From the Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, August 4, 2000, and in revised form, November 14, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glucocorticoids (GCs) function, in part, through
the ability of the glucocorticoid receptor (GR) to activate gene
expression and in part through the transrepression of AP-1 and NF- Glucocorticoids (GCs)1
have long been used as anti-inflammatory, immunosuppressive, and
chemotherapeutic drugs. Initially, GCs were thought to mediate their
therapeutic actions through the transcription of GC-responsive genes
(1). This mechanism, however, did little to explain how GCs could
inhibit the transcription of many cytokine genes, a critical part of
its anti-inflammatory and immunosuppressive actions (2). Subsequently,
it was discovered that GR could inhibit the activity of the
transcription factor, AP-1, in the absence of a GC-responsive element
(GRE) (3-6). This ability to inhibit directly a transcription factor
activity in the absence of a GRE was termed transrepression and was
shown to involve a direct physical interaction between GR and AP-1. GCs
were also found to inhibit the activity of the transcription factor
NF- Footprinting studies of the AP-1-driven collagenase promoter
after GC treatment showed no change in protein occupancy, suggesting that the GR/AP-1 interaction functions to promote a restructuring of
the composition of factors binding to the promoter rather than a simple
inhibition of promoter occupancy (14). Domain mapping studies
identified the zinc finger DNA binding domain (DBD) of GR as essential
for transrepression of AP-1, suggesting that the GR DBD is responsible
for both transrepression and activation (4-6). DNA binding and the
subsequent activation of gene expression require the dimerization of GR
and its binding to a palindromic GRE (15). Careful dissection of the GR
DBD, through mutagenesis, demonstrated that disruption of the D loop
within the second zinc finger could abolish the ability of GR to
dimerize and, consequently, inhibit GC-mediated activation (16). Others
(17, 18) have shown that mutations in this region, while inhibiting
GR-mediated activation, did not affect transrepression of AP-1. Further
mutations within the first zinc finger of the GR DBD were identified
that disrupted transrepression of AP-1 but did not inhibit the ability of GR to transactivate through a GRE (18), suggesting that activation and transrepression are separable phenomena.
Evidence in support of GR-mediated inhibition of NF- Here, we have begun to address this problem by introducing mutations
into the GR DNA binding domain and analyzing the effect of these
mutations on protein conformation and on transrepression of
NF- Plasmid Construction--
Mutations in the GR DBD (S425G, L436V,
and N454D/A458T) were constructed from our human GR expression
construct, pYCGR (10), by in vitro site-directed mutagenesis
using the QuikChange site-directed mutagenesis kit (Stratagene)
with the following oligonucleotides: GR S425G,
5'-CAAACTCTGCCTGGTGTGCGGTGATGAAGCTTCAGGATG-3' and
5'-CATCCTGAAGCTTCATCACCGCACACCCGGCAGAGTTTG-3'; GR L436V,
5'-CAGGATGTCATTATGGAGTCGTAACTTGTGGAAGCTGTAAAG-3' and 5'-CTTTACAGCTTCCACAAGTTACGACTCCATAATGACATCCTG-3'; GR N454D/A458T, 5'-GTGGAAGGACAGCACGATTACCTATGTACTGGAAGGAATGATTG-3' and
5'-CAATCATTCCTTCCAGTACATAGGTAATCGTGCTGTCCTTCCAC-3'. GST fusion proteins
of wild type GRDBD, S425GDBD, L436VDBD, and N454D/A458TDBD were
constructed by polymerase chain reaction amplification of the above
wild type or mutant GRDBD using Pfu (Stratagene) and then
subcloned in-frame into the BamHI/NotI site of
pGEX 4T-3 (Amersham Pharmacia Biotech). The primers used were:
GR416, 5'-GGGGGATCCCCACCTCCCAAACTCTGCCTG-3'; and GR504,
5'-GGGGCGGCCGCAGTGGCCTGCTGAATTCCTTTTAT-3'. All clones were
sequenced before use.
