibodies (BuGR-2), preimmune sera (Preimmune), 15 µl unprogrammed
lysate (Un. lysate), 5 µl and 15 µl of wt p53 (wt p53), with 5 µl
wt p53 and 20 µl of anti-p53 antibody PAb421 (wt p53+PAb421), or with
wt p53 inactivated by heating at 90 C for 10 min (wt p53, heat treated)
before addition of the radiolabeled probes. As negative controls,
unprogrammed lysate alone or wt p53 alone without added extracts were
incubated with the sgkGRE or consensus GRE-labeled
probes. Each binding reaction was adjusted to equal amounts of
reticulocyte lysate by adding appropriate amounts of the unprogrammed
lysate. All of the shifted protein-DNA complexes were shown to be
specific by competition with unlabeled DNA (data not shown). In the
lower panel, extracts were incubated with increasing
amounts of reticulocyte lysate containing wt p53 (0.5 µl, 1.0 µl,
2.5 µl, 5.0 µl, 10.0 µl, and 15 µl) or 15 µl of unprogrammed
lysate (Un. lysate), followed by addition of labeled consensus GRE
probe. Arrows indicate position of specific protein-DNA
complexes and free probe.
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Preincubation of extracts with increasing amounts of in
vitro synthesized p53 (0.5 µl to 15 µl) prevented GR binding
to the consensus GRE probe in a dose-dependent fashion (Fig. 8
, lower panel) whereas addition of unprogrammed lysate (15
µl) did not affect GR binding to the DNA probe (Un. lysate). Similar
dose-dependent inhibition of GR DNA binding by p53 was observed with
the sgkGRE probe (data not shown). Take together, our data
show that p53 inhibits the specific binding of GR to the
sgk-derived GRE or to a consensus GRE, probably by
physically interacting with GR and thereby rendering it incapable of
DNA binding.
Inhibition of GR-Dependent Transactivation by UV Activation of
Endogenous p53
The repression of GR transactivation by ectopic expression of wild
type p53 suggests that one of the functions of endogenous p53 is to
inhibit or modulate GR activity. It is well established that the
transient induction of p53 protein is one of the key cellular responses
to DNA-damaging agents such as UV radiation (46, 47, 48). Therefore, to
test whether the p53 repression of GR transactivation is a
physiologically relevant response, the ability of activated endogenous
p53 to alter GR transactivation was examined during a time course of UV
treatment of mammary epithelial cells. The optimal time of induction of
endogenous p53 in response to UV treatment was first ascertained in the
untransformed mammary cells (NMuMG). At various time points after
exposure to UV at a dose of 40 J/m2 the level of nuclear
p53 was examined by Western analysis. The accumulation of p53 protein
was first observed at 3 h after UV treatment, peaking at
approximately 68 h, and then declining beyond 14 h after UV
exposure (Fig. 9
, upper panel). To directly
test whether endogenously activated p53 can repress GR-dependent
transactivation, NMuMG mammary cells were transfected with the
consensus GRE-CAT reporter plasmid and 24 h later one set of cells
was exposed to UV treatment and a second set maintained as a nonexposed
control. Cells were treated with dexamethasone either during the
transient peak in p53 (from 3 h to 10 h post-UV treatment) or
after the level of p53 protein subsides to near basal levels (22 to
29 h post-UV treatment). Analysis of CAT activities revealed that
the GR-mediated transactivation of the consensus GRE-CAT reporter
plasmid was strongly inhibited when dexamethasone was added at a time
coincident with the peak induction of p53 protein after UV treatment
(Fig. 9
, lower panel). In contrast, the fold activation of
the GRE-CAT reporter plasmid was unaffected when dexamethasone was
added after the p53 protein level had returned to near basal levels
(Fig. 9
, lower panel). Thus, endogenously activated p53 is
capable of inhibiting GR-mediated transactivation of a consensus
GRE-CAT reporter plasmid in a manner similar to that observed with
ectopically expressed p53, suggesting that the inhibition of GR
activity by p53 is biologically important.

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Figure 9. Time Course of Induction of p53 by UV Radiation and
Concomitant Suppression of Dexamethasone-Dependent Transactivation of
the GRE-CAT Reporter Plasmid in NMuMG Mammary Cells
NMuMG mammary epithelial cells were exposed to 40 J/m2 UV
radiation and harvested at the indicated times (upper
panel). Nuclear protein extracts (30 µg) were isolated,
electrophoretically fractionated, blotted onto nitrocellulose membrane,
and analyzed for p53 protein by Western blotting as described in
Materials and Methods. The protein molecular mass
standards shown on the left correspond to albumin (69
kDa), ovalbumin (46 kDa), and carbonic anhydrase (28 kDa),
respectively. The arrow indicates the position of the
p53-specific band. NMuMG cells were transfected with the GRE-CAT
reporter plasmid (lower panel) and 24 h later
exposed to UV (40 J/m2) (+) or remained untreated (-).
Cells were then either treated with or without dexamethasone from
3 h to 10 h post UV (during the peak in p53 protein levels)
or 22 to 29 h post UV (after p53 protein returns to basal levels)
and harvested for monitoring CAT activity. Results are expressed as
fold activation by dexamethasone, which is ratio of
dexamethasone-induced CAT activity to the uninduced CAT activity. Data
presented represent mean ± SD of at least three
independent transfections done in triplicate.
