(Received for publication, September 12, 1995; and in revised form, December 26, 1995)
From the
Interactions between transcription factors are an important
means of regulating gene transcription. The present study describes the
mutual repression of two transcription factors, the RelA(p65) subunit
of NF-B and the progesterone receptor (PR). This trans-repression is shown to occur independent of PR isoform,
reporter construct, or cell type used. Together with the demonstration
of an interaction between PR and RelA in vitro, these findings
suggest that the mutual repression is due to a direct interaction
between these proteins. Furthermore, activation of NF-
B by tumor
necrosis factor-
also results in repression of PR, while PR is
able to repress tumor necrosis factor-
-induced NF-
B activity.
Since NF-
B-regulating cytokine receptors are expressed in
progesterone target tissues, like breast and endometrium, the mutual
repression of PR and RelA could play an important role in a wide
variety of physiological processes in these tissues, including
maintenance of pregnancy, immunosuppression, and tumorigenesis.
The human progesterone receptor (PR) ()belongs to the
superfamily of steroid/thyroid hormone
receptors(1, 2, 3) . The transcription
factors of this family share (at least) two structurally related
functional domains, the DBD, which contains the so-called zinc finger
motif, and the more C-terminally located HBD. Two transactivation
domains have been mapped in the PR, of which one is located N-terminal
to the DBD. This transactivation domain, named AF-1, functions
autonomously, but the level of activity depends on the cell type and
reporter construct used(4, 5) . The second
transactivation domain AF-2 lies within the HBD, and its activity is
strictly dependent on the presence of ligand(4) . Detailed
analysis of the function of these transactivation domains of the PR has
also led to more insight in anti-hormone action. Anti-progestins bind
to the receptor without activating AF-2, and the partial agonistic
activity evoked by the anti-progestin RU486 is therefore the result of
its stimulation of AF-1 activity(4, 5) .
Within the
superfamily of steroid receptors, PR is unique in that it exists in two
isoforms, named A and B(6) , which differ in their N terminus.
Differences in transcriptional activity between the PR (amino acids 1-933) and PR
(amino acids
164-933) isoforms have been observed, depending on the cell type
and the reporter construct used(4, 5) .
Functioning
of transcription factors can be modified by interplay with
transcription factors of a different type, resulting in either an
inhibitory or stimulatory effect. In particular, interactions between
steroid receptors and members of the AP1 family of transcription
factors have been studied extensively. AP1 family members, like c-Jun
and c-Fos, and GR have been shown to repress each others functioning (7, 8, 9, 10, 11) , but the
actual mechanism of repression is currently controversial. In
vitro, a direct interaction between GR and AP1 was shown,
resulting in impaired DNA binding(7, 9) , while others
failed to detect a direct interaction (10, 11) or
found the two proteins to interact without DNA binding being
affected(8, 12) . It has clearly been demonstrated
that the magnitude of the repression of AP1 by GR and PR is cell type-
and promoter-specific(11, 13, 14) ,
suggesting that intermediary proteins are likely to be involved, the
expression of which can vary between different cell types. The
transcriptional activity of PR was shown to be affected by c-Jun in a
stimulatory or an inhibitory fashion, depending on the cell
type(11) , while c-Fos was shown to inhibit PR in all
cases(11) . Recently, GR (15, 16, 17, 18) , and also estrogen
receptor(19, 20) , have been shown to be inhibited by
members of a second family of transcription factors, the NF-B
family. These repressions were found to be mutual, and GR (15, 17, 18) and estrogen receptor (20) were shown to interact physically with NF-
B proteins in vitro. Several studies have indicated that, in the case of
GR, this interaction was shown to result in impaired DNA
binding(15, 16, 17) , but others failed to
find this (21) .
The NF-B family consists of
DNA-binding proteins that share homology in an N-terminal region of 300
amino acids, termed the Rel homology region. Amongst others, this
family includes RelA(p65), NFKB1(p50), and
c-Rel(22, 23) . NF-
B exists as a dimer, typically
a heterodimer of RelA and NFKB1(24) , but homodimers as well as
heterodimers of different composition are also possible. NF-
B is
present in an inactive form in the cytoplasm, where it is associated to
an inhibitory protein, I
B. Exposure of cells to a plethora of
stimuli, including cytokines (TNF-
and IL-1), lipopolysaccharide,
UV radiation, and oxidative stress, results in the dissociation of
I
B from the NF-
B complex, probably through a phosphorylation
event(25) , upon which NF-
B translocates to the nucleus.
