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Department of Biochemistry and Molecular Biology and Stanley S. Scott Cancer Center Louisiana State University Medical Center New Orleans, Louisiana 70112-1393
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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40%, which accounts for
two-thirds of the overall down-regulation observed at the mRNA level.
This reduction of c-jun promoter activity was abolished
after deletion of the region between -1780 to -63, where two AP-1
sites (-182 and -64) are located. Site-directed deletion of these
AP-1 sites reduced the basal activity of the c-jun promoter
and prevented repression by DEX. Repression of the c-jun
gene is due to the transrepression activity of the glucocorticoid
receptor (GR), as determined using GR mutants lacking this activity.
Overexpression of cJun overcame the negative effect of DEX, suggesting
that down-regulation of the c-jun gene by hormone is
mediated by the interaction between the GR and the cJun protein. These
studies are the first to show that glucocorticoids can repress
c-jun promoter activity through the AP-1 sites in the
c-jun promoter in mouse fibroblast cells. They also suggest
that inhibition of cell proliferation by glucocorticoids may be due not
only to the interference with AP-1 activity on other cellular genes,
but also because of a direct transcriptional suppression of
c-jun gene expression by the GR. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Glucocorticoid treatment causes a decrease in c-jun mRNA levels in many cell lines, although the degrees of inhibition and kinetics are different (9, 10, 11). The effect of glucocorticoids on the c-jun gene is a primary effect, as it occurs rapidly and is not inhibited by cycloheximide (9). Furthermore, nuclear run-on transcription assays revealed a rapid decrease in c-jun gene transcription rates (9), suggesting that down-regulation of the c-jun gene expression occurs at the transcriptional level.
Among important regulatory elements previously identified in the c-jun promoter are two AP-1 sites, a proximal AP-1 site (pAP-1) located between -71 and -64 in the c-jun promoter (5) and a distal AP-1 site (dAP-1) located between -190 and -183 (12). Preexisting cJun homodimers and cJun/ATF-2 heterodimers can bind, respectively, to these two AP-1 sites and activate transcription (5, 12). Both AP-1 sites are involved in transcriptional regulation in response to UV irradiation (8, 12), phorbol esters (5), or the E1A product of adenovirus (7, 13). It has been well established that glucocorticoids repress genes that are under the positive control of the AP-1 transcription factors (reviewed in Refs. 14, 15). The presence of these AP-1 sites within the c-jun promoter suggested that they may be the key elements involved in the response of the c-jun gene to glucocorticoids.
Based on previous studies, a transcriptional interference model has been proposed for hormone-mediated down-regulation of c-jun gene transcription, in which interference with AP-1 activity by the glucocorticoid receptor (GR) caused inhibition of c-jun gene expression (9). In this paper we show that the inhibitory effects of glucocorticoids on basal c-jun gene expression in mouse fibroblast cells are mediated via both the distal and proximal AP-1 sites in the promoter. Specific mutation of these AP-1 sites correlated with a significant reduction of basal promoter activity and with the loss of the glucocorticoid-mediated down-regulation. We provide evidence suggesting that repression of c-jun gene expression by glucocorticoids is due to the transrepression activity of the GR protein. In addition, overexpression of the cJun protein blunts the response of c-jun to glucocorticoids. Finally, given the role of cJun in cell proliferation (3, 4, 6, 16, 17, 18), we propose that repression of c-jun transcription represents an important mechanism for the antiproliferative effects of glucocorticoids.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The c-jun mRNA levels decreased to approximately 60% of
those observed in the control cells 2 h after hormone treatment
(Fig. 1
). They were reduced further as
the time of hormone treatment increased. This biphasic curve suggests
the interesting possibility that there are two kinetic processes
involved in DEX-mediated inhibition of c-jun gene
expression. Twenty-four hours after the addition of hormone, the
c-jun mRNA levels were only 30–40% of the control cells.
Thus, DEX treatment rapidly and dramatically inhibited c-jun
gene expression in a time-dependent manner.