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GRs Antagonize p53 Transactivation without Affecting the p53
Transrepression Function
To determine whether activated GR, in a reciprocal manner, affects
either the transactivation or transrepression functions of p53, NMuMG
mammary cells were transfected either with a p53-responsive reporter
plasmid (sgk p53tk-CAT) that is induced by wild type p53
(22) or with CMV-CAT reporter that is known to be transcriptionally
repressed by p53 (49) alone or along with expression plasmids encoding
wild type 53, mutant p53, or GR. The p53 expression plasmids were
transfected individually or in combination with the GR-encoding
expression vector. Reporter gene activity was monitored in cell lysates
after treatment with or without dexamethasone for 24 h. As shown
in Fig. 10
(left panel), the p53 activation
of the sgk p53tk-CAT reporter was significantly reduced by
activated GR in cells expressing endogenous GR as well as in cells
exogenously transfected with GR expression vector (p53wt
and p53wt+GR). Negligible activation of this p53-responsive
reporter plasmid was observed in cells transfected with the mutant p53
gene alone or along with GR vector (p53mt and
p53mt+GR). Similarly, endogenously expressed or exogenously
transfected GR had no effect on the p53-inducible reporter plasmid in
the absence of wild type p53 (None and GR). The reporter plasmid
lacking the p53- responsive element (tk-CAT) displayed low basal
activity under all conditions tested and was not p53 or GR responsive
(data not shown). In contrast to the GR inhibition of p53
transactivation, the p53-dependent repression of CMV-CAT activity was
completely retained and unaffected by dexamethasone activation of
either the endogenous or exogenous GR (Fig. 10
, right
panel). Mutant p53 exhibited a mild repression of CMV-CAT activity
regardless of dexamethasone treatment or the presence of the
transfected GR expression vector, whereas dexamethasone treatment in
the absence of either wild type or mutant p53 had little effect on the
CMV-CAT activity. These results demonstrate that activated GR is
capable of attenuating the p53-dependent transactivation function but
not its transrepression activity and that both GR and p53 display a
mutual inhibition of their respective transactivation functions.

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Figure 10. Activated GR Inhibits p53-Dependent
Transactivation Function but not the Transrepression Activity
NMuMG mammary epithelial cells were transiently transfected with
sgkp53tk-CAT reporter (left panel), or
with CMV-CAT reporter construct (right panel), either
alone or cotransfected with expression vectors encoding GR, wild type
p53 (p53wt), mutant p53 (p53mt) individually or
as combinations (p53wt+GR), (p53mt+GR). Cells
were either treated (+) or untreated (-) with dexamethasone for
24 h and harvested for assaying CAT activity. The mean levels of
CAT activity with SD determined in at least three separate
transfections are presented normalized to protein levels.
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DISCUSSION
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Our previous studies have demonstrated that the sgk
serine/threonine protein kinase gene displays unique transcriptional
stimulation by glucocorticoids (13) and is the second of four known
protein kinases to be under transcriptional control by serum or
mitogens (18, 19, 21). By functional dissection of
cis-acting regulatory elements in the sgk
promoter, we now demonstrate that GRs stimulate sgk
transcription through a GRE located at -1.0 kb of the sgk
promoter. The existence of this GRE provides a direct molecular basis
for the transcriptional activation of sgk by glucocorticoid
hormones (13, 14) and establishes a direct link between GR-dependent
signaling and control of cellular phosphorylation/dephosphorylation
cascades. We have also identified the p53 transcription factor as an
important modulator of glucocorticoid-dependent transcriptional control
of the sgk gene promoter. Transient transfection studies
revealed a mutual antagonism of transactivation functions between p53
and GR. In addition, p53 specifically abrogates the ability of GR to
bind to either the sgkGRE or to a consensus GRE. Taken
together, our findings demonstrate functional interactions between the
p53 protein and GRs and implicate an interplay of disparate
transcription factor families in the control of cellular processes.
A functional GRE within the sgk promoter was identified
within a 25-bp segment spanning nucleotides -1000 bp to -975 bp
which, when linked to a reporter gene, confers glucocorticoid
inducibility of sgk transcription, both in the context of
the natural promoter and when placed upstream of a heterologous
promoter in a variety of GR-positive cell lines. The sgkGRE
sequence 5'-AGGACAgaaTGTTCT-3', although not a perfect palindrome,
bears significant homology with the consensus GRE sequence
5'-GGTACAnnnTGTTCT-3', which is well established by previous studies to
mediate glucocorticoid enhancement of gene transcription (6).
Importantly, gel shift analysis demonstrated that the GR can
specifically recognize the sgkGRE sequence in
vitro. The receptor/DNA complexes can be supershifted by
GR-specific antibodies and efficiently competed by unlabeled consensus
GRE for binding the sgkGRE probe, establishing that the GR
transactivation involves direct interaction with the sgkGRE.
The precise functions and physiological substrates of this novel
glucocorticoid-regulated kinase are not known. However, preliminary
studies suggest the involvement of sgk in the control of
mammary cell growth. Consistent with this concept, dexamethasone has
recently been shown to inhibit the activity of the mitogen-activated
pp70 S6 kinase, by mechanisms requiring ongoing transcription (50).
In addition to the regulation of the sgk promoter by
glucocorticoids, our studies demonstrate a role for the p53 protein in
the GR-dependent transcriptional activation of sgk promoter.
The ectopic expression of wild type p53 strongly disrupted the
GR-mediated transactivation of the sgk promoter, which
contains several p53-binding sites. Strikingly, the suppression of
glucocorticoid-responsive sgk promoter activity by wild type
p53 was observed regardless of whether the p53 DNA binding sites were
present or not in the reporter plasmid. In fact, this suppression was
discernible using reporter plasmids containing only the
sgkGRE, or a consensus GRE, transfected into
glucocorticoid-responsive epithelial cell lines as well as Rat2
fibroblasts, which suggests a generality of the response and a minimal
requirement for the presence of a GRE. The p53-dependent repression of
another GRE-containing natural promoter, such as the AGP gene promoter,
but lacks any p53-binding sites (43), further substantiates the view
that the repression of GR transactivation by p53 does not entail
binding to specific p53 recognition elements, but instead likely
involves regulation at the protein-protein level. Our results also
exclude the possibility of p53 evoking repression via affecting general
transcription machinery, since basal promoter activity of the synthetic
GRE-CAT construct was unaltered by coexpression of p53. Cotransfection
of mutated human p53 genes revealed that the DNA-binding and
transactivation domains of p53 are important for the inhibition of GRE
activity. Receptor reconstitution experiments using a GR-negative cell
line, demonstrated that the GR is required for the p53-dependent
repression of transactivation (data not shown).