Circular Dichroism--
Wild type and mutant GRDBD GST fusion
proteins were expressed in DH5
Far-UV CD spectra were recorded at 20 °C in a
thermostat-controlled cell holder using an 0.1-cm strain-free quartz
cell in an Aviv model 62DS circular dichroism spectropolarimeter. Data was collected from 260 to 180 nm at 0.5-nm intervals using a bandwidth of 1.5 nm and an averaging time of 5 s for each point. The
protein concentration was determined by measuring absorbency at 280 nm (A280). The final spectrum for each mutant was
generated by averaging the spectra collected from several independent
purifications of each protein. Thermal melting curves for each mutant
were generated by monitoring ellipticity at 222 nm as a function of
increasing temperature between 20 and 95 °C at a rate of
1.5 °C/min, using a step size of 1 °C and an averaging time of
5 s at each temperature.
Cell Culture, Transfection of Cell Lines, and Reporter and
Apoptosis Assays--
COS-7, CEM C7-14 (GC-sensitive), and CEM
C1-15 (GC-resistant) cells were maintained at 37 °C in a 5%
CO2 humidified atmosphere in Dulbecco's minimal essential
medium (DMEM) supplemented with 10% fetal calf serum, 50 µg/ml penicillin, and 50 µg/ml streptomycin. Transient transfection
assays for transrepression were carried out using the LipofectAMINE
Plus reagent according to the manufacturer's instructions. For the
NF-
CEM C1-15 cells were stably transfected by electroporation.
Aliquots of 2 × 106 cells were suspended in 0.5 ml of
DMEM containing 1 mM sodium pyruvate (Life Technologies,
Inc.). Using 0.4-cm electroporation cuvettes (Invitrogen), samples were
electroporated at a capacitance of 960 microfarads and 50 volts
with 5 µg of pCI-neo (Promega) and 20 µg of wild type GR or GR
mutants. Cells were then removed and split into three parts, and each
part was cultured in 2 ml of supplemented DMEM for 72 h. The cells
were then fed with medium supplemented with G418 (450 µg/ml or 900 µg/ml, Gemini Bio-Products) for 3 month. The medium was changed every
3-4 days. Apoptosis was assayed morphologically using the acridine
orange/ethidium bromide double dye method (29).
Northern Blot Analysis--
Total RNA was isolated from cells
with TRIzol Regent (Life Technologies, Inc.) per the manufacturer's
instruction. RNA integrity and loading consistency were assessed by
visual inspection of ribosomal RNA bands after agarose gel
electrophoresis and ethidium bromide staining. 20 µg of RNA was
separated on a 1% formaldehyde-containing agarose gels, transferred to
Zeta-probe membranes (Bio-Rad) by standard capillary blotting, and
cross-linked by UV irradiation. The cDNA probe for I Western Blot Analysis--
Whole cell lysates were prepared, and
12.5 µg of protein from each lysate was electrophoresed through 10%
SDS-PAGE and transferred to nitrocellulose by electroblotting (Life
Technologies, Inc.). Blocking was performed overnight at 4 °C in CTT
buffer (10 mM Tris, 15 mM NaCl, pH 7.4) plus
10% nonfat dry milk and 0.1% Tween 20. The membrane was rinsed and
incubated for 2 h. at room temperature in CTT buffer containing
1% nonfat dry milk plus anti-GR antibody PA1-511 (Affinity
Bioreagents) at a 1:5 dilution. After 5 washes in CTT plus 0.1% Tween
20, the blot was incubated for 2 h with the secondary antibody
(horseradish peroxidase-conjugated anti-rabbit IgG from Promega,
diluted 1:5000 in CTT plus 1% dried milk). After 5 additional
washes, GR immunoreactivity was visualized by chemiluminescence (ECL,
Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Mutations in the GR DBD Do Not Cause Major Changes in
Conformation--
Previously, we showed that GC-mediated
transrepression of NF-
The structures of wild type and mutant GR DBDs were studied by CD
spectroscopy. Fig. 1 shows representative
far-UV CD spectra of the DBDs. The strong negative maxima at 222 and
208 nm indicate that the DBDs possess an amount of Analysis of Transrepression--
The mutations S425G, L436V, and
N454D/A458T were introduced into our GR expression vector, pYCGR, as
described under "Experimental Procedures." We then tested their
ability to transrepress NF- DEX-induced Apoptosis Is Restored Both by GR Mutants Deficient in
Dimerization and by GR Mutants Deficient in Transrepression--
GCs
induce apoptosis in both thymocytes and transformed T cells. To analyze
the properties of these GR mutants in the context of T cell apoptosis,
we used previously described sister human acute lymphoblastic leukemic
T cell lines, GC-sensitive CEM C7-14 and GC-resistant CEM C1-15 (33).