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To identify functional elements involved in regulating c-jun
gene expression, we constructed a series of deletion mutations of the
c-jun promoter with variable 5'-ends. These promoter/LUC
constructs were transiently transfected into L929 cells. The activity
of the full-length promoter was arbitrarily defined as 100%. Deletion
from -1780 to -345 did not affect the basal transcriptional activity
of the promoter (Table 1). By contrast,
70% of the basal promoter activity was lost after deletion of the
region between -345 and -180. The region between -180 and -63
contributed the rest of the basal promoter activity. Therefore, the
elements responsible for the basal promoter activity seem to be located
between -345 and -63. We next studied DEX inhibition of the
c-jun promoter activity. Cells transfected with the
promoter/LUC constructs were treated with either ETOH or 1
µM DEX for 24 h. The promoter activity of each DNA
construct in ETOH-treated cells was defined as 100%. A
glucocorticoid-dependent decrease in c-jun promoter-driven
luciferase activity was observed (Fig. 2). The reporter construct containing a
full-length promoter sequence exhibited about a 40% reduction of the
luciferase activity. Deletion from -1780 to -180 did not
significantly change the DEX effect on the c-jun promoter,
while further deletion to -63 abolished down-regulation of the
c-jun promoter by DEX. Thus, it appears that the critical
elements for glucocorticoid responsiveness reside in the region from
-180 to -63 in the c-jun promoter.
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To study how these two AP-1 sites contribute to the basal promoter
activity and the hormonal response, we specifically mutated or deleted
the AP-1 sites in the context of the full-length promoter. Again, the
basal promoter activity of the full-length, wild-type c-jun
promoter was defined as 100%. Mutation of the pAP-1 site reduced the
basal activity to about 60%, while deletion of this site reduced it to
about 40%, of the control levels (Table 1
). The dAP-1 site is also
required for basal promoter activity since the c-jun
promoter lost 40% of its activity after deletion of this site.
Finally, only 22% of the promoter activity remained after both AP-1
sites were deleted. Taken together, these results demonstrate that both
AP-1 sites are essential in contributing to the basal activity of the
c-jun promoter (Table 1).
The GR represses a number of different genes by interacting with
transcription factors, including the AP-1 transcription factor (14, 15). Since the c-jun AP-1 sites play important roles in
basal promoter activity, it seemed feasible that they also mediate the
down-regulation of the c-jun gene expression by DEX. To
examine whether the AP-1 sites are also the targets for repression by
glucocorticoids, we transiently transfected L929 cells with
promoter/LUC constructs containing either mutated or deleted AP-1
sites. Again, incubation of the transfected cells with DEX led to a
significant decrease in the activity of the full-length, wild-type
promoter (Fig. 3). Deletion or mutation
of either the pAP-1 or the dAP-1 site alone did not significantly alter
the DEX responsiveness of the promoter. By contrast, the promoter/LUC
construct (dpAP-1 Dels) from which both AP-1 sites had been deleted was
not repressed at all by DEX (Fig. 3). Thus, either AP-1 site within the
c-jun promoter is sufficient to mediate DEX inhibition of
c-jun gene expression. Only when both sites are mutated is
DEX repression on c-jun promoter activity lost.
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Repression of c-jun Gene Expression by DEX Is Strictly
Dependent on the Presence of the Functional GR Protein
To study the role of the GR protein in down-regulation of
c-jun gene expression, we analyzed the c-jun mRNA
in the E8.2 cell, a mouse L929 fibroblast variant that does not express
any endogenous GR protein (23). DEX treatment did not decrease
c-jun mRNA levels in these cells (Fig. 4), suggesting that the functional GR
protein is necessary for this process. A rat GR expression plasmid was
then stably transfected into these cells to reconstitute the GR protein
level. A derivative cell line, E8.2/GR3, was obtained from a single
transfected cell; the expression of the rat GR protein in this line is
controlled by tetracycline via the tetracycline-regulated expression
system (24). Therefore, the GR protein levels can be modulated by
tetracycline (Tc) (25). Forty-eight hours after removal of Tc, E8.2/GR3
cells express rat GR protein equivalent to that in wild-type L929 cells
(25). In these cells, we observed a rapid down-regulation of the
c-jun mRNA 2 h after the addition of hormone (Fig. 4).