Subsequently, binding to specific DNA sequences and activation of
transcription can occur. For some members of the NF-
B family, like
RelA (26) and c-Rel(27, 28) , C-terminally
located transactivation functions have been found.
Since NF-B
sites have been identified in the promoters of numerous genes that play
a role in cell proliferation and immune
response(22, 23) , and since progestins also play a
role in these processes(29, 30) we have investigated
the effects of NF-
B family members and PR on each others
transcriptional activity. Here we show the RelA subunit to specifically
inhibit the hormone-induced transactivation of PR, independent of
receptor isoform, reporter construct, or cell type used. Furthermore,
the repression is mutual, since PR is shown to repress the
transcriptional activity of RelA. Both the DBD and the HBD of PR are
shown to contribute to the repression. This trans-repression
between RelA and PR could play an important role in a large variety of
processes like maintenance of pregnancy, immunosuppression, and
tumorigenesis.
The CMV-4
expression vectors containing the cDNAs encoding human RelA, NFKB1, and
c-Rel have been described before(18) . The TA1 construct
of RelA, lacking amino acids 515-550 (numbering refers to
original cDNA(37) ), was made by cutting the cDNA at a unique SmaI site and ligating it into CMV-4.
The reporter plasmids
PREtkCAT(38) , MMTVCAT(39) , and
PRE
TATACAT (40) were kind gifts of Drs. Muller
(Martinsried, Germany), Evans (San Diego, CA), and Gronemeyer
(Strasbourg, France), respectively. The luciferase reporter containing
three NF-
B sites from the ICAM-1 promoter(21) , and
PDMLacZ (41) have been described before.
GST-PR (amino acids
457-933) was made by cutting the PR cDNA at an
internal HincII site and a BamHI site in the pSG5
vector and, after filling-in, ligated into SmaI-cut PGEX-2T
vector (Pharmacia, Uppsala, Sweden). GST-PR
C was cloned similarly,
using pSG5-PR
C to isolate the HincII-BamHI fragment. To create GST-NFKB1, PCR was
performed with 5`-tcccccgggcaccATGGCAGAAGATGATCC-3` as forward primer
and T3 as reverse primer on the SK
-NFKB1 vector. This
fragment was cut with SmaI and ligated into SmaI-cut
PGEX-2T. For in vitro translation, the coding region of RelA
was cut from the CMV4-RelA vector, using XbaI and HindIII sites outside the coding region, and cloned into XbaI-HindIII-cut pBluescript SK
.
Figure 1:
Repression of the hormone-induced
activity of the human PR by RelA. A, HeLa cells were
transiently transfected with 100 ng of PR (white
bars) or PR
(black bars) and 1.0 µg of
empty expression vector (CMV-4; -), 0.5 µg of empty vector and 0.5
µg of RelA or 1.0 µg of RelA. PRE
tkCAT (5 µg)
was used as reporter construct. Fold induction indicates
reporter activity in cells treated with 10 nM Org2058 over
untreated cells. Bars represent the mean of four to five
independent experiments ±S.E. B, HeLa cells were
transfected with PR
(white bars) or PR
(black bars), as under A, but now with 1.0
µg of empty expression vector (CMV-4; -) or the same vector
containing the RelA, NFKB1, or c-Rel cDNAs, as indicated. C,
cells were transiently transfected as under A, using MMTV-CAT
(5 µg) as reporter construct.
Next, transient
transfections were performed with a reporter construct containing the
MMTV promoter, which contains numerous PREs(38) . As shown
before(5) , a marked difference between the activities of
PR and PR
is observed, with PR
being a weak activator of this reporter construct (Fig. 1C). Again, hormone-induced transactivation by
both PR
and PR
is repressed by RelA (Fig. 1C). Similar results were obtained when a
reporter construct was used in which two PREs are located directly
upstream of a TATA box (data not shown). We conclude therefore that the
RelA subunit of NF-
B is capable of repressing both PR
and PR
, independent of the context of the PRE.
To
examine whether PR is also able to repress RelA, HeLa cells were
transfected with a reporter construct containing three NF-B sites
from the human ICAM promoter(40) , coupled to the thymidine
kinase promoter in front of luciferase(21) . Co-transfection
with RelA expression vector (100 ng) resulted in a modest but
significant activation of this construct (Fig. 2). When
expression vectors containing PR
or PR
(1
µg) were added, the RelA-mediated activity was already reduced in
the absence of hormone, while hormone addition resulted in a further
repression (Fig. 2). Similar results were obtained when 10-fold
lower amounts of RelA and PR expression vectors were used (data not
shown). Taken together, these results show that the repression between
PR and RelA is mutual and occurs on a variety of promoters.