We cotransfected CEM C1-15 cultures with pCI-neo along with an
expression plasmid encoding either wild type GR or GR, containing one
of the various GR mutants described above, and selected stable
transfectants by culturing in G418. Although endogenous (nonfunctional)
GR migrated identically to transfected GR on SDS-PAGE, we found that GR
immunoreactivity was significantly higher in transfected lines,
indicating that our transgenes were expressed (Fig.
3A, inset). To investigate whether any of our GR mutants could restore GC sensitivity to apoptosis, our transfected CEM cell lines were treated with DEX for
48 h, and apoptosis was determined morphologically as described under "Experimental Procedures." As expected, DEX induced apoptosis in ~25% of the C14 line after 48 h, with little apoptosis in
the C15 line (Fig. 3A). Wild type GR restored DEX
sensitivity to the C15 line as did the dimerization mutant,
N454D/A458T, a result consistent with the results of Helmberg et
al. (17). Interestingly, both the S425G and L436V mutants also
restored DEX sensitivity to the C15 line. Statistical analysis of
apoptosis levels indicated no significant differences in apoptosis
between wild type GR transfected C15 cells and C15 cells transfected
with S425G, L436V, or N454D/A458T GR mutants.
These data can be interpreted in several ways. As S425G is unable to
transrepress NF-
If, however, N455D/A458T is capable of activating the expression
of a subset of GC-responsive genes, an alternative explanation for the
data would be that it is this subset of genes that regulates apoptosis.
As S425G and L436V are capable of activating gene expression, this
alternative hypothesis is consistent with our observations. Evidence
supporting this idea was generated by Rogatsky et al. (34),
who showed that when a GR dimerization mutant is introduced into GR
negative SAOS cells, proliferation is blocked through induction of p21
gene expression. However, evidence against this idea was also generated
by directing a D loop mutation to murine embryonic stem cells and
establishing a dimerization-deficient GR-expressing mouse (35).
This group found that thymocytes derived from these mice were
insensitive to DEX-mediated apoptosis.
Induction of I
To our knowledge, we are the first group to analyze the structure of
our GR mutants and systematically compare NF-B.
Here we characterize the effect of GR DNA binding domain (DBD)
mutations, previously analyzed for changes in the ability to activate
gene expression or transrepress AP-1. We have identified a GR mutant capable of distinguishing between transrepression of NF-
B and AP-1.
Using circular dichroism spectroscopy, we show that this mutation does
not appreciably alter GR DBD conformation, suggesting that functional
differences between the mutant and wild type protein are the
result of an alteration of a specific interaction surface. These data
suggest that transrepression of NF-
B and AP-1 occurs through
distinct protein-protein interactions and argue against the hypothesis
that transrepression occurs through competition for a single
coactivator protein. Introduction of these mutations into GC-resistant
CEM lymphoblastic T cells restored dexamethasone (DEX)-mediated
apoptosis as did wild type GR regardless of whether these mutants were
transrepression or activation defective. Thus, DEX-mediated apoptosis
in transformed T cells is more complex than originally appreciated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B (7-11). As NF-
B and AP-1 together regulate genes involved
in inflammation and immunosuppression, these discoveries allowed the
construction of a model for the therapeutic properties of GCs involving
both activation and transrepression (12, 13).