The c-jun mRNA levels were suppressed about 40% by DEX
treatment. This pattern of down-regulation resembles that seen in the
wild-type L929 cells (Fig. 1). These results indicate that functional
GR protein is required for the hormone-mediated inhibition of
c-jun gene expression.
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and
phGRSB), GR mutants (S425G, L436V) that lack transrepression activity
(32), and GR
463–473, in which 11 amino acids from the DNA
binding domain were deleted. Using DNA sequence analysis, we discovered
that the original S425G mutant was in fact a double mutant
(S425G/E427G). In our transfection experiments, expression of the GR
protein was again under the control of Tc. In the presence of 1 µg/ml
Tc, no GR protein was expressed in these stably transfected cells.
Removal of Tc from the culture medium stimulated expression of
the GR protein (Fig. 5A). In the
cells expressing wild-type GR protein (GR3, pRShGR, and phGRSB), we
observed a 35–40% decline in c-jun mRNA levels after
hormone treatment (Fig. 5B). The mutant GR containing a conservative
amino acid substitution (L436V), which did not transrepress the 5x TRE
TATA CAT reporter gene (32), still gave the same transrepression of the
c-jun gene as wild-type GRs (Fig. 5B). This suggests that
the repression of the endogenous c-jun gene promoter is
opposite from that of the 5x TRE TATA CAT reporter gene, which
contains a highly artificial promoter. Similar to the c-jun
promoter, cadmium-induced expression of the heme oxygenase promoter was
transrepressed by the GR (L436V) mutant (J. Alam, unpublished data),
which further suggests that transrepression mutants of the GR may or
may not be effective, depending upon the actual AP-1 site sequence in
the DNA and the promoter context in which the AP-1 site resides.
However, the c-jun mRNA level was not affected by DEX in the
cells expressing the double-mutant GR (S425G/E427G) and the deletion
mutant GR463–473. The double mutant still exhibited
transactivation activity, as confirmed by transient transfection
experiments with an MMTV-CAT reporter gene (J. Alam, unpublished data).
On the other hand, the GR463–473 deletion mutant completely lost
both transactivation and transrepression activities. Thus, these
results clearly demonstrate that the repression of
c-jun gene expression by glucocorticoids requires GR that is
functional for its transrepression function. The prediction that this
is due to interference of the AP-1 activity by the GR protein was
tested next.
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If the cJun protein is the molecular target in the repression of
c-jun gene expression by DEX, then overexpression of cJun
should relieve the repression. We used a cJun expression vector to
examine this possibility. A full-length c-jun promoter/LUC
plasmid was transiently transfected alone or together with a cJun
expression plasmid (CMV-cJun) into mouse fibroblast NIH 3T3 cells. DEX
treatment reduced the promoter activity by 35% in the control cells
(Fig. 6). Furthermore, overexpression of
the cJun protein alleviated the repressive effect of GR on the
c-jun promoter. The cJun effect is dose-dependent; a slight
relief of inhibition was seen with 0.5 µg of the cJun expression
vector, whereas a complete prevention of inhibition was attained when a
larger amount of plasmid (1.5 µg) was used (Fig. 6). Thus, production
of excess cJun protein can overcome the DEX effect, suggesting that GR
interferes directly with cJun activity to inhibit c-jun gene
expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The expression of c-jun is rapidly and dramatically
down-regulated by glucocorticoids in many cell lines (9, 10, 11). Here we
report that repression of c-jun gene expression by DEX is a
direct result of decreased c-jun promoter activity. The 60%
decrease in steady-state c-jun mRNA levels (Fig. 1) is at
least partially responsible for the 75–90% decrease in cJun protein
levels (10) seen in glucocorticoid-treated L929 cells. Deletion of
proximal and distal AP-1 sites in the c-jun promoter
abolishes the responsiveness of the c-jun gene to
glucocorticoids, indicating that both are responsible for the
down-regulation. Inhibition of the c-jun gene requires