The repression of GR transactivation by p53 is not due to the unusually
high levels of p53 protein that occurs after its ectopic expression
since the same functional antagonism of GR function was observed in
cells after the activation of endogenous p53 protein by exposure to UV
stress. The transient induction of stable p53 protein is a well
characterized protective cellular response to genotoxic agents
including UV radiation (46, 47, 48). Inhibition of dexamethasone-induced
GRE-CAT activity was observed at the time frame in which p53 protein is
induced by UV, whereas, no effects on GR transactivation were observed
when dexamethasone was added either preceding or subsequent to the
expression of UV-induced p53. The ability of endogenously activated p53
to repress glucocorticoid-stimulated transactivation of the consensus
GRE in transfected cells suggests that this transcriptional repression
may be a widely used cellular pathway to modulate glucocorticoid
responsiveness in a physiologically appropriate context.
Although the detailed molecular mechanism of p53-mediated repression of
GR activity remains unknown, our data indicate that p53 impairs GR DNA
binding capacity either to the sgkGRE or to a consensus GRE
oligomer. One possible explanation for such inhibition of GR DNA
binding by p53 may be competition for response element binding, which
has been suggested as a likely mechanism for GR ß-mediated repression
of GR activity (51). However, in our studies, data from both
transfection experiments and DNA binding assays demonstrate that the
inhibition of GR function by p53 was not due to a direct competition of
p53 with GR for binding to the GRE because p53 neither bound to nor
transactivated a GRE. In addition, the fact that the position of the
GR- specific gel shifted band was unaltered in the presence of p53
suggest that any physical interaction of p53 with GR likely precludes
GR from binding to DNA but does not alter the GR-DNA interaction once
the receptor is bound to DNA. Although p53 could indirectly influence
functionality of the receptor-DNA complex by altering the expression or
activity of another protein, the strong inhibition of GR DNA binding
in vitro by p53 argues against an indirect effect of p53. In
this regard, p53 has been recently shown to physically interact with
another member of the zinc finger family of hormone receptors, the
human thyroid receptor ß-1, causing a repression of thyroid
receptor-dependent transactivation (39). In another study,
transcription of the orphan receptor TR2, which is also a member of the
steroid receptor superfamily of transcription factors, was strongly
suppressed by p53 (37), implying functional interactions between p53
and members of steroid receptor superfamily. It is interesting to note
that in previous studies, functional interactions between p53 and other
zinc finger proteins such as Sp1, WT1, and mdm2 have been reported,
resulting in either transcriptional synergy or inhibition (35, 38, 41).
Compatible with these findings, our results demonstrate another example
of p53 modulating the transcriptional activity of the GR, which is a
well characterized zinc finger transcription factor.
The strong inhibition of p53-dependent transactivation by
overexpression of GR or endogenous activation of GR shows that p53 and
GR mutually antagonize each others function, implicating a direct
interaction between the two proteins. Conceivably, GR may be affecting
DNA binding and/or transactivation function of p53. One of our future
approaches will be to functionally define the specific structural
domains within the GR protein that are required for its interaction
with p53. In this context, GRs have been shown to interact with a
select number of transcription factors resulting in either synergistic
(such as C/EBP family members) or antagonistic (such as members of the
AP-1 family or NF-KB) regulation of GR responsiveness (8, 10, 52, 53, 54, 55, 56). In addition, the tumor suppressor protein Rb has also been
shown to potentiate transactivation by the GR (57). Interestingly, our
results have further shown that activated GR had no significant
influence on the transrepression function of p53. The reasons for this
lack of effect of GR are not clear, especially since the mechanisms of
p53-dependent transcriptional repression are poorly understood.
Transcriptional repression by p53 requires the carboxy-terminal end of
the protein and is believed to occur by interaction with components of
the core transcriptional machinery (58, 59). On the other hand,
p53-dependent transactivation involves specific DNA binding and the
presence of an intact transactivation and DNA-binding domains, within
the p53 molecule (29, 30). Therefore, it is likely that the selective
inhibition of transactivation function of p53 by GR may be related to
distinct differences in the mechanisms underlying p53-dependent
transactivation vs. transrepression.
The repression of GR-dependent transactivation by p53 implicates, at a
functional level, a new pathway by which this tumor suppressor can
elicit its pleiotropic effects on mammalian cells. Both glucocorticoid
hormones and p53 play important physiological roles in the control of
cellular growth and differentiation in a variety of tissue and cell
types (24, 25, 26, 27, 60, 61, 62, 63). The functional interactions between a growth
regulator such as p53 and the GR, also implicated in growth control,
may serve to provide a framework for understanding how transcription
factor interactions affect diverse growth- regulatory networks. For
instance, the degree of responsiveness to glucocorticoids varies widely
not only between different tissues but also within specific cell types
(5). Modulation of GR activity by p53 may partly account for such
differences in tissue sensitivity to glucocorticoids, reflecting
another level of receptor regulation. The inhibited expression of a
glucocorticoid-responsive protein kinase gene, sgk, by p53
likely represents a possible target of cross-talk between two distinct
signaling pathways that regulate cellular phosphorylation cascades as
an appropriate response to specific extracellular signals. In addition
to the multiple p53 DNA-binding sites and a GRE, the sgk
promoter also contains potential binding sites for several other
important regulatory factors implicated in growth and differentiation.
These putative regulatory sites include the AP-1 complex recognition
sequence, a consensus C/EBP site, which is recognized by a family of
transcription factors involved in growth control, and a serum response
element as well as DNA sites for factors involved in developmental
programs, such as the kruppel and hunch-back
proteins. Thus, important future approaches will attempt to determine
the critical combinatorial effects, functional roles, and biological
significance of the multiple cis-acting promoter elements
that regulate transcription of the sgk gene in a cell
type-specific manner.