B as one
mechanism for the clinical actions of GCs has been generated in various
systems (19, 20). Transrepression of NF-
B by GCs has turned out to
be mediated by a number of separate mechanisms, however. NF-
B
comprises dimers of a protein family that shares homology with
c-Rel, including relA (p65), RelB, c-Rel, p50, and p52. Classic
NF-
B is a dimer of RelA and p50. Most forms of NF-
B are held in
the cytoplasm by members of the I
B family. NF-
B can be activated
through multiple signaling pathways through the activation of the I
B
kinase (IKK) and the phosphorylation of I
Bs. Initial reports of
GC-mediated transrepression of NF-
B included data demonstrating a
physical interaction between NF-
B subunits and GR similar to that
shown for AP-1 (7, 8, 10). Subsequently, we and others (9, 11) have
demonstrated that GCs could induce transcription of the NF-
B
inhibitor, I
B
. Most recently, it was demonstrated that both
NF-
B and GR can compete for interactions with coactivators such as
p300 and CREB-binding protein (CBP), which are present in
limited amounts (21-23). Interestingly, however, a number of
conflicting reports have emerged demonstrating systems in which GCs
transrepress NF-
B in the absence of I
B
induction (24-26). In
addition, others have reported that GC-mediated transrepression can
occur also when the coactivators p300 and SRC-1 are overexpressed (27).
Thus, the molecular basis for GC-mediated transrepression is
controversial and may, to some extent, be tissue-specific.
B. Using this structural information, we are able to propose that
transrepression of NF-
B and AP-1 is mediated through different interaction surfaces within the GR DBD. Our data argue against the
hypothesis that transrepression of both AP-1 and NF-
B is mediated by
competition with GR for a common factor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and purified by binding to
glutathione-Sepharose 4B beads, and the GST domain was cleaved by
thrombin digestion (Amersham Pharmacia Biotech) according to the
manufacturer's instructions. To remove contaminating bacterial DNA,
the GR DBDs were then incubated with 0.2 volumes of Q-Sepharose
(Amersham Pharmacia Biotech) at room temperature for 4 h and
dialyzed against 2 liters of phosphate-buffered saline overnight.
B transrepression assays, 100-mm plates of 80% confluent COS-7
cells were transfected with 23 µg of DNA including 10 µg of
3XMHC-luc NF-
B reporter construct, 1 µg of
-galactosidase expression plasmid along with 1 µg of RelA and either 10 µg of wild type GR or mutant GR expression plasmid. DEX was
added to a final concentration of 10
6
M at 3 h after the transfection. The cells were
harvested 48 h later, and luciferase activity was measured by
combining 40 µl of cell extract with 160 µl of luciferase assay
buffer (100 mM KPO4, pH 7.8, 15 mM
MgSO4, 5 mM ATP, 1 mM
dithiothreitol). The
-galactosidase assay was carried out as
described by Rosenthal (28). For the AP-1 transrepression assays,
6-well plates of 80% confluent COS-7 cells were transfected with 1 µg of 5XAP1-TATAcat, 100 ng of
-galactosidase expression plasmid
along with 100 ng of either wild type GR or mutant GR expression
plasmid. Endogenous AP-1 activity was induced 3 h
post-transfection with 80 ng/ml phorbol 12-myristate 13-acetate in the
presence or absence of 10
6 M DEX.
CAT activity was measured using a CAT enzyme-linked immunosorbent assay
kit according to the manufacturer's instructions (Roche Molecular Biochemicals). Relative transrepression was calculated as the
percent of decrease in
-galactosidase normalized reporter activity
after DEX treatment and then normalized to transrepression of either
NF-
B or AP-1 activity, respectively, by wild type GR. The average
transrepression mediated by wild type GR is defined as 1.0.