functional GR protein, and it is due to the transrepression activity of
the GR. The inhibitory effect of glucocorticoids on the
c-jun promoter is blocked by overexpression of the cJun
protein. These data support a transcriptional interference model (Fig. 7), in which binding of the AP-1 proteins
and other transcription factors to the c-jun promoter drives
the basal transcription of the c-jun gene. In the presence
of hormone, GR is activated by hormone binding and is released from
associated proteins, such as hsp90 and hsp56 and p23. GR monomers then
could form heteromeric complexes with cJun proteins, which may be
prebound to the promoter. Thus, GR/cJun protein-protein interactions
may modulate the AP-1 activity, finally causing down-regulation of the
c-jun gene. It must be emphasized that glucocorticoid
treatment does not universally down-regulate c-jun gene
expression. Indeed, we (10) and others (36) have shown that the hormone
increases c-jun mRNA and protein levels in the human CEM-C7
T-lymphoblast cell line, and cJun up-regulation is necessary for
hormone-mediated apoptosis in these cells (36). Thus, it may be that in
cells that respond homeostatically to the GR and cJun pathways, the
hormone suppresses c-jun gene expression, while in those
that terminally differentiate and undergo apoptosis there is a
positive, reinforcing cross-talk between the two signal transduction
pathways. Whether this latter situation extends past the T lymphoblast
cell remains to be determined, as does the molecular mechanism for
hormone-induced up-regulation of c-jun gene expression.
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It was found that the occupancy of AP-1 sites was unchanged during induction and subsequent repression of the c-jun promoter by TPA and UV irradiation (44) or serum growth factors (45). Further, in vivo deoxyribonuclease footprinting showed that occupancy of the collagenase gene promoter AP-1 site is unaltered by DEX treatment, even though expression of the collagenase gene is strongly suppressed (46). Finally, DEX-mediated inhibition of the c-jun gene does not alter DNA-protein interactions at the AP-1 site in vitro (47). Therefore, it is likely that the GR interacts with the cJun protein while it is bound to DNA in an AP-1/DNA complex. This represents a refinement of the transcriptional interference model we presented previously for GR repression of c-jun gene expression, which suggested a disruption of the AP-1 complex from the AP-1 site in the promoter (9). An alternative hypothesis is that GR competes for a common coactivator that is required for the activity of other transcription factors. For example, recent studies showed that P300/CBP (CREB binding protein) is required for transcriptional activation by both the GR and the AP-1 transcription factor (48). It was proposed that competition for limiting amounts of CBP might account for inhibitory actions of the GR. Whether or not there is a role for CBP in DEX-mediated transrepression of c-jun gene expression remains to be determined. Our studies clearly show that cJun overexpression is sufficient to overcome DEX-mediated transrepression of the c-jun gene. This strongly suggests that GR/cJun protein-protein interactions are important in the transrepression mechanism.
Glucocorticoids inhibit proliferation of a variety of cultured cell lines, including L929 fibroblasts (49, 50), and they are also used as antineoplastic agents (51, 52). Although antagonism between the proliferative function of AP-1 factors and the differentiative function of various nuclear receptors has been frequently noted, little is known about the mechanism by which glucocorticoids inhibit the proliferation of cells. The antiproliferative effects of glucocorticoids are believed to be mediated by the GR (50), and it could be due to inhibition of AP-1 activity (29).
Many studies of cJun function suggest that it plays an important role
in cellular growth. First, the c-jun gene is an
early-response gene that is rapidly induced in many cell types in
response to mitogens such as serum, epidermal growth factor,
transforming growth factor-, and platelet-derived growth factor
(3, 4, 5, 6, 16). Second, higher c-jun mRNA levels were observed
in logarithmically growing cells than in serum-starved cells (5).