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MATERIALS AND METHODS
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Cells and Materials
Rat2 cells are an untransformed rat fibroblast cell line
generously provided by G. S. Martin (Department of Molecular and Cell
Biology, University of California, Berkeley, CA). Con8Hd6 mammary
epithelial tumor cells and Rat2 fibroblasts were maintained in
DMEM/Hams F-12 medium (50:50) supplemented with 10% calf serum. The
BDS1 cell line is an epithelial tumor cell line derived from the
minimal deviation rat Reuber hepatoma (64) and cultured in DMEM/F-12
supplemented with 10% FBS. NMuMG cells are a nontransformed mouse
mammary epithelial cell line originally derived from normal glandular
tissue of an adult NAMRU mouse (65). NMuMG cells were cultured in DMEM
supplemented with 10% FBS and insulin (10 µg/ml). All cell lines
were routinely grown at 37°C in humidified air containing 5%
CO2. Cell culture media were changed every 48 h, and
dexamethasone was added to a final concentration of 1 µM
as indicated. DMEM/F12 (1:1), the calf serum, and fetal bovine serum,
the calcium- and magnesium-free PBS, and trypsin-EDTA were supplied by
BioWhittaker (Walkersville, MD). Dexamethasone was obtained from Sigma
Chemical Co. (St. Louis, MO). [3H]Acetyl coenzyme A (200
mCi/mmol) was purchased from DuPont/NEN (Wilmington, DE) and
[
-32P]dATP was procured from ICN Biomedicals Inc.
(Costa Mesa, CA). The BuGR-2 GR-specific antibody (66) was generously
provided by Dr. John Forte (Department of Molecular and Cell Biology,
University of California, Berkeley, CA). The anti-p53 monoclonal
antibody PAb421 was purchased from Oncogene Science Inc. (Cambridge,
MA). The GRE-CAT chimeric reporter plasmid containing six copies of
GREs from the mouse mammary tumor virus (MMTV) promoter linked to the
CAT reporter gene and the 6RGR expression plasmid containing the GR
cDNA under the control of the RSV promoter were generous gifts from Dr.
Keith R. Yamamoto (Department of Biochemistry & Biophysics, University
of California, San Francisco, CA). The RSV-CAT reporter plasmid encodes
the bacterial CAT gene constitutively driven by the long terminal
repeat of the RSV promoter and was a gift from Dr. Marc Montimony (Salk
Institute, La Jolla, CA). In the CMV-CAT plasmid construct, the CAT
gene is driven by the CMV promoter. The sgk p53tk-CAT
reporter plasmid contains the p53-responsive element from the
sgk gene fused upstream of tk minimal promoter linked to the
CAT gene (22). The AGP reporter plasmid designated as AGP3x(GRE)-CAT
was kindly provided by Dr. Heinz Baumann (Department of Molecular and
Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY) and
comprises 127 bp of the native AGP promoter sequences containing three
copies of GRE linked to a CAT reporter (43). The murine wild type p53
expression plasmid driven by the CMV promoter, CMVp53wt, and the
transforming mutant p53 expression plasmid, CMVp53mt, derived from Meth
A fibrosarcoma cells bearing point mutations at positions corresponding
to residues 168 and 234 of the protein (67) and incapable of DNA
binding (68) were obtained from Dr. Moshe Oren (Weisman Institute for
Science, Rehevot, Israel). The human p53 expression plasmids
CMV-p53wt, CMV-p53179, and
CMV-p53
43 were generous gifts of Dr. Peter Howley
(National Cancer Institute, Rockville, MD). CMV-p53wt
encodes the wild type p53 protein. CMV-p53179 was isolated
from a human lung tumor and has a His-to Gln substitution at codon 179,
and CMV-p53
43 has the first 43 amino acids from the N
terminus of the protein deleted (45). All other reagents were of
highest available purity.
Plasmid Constructions
Construction of the plasmid p-4.0sgk-CAT, which
contains -4.0 kb of 5'-flanking sequences of rat sgk gene,
ending at +51(-4000/+51), relative to transcriptional start site and
fused to the coding region of bacterial CAT gene in the vector
pBLCAT3, has been previously described (13). Unilateral 5'-deletions of
p-4.0sgk-CAT were generated by ExonucleaseIII/S1
nuclease digestion of the HindIII and
AflII-double-digested p-4.0sgk-CAT construct
according to manufacturers instructions (Promega, Madison, WI). The
5'-protruding end of the HindIII site was protected from
ExoIII by filling in the 3'-recessed end with
-phosphorothioate deoxynucleoside triphosphate. These deleted
fragments were filled in using Klenow enzyme and religated and
sequenced to determine endpoints of deletion. The
sgkGREtk-CAT reporter plasmid was created by inserting
double-stranded annealed oligonucleotide encompassing 5'-flanking
sgk gene sequences from -1000 to -975 (5'-
tgcggAGGACAgaaTGTTCTcggag-3') with the
HindIII site at the 5'-end and the BamHI site at
the 3'-end into HindIII/BamHI sites of the
plasmid HSVtk-CAT, which contains the tk promoter (-105/+57) upstream
of CAT gene (69). The plasmid was confirmed by DNA sequencing.
Transfection and UV Treatment of Cells
Logarithmically growing Rat2 fibroblasts and Con8Hd6 mammary
epithelial tumor cells were incubated in DMEM/F12 (1:1)/10% calf serum
and transfected by electroporation (13). Briefly, single-cell
suspensions were generated by trypsinization, washed twice with sterile
1x PBS, and resuspended in an electroporation buffer containing 270
mM sucrose, 7 mM sodium phosphate buffer, pH
7.4, 1 mM MgCl2. The cells (12 x
107 cells per sample) contained in 250 µl of
electroporation buffer were dispensed into sterile cuvettes. In most
experiments, cells were transfected with 10 µg of reporter plasmid
and 10 µg of appropriate expression plasmid. In all transfections,
the total amount of DNA was adjusted to 30 µg with the promoterless
pBLCAT3 vector DNA. All plasmids used for DNA transfections were
purified twice by a CsCl banding method. The cells and DNA were gently
mixed and electrically pulsed five times (700 V square wave pulse for
99 µsec for Rat2 cells and 400 V square wave pulse for 99 µsec for
Con8Hd6 cells) using a BTX 800 Transfector apparatus (BTX Inc., San
Diego, CA), and the cells were incubated on ice for 10 min. Transfected
cells were plated into prewarmed DMEM-F12 (1:1)/10% calf serum in
100-mm Corning plastic tissue culture dishes and incubated at 37 C.