Figure 2:
PR represses RelA-mediated
transactivation. HeLa cells were transiently transfected with 100 ng of
RelA, 1.0 µg of empty expression vector (pSG5; -), 0.5 µg
of empty vector, and 0.5 µg of PR or PR
, or
1 µg of the PR constructs. 3xNF-
BtkLuc (2 µg) was used as
reporter construct. Cells were treated with vehicle (white
bars) or with 10 nM Org2058 (black bars),
harvested and luciferase activity was measured. Depicted is the
induction of luciferase activity evoked by RelA over cells transfected
with empty expression vector.
In addition, the effects of PR and PR
on RelA-induced reporter activity were studied in the same cell
lines. The activation by RelA was much greater in COS-1 cells than the
modest activation in HeLa cells (Fig. 2), but also in these
cells both PR isoforms inhibited RelA transactivation and progestin
treatment resulted in a further repression of RelA-induced reporter
activity (Table 1). Furthermore, endogenous PR also represses
RelA, as shown in T47D cells. Although the transactivation and
repression potential of RelA and the PR isoforms differ between cell
lines, the mutual repression between these proteins is observed clearly
in all cell lines tested.
Figure 3:
Domains of PR and RelA involved in
repression. A, schematic representation of PR deletion
constructs. Numbers refer to original amino acid
sequence(35, 36) . B, HeLa cells were
transfected with 100 ng of expression vector containing
PRE or PR
AB1 as indicated in the absence or
presence of 1.0 µg of RelA expression vector. Assays were performed
as described in Fig. 1A. Fold induction indicates reporter activity in cells transfected with expression
vector containing PR
E over empty expression vector or
reporter activity in cells treated with 10 nM Org2058 over
untreated cells, in the case of PR
AB1. C, COS-1 cells
were transfected with 100 ng of RelA expression vector and 2 µg of
3xNF-
BtkLuc reporter, in the presence of various PR deletion
constructs (1 µg). Cells were treated with vehicle (white
bars) or with Org2058 (10 nM) and assayed for luciferase
activity. D, HeLa cells were transiently transfected with 100
ng of PR
(white bars) or PR
(black
bars) in the presence of 1 µg of empty expression vector
(CMV-4; -), RelA, or RelA
TA1, using PRE
tkCAT as
a reporter (5 µg). Depicted is the transactivation evoked by 10
nM Org2058.
To examine whether the transactivation function of
RelA is essential for the repression of PR, a deletion construct of
RelA was used that lacks the 30 most C-terminal amino acids, which
encode transactivation domain TA1(26) . Only a marginal
difference between the repressive activity on PR and
PR
of this construct compared with the full-length RelA
protein was observed (Fig. 3D). Similar results were
obtained in COS-1 and T47D cells (data not shown). Therefore, TA1 is
not essential for the repressive activity of RelA.
Figure 4:
Effects of anti-progestins on PR-RelA
interaction. A, HeLa cells were transfected with full-length
PR in the presence of CMV-4 or CMV-4/RelA as in Fig. 1A and treated with 1 µM RU486.
PRE
tkCAT (5 µg) was used as reporter construct. B, COS-1 cells were transfected with RelA vector in the
presence of empty expression vector (pSG5; -), PR
,
PR
, or PR
AB1, as in Fig. 3C, and
treated with 1 µM RU486 (black bars) or 1
µM ZK98299 (striped bars). 3xNF-
BtkLuc was
used as reporter construct.
To investigate whether
antagonist-occupied PR could also inhibit RelA-mediated transcription,
transfections in COS-1 cells were performed with the 3xNF-B
reporter described above. RU486 was able to induce PR
- or
PR
-mediated repression of RelA (Fig. 4B),
albeit to a lesser extent than the agonist Org2058 (Table 1). The
RU486-evoked repression was more easily observed when the PR
AB
construct of PR is used (Fig. 4B), which lacks the
hormone-independent repression observed with PR
and
PR
(Fig. 3C). While RU486 only exhibits
weak agonistic effects under certain specific conditions (Fig. 4A), the ``pure'' antagonist ZK98299 is
unable to evoke PR-mediated transactivation (data not shown). Like
RU486, this anti-progestin was able to repress RelA (Fig. 4B). Together with RelA-repression by
RU486-occupied PR
AB, which lacks AF-1 through which RU486 is
thought to exert its agonistic action(5) , these results
indicate that the enhancer and repressor functions of the two PR
isoforms are separate functions. This hypothesis is confirmed by our
findings that the transcriptional activity of the two PR isoforms
differs in HeLa and COS-1 cells, while the repressive effect on RelA is
independent of receptor-isoform and cell type (Table 1).