Third, the c-jun gene is rapidly increased during transition
of fibroblasts from G0 to G1 (3, 16, 53, 54).
Fourth, the expression of c-jun appears to be required for
cell cycle progression in fibroblasts (4, 17, 55), and its inhibition
causes a reversible cell cycle arrest (56). Finally, expression of
c-jun in retinal tissue is high at early embryonic ages, and
it decreases during development as cell proliferation declines and
ceases (57). These observations support the idea that cJun may control
the expression of genes involved in cellular proliferation.
The potent effects of glucocorticoids on cell proliferation may occur by regulating the expression of the AP-1-containing genes, including c-jun. GR interferes with the activity of the AP-1 transcription factor (26, 28, 29, 35). This interference, in turn, causes repression of the c-jun gene itself via the AP-1 sites within the c-jun promoter. Thus, in addition to proliferative genes located downstream of c-jun, expression of the c-jun gene itself may be a primary target for the antiproliferative effect of glucocorticoids. This cross-talk could represent one mechanism by which the proliferative effects of cJun are homeostatically counterbalanced by the antiproliferative effects of the glucocorticoid/GR complex, and vice versa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, and phGRSB) or mutant GR protein
(S425G/E427G, L436V, and GR463–473) were maintained in the
presence of 1 µg/ml Tc (Sigma Chemical Co., St. Louis, MO), 200
µg/ml of G418 (Geneticin, GIBCO, Grand Island, NY), and 200 µg/ml
of Hygromycin B (Sigma). All cells were grown at 37 C in a humidified
incubator under 6% CO2.
Plasmid Constructs
JAC. 1 (provided by Dr. Daniel Nathans, the Johns Hopkins
University School of Medicine, Baltimore, MD) and pL7Bgl200 (provided
by Dr. Robert P. Perry, the Institute for Cancer Research, Fox Chase
Cancer Center, Philadelphia, PA) were used to generate riboprobes to
determine the abundance of the c-jun and the mouse L7
ribosomal protein mRNA transcripts, respectively. pBL3 c-jun
-1.1/+740 and pTATACAT c-jun -1.6/-132
HindIII, NCO I were kindly provided by Dr. Andrew S. Kraft
(University of Colorado Health Sciences Center, Denver, CO). The tTA
(tet repressor) expression plasmid, pUHD15.1 neo, and the target
vector, pUHD10–3, were provided by Dr. H. Bujard (University of
Heidelberg, Heidelberg, Germany). Wild-type human GR (pRShGR
and phGRSB) and the mutants (L436V and S425G), provided by Dr. Andrew
C. B. Cato (Forschungszentrum Karlsruhe GmbH, Karlsruhe,
Germany), were digested with KpnI and then filled in
with T4 DNA polymerase. The DNAs were further digested with
DraI. The 2.5-kb fragment was cloned into pUHDBG (25), which
had been digested with BamHI, treated with calf intestinal
alkaline phosphatase, and then filled in with the Klenow fragment of
DNA polymerase. Construction of the GR463–473 plasmid will be
described elsewhere (J. Alam, in preparation). The cJun expression
plasmid, CMV-jun, was provided by Dr. Tom Curran (St. Jude Children’s
Research Hospital, Memphis, TN). CMV-ßGal, a ß-galactosidase
expression plasmid, was provided by Dr. Grant R. MacGregor (Baylor
College of Medicine, Houston, TX). The luciferase reporter gene,
pGL3-Basic vector, was purchased from Promega (Madison, WI), and
pBluescript II SK- (pBSSK-) was purchased
from Stratagene (La Jolla, CA). The puromycin-N-acetyl
transferase expression plasmid, pPUR, was purchased from CLONTECH (Palo
Alto, CA).