Typically, 5 h after transfection, the medium was removed by
aspiration, and the cells were washed in PBS and then incubated with
fresh medium in the presence or absence of 1 µM
dexamethasone. After 24 h, cells were harvested for CAT assays,
and the protein contents of the resulting cell extracts were determined
by the Bradford protein assay (70). NMuMG mammary epithelial cells and
BDS1 hepatoma cells from a logarithmically growing culture were
transfected by the calcium phosphate precipitation method (71).
Briefly, cells were plated into 100-mm tissue culture plates after
dispersion by treatment with trypsin-EDTA 1620 h before
transfections. Four hours before transfection, cells were replenished
with fresh medium. Typically 10 µg of reporter plasmid, along with 10
µg of appropriate expression plasmid, were used to prepare the
DNA-CaPO4 precipitates for each plate and added
individually while rocking the plate gently. The total amount of DNA
used in CaPO4 transfections for CAT assays was held
constant at 20 µg and, in appropriate transfections, the total DNA
adjusted to this amount using the empty CAT vector plasmid, pBLCAT3.
After 4 h of incubation at 37 C, the cells were briefly shocked
with 15% glycerol solution for 3 min at 37 C, followed by two PBS
washes and subsequently incubated with fresh medium. UV treatment of
cells was carried out using a UV Stratalinker (1800 series, Stratagene,
La Jolla, CA) where the energy delivered was precisely controlled by
the cross-linker. The medium was aspirated, and cells were rinsed in
PBS twice and exposed to 40 J/m2 of UV energy. After UV
treatment, cells were grown in fresh serum-replete medium for defined
periods of time.
CAT Reporter Gene Assays
A quantitative nonchromatographic assay (72) was used to measure
CAT activity in the cell extracts as detailed elsewhere (13). Briefly,
cells were harvested by washing twice in PBS, resuspended in 0.1
M Tris-HCl, pH 7.8, and lysed by three cycles of
freeze-thawing (alternating between an ethanol-dry ice bath and a
37°C water bath, 5 min per cycle). Cell lysates were heated at 68°C
for 15 min and centrifuged at 12,000 x g for 10 min,
and supernatants were recovered. CAT enzymatic assay was monitored in
5075 µg of cell extracts and was carried out in 0.1 M
Tris-HCl, pH 7.8, 1 mM aqueous chloramphenicol, and 1 µCi
[3H]acetyl-coenzyme A (final reaction volume of 250 µl)
at 37°C for 4 h. The reaction mixture was gently overlaid with 4
ml of Econofluor (DuPont/NEN), and the production of
[3H]acetylated chloramphenicol was quantitated by liquid
scintillation counting. The enzyme activity was expressed as
[3H]acetylated chloramphenicol produced per µg protein
present in corresponding cell lysates (counts per min/µg protein/4
h). Transfections were done in triplicate and repeated at least three
times.
Gel Mobility Shift Assays
Cytoplasmic extracts from either NMuMG cells or Rat2 fibroblasts
transiently transfected with GR-encoding plasmids were prepared
essentially by the method of Dignam et al. (73). Protein
contents were evaluated by the Bradford procedure (70). In assays
utilizing in vitro synthesized protein, the TNT system
(Promega) was used. The CMV-p53wt expression plasmid engineered for
in vitro expression was used to synthesize wild type p53
either from labeled or unlabeled amino acids according to the
manufacturers instructions. The purity of the synthesized wild type
p53 from labeled amino acids was confirmed by resolving the product on
7.8% SDS polyacrylamide gels. Subsequently, unlabeled amino acids were
used to generate in vitro synthesized protein for gel shift
analysis. The sense strands of the oligonucleotides used to synthesize
32P-labeled DNA probes for gel shift assays included: 1)
sgkGRE,
5'-tgcggAGGACAgaaTGTTCTcggag-3' derived from
sequences within -1000 to -975 of sgk promoter. 2) Con GRE
5'-tcgacGGTACAggaTGTTCTagctact-3' has consensus
GRE sequence and has been shown to specifically interact with the GR
(74). In addition, unlabeled oligonucleotides corresponding to the
sgkGRE, Con GRE, and a nonspecific sequence were utilized as
competitor DNA in the gel shift assays. All oligonucleotides were
synthesized by model 394 synthesizer in the University of California at
Berkeley Cancer Research Laboratory Microchemical facility.
Radiolabeling of 5'-ends of the oligonucleotides involved incubation of
equal amounts (10 pm), of sense and antisense strands with
[
-32P]ATP, 7000 Ci/mmole (ICN), and T4 polynucleotide
kinase (Boeringer Mannheim, Indianapolis, IN) for 30 min, at 37 C.
Annealing of complementary strands either for labeled or unlabeled DNA
probes was carried out by mixing equal amounts of sense and antisense
oligonucleotides in 0.1 M NaCl, heated at 70 C for 10 min
followed by gradual cooling to room temperature. Double-stranded
labeled oligonucleotides were purified from single-stranded DNA, and
unincorporated nucleotides were purified by electrophoretic
fractionation on a 8% native polyacrylamide gel. The radioactive bands
were excised, eluted in 400 µl TE buffer (10 mM Tris, 1
mM EDTA, pH 7.4) and 40 µl 3 M sodium
acetate, pH 5.0, ethanol precipitated, washed in 70% ethanol,
resuspended in TE buffer, and stored at -70 C.
The binding reactions of cell extracts with DNA were carried out in a
20 µl reaction volume, containing 10 µg of cytoplasmic extracts,
0.5 ng of [32P]labeled (5 x 104 cpm)
DNA probe, 500 ng of poly(deoxyinosinic-deoxycytidylic)acid, 7 µl of
2x binding buffer (20% glycerol, 20 mM HEPES, pH 7.9, 50
mM KCl, 6.25 mM MgCl2, 0.5%NP-40,
0.2 mM EDTA, 4 mM spermidine) and incubated for
20 min on ice. In specific competition experiments, 100-fold molar
excess of annealed double stranded unlabeled competing oligonucleotides
were added before the addition of radiolabeled DNA probes. In some
cases, the extracts were preincubated with either specific antibodies
or in vitro translated proteins for 60 min before addition
of radiolabeled probes. Antibodies used in the binding reactions
included anti-GR antibody, BuGR-2, or the anti-p53 antibody, PAb421.