B
was
labeled with [
-32P]dCTP using a random primer DNA
labeling kit (Bio-Rad). The filter was washed twice in 40 mM NaPO4, 1 mM EDTA, 1% SDS, and
data visualized by PhosphorImager.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B requires the DBD but not the
1
activation domain (10). To further dissect this requirement, we tested
the DBD mutants S425G and L436V, shown to block transrepression of AP-1
(18), and N454D/A458T, a D loop mutation that blocks GR dimerization and activation of transcription. We began by analyzing the effect of
these mutations on GR conformation to determine whether the mutations
affected one specific region of the GR DBD or whether the effects of
these mutations were spread throughout the domain. This control
experiment was critical for the appropriate structural interpretation
of the subsequent functional data; however, to our knowledge, it had
never been performed in the context of studying GR-mediated
transrepression. To this end, we expressed wild type and mutant GR DBD
domains as GST fusion proteins in Escherichia coli. The GR
DBD proteins were purified, GST domains cleaved, and contaminating DNA
removed as described under "Experimental Procedures." The
extinction coefficient (
280) of 5470 M
1 cm
1
was determined by the method of Gill and von Hipple (30). SDS-PAGE analysis of the mutants generated an apparent molecular mass of 10 kDa, consistent with the theoretical molecular weights of our GR DBD
proteins (data not shown).
-helical
structure consistent with earlier studies of this domain done by others
using NMR spectroscopy and x-ray diffraction (31, 32). By visual
inspection, the spectra for the S425G mutant appears to deviate
slightly in intensity at the wavelengths representing
-helix, 208 and 222 nm. However, measurements of the percentage of
-helical
structure from three independent preparations, as measured by
deconvolution analysis of the spectra, showed that these deviations
were not statistically significant (Fig. 1, inset). These
data indicate that within the error of the measurement, there are no
obvious secondary structural differences between the wild type DBD and
any of the mutants. To further test the structural similarity of these
DBDs, thermal melting scans were collected on the wild type and each of
the mutants. Although the transition was not thermodynamically
reversible, the apparent melting temperatures (Tm)
for all of the mutants were equivalent within error, indicating that
the relative stability for the wild type DBD and the mutants are
indistinguishable (data not shown). In conclusion, CD spectroscopy
indicates that no global conformational changes exist in any of the
mutants, and any functional changes that result from these mutations
must be caused by either specific changes in the nature of the amino acids in an interaction interface region or by subtle local
conformational changes of involving only several residues.
View larger version (24K):
[in a new window]
Fig. 1.
Structural analysis of GR DBD mutants.
Far-UV CD spectra of the wild type GR DBD and mutants. Data presented
are the averages of three (two for N454D/A458T) independent
experiments. Open triangles, wtGR; filled
triangles, S425G; open circles, L436V; filled
circles, N454D/A458T. Inset, deconvolution analysis of
average spectra to show % of -helix. Standard errors are shown in
parentheses.
B and AP-1 by transient transfection
followed by treatment with DEX. The ability of these GR mutants to
transrepress NF-
B and AP-1 reporter constructs is shown in Fig.
2. We found that the mutations L436V and
N454D/A458T had no effect on the ability of GR to transrepress NF-
B,
whereas the S425G mutation greatly reduced the ability of GR to
transrepress NF-
B. N454D/A458T-mediated transrepression of AP-1 is
consistent with the observation that GR can transrepress AP-1 as a
monomer. Here we show that this property of GR can be extended to
transrepression of NF-
B. In our hands, S425G and L436V both
transrepress AP-1. These results are different from that reported by
Heck et al. (18), which may be because of differences in the
cell lines used by our respective laboratories. Additionally, these
differences may be due to amino acid differences caused by the
introduction of restriction sites flanking the DBD in their human GR
expression plasmid (18). Given that our experiments were performed in
one cell line with the same GR constructs, we can state that the
requirements for transrepression of AP-1 and NF-
B are not identical
in that the S425G mutation can discriminate between these two
functions. The serine to glycine substitution at position 425 removes
an hydroxyl group, possibly altering hydrogen bonding patterns between
the zinc finger domain and other proteins, suggesting that this
hydrogen bonding pattern is important for the transrepression of
NF-
B but not AP-1. Serine 425 is not thought to hydrogen bond either with DNA or within the zinc finger. X-ray crystallography data indicate
that this amino acid is solvent-accessible (32), consistent with the
possibility of its contribution to an intermolecular interaction
interface. These data indicate that multiple elements within the GR DBD
contribute to transrepression and argue against the hypothesis that
transrepression primarily involves competition for a common factor, as
this would predict that transrepression of NF-
B and AP-1 would not
be separable phenomena.