PCR
For PCR, 100 ng human genomic DNA were used. The sense
oligonucleotide was: 5'-GAGAATTCCAAGTTCAGAAGCAG-3'; the antisense
oligonucleotide was: 5'-GAGCTACCCGGCTTTGAAAAGT-3'. An XhoI
half-site was added to the 5'-end of each oligonucleotide. The genomic
DNA was denatured at 94 C for 2 min. Amplification was performed at 94
C for 10 sec, at 65 C for 30 sec, at 68 C for 2 min for 10 cycles; and
then at 94 C for 10 sec, at 65 C for 30 sec, at 68 C for 2 min plus
cycle elongation of 20 sec for each cycle (e. g. cycle
no. 11 has in addition 20 sec; cycle no. 12 has in addition 40 sec,
etc.) for 20 cycles; and, finally, at 68 C for 7 min. The polymerase
from the Expand Long Template PCR System (Boehringer Mannheim,
Indianapolis, IN) and GeneAmp 10x PCR Buffer II and MgCl2
Solution (Perkin Elmer, Foster City, CA) were used to perform the
reaction. The resulting fragment (-1780 to +731) was ligated, digested
with XhoI, and then cloned into the XhoI site of
the pGL3 basic vector (Promega, Madison, WI). The entire promoter was
sequenced using the Thermo Sequenase radiolabeled terminator cycle
sequencing kit (Amersham, Arlington Heights, IL).
Construction of Progressive Promoter-Luciferase Plasmid
Deletions
The -1780/+730 promoter/LUC plasmid was digested with
Mlu I. The 5'-overhang was filled in with deoxy
thioderivatives by Klenow polymerase. The DNA was then digested with
Avr II. The double digested DNA was treated with Exonuclease
III (Stratagene, La Jolla, CA) for 1–8 min. Mung Bean nuclease
(Stratagene) was used to create blunt ends. The DNA was ligated and the
deletion promoter/LUC constructs were confirmed by DNA sequence
analysis.
Site-Directed Mutagenesis
Site-directed mutagenesis was performed using the Muta-Gene
in vitro Mutagenesis Kit (Bio-Rad Laboratories, Hercules,
CA). The oligonucleotides used were as follows: pAP-1 mutation:
5'-ATAGCCCATGGTGGATCCCCAAGGCCT-3'; pAP-1 deletion:
5'-CCTAAAAATAGCCCACCCCAAGGCCTTCCC-3'; dAP-1 deletion:
5'-GGAGGCTCACGGGTCGTCCGCTGCCCTC-3'. All mutations were confirmed by DNA
sequence analysis.
Transfection
Transient transfections were performed by using LipofectAmine
(GIBCO, Grand Island, NY) in six-well plates. L929 cells were
transiently transfected with 3 µg c-jun promoter construct
and 1 µg CMV-ßGal plasmid per well of the six-well plate. Twenty
hours after transfection, DEX was added to the cells to a final
concentration of 1 µM for an additional 24 h.
Promoter activities were determined by measuring the luciferase
activity, which was assayed with a luminometer (Microlite 2250, Dynex
Technologies, Chantilly, VA) following the protocol provided by
Analytical Luminescence Laboratory (San Diego, CA). Variations in
transfection efficiency were normalized by assaying ß-galactosidase
activity with Galacto-Light (TROPIX, Inc., Bedford, MA) (58). In NIH
3T3 cells, a total of 4 µg DNA was used, which consisted of 2 µg
promoter-luciferase plasmid, 0.5 µg CMV-ßGal, and 0.5 or 1.5 µg
CMV-cJun expression plasmid. pBluescript II SK-
(pBSSK-, Stratagene) was used as carrier DNA to keep the
amount of total DNA constant. The transfected cells were treated with
DEX and the luciferase activity was assayed to determine the promoter
activity.
Using the CaPO4 precipitation technique (59), 13 µg of
the pUHDBG/pRShGR, pUHDBG/phGRSB, pUHDBG/S425G, or pUHDBG/L436V
plasmids were introduced into a 10-cm dish of E8.2 T4 cells, which
express an appropriate amount of tTA protein (25). Two micrograms of
the puromycin-N-acetyl transferase expression plasmid, pPUR,
was also cotransfected into the cells. Puromycin-resistant clones were
screened for the expression of the GR protein using Western blot
analysis.