The DNA- binding reactions were analyzed by electrophoretic
fractionation on a 4% nondenaturing polyacrylamide gel (80:1
acrylamide/bis-acrylamide) in 0.25x TAE buffer (0.04 M
Tris-acetate, 1 mM EDTA, pH 7.4) and 1 mM EDTA
and 0.05% NP-40, at 180 V at 4 C. Gels were routinely prerun for
2 h, at 180 V at 4 C. After electrophoresis, gels were dried and
protein-DNA complexes were visualized by autoradiography using Amersham
Hyperfilm.
Western Blotting
Nuclear extracts were prepared according to previously described
procedures (73). Thirty micrograms of nuclear protein extracts from
UV-treated NMuMG cells were resolved on 7.8% SDS-polyacrylamide gel
and transferred to nitrocellulose membrane (Micron Separations,
Westborough, MA). The membrane was blocked overnight at 4 C with TBST
blocking solution (50 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk,
and then incubated for 2 h at room temperature with primary
anti-p53 monoclonal antibody PAb421 at a 1:100 dilution in TBST
blocking solution. The secondary antibody used was directed against
mouse IgG conjugated to horse radish peroxidase (Zymed Laboratories,
Inc., South San Francisco, CA) at a 1:10,000 dilution in TBST blocking
solution containing 1% nonfat dry milk and incubated for 1 h. The
signal was detected by enhanced chemiluminescence on Hyperfilm ECL
(Amersham Corp.) in accordance with the manufacturers instructions.
Equivalent protein loading was verified in parallel sets of samples by
Coomassie Blue staining of the protein gel.
 |
ACKNOWLEDGMENTS
|
---|
We express our appreciation to Paul Woo, Helen Cha, Yukihiro
Nishio, and Ross Ramos, for critical reading of the manuscript. We also
wish to thank Marina Chin, Althaea Yronwode, Khanh Tong, Thai Truong,
Vinh Trinh, Charles C. Jackson, and William J. Meilandt for their
technical assistance. We are grateful to Jerry Kapler for his excellent
photography.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Gary L. Firestone, Department of Molecular and Cell Biology, Box 591 LSA, University of California at Berkeley, Berkeley, California 94720.
This work was supported by NIH Grant CA-71514 awarded by the National
Cancer Institute. In the initial stages of this work, A.C.M. was a
postdoctoral trainee supported by National Research Service Grant
CA-09041 awarded by the NIH.
1 Supported by a summer research fellowship from the Biology
Fellows Program at the University of California at Berkeley, sponsored
by the Howard Hughes Medical Institute. 
Received for publication January 31, 1996.
Revision received October 23, 1996.
Accepted for publication December 4, 1996.
 |
REFERENCES
|
---|
-
Beato M, Truss M, Chavez S 1996 Control of transcription
by steroid hormones. Ann NY Acad Sci 784:93123[Medline]
-
Gustafsson JA, Carlstedt DJ, Poellinger L, Okret S,
Wikstrom AC, Bronnegard M, Gillner M, Dong Y, Fuxe K, Cintra A 1987 Biochemistry, molecular biology, and physiology of the
glucocorticoid receptor. Endocr Rev 8:185234[Medline]
-
Tsai MJ, OMalley BW 1994 Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
-
Yamamoto KR 1985 Steroid receptor regulated transcription of
specific genes and gene networks. Annu Rev Genet 19:209252[CrossRef][Medline]
-
Bamberger CM, Schulte HM, Chrousos GP 1996 Molecular
determinants of glucocorticoid receptor function and tissue sensitivity
to glucocorticoids. Endocr Rev 17:245261[Abstract]
-
Truss M, Beato M 1993 Steroid hormone receptors: interaction
with deoxyribonucleic acid and transcription factors. Endocr Rev 14:459479[Abstract]
-
Reichel RR, Jacob ST 1993 Control of gene expression by
lipophilic hormones. FASEB J 7:427436[Abstract/Free Full Text]
-
Pfahl M 1993 Nuclear receptor/AP-1 interaction. Endocr Rev 14:651658[Medline]
-
Parker MG 1993 Steroid and related receptors. Curr Opin Cell
Biol 5:499504[Medline]
-
Schule R, Evans RM 1991 Cross-coupling of signal transduction
pathways: zinc finger meets leucine zipper. Trends Genet 7:377381[Medline]
-
Wahli W, Martinez E 1991 Superfamily of steroid nuclear
receptors: positive and negative regulators of gene expression. FASEB J 5:22432249[Abstract/Free Full Text]
-
Karin M 1994 Signal transduction from the cell surface to the
nucleus through the phosphorylation of transcription factors. Curr Opin
Cell Biol 6:415424[Medline]
-
Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL 1993 Characterization of sgk, a novel member of the serine/threonine protein
kinase gene family which is transcriptionally induced by
glucocorticoids and serum. Mol Cell Biol 13:20312040[Abstract]
-
Webster MK, Goya L, Firestone GL 1993 Immediate-early
transcriptional regulation and rapid mRNA turnover of a putative
serine/threonine protein kinase. J Biol Chem 268:1148211485[Abstract/Free Full Text]
-
McMahon SB, Monroe JG 1992 Role of primary response genes in
generating cellular responses to growth factors. FASEB J 6:27072715[Abstract/Free Full Text]
-
Richards JS, Fitzpatrick SL, Clemens JW, Morris JK, Alliston
T, Sirois J 1995 Ovarian cell differentiation: a cascade of multiple
hormones, cellular signals, and regulated genes. Recent Prog Horm Res 50:223254[Medline]
-
Imaizumi K, Tsuda M, Wanaka A, Tohyama M, Takagi T 1994 Differential expression of sgk mRNA, a member of the Ser/Thr protein
kinase gene family, in rat brain after CNS injury. Brain Res Mol Brain
Res 26:189196[Medline]
-
Clay FJ, McEwen SJ, Bertoncello I, Wilks AF, Dunn AR 1993 Identification and cloning of a protein kinase-encoding mouse gene,
Plk, related to the polo gene of Drosophila. Proc Natl Acad Sci USA 90:48824886[Abstract]
-
Donohue PJ, Alberts GF, Guo Y, Winkles JA 1995 Identification
by targeted differential display of an immediate early gene encoding a
putative serine/threonine kinase. J Biol Chem 270:1035110357[Abstract/Free Full Text]
-
Golsteyn RM, Schultz SJ, Bartek J, Ziemiecki A, Ried T, Nigg
EA 1994 Cell cycle analysis and chromosomal localization of human Plk1,
a putative homologue of the mitotic kinases Drosophila polo
and Saccharomyces cerevisiae Cdc5. J Cell Sci 15091517
-
Simmons DL, Neel BG, Stevens R, Evett G, Erikson RL 1992 Identification of an early-growth-response gene encoding a novel
putative protein kinase. Mol Cell Biol 12:41644169[Abstract]
-
Maiyar AC, Huang AJ, Phu PT, Cha HH, Firestone GL 1996 p53
stimulates promoter activity of the sgk serum/glucocorticoid-inducible
serine/threonine protein kinase gene in rodent mammary epithelial
cells. J Biol Chem 271:12414- 12422[Abstract/Free Full Text]
-
Cox LS, Lane DP 1995 Tumour suppressors, kinases and clamps:
how p53 regulates the cell cycle in response to DNA damage. Bioessays 17:501508[Medline]
-
Haffner R, Oren M 1995 Biochemical properties and biological
effects of p53. Curr Opin Genet Dev 5:8490[Medline]
-
Lee JM, Bernstein A 1995 Apoptosis, cancer and the p53 tumour
suppressor gene. Cancer Metastasis Rev 14:149161[Medline]
-
Ko LJ, Prives C 1996 p53: puzzle and paradigm. Genes Dev 10:10541072[CrossRef][Medline]
-
Gottlieb TM, Oren M 1996 p53 in growth control and neoplasia.