Figure 5:
RelA and PR are capable of direct
interaction. GST, GST-PR, GST-PRC, or GST-NFKB1, bound to
glutathione-agarose beads, were incubated with radiolabeled in
vitro translated RelA in the absence or presence of Org2058 (1
µM), as indicated. After extensive washing, proteins were
loaded on a 10% SDS-PAA gel. In lane 1, one-tenth of the total
input of in vitro translated RelA was loaded. Numbers on the right indicate molecular weight markers; arrowhead on
the left indicates the position of radiolabeled
RelA.
Figure 6:
Effects of RelA on PR phosphorylation.
COS-1 cells were transfected with PR or PR
expression vectors (1 µg) in combination with CMV-4 or
CMV-4/RelA (9 µg), as indicated. Subsequently, cells were treated
with vehicle(-) or 10 nM Org2058 (P),
harvested, and loaded on a 8% SDS-polyacrylamide gel. PR and RelA were
visualized on Western blot as described under ``Experimental
Procedures.''
To study if the PR/NF-B interaction could have
relevance for the in vivo situation, the influence of
NF-
B inducing reagents on the expression of a PR target gene was
examined. To this end, T47D cells were treated with Org2058 (10
nM) for 16 h in the absence or presence of TNF-
(250
units/ml) or H
O
(150 µM), and
total RNA was isolated. Northern blot analysis showed that the
induction of the progestin-induced fatty acid synthetase mRNA was
decreased both by TNF-
and H
O
(Fig. 7A), with TNF-
treatment resulting in a 30% decrease and H
O
in a 60% decrease of induction (Fig. 7B). Although the reduction by TNF-
was
relatively small, it is in line with the repression of PR mediated
transactivation by TNF-
in these cells (Table 2), which is
probably caused by their inherent low sensitivity to TNF-
. From
these results, we conclude that TNF-
-induced NF-
B activity
can repress PR functioning and vice versa and that this trans-repression is likely to be operational in vivo.
Figure 7:
Repression of progestin-induced fatty acid
synthetase mRNA expression by RelA inducing agents in T47D cells. A, cells were treated with the progestin Org2058 (P;
10 nM) in the absence or presence of TNF- (250 units/ml)
or H
O
(150 µM) for 16 h. Total RNA
was isolated, and Northern blotting was performed. Blots were probed
with a cDNA encoding the fatty acid synthetase gene and subsequently
with a glyceraldehyde 3-phosphate dehydrogenase probe as a control for
equal loading (10 µg/lane). B, graphic representation of
the repression of progestin-induced (P) fatty acid synthetase
mRNA expression by TNF-
(T) and H
O
(H), as determined by PhosphorImager quantification
after correction for the internal glyceraldehyde 3-phosphate
dehydrogenase control. The results are presented as the level of
induction with reference to the induction by progestin (100%). The bar diagram represents the average of two independent
experiments.
In the present study, we show mutual repression of the
hormone-activated PR and the RelA(p65) subunit of NF-B. This
mutual repression could be caused by several mechanisms. The first
possibility is that RelA and PR are able to bind to their respective
cognate DNA elements. However, treatment of T47D cells with progestins
did not result in specific complex formation on the NF-
B site from
the ICAM-1 promoter. (
)Also in the case of the mutual
inhibition of GR and AP1, no evidence was found for this
mechanism(7, 9, 10, 11) . Second, PR
and RelA could compete for common co-activators or transcription
intermediary factors, a process referred to as transcriptional
interference or squelching(53, 54) . This is unlikely,
since we found the repression of PR by RelA to occur independent of
receptor isoforms, transactivation functions of PR, reporter construct,
and cell type and since repression of PR by RelA was independent of the
main transactivation domain TA1 of RelA. A third possibility is that a
direct interaction between the two proteins, resulting in a
heterodimer, could account for the repression. In vitro association assays showed that the two proteins are indeed capable
of a direct physical interaction. Such a complex could either (i) be
unable to bind DNA or (ii) result in the formation of inactive
complexes on the DNA by preventing the interaction with essential
co-factors or the basal transcription machinery. Our data are in line
with the second mechanism, since TNF-
-induced DNA binding of
NF-
B was found to be unaffected by the presence of PR in gel
retardation assays.
Furthermore, we also found the
hormone-induced change in mobility of PR, which is caused by
DNA-dependent phosphorylation events(51, 52) , to
occur irrespective of the presence of RelA, indicating that binding of
PR to DNA is not prevented by RelA. Similarly, GR was shown to
interfere with AP1 activity without altering its DNA binding, as shown
by in vivo footprinting (12) .
As was also shown for the repression of AP1 by GR(7, 8, 9) , we found the C and E domains of PR to be essential for repression of RelA. With respect to the HBD, this domain is likely to function differently with respect to the enhancer and repressor functions of PR. The ability of this domain to enhance transcription is probably not essential for the repression of PR, since anti-progestin-occupied receptors, which are unable to evoke transactivation, still induced repression of RelA. Together with the cell type-specific differences between the two PR isoforms in transcriptional activity, but not in repressive function, these findings show that the ability to enhance or repress transcription are separate functions within the steroid receptor proteins.
Although the interactions of steroid receptors with
transcription factors of the AP1 and NF-B families share several
features, notable differences are also evident. The repressive action
of c-Fos was mainly directed against AF-2 of PR(11) , while we
show RelA to inhibit the transactivation of a PR construct lacking this
domain (PR
E). Second, the repression of AP1 proteins
by PR was shown to be cell type- and promoter-specific(11) ,
indicating that additional, promoter-specific proteins are involved
also, the expression of which could be different in various cell types.
While the transactivation potential of RelA and the PR isoforms was
shown to differ, the repression of RelA by PR appeared to be
independent of cell type and promoter context. Together with the
association between PR and RelA in vitro, these data indicate
that the mutual repression between PR and RelA could be due to a direct
interaction between the proteins, without the additional involvement of
other proteins.
Deletion analysis of RelA showed that the region
corresponding to TA1 (amino acids 515-550; (26) ) is not
required for the repression of PR, indicating that other regions of the
protein are involved in the interaction with PR. Preliminary results
indeed indicate that regions outside the TA1 domain of RelA are
essential for repression of PR and GR. ()This could explain
why the NFKB1(p50) and c-Rel proteins, which are substantially
different from RelA outside the Rel homology region, are unable to
repress PR (this study) and GR (18) .
The interaction
between PR and RelA is potentially important in organs in which the PR
together with NF-B-regulating cytokine receptors is expressed,
like mammary gland, ovarium, and endometrium. During pregnancy,
progesterone levels are high, and the presence of progesterone is
essential for the maintenance of pregnancy(30) . In endometrial
cells, cytokines, which induce NF-
B, like TNF-
and IL-1, and
their receptors are expressed, as well as PR(55) .
Progesterone-induced decidualization of endometrial cells, which is
thought to be important in maintaining pregnancy, is inhibited in
vitro by TNF-
and IL-1 in endometrial
cells(56, 57) . Second, the expression of IL-8, which
is both a chemotactic factor for neutrophils and causes them to secrete
lysosomal enzymes, is repressed by progesterone(58) . Since
these effects of IL-8 may, besides playing a role in inflammation, also
be an early step in the initiation of labor(59) , suppression
of its secretion prevents premature birth. The recent finding that in
mice that carry a null mutation of the PR, uterine inflammations occur
frequently (60) is in line with this hypothesis. Recently,
NF-
B-induced expression of IL-8 was shown to be repressed by GR (16) in a mechanism similar to what we propose for PR. Another
important function of progesterone during pregnancy is its
immunosuppressive effect, to prevent activation of an immune response
directed against the embryo(30) . A number of genes that are
important in the immune system have been shown to be regulated by
NF-
B(22, 23) . It is therefore possible that the
immunosuppressive action of progesterone during pregnancy is partly due
to the inhibition of the transcriptional activity of NF-
B.
The
negative cross-talk between RelA and PR could also play a role in cell
proliferation, since both NF-B (23) and progestins (29) have been implicated in this process. Several lines of
evidence suggest that constitutive activation of NF-
B contributes
to the malignant phenotype of tumor cells. First, a naturally occurring
splice variant of RelA, named p65
, was shown to transform Rat-1
cells(61) . Higgins et al.(62) have shown
proliferation and tumorigenicity of several tumor cell lines, including
the human breast tumor cell lines MCF7 and T47D, to be inhibited by
antisense oligonucleotides to RelA. In addition, activation of
NF-
B through the disruption of I
B
regulation, was shown
to result in malignant transformation(63) . In contrast to
their different ability to transactivate, both progestins and
anti-progestins can be used to treat breast tumors(64) .
Therefore, it seems reasonable to propose that trans-repression of NF-
B (this study) and
AP1(11) , which is induced by both types of ligand, could be
relevant for tumor inhibition.