Western Blot Analysis
Cells were cultured in the absence of Tc for 24 h (GR3),
48 h (pRShGR, phGRSB, L436V, and GR463–473), or 96
h (S425G/E427G). They were then treated with either ETOH only or 1
µM DEX for an additional 24 h. Whole-cell extracts
were prepared from the same flask of cells treated with ETOH.
Additionally, whole-cell extracts were also prepared from cells
cultured in the presence of Tc. The protein samples were subjected to
Western blot analysis as described previously (9). The PA1–512
antibody (Affinity BioReagents, Golden, CO) and the BuGR2 antibody (60)
were used to detect the human and rat GR proteins, respectively.
RNA Purification and Ribonuclease Protection Assay
Total cellular RNA was isolated using TRI Reagent (Molecular
Research Center, Inc.). To generate riboprobes, JAC.1 was linearized
with PvuII, and pL7Bgl200 was linearized with
XbaI. The linearized DNA templates were used to perform
in vitro transcription using a MAXIscript kit (Ambion, Inc.,
Austin, TX). T7 RNA polymerases were used to generate both the
c-jun and L7 probes. The specific activity of the L7 probe
was 0.6% of that of the c-jun probe because of the
difference in the expression levels of these two RNAs, which were
quantified in the same gel lanes. 32P-labeled RNA probes
were then hybridized with 15–20 µg of total RNA. Free probes were
removed using 100 U/ml RNase T1 (37 C, 30 min) (Ambion, Inc.). The
probes that hybridized to complementary RNA in the sample mixture were
protected from ribonuclease digestion, and the reaction products were
analyzed on a 6% polyacrylamide/7 M urea gel as described
elsewhere (61).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by NIH Grant DK-47211 (to W.V.V.) and a Student Research Grant from the Cancer Association of Greater New Orleans (to P.W.).
Received for publication January 28, 1998. Revision received May 6, 1998. Accepted for publication May 8, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 E. H. RABBITT, G. G. LAVERY, E. A. WALKER, M. S. COOPER, P. M. STEWART, and M. HEWISON Prereceptor regulation of glucocorticoid action by 11{beta}-hydroxysteroid dehydrogenase: a novel determinant of cell proliferation FASEB J, January 1, 2002; 16(1): 36 - 44. [Abstract] [Full Text] [PDF]
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K. De Bosscher, W. Vanden Berghe, and G. Haegeman Glucocorticoid Repression of AP-1 Is Not Mediated by Competition for Nuclear Coactivators Mol. Endocrinol., February 1, 2001; 15(2): 219 - 227. [Abstract] [Full Text]
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 C. Lethimonier, G. Flouriot, Y. Valotaire, O. Kah, and B. Ducouret Transcriptional Interference Between Glucocorticoid Receptor and Estradiol Receptor Mediates the Inhibitory Effect of Cortisol on Fish Vitellogenesis Biol Reprod, June 1, 2000; 62(6): 1763 - 1771. [Abstract] [Full Text]
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 A. Meissner, I. Luss, N. Rolf, P. Boknik, U. Kirchhefer, V. Kehm, J. Knapp, B. Linck, H. Luss, F. U. Muller, et al. THE EARLY RESPONSE GENES C-JUN AND HSP-70 ARE INDUCED IN REGIONAL CARDIAC STUNNING IN CONSCIOUS MAMMALS J. Thorac. Cardiovasc. Surg., April 1, 2000; 119(4): 820 - 825. [Abstract] [Full Text] [PDF]
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 J. P. Tuckermann, H. M. Reichardt, R. Arribas, K. H. Richter, G. Schutz, and P. Angel The DNA Binding-Independent Function of the Glucocorticoid Receptor Mediates Repression of Ap-1-Dependent Genes in Skin J. Cell Biol., December 27, 1999; 147(7): 1365 - 1370. [Abstract] [Full Text] [PDF]
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