Biochim Biophys Acta 1287:77102[CrossRef][Medline]
-
Cho Y, Gorina S, Jeffrey PD, Pavletich NP 1994 Crystal
structure of a p53 tumor suppressor-DNA complex: understanding
tumorigenic mutations [see comments]. Science 265:346355[Medline]
-
Subler MA, Martin DW, Deb S 1994 Overlapping domains on the
p53 protein regulate its transcriptional activation and repression
functions. Oncogene 9:13511359[Medline]
-
Sang BC, Chen JY, Minna J, Barbosa MS 1994 Distinct regions of
p53 have a differential role in transcriptional activation and
repression functions. Oncogene 9:853859[Medline]
-
Truant R, Xiao H, Ingles CJ, Greenblatt J 1993 Direct
interaction between the transcriptional activation domain of human p53
and the TATA box-binding protein. J Biol Chem 268:22842287[Abstract/Free Full Text]
-
Martin DW, Munoz RM, Subler MA, Deb S 1993 p53 binds to the
TATA-binding protein-TATA complex. J Biol Chem 268:1306213067[Abstract/Free Full Text]
-
Seto E, Usheva A, Zambetti GP, Momand J, Horikoshi N, Weinmann
R, Levine AJ, Shenk T 1992 Wild-type p53 binds to the TATA-binding
protein and represses transcription. Proc Natl Acad Sci USA 89:1202812032[Abstract]
-
Agoff SN, Hou J, Linzer DI, Wu B 1993 Regulation of the human
hsp70 promoter by p53. Science 259:8487[Medline]
-
Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW,
Vogelstein B 1993 Oncoprotein MDM2 conceals the activation domain of
tumour suppressor p53. Nature 362:857860[CrossRef][Medline]
-
Pietenpol JA, Vogelstein B 1993 Tumour suppressor genes. No
room at the p53 inn [news; comment]. Nature 365:1718[CrossRef][Medline]
-
Lin DL, Chang C 1996 p53 is a mediator for radiation-repressed
human TR2 orphan receptor expression in MCF-7 cells, a new pathway from
tumor suppressor to member of the steroid receptor superfamily. J
Biol Chem 271:1464914652[Abstract/Free Full Text]
-
Maheswaran S, Park S, Bernard A, Morris JF, Rauscher F3, Hill
DE, Haber DA 1993 Physical and functional interaction between WT1 and
p53 proteins. Proc Natl Acad Sci USA 90:51005104[Abstract]
-
Yap N, Yu CL, Cheng SY 1996 Modulation of the transcriptional
activity of thyroid hormone receptors by the tumor suppressor p53. Proc
Natl Acad Sci USA 93:42734277[Abstract/Free Full Text]
-
Sorensen TS, Girling R, Lee CW, Gannon J, Bandara LR, La
Thangue NB 1996 Functional interactions between DP-1 and p53. Mol Cell
Biol 16:58885995[Abstract]
-
Gualberto A, Baldwin AJ 1995 p53 and Sp1 interact and
cooperate in the tumor necrosis factor-induced transcriptional
activation of the HIV-1 long terminal repeat. J Biol Chem 270:1968019683[Abstract/Free Full Text]
-
Nordeen SK, Suh BJ, Kuhnel B, Hutchison C 1990 Structural
determinants of a glucocorticoid receptor recognition element. Mol
Endocrinol 4:18661873[Abstract]
-
Baumann H, Jahreis GP, Morella KK, Won KA, Pruitt SC, Jones
VE, Prowse KR 1991 Transcriptional regulation through cytokine and
glucocorticoid response elements of rat acute phase plasma protein
genes by C/EBP and JunB. J Biol Chem 266:2039020399[Abstract/Free Full Text]
-
Osifchin NE, Jiang D, Ohtani FN, Fujita T, Carroza M, Kim SJ,
Sakai T, Robbins PD 1994 Identification of a p53 binding site in the
human retinoblastoma susceptibility gene promoter. J Biol Chem 269:63836389[Abstract/Free Full Text]
-
Unger T, Mietz JA, Scheffner M, Yee CL, Howley PM 1993 Functional domains of wild-type and mutant p53 proteins involved in
transcriptional regulation, transdominant inhibition, and
transformation suppression. Mol Cell Biol 13:51865194[Abstract]
-
Kastan MB, Zhan Q, El Diery W, Carrier F, Jacks T, Walsh WV,
Plunkett BS, Vogelstein B, Fornace AJ 1992 A mammalian cell cycle
checkpoint pathway utilizing p53 and GADD45 is defective in
ataxia-telangiectasia. Cell 71:587597[Medline]
-
Lu X, Lane DP 1993 Differential induction of transcriptionally
active p53 following UV or ionizing radiation: defects in chromosome
instability syndromes? Cell 75:765778[Medline]
-
Nelson WG, Kastan MB 1994 DNA strand breaks: the DNA template
alterations that trigger p53-dependent DNA damage response pathways.
