A Dual Mechanism Mediates Repression of NF-
B Activity by Glucocorticoids
S. Wissink,
E. C. van Heerde,
B. van der Burg and
P. T. van der Saag
Hubrecht Laboratory Netherlands Institute for Developmental
Biology 3584 CT Utrecht, The Netherlands
 |
ABSTRACT
|
---|
Repression of nuclear factor (NF)-
B-dependent
gene expression is one of the key characteristics by which
glucocorticoids exert their antiinflammatory and immunosuppressive
effects. In vitro studies have shown protein-protein
interactions between NF-
B and the glucocorticoid receptor, possibly
explaining their mutual repression of transcriptional activity.
Furthermore, glucocorticoid-induced transcription of I
B
was
presented as a mechanism in mediation of immunosuppression by
glucocorticoids. At present, the relative contribution of each
mechanism has not been investigated. We show that dexamethasone induced
I
B
gene transcription in human pulmonary epithelial A549 cells.
However, this enhanced I
B
synthesis did not cause repression of
NF-
B DNA-binding activity. In addition, dexamethasone was still
able to inhibit the expression of NF-
B target genes
(cyclooxygenase-2, intercellular adhesion molecule-1) in the absence of
protein synthesis. Furthermore, we show that the antihormone RU486 did
not induce I
B
expression. However, RU486 was still able to
induce, albeit less efficiently, both glucocorticoid- and progesterone
receptor-mediated repression of endogenous NF-
B target gene
expression in A549 cells and the breast cancer cell line T47D,
respectively. Taken together, these results indicate that induced
I
B
expression accounts for only part of the repression of NF-
B
activity by glucocorticoids and progestins. In addition,
protein-protein interactions between NF-
B and the glucocorticoid or
progesterone receptor, resulting in repression of NF-
B activity,
seem also to be involved. We therefore conclude that NF-
B activity
is repressed via a dual mechanism involving both protein-protein
interactions and induction of I
B
.
 |
INTRODUCTION
|
---|
Glucocorticoids are widely used as immunosuppressive and
antiinflammatory agents. They have been shown to inhibit the expression
of cytokines, adhesion molecules, and enzymes involved in the
inflammatory process (1). Glucocorticoids mediate these effects through
an intracellular receptor, the glucocorticoid receptor (GR), a member
of the steroid/thyroid hormone receptor superfamily. Upon hormone
binding, the cytoplasmic GR can enter the nucleus, dimerize, and bind
to specific DNA sequences, the glucocorticoid response elements
(GREs), and activate transcription of target genes (2). However,
the antiinflammatory and immunosuppressive effects of
glucocorticoids are achieved by inhibition rather than by activation of
target gene expression. Many negatively regulated genes involved in the
inflammatory response do not contain GREs in their promoter and
therefore must be negatively regulated by a different mechanism,
e.g. through transcriptional interference between GR and
other transcription factors, such as AP-1 and nuclear factor (NF)-
B
(3, 4, 5).
The NF-
B/Rel family of transcription factors regulates the
expression of many genes involved in immune and inflammatory responses.
NF-
B was originally identified as a heterodimer of NF-
B1 and RelA
(6), but a variety of other
B/Rel homo- and heterodimers have now
been described. NF-
B is present in an inactive state in the
cytoplasm, sequestered by an inhibitor protein, designated I
B. After
stimulation of the cells, I
B becomes phosphorylated, ubiquitinated,
and subsequently degraded (7). As a result, NF-
B is free to
translocate to the nucleus and activate transcription of target genes.
In the nucleus, NF-
B can also induce the synthesis of I
B
,
which terminates the NF-
B response, explaining its transient nature
(8).
Glucocorticoids were shown to be potent inhibitors of NF-
B
activation. In addition, the NF-
B subunit, RelA, has been shown to
physically interact with GR in vitro (9, 10, 11, 12) as well as with
other steroid receptors, such as the estrogen receptor (ER; Ref.13),
the progesterone receptor (PR; Ref.14), and the androgen receptor (AR;
Ref.15). Since it has been demonstrated that NF-
B was also able to
repress ligand-dependent activation of steroid receptor-regulated
promoters in vitro, a mutually inactive complex formed
either by direct protein-protein interaction of the receptor and RelA
or via a third partner has been proposed (9, 10, 11, 12, 13, 14, 15).
A second independent mechanism through which glucocorticoids could
repress NF-
B activity has been described (16, 17). Glucocorticoids
were shown to induce transcription of the I
B
gene in HeLa cells,
monocytic cells, and T-lymphocytes. This induction resulted in
increased synthesis of I
B
protein, which is able to interact with
activated NF-
B, thereby terminating the NF-
B response. However,
Brostjan et al. (18) reported that glucocorticoid-mediated
repression of NF-
B activity did not involve induction of I
B
synthesis in endothelial cells.
The physiological relevance of both these mechanisms has not been
clearly established, and it remains unclear which pathway represents
the major mechanism. Therefore, we investigated the contribution of
each mechanism to the antiinflammatory and immunosuppressive activity
of glucocorticoids. Here we show that dexamethasone (Dex) induces
expression of I
B
in human pulmonary epithelial A549 cells.
Furthermore, we show that Dex is able to inhibit the expression of two
endogenous NF-
B target genes, cyclooxygenase-2 (COX-2) and
intercellular adhesion molecule-1 (ICAM-1) partially independent of
newly synthesized I
B
. On the basis of these results, we conclude
that glucocorticoids repress NF-
B activity in A549 cells via a dual
mechanism, which involves both protein-protein interaction and
induction of I
B
.
 |
RESULTS
|
---|
Glucocorticoids Induce I
B
in A549 Cells
Glucocorticoids have been described to induce I
B
synthesis
in monocytic and lymphocytic cells (16, 17), but not in endothelial
cells (18). To determine whether glucocorticoids increased I
B
mRNA in human pulmonary epithelial A549 cells, Northern blotting
analysis was performed on mRNA derived from these cells treated with
Dex for increasing periods of time. As shown in Fig. 1
, Dex induced an increase in I
B
mRNA in these cells, which peaked by 28 h (3- to 4-fold) and remained
elevated to 24 h (2-fold).
Repression of NF-
B-Regulated Genes Is Not Only Mediated by
I
B
Induction
To investigate the mechanism(s) involved in repression of NF-
B
activity by glucocorticoids, we determined the repression by Dex of
NF-
B-regulated genes in the absence of I
B
protein synthesis.
Treatment of A549 cells with interleukin (IL)-1ß resulted in a 5-fold
increase in I
B
mRNA expression, and a 3-fold induction was
observed after treatment with Dex. The combination of IL-1ß and Dex
showed a similar induction as IL-1ß treatment alone (Fig. 2A
, lanes 14). Dex-mediated I
B
induction and I
B
resynthesis after IL-1ß-induced degradation
can be observed for I
B
protein (Fig. 2B
, lanes 14), indicating
that IL-1ß and Dex can induce both I
B
transcription and protein
synthesis in A549 cells. Cycloheximide (CHX), an inhibitor of protein
synthesis, also induced I
B
mRNA expression (5-fold; Fig. 2A
, lane
5), as has been observed for other NF-
B target genes,
e.g. ICAM-1 (19). CHX together with IL-1ß superinduced
I
B
mRNA (29-fold; Fig. 2A
, lane 6), whereas CHX in combination
with Dex resulted in a weaker induction (9-fold; Fig. 2A
, lane 7). No
resynthesis of I
B
protein could be observed in the presence
of CHX (Fig. 2B
, lanes 6 and 8).

View larger version (37K):
[in this window]
[in a new window]
|
Figure 2. Repression of IL-1ß-Induced COX-2 and ICAM-1
Expression by Dex in the Absence of Protein Synthesis
A549 cells were treated with IL-1ß (I; 100 U/ml) and Dex (D; 1
µM) in the absence or presence of CHX (10 µg/ml) for
6 h. A, Total RNA was isolated and Northern blotting analysis was
performed. Left panel shows blot sequentially hybridized
with a probe containing I B and GAPDH cDNA, which serves as a
control for the amount of RNA loaded in each lane. The positions of the
transcripts of I B and GAPDH are indicated. Right
panel shows quantification of I B hybridization signal.
Fold induction indicates hybridization signal for cells treated with
IL-1ß, Dex, and/or CHX over untreated cells. B, Whole cell extracts
were prepared and fractionated on a 12.5% SDS-PAGE, and Western
blotting analysis was performed. Blots were immunostained with a
polyclonal antibody to I B . I B was visualized after
incubation with a peroxidase-conjugated second antibody and ECL. C,
Total RNA was isolated and Northern blotting analysis was performed.
Left panel shows blots sequentially hybridized with a
probe containing COX-2, ICAM-1, or GAPDH cDNA, which serves as a
control for the amount of RNA loaded in each lane. The positions of the
transcripts of COX-2, ICAM-1, and GAPDH are indicated. Right
panel shows PhosphoImager quantification of COX-2 (upper
panel) and ICAM-1 (lower panel) hybridization
signal. The relative induction, indicating hybridization signal of
cells treated with IL-1ß over untreated cells, in the absence
(black bars) and presence (white bars) of
CHX is set at 100%. Error bars indicate
SE.
|
|
To study whether protein synthesis was necessary for the repressive
effect of Dex on endogenous NF-
B target gene expression, COX-2 (20)
and ICAM-1 (21) mRNA expression was investigated. As shown in Fig. 2C
, IL-1ß induced the expression of both COX-2 and ICAM-1, which could be
strongly repressed by Dex (lanes 2 and 4). Interestingly, in the
absence of protein synthesis and I
B
protein induction, Dex was
still able to repress IL-1ß-induced COX-2 and ICAM-1 expression
(lanes 6 and 8), although the repression was less strong than in the
absence of CHX (Fig. 2C
, right panel). This suggests that
induction of I
B
plays a role, but is obviously not the only
mechanism mediating the repressive effect of Dex.
NF-
B DNA-Binding Activity Is Not Inhibited by Dex
It has been shown that Dex-induced I
B
was able to
inhibit NF-
B activity by preventing nuclear translocation and DNA
binding (16, 17). To determine whether Dex-induced I
B
could block
NF-
B DNA-binding activity in A549 cells, the cells were pretreated
with Dex for 15 h and subsequently treated with IL-1ß for 1
h. NF-
B binding to the radiolabeled probe containing the human
immunodeficiency virus (HIV) long terminal repeat (LTR) was observed
with nuclear extracts from cells treated with IL-1ß (Fig. 3
, lane 3). Pretreatment with Dex did not
result in inhibition of binding (lane 5). The same results were
obtained after pretreatment with Dex for 5 h (data not shown). The
observed binding activity was specific because it could be competed
with a 100-fold excess of unlabeled
B probe but not with a mutant
B probe (lanes 6 and 7). The
B-binding activity was composed
mostly of NF-
B1 and RelA heterodimer (NF-
B) as determined by
supershift analysis (lanes 8 and 9). The faster migrating complexes
appeared to contain NF-
B1 protein in other combinations.
These results show that, in A549 cells, Dex-induced I
B
is not
able to prevent nuclear translocation or DNA binding of NF-
B,
suggesting a minor contribution of I
B
in the mechanism of
repression.
Antihormones Repress NF-
B Activity without Induction of
I
B
We recently showed that the antiglucocorticoid/antiprogestin
RU486 was able to induce PR-mediated repression of RelA activity (14).
To examine the repression of NF-
B target genes by RU486-occupied GR,
we transiently transfected COS-1 cells with a reporter construct
containing four NF-
B sites from the ICAM-1 promoter. Cotransfection
with expression vector encoding RelA (20 ng) resulted in an induction
of luciferase activity, which could be repressed by cotransfection of
an expression vector for GR (200 ng) and treatment of the cells with
RU486 (Fig. 4A
). The repressive activity
of GR was only slightly reduced with an RU486-occupied receptor
(
65% repression) as compared with a receptor occupied with the
agonist Dex (
85% repression).

View larger version (17K):
[in this window]
[in a new window]
|
Figure 4. Effects of the Antiglucocorticoid RU486 on GR-RelA
Interaction
A, COS-1 cells were transiently transfected with 0.4 µg
4xNF-kB(IC)tkluc reporter, 20 ng RelA, and 200 ng GR expression
constructs and treated with Dex or RU486 for 24 h. The
concentration of Dex or RU486 used was 1 µM (black
bars) or 0.1 µM (hatched bars).
Depicted is the induction of luciferase activity evoked by RelA over
cells transfected with empty expression vector. B, COS-1 cells were
transiently transfected with 0.4 µg 2xGREtkluc reporter and 20 ng GR
expression construct and treated with Dex or RU486 for 24 h. The
concentration of Dex or RU486 used was 1 µM (black
bars) or 0.1 µM (hatched bars).
Fold induction indicates reporter activity in cells treated with Dex or
RU486 over untreated cells. Error bars indicate
SE.
|
|
In addition to being antagonistic, RU486 has also partial agonistic
activity with respect to PR- and GR-mediated transcription (22). To
investigate the partial agonistic activity of RU486, COS-1 cells were
transfected with a reporter construct containing two GREs. As shown in
Fig. 4B
, cotransfection of an expression vector encoding GR resulted in
a hormone-dependent induction of luciferase activity after treatment of
the cells with Dex (65-fold) and very low induction upon RU486
treatment (3-fold). This indicates that repression by RU486 is not
correlated with transcriptional activation mediated by RU486.
In A549 cells, both Dex and RU486 were able to repress IL-1ß-induced
COX-2 mRNA expression, although the anti-hormone was less efficient
(Fig. 5A
, lanes 14). As expected, the
antagonist RU486 was unable to induce I
B
mRNA (Fig. 5A
, lane 6)
or I
B
protein (Fig. 5C
, lanes 4 and 6) in these cells, indicating
that I
B
synthesis is not necessary for repression of NF-
B
activity by RU486.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 5. Repression of Il-1ß-Induced NF- B Target Gene
Expression by RU486
A, A549 cells were treated with Il-1ß (I; 100 U/ml) and Dex (D; 1
µM) or RU486 (R; 1 µM) for 24 h. Total
RNA was isolated and Northern blotting analysis was performed.
Left panel shows blot sequentially hybridized with a
probe containing COX-2 or I B and GAPDH cDNA, which serves as a
control for the amount of RNA loaded in each lane. The positions of the
transcripts of COX-2, I B , and GAPDH are indicated. Right
panel shows quantification of COX-2 (black bars)
and I B (white bars) hybridization signal. Fold
induction indicates hybridization signal for cells treated with
IL-1ß, Dex, and/or RU486 over untreated cells. B, T47D cells were
treated with Il-1ß (I; 100 U/ml) and Org2058 (O; 10 nM)
or RU486 (R; 1 µM) for 24 h. Total RNA was isolated
and Northern blotting analysis was performed. Left panel
shows blot sequentially hybridized with a probe containing ICAM-1 or
I B and GAPDH cDNA, which serves as a control for the amount of
RNA loaded in each lane. The positions of the transcripts of ICAM-1,
I B , and GAPDH are indicated. Right panel shows
PhosphoImager quantification of ICAM-1 (black bars) and
I B (white bars) hybridization signal. Fold
induction indicates hybridization signal for cells treated with
IL-1ß, Org2058, and/or RU486 over untreated cells. Error
bars indicate SE. C, A549 cells were treated as in
panel A. Whole cell extracts were prepared and fractionated on a 12.5%
SDS-PAGE, and Western blotting analysis was performed. Blots were
immunostained with a polyclonal antibody to I B . I B was
visualized after incubation with a peroxidase-conjugated second
antibody and ECL. D, T47D cells were treated as in panel B, and Western
blotting was performed as in panel C.
|
|
Previously, we described mutual repression between progesterone
receptor (PR) and RelA in the breast tumor cell line T47D (14). To
investigate whether RU486 could also repress NF-
B target genes in
these cells containing endogenous PR, the same experiment was performed
in T47D cells. Both the progestagen Org2058 and the progesterone
antagonist RU486 were able to repress IL-1ß-induced ICAM-1 expression
(Fig. 5B
, lanes 3 and 4). Whereas Org2058 induced I
B
mRNA
expression, RU486 was unable to induce I
B
mRNA in these cells
(Fig. 5B
, lanes 5 and 6), although a small increase in I
B
protein
could be observed (Fig. 5D
, lanes 4 and 6). The fact that
RU486-occupied receptors are able to repress NF-
B target gene
expression in the absence of induced I
B
expression indicates that
the repression of endogenous NF-
B target genes by GR and PR is at
least partially mediated by an I
B
-independent mechanism.
 |
DISCUSSION
|
---|
NF-
B plays a pivotal role in the regulation of a variety of
genes involved in immune and inflammatory responses. Therefore,
inhibition of NF-
B activity can account for many of the
immunosuppressive and antiinflammatory activities of glucocorticoids.
In the present study, we show that glucocorticoids can control immune
response and inflammation by repressing NF-
B activity via a dual
mechanism.
First, Dex was shown to induce I
B
mRNA expression in A549 cells,
which has also been reported for HeLa cells, monocytic cells, and T
lymphocytes (16, 17). The fact that this induction occurs in the
presence of CHX suggests that glucocorticoids activate I
B
gene
transcription directly. For these cells it has been shown that
Dex-induced I
B
was able to inhibit NF-
B activity by preventing
nuclear translocation and DNA binding of NF-
B (16, 17). However, in
A549 cells we observed no inhibition of NF-
B DNA-binding activity by
Dex, suggesting that in this case the Dex-induced I
B
was not able
to efficiently sequester NF-
B in the cytoplasm and to prevent DNA
binding. Similar results have been described for endothelial cells
(18). Nevertheless, repression of NF-
B activity by protein-protein
interaction can occur via tethering of GR to NF-
B in its DNA-bound
form, without affecting DNA binding.
Second, we showed that in the absence of I
B
protein synthesis,
Dex was still able to repress IL-1ß-induced expression of the NF-
B
target genes, COX-2 and ICAM-1. The repressive activity of Dex in the
presence of CHX was less strong than in the absence of CHX, providing
evidence for more than one mechanism involved in Dex-mediated
repression of NF-
B activity. In contrast to this observation, Auphan
et al. (16) and Scheinman et al. (17) showed that
in the presence of CHX, inhibition of NF-
B DNA binding activity
could no longer be observed, suggesting a requirement of I
B
for
repression of NF-
B activity. However, we showed that in A549 cells,
I
B
was unable to prevent NF-
B DNA binding, suggesting that
inhibition of NF-
B DNA binding is not essential for repression of
NF-
B target genes in these cells.
As we showed previously for the repression of RelA activity by PR
(14), we found that the antiprogestin/antiglucocorticoid RU486 was also
able to induce GR-mediated repression of RelA activity. In addition,
RU486 could repress IL-1ß-induced expression of COX-2 in A549 cells,
albeit less efficiently than the agonist, Dex. Furthermore, RU486 was
able to induce PR-mediated repression of the IL-1ß-induced
expression of the NF-
B target gene, ICAM-1, in T47D cells. In
contrast to the agonists, Dex and Org2058, RU486 was not able to induce
I
B
synthesis in both cell lines. Taken together, these findings
demonstrate that in addition to Dex- and Org2058-induced I
B
synthesis, a second mechanism must be involved in the repression of
NF-
B activity by both glucocorticoids and progestins. Furthermore,
Dex-mediated repression of NF-
B activity has been shown to be
independent of I
B
synthesis in endothelial cells (18) and in rat
kidney epithelial cells (23), which again suggests that the induction
of I
B
is not a universal mechanism explaining NF-
B repression
by glucocorticoids in all cell types. In addition to the induction of
IkB
synthesis, glucocorticoids have been shown to repress
transcription of target genes by transcriptional interference, a
mechanism likely to involve protein-protein interactions between GR and
NF-
B (9, 10, 11, 12). In this way, GR can interfere with the transcriptional
activity of NF-
B by 1) forming a complex with NF-
B and inhibiting
its DNA-binding activity or by 2) forming a complex with NF-
B in its
DNA-bound form without affecting DNA binding, or by 3) contacting a
cofactor that is bound to NF-
B and thereby inhibiting the
transactivation potential of NF-
B. Further experiments will have to
be carried out to determine which of the mechanisms is involved. GR has
been found to associate in vitro with NF-
B either in a
manner leading to inhibition of DNA binding (9, 10) or without
affecting DNA binding (18, 21). However, previous reports regarding a
decreased AP-1 DNA-binding activity in the presence of GR in
vitro (24) could not be confirmed by in vivo
footprinting studies (25). Therefore in vivo footprinting
analysis could be used to study NF-
B binding to DNA in the presence
of GR. Similar to GR, other steroid receptors, such as ER (13), PR
(14), and AR (15), have also been shown to physically interact with
NF-
B in vitro and inhibit its transcriptional activity,
suggesting an important role for protein-protein interactions in
repression of NF-
B activity by steroids.
In contrast to the previously described mechanism, which indicates that
inhibition of NF-
B activity does not rely on interaction between GR
and NF-
B but is predominantly based on induction of I
B
expression, we provide evidence that both mechanisms, resulting in
repression of NF-
B activity, contribute to the antiinflammatory
action of glucocorticoids. The involvement of both these mechanisms
emphasizes the importance of multiple levels of regulation of NF-
B
activity by glucocorticoids in modulation of the antiinflammatory
response. This sustains the possibility of developing ligands that
specifically activate the repression function of GR and that may
therefore be more efficient in the treatment of inflammatory diseases
without undesirable side effects.
 |
MATERIALS and METHODS
|
---|
Special Reagents and Antibodies
Dexamethasone and cycloheximide were obtained from Sigma
Chemical Co. (St. Louis, MO). The progestin Org2058 was provided by
Organon International (Oss, The Netherlands). IL-1ß was obtained from
NCI Biological Resources Branch (Frederick, MD). RU486 was obtained
form Roussel-Uclaf (Romainville, France). Polyclonal antibody against
I
B
was purchased from Upstate Biotechnology Inc. (Lake Placid,
NY). The polyclonal antibody against the N terminus of RelA was from
Santa Cruz Biotechnology (Santa Cruz, CA). Antiserum 2 against NF-
B1
was a kind gift of Dr. A. Israël (Paris, France).
Cell Culture
Human pulmonary epithelial A549 cells were obtained from
American Type Culture Collection (ATCC; Rockville, MD). Cells were
cultured in DMEM from Life Technologies Inc. (Gaithersburg, MD),
buffered with bicarbonate, and supplemented with 7.5% FCS from Integro
(Linz, Austria). Monkey COS-1 cells (ATCC) and human breast tumor T47D
cells, originally provided by Dr. R. L. Sutherland (Sydney,
Australia), were cultured in a 1:1 mixture of DMEM and Hams F-12
medium (DF; Life Technologies Inc.), buffered with bicarbonate, and
supplemented with 7.5% FCS. Dextran-coated charcoal-FCS was prepared
by treatment of FCS with dextran-coated charcoal to remove steroids, as
described previously (26).
Plasmids and Transient Transfections
The luciferase reporter plasmid (4xNF-
B(IC)tkluc)
containing four NF-
B sites from the ICAM-1 promoter was
constructed by ligating two copies of the annealed oligonucleotides
(5'-AGCTTATGGAAATTCCGAGATCATGGAAATTCCGAC-3') and
(5'-AGCTGTCGGAATTT-CCATGATCTCGGAATTTCCATA-3'), containing two NF-
B
sites from the ICAM-1 promoter and HindIII linkers, into the
HindIII site of ptkluc. The reporter plasmid 2xGREtkluc has
been described elsewhere (27). The CMV4 expression vectors containing
full-length cDNAs encoding human RelA and GR have been described
previously (11). For transient transfections, COS-1 cells were cultured
in 24-well plates and transfected using calcium-phosphate
coprecipitation with 0.4 µg luciferase reporter, 0.6 µg PDMlacZ,
and the indicated amount of expression plasmids. pBluescript was added
to obtain a total amount of 2 µg DNA/well. After 16 h, the
medium was refreshed and hormone was added. Cells were harvested
24 h later and assayed for luciferase activity using the Luclite
luciferase reporter gene assay kit (Packard Instruments, Meriden, CT)
according to the manufacturers protocol and the Topcount liquid
scintillation counter (Packard Instruments). Values were corrected
for transfection efficiency by measuring ß-galactosidase
activity (28).
Northern Blotting Analysis
A549 cells were cultured in 10-cm dishes, treated as indicated,
and harvested. T47D cells were cultured in DF+, supplemented with 5%
dextran-coated charcoal-FCS, and treated as A549 cells. Total RNA was
isolated using the acid-phenol method of Chomczynski and Sacchi (29).
Twenty micrograms of RNA were fractionated on a 0.8% agarose gel and
transferred to Hybond C-extra membranes by capillary transfer using
10xstandard sodium citrate (SSC). The blots were baked under vacuum
for 2 h at 80 C. The blots were hybridized to cDNA probes
overnight at 42 C in hybridization buffer. Subsequently, blots were
washed with 2xSSC/0.1%SDS, 1xSSC/0.1%SDS, 0.2xSSC/0.1%SDS, and
0.1xSSC/0.1%SDS when necessary. cDNA probes were labeled with
[
32P]dCTP by random priming according to the
manufacturers protocol (Amersham Pharmacia Biotech., Rainham, Essex,
UK). As probes for Northern blotting, a 1-kb HindIII
fragment of the I
B
cDNA, a 1.8-kb XbaI fragment of the
ICAM-1 cDNA, a 1-kb EcoRI/XhoI fragment of the
murine COX-2 cDNA, a kind gift from Dr. H. Herschman, and a 1.4-kb
PstI fragment of the glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were used.
Western Blotting Analysis
For isolation of whole cell extracts A549 cells were cultured in
5-cm dishes, treated as described, and harvested in buffer containing
50 mM Tris (pH 7.4), 50 mM NaCl, 0.5% nonidet
P-40, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 1 µg/ml
leupeptin at 4 C. Subsequently, cells were centrifuged for 15 min at 4
C, and protein concentration of the supernatant was determined by the
Bio-Rad (Richmond, CA) protein assay according to the manufacturers
protocol. Twenty five micrograms of extract were separated on SDS-PAGE
gels and transferred to Immobilon (Milipore, MA). For the polyclonal
antibody against I
B
(Upstate Biotechnology Inc.), all incubations
were carried out according to the manufacturers protocol.
Immunoreactive bands were visualized with enhanced chemiluminescence
(ECL) (Amersham).
Electrophoretic mobility shift assay (EMSA)
A549 cells were cultured in 10-cm dishes and pretreated with Dex
(1 µM) for 15 h and with IL-1ß (100 U/ml) for
1 h. Cells were harvested and nuclear extracts were prepared
according to Lee et al. (30). A double-stranded
oligonucleotide containing the
B site from the HIV LTR
(5'-agcttcagaGGGGACTTTCCgagagg-3') was labeled with
[32P]dCTP using the Klenow fragment of DNA polymerase I.
Labeled probe was separated from unincorporated nucleotides by gel
filtration on Sephadex G-50 spin columns and eluted overnight from 5%
polyacrylamide gels in 0.5 M
CH3COONH4/1 mM EDTA at 37 C.
Nuclear extracts of A549 cells (10 µg per assay) were incubated with
10.000 cpm of probe (0.1 to 0.5 ng) and 1 µg poly(dI-dC),
respectively, for 30 min at room temperature in a total reaction
mixture of 20 µl containing 20 mM HEPES, pH 7.5, 100
mM KCl, 0.2 mM EDTA, 20% glycerol, 1
mM dithiothreitol, 1 µg/µl BSA. Samples were loaded on
a 5% polyacrylamide (29:1) gel, containing 0.25 x TBE as running
buffer, and the gel was run at room temperature at 150 V for 22.5 h.
Excess unlabeled competitor oligonucleotide, containing the HIV
B
site or a mutant
B site (5'-AGCTTGTAAATTGTGGAGC-3') or antisera to
NF-
B1 and RelA, was added to the reaction mixture before addition of
labeled probe. After electrophoresis, gels were dried and
autoradiographed for 1 day at -80 C.
 |
ACKNOWLEDGMENTS
|
---|
We thank J. Heinen and F. Vervoordeldonk for photographic
reproductions.
Note added in Proof. Recently two papers have
appeared reporting findings similar to those reported here: Heck
et al. (1997) EMBO J 16:46984707; de Bosscher et
al. (1997) Proc Natl Acad Sci USA 94:1350413509.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. P. T. van der Saag, Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
This research was supported by grants from the Netherlands Asthma
Foundation (92.96), the European Community (BIOMED. 2, PL 951358),
and Boehringer Ingelheim GmbH.
Received for publication June 2, 1997.
Revision received November 11, 1997.
Accepted for publication December 18, 1997.
 |
REFERENCES
|
---|
-
Barnes PJ, Adcock I 1993 Anti-inflammatory actions of
steroids: molecular mechanisms. Trends Pharmacol Sci 14:436441[Medline]
-
Bamberger CM, Schulte HM, Chrousos GP 1996 Molecular
determinants of glucocorticoid receptor function and tissue sensitivity
to glucocorticoids. Endocr Rev 17:245261[Abstract]
-
Cato ACB, Wade E 1996 Molecular mechanisms of
anti-inflammatory action of glucocorticoids. BioEssays 18:317378[Medline]
-
McEwan IJ, Wright, APH, Gustafsson J-Å 1997 Mechanism of
gene expression by the glucocorticoid receptor: role of protein-protein
interactions. BioEssays 19:153160[Medline]
-
Van der Burg B, Okret S, Liden J, Wissink S, Van der Saag PT,
Gustafsson J-Å 1997 Nuclear factor-kappa-B repression in
anti-inflammation and immunosuppression by glucocorticoids. Trends
Endocrinol Metab 8:152157[CrossRef]
-
Urban MB, Schreck R, Baeuerle PA 1991 NF-
B contacts DNA by
a heterodimer of the p50 and p65 subunit. EMBO J 10:18171825[Abstract]
-
Baldwin Jr AS 1996 The NF-
B and I
B proteins: new
discoveries and insights. Annu Rev Immunol 14:649683[CrossRef][Medline]
-
Beg AA, Baldwin Jr AS 1993 The I
B proteins:
multifunctional regulators of Rel/NF-
B transcription factors. Genes
Dev 7:20642070[CrossRef][Medline]
-
Ray A, Prefontaine KE 1994 Physical association and
functional antagonism between the p65 subunit of transcription factor
NF-
B and the glucocorticoid receptor. Proc Natl Acad Sci USA 91:752756[Abstract]
-
Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin Jr
AS 1995 Characterization of mechanisms involved in transrepression of
NF-
B by activated glucocorticoid receptors. Mol Cell Biol 15:943953[Abstract]
-
Caldenhoven E, Liden J, Wissink S, Van de Stolpe A,
Raaijmakers J, Okret S, Gustafsson J-Å, Van der Saag PT 1995 Negative
crosstalk between RelA and the glucocorticoid receptor: a possible
mechanism for the anti-inflammatory action of glucocorticoids. Mol
Endocrinol 9:401412[Abstract]
-
Wissink S, van Heerde EC, Schmitz LM, Kalkhoven E, van der
Burg B, Baeuerle PA, van der Saag PT 1997 Distinct domains of the RelA
NF-
B subunit are required for negative crosstalk and direct
interaction with the glucocorticoid receptor. J Biol Chem 272:2227822284[Abstract/Free Full Text]
-
Stein B, Yang MX 1995 Repression of the interleukin-6 promoter
by estrogen receptor is mediated by NF-
B and C/EBPß. Mol Cell Biol 15:49714979[Abstract]
-
Kalkhoven E, Wissink S, Van der Saag PT, Van der Burg B 1996 Negative interaction between the RelA(p65) subunit of NF-
B and the
progesterone receptor. J Biol Chem 271:62176224[Abstract/Free Full Text]
-
Palvimo JJ, Rinikainen P, Ikonen T, Kallio PJ, Moilanen A,
Jänne OA 1996 Mutual transcriptional interference between RelA
and androgen receptor. J Biol Chem 271:2415124156[Abstract/Free Full Text]
-
Auphan N, Didonato JA, Rosette C, Helmberg A, Karin M 1995 Immunosuppression by glucocorticoids: inhibition of NF-
B activity
through induction of I
B synthesis. Science 270:286290[Abstract]
-
Scheinman RI, Cogswell PC, Lofquist AK, Baldwin Jr AS 1995 Role of transcriptional activation