View larger version (21K):
[in a new window]
Fig. 2.
Effect of amino acid exchanges in the DNA
binding domain of human GR on transrepression of
NF- B and AP-1. COS-7 cells were
transfected as described under "Experimental Procedures" to assess
transrepression of either NF-
B activity (white bars) or
AP-1 activity (black bars). Cells were treated with
10
6 M DEX for 48 h, after
which the cells were harvested for luciferase (NF-
B) or CAT (AP-1)
and
-galactosidase assays. Luciferase or CAT activities were
normalized to
-galactosidase activity. To facilitate comparison,
relative transrepression was calculated as described under
"Experimental Procedures." Data presented here are averages of at
least three independent experiments. Error bars denote
S.D.
View larger version (34K):
[in a new window]
Fig. 3.
Stable transfection of GR DBD mutants into
GR-deficient CEM T cells. CEM C1-15 (GC-resistant) cells were
stably transfected with wild type GR or mutants. CEM C7-14 is a
GC-sensitive cell line. A, apoptosis was determined
morphologically using the ethidium bromide/acridine orange technique.
Open columns, untreated; filled columns,
DEX-treated; wt, wild type GR; SG, S425G;
LV, L436V; dbl, N454D/A458T. Inset,
Western blot of GR immunoreactivity in untransfected and transfected
C1-15 cells. Nonfunctional (endogenous) and transfected GR run at the
same molecular size. The lanes were scanned, background subtracted, and
data presented as the fold increase over the GR signal from
untransfected cells above each lane.
B, Northern blot analysis of I B
gene transcription.
Cell lines were either treated with DEX for 48 h (+) or left
untreated (
) and harvested for total RNA. Equal amounts of total RNA
(20 µg) were separated by formaldehyde-agarose gel electrophoresis.
RNA was transferred to Zeta-probe membranes and probed with a
MAD3/I
B
cDNA labeled by random priming.
B, and yet restores apoptosis, it would suggest that
transrepression of NF-
B is not required for this process.
Conversely, as N454D/A458T is unable to interact efficiently with DNA,
and yet restores apoptosis, this would argue that GR-mediated gene
expression is not required for this process. One potential resolution
of this apparent paradox is to consider the possibility that several
independent pathways leading to apoptosis are initiated by GCs.
B
Is Independent of GR-mediated
Transrepression in CEM C1-15 T Cells--
Given the above arguments,
we wanted to test the ability of our mutant GR lines to activate
transcription of an endogenous gene. Because of the interest in
GC-mediated regulation of I
B
expression, we chose to examine the
ability of these mutants to regulate I
B
mRNA abundance by
Northern blot analysis. Endogenous I
B
mRNA expression was
unresponsive to DEX treatment in CEM C1-15 cultures lacking
functional GR (Fig. 3B). In turn, I
B
mRNA
expression was highly responsive to DEX treatment in CEM C7-14
cultures that contained functional GR. Expression of the wild
type GR transgene in CEM C1-15 cells restored the ability of DEX to
induce I
B
mRNA, as expected. In addition, the S425G and L436V
mutant transgenes were capable of restoring the ability of DEX
induction of I
B
mRNA. In comparison, the N454D/A458T double
mutant was unable to restore DEX induction of I
B
mRNA, indicating that the I
B
gene is not part of the subset of
GC-responsive genes activated by this mutant.
B and AP-1 in the study
of GR-mediated transrepression. Through this work, we have identified a
region of the first zinc finger, which is differentially involved in
transrepression of NF-
B and with AP-1. Furthermore, we have shown
that DEX-mediated apoptosis is dependent solely neither on
transrepression nor on activation. Rather, our data suggest that
GC-mediated apoptosis in CEM cells is mediated potentially through
several independent pathways.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael Karin for supplying the AP-1 reporter construct, Dr. Carilee Lamb for supplying CEM C14 and C15 lines, and Maureen McHugh for expert technical assistance. CD spectroscopy and data analysis were performed under the direction of Dr. Mark Manning and Jeff Meyer in the Manning laboratory (University of Colorado Health Sciences Center). In addition we thank Drs. David Ross, Andrew Kraft, and Carlos Catalano for critical readings of the manuscript and valuable suggestions.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported through grants from the Arthritis Foundation and the
American Diabetes Association. To whom correspondence should be
addressed. Tel.: 303-315-194; Fax: 303-315-0274; E-mail: robert. scheinman{at}uchsc.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.C000526200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GC, glucocorticoids; GR, GC receptor; GRE, GC-responsive element; AP-1, activating protein 1; DBD, DNA binding domain; GST, gluthathione S-transferase; DMEM, Dulbecco's minimal essential medium; DEX, dexamethasone; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Giguere, V., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1986) Cell 46, 645-652[Medline] [Order article via Infotrieve] |
2. | Barnes, P. J., and Adcock, I. (1993) Trends Pharmacol. Sci. 14, 436-441[Medline] [Order article via Infotrieve] |
3. | Diamond, M. I., Miner, J. N., Yoshinaga, S. K., and Yamamoto, K. R. (1990) Science 249, 1266-1272[Medline] [Order article via Infotrieve] |
4. | Jonat, C., Rahmsdorf, H. J., Park, K. K., Cato, A. C., Gebel, S., Ponta, H., and Herrlich, P. (1990) Cell 62, 1189-1204[Medline] [Order article via Infotrieve] |
5. | Schule, R., Rangarajan, P., Kliewer, S., Ransone, L. J., Bolado, J., Yang, N., Verma, I. M., and Evans, R. M. (1990) Cell 62, 1217-1226[Medline] [Order article via Infotrieve] |
6. | Yang-Yen, H. F., Chambard, J. C., Sun, Y. L., Smeal, T., Schmidt, T. J., Drouin, J., and Karin, M. (1990) Cell 62, 1205-1215[Medline] [Order article via Infotrieve] |
7. | Caldenhoven, E., Liden, J., Wissink, S., Van de Stolpe, A., Raaijmakers, J., Koenderman, L., Okret, S., Gustafsson, J. A., and Van der Saag, P. T. (1995) Mol. Endocrinol. 9, 401-412[Abstract] |
8. | Ray, A., and Prefontaine, K. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 752-756[Abstract] |
9. | Scheinman, R. I., Cogswell, P. C., Lofquist, A. K., and Baldwin, A. S., Jr. (1995) Science 270, 283-286[Abstract] |
10. | Scheinman, R. I., Gualberto, A., Jewell, C. M., Cidlowski, J. A., and Baldwin, A. S., Jr. (1995) Mol. Cell. Biol. 15, 943-953[Abstract] |
11. | Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A., and Karin, M. (1995) Science 270, 286-290[Abstract] |
12. | Cato, A. C., and Wade, E. (1996) Bioessays 18, 371-378[Medline] [Order article via Infotrieve] |
13. |
Barnes, P. J.,
and Karin, M.
(1997)
N. Engl. J. Med.
336,
1066-1071 |
14. | Konig, H., Ponta, H., Rahmsdorf, H. J., and Herrlich, P. (1992) EMBO J. 11, 2241-2246[Abstract] |
15. |
Wrange, O.,
Eriksson, P.,
and Perlmann, T.
(1989)
J. Biol. Chem.
264,
5253-5259 |
16. |
Dahlman-Wright, K.,
Wright, A.,
Gustafsson, J. A.,
and Carlstedt-Duke, J.
(1991)
J. Biol. Chem.
266,
3107-3112 |
17. | Helmberg, A., Auphan, N., Caelles, C., and Karin, M. (1995) EMBO J. 14, 452-460[Abstract] |
18. | Heck, S., Kullmann, M., Gast, A., Ponta, H., Rahmsdorf, H. J., Herrlich, P., and Cato, A. C. (1994) EMBO J. 13, 4087-4095[Abstract] |
19. | Brostjan, C., Anrather, J., Csizmadia, V., Natarajan, G., and Winkler, H. (1997) J. Immunol. 158, 3836-3844[Abstract] |
20. |
Brack, A.,
Rittner, H. L.,
Younge, B. R.,
Kaltschmidt, C.,
Weyand, C. M.,
and Goronzy, J. J.
(1997)
J. Clin. Invest.
99,
2842-2850 |
21. |
Sheppard, K. A.,
Phelps, K. M.,
Williams, A. J.,
Thanos, D.,
Glass, C. K.,
Rosenfeld, M. G.,
Gerritsen, M. E.,
and Collins, T.
(1998)
J. Biol. Chem.
273,
29291-29294 |
22. | Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414[Medline] [Order article via Infotrieve] |
23. |
Lee, S. K.,
Kim, H. J.,
Na, S. Y.,
Kim, T. S.,
Choi, H. S.,
Im, S. Y.,
and Lee, J. W.
(1998)
J. Biol. Chem.
273,
16651-16654 |
24. |
Heck, S.,
Bender, K.,
Kullmann, M.,
Gottlicher, M.,
Herrlich, P.,
and Cato, A. C.
(1997)
EMBO J.
16,
4698-4707 |
25. | Unlap, M. T., and Jope, R. S. (1997) Brain Res. Mol. Brain Res. 45, 83-89[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Vanden Berghe, W.,
De Bosscher, K.,
Boone, E.,
Plaisance, S.,
and Haegeman, G.
(1999)
J. Biol. Chem.
274,
32091-32098 |
27. |
De Bosscher, K.,
Vanden Berghe, W.,
Vermeulen, L.,
Plaisance, S.,
Boone, E.,
and Haegeman, G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3919-3924 |
28. | Rosenthal, N. (1987) Methods Enzymol. 152, 704-720[Medline] [Order article via Infotrieve] |
29. | Duke, R. C., and Cohen, J. J. (1991) in Current Protocols in Immunology (Sevach, E. M. , and Strober, W., eds) , pp. 3.17.1-3.17.3, Green Publishing/Wiley-Interscience, New York |
30. | Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve] |
31. | Baumann, H., Paulsen, K., Kovacs, H., Berglund, H., Wright, A. P., Gustafsson, J. A., and Hard, T. (1993) Biochemistry 32, 13463-13471[Medline] [Order article via Infotrieve] |
32. | Luisi, B. F., Xu, W. X., Otwinowski, Z., Freedman, L. P., Yamamoto, K. R., and Sigler, P. B. (1991) Nature 352, 497-505[CrossRef][Medline] [Order article via Infotrieve] |
33. | Norman, M. R., and Thompson, E. B. (1977) Cancer Res. 37, 3785-3791[Abstract] |
34. |
Rogatsky, I.,
Hittelman, A. B.,
Pearce, D.,
and Garabedian, M. J.
(1999)
Mol. Cell. Biol.
19,
5036-5049 |
35. | Reichardt, H. M., Kaestner, K. H., Tuckermann, J., Kretz, O., Wessely, O., Bock, R., Gass, P., Schmid, W., Herrlich, P., Angel, P., and Schutz, G. (1998) Cell 93, 531-541[Medline] [Order article via Infotrieve] |