Mol Cell Biol 14:18151823[Abstract]
-
Subler MA, Martin DW, Deb S 1992 Inhibition of viral and
cellular promoters by human wild-type p53. J Virol 66:47574762[Abstract]
-
Monfar M, Blenis J 1996 Inhibition of p70/p85 S6 kinase
activities in T cells by dexamethasone. Mol Endocrinol 10:11071115[Abstract]
-
Bamberger CM, Bamberger AM, de Castro M, Chrousos GP 1995 Glucocorticoid receptor beta, a potential endogenous inhibitor of
glucocorticoid action in humans. J Clin Invest 95:24352441[Medline]
-
Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin AJ 1995 Characterization of mechanisms involved in transrepression of
NF-kappa B by activated glucocorticoid receptors. Mol Cell Biol 15:943953[Abstract]
-
Ray A, Prefontaine KE 1994 Physical association and functional
antagonism between the p65 subunit of transcription factor NF-kappa B
and the glucocorticoid receptor. Proc Natl Acad Sci USA 91:752756[Abstract]
-
Nishio Y, Isshiki H, Kishimoto T, Akira S 1993 A nuclear
factor for interleukin-6 expression (NF-IL6) and the glucocorticoid
receptor synergistically activate transcription of the rat alpha 1-acid
glycoprotein gene via direct protein-protein interaction. Mol Cell Biol 13:18541862[Abstract]
-
Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: selectors of positive or negative
regulation from a single DNA element. Science 249:12661272[Medline]
-
Trapp T, Holsboer F 1996 Nuclear orphan receptor as a
repressor of glucocorticoid receptor transcriptional activity. J
Biol Chem 271:98799882[Abstract/Free Full Text]
-
Singh P, Coe J, Hong W 1995 A role for retinoblastoma protein
in potentiating transcriptional activation by the glucocorticoid
receptor. Nature 374:562565[CrossRef][Medline]
-
Shaulian E, Haviv I, Shaul Y, Oren M 1995 Transcriptional
repression by the C-terminal domain of p53. Oncogene 10:671680[Medline]
-
Horikoshi N, Usheva A, Chen J, Levine AJ, Weinmann R, Shenk T 1995 Two domains of p53 interact with the TATA-binding protein, and the
adenovirus 13S E1A protein disrupts the association, relieving
p53-mediated transcriptional repression. Mol Cell Biol 15:227234[Abstract]
-
Sanchez I, Goya L, Vallerga AK, Firestone GL 1993 Glucocorticoids reversibly arrest rat hepatoma cell growth by inducing
an early G1 block in cell cycle progression. Cell Growth Differ 4:215225[Abstract]
-
Zettl KS, Sjaastad MD, Riskin PM, Parry G, Machen TE,
Firestone GL 1992 Glucocorticoid-induced formation of tight junctions
in mouse mammary epithelial cells in vitro. Proc Natl Acad
Sci USA 89:90699073[Abstract]
-
Goya L, Maiyar AC, Ge Y, Firestone GL 1993 Glucocorticoids
induce a G1/G0 cell cycle arrest of Con8 rat mammary tumor cells that
is synchronously reversed by steroid withdrawal or addition of
transforming growth factor-alpha. Mol Endocrinol 7:11211132[Abstract]
-
Ramos RA, Nishio Y, Maiyar AC, Simon KE, Ridder CC, Ge Y,
Firestone GL 1996 Glucocorticoid-stimulated CCAAT/enhancer-binding
protein
expression is required for steroid-induced G1 cell cycle
arrest of minimal-deviation rat hepatoma cells. Mol Cell Biol 16:52885301[Abstract]
-
Cook PW, Swanson KT, Edwards CP, Firestone GL 1988 Glucocorticoid receptor-dependent inhibition of cellular proliferation
in dexamethasone-resistant and hypersensitive rat hepatoma cell
variants. Mol Cell Biol 8:14491459[Medline]
-
Owens RB 1974 Glandular epithelial cells from mice: a method
for selective cultivation. J Natl Cancer Inst 52:13751378[Medline]
-
Hendry III WJ, Danzo BJ, Harrison III RW 1987 Analysis of the
disruptive action of an epididymal protease and the stabilizing
influence of molybdate on nondenatured and denatured glucocorticoid
receptor. Endocrinology 120:629639[Abstract]
-
Eliyahu D, Goldfinger N, Pinhasi KO, Shaulsky G, Skurnik Y,
Arai N, Rotter V, Oren M 1988 Meth A fibrosarcoma cells express two
transforming mutant p53 species. Oncogene 3:313321[Medline]
-
Halazonetis TD, Davis LJ, Kandil AN 1993 Wild-type p53 adopts
a mutant-like conformation when bound to DNA. EMBO J 12:10211028[Abstract]
-
Luckow B, Schutz G 1987 CAT constructions with multiple unique
restriction sites for the functional analysis of eukaryotic promoters
and regulatory elements. Nucleic Acids Res 15:5490[Medline]
-
Bradford MM 1976 A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
-
Chen CA, Okayama H 1988 Calcium phosphate-mediated gene
transfer: a highly efficient transfection system for stably
transforming cells with plasmid DNA. Biotechniques 6:632638[Medline]
-
Neumann JR, Morency CA, Russian KO 1987 A novel rapid assay
for chloramphenicol acetyl transferase gene expression. Biotechniques 5:444447
-
Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]
-
Beato M, Chalepakis G, Schauer M, Slater EP 1989 DNA
regulatory elements for steroid hormones. J Steroid Biochem 32:737747[CrossRef][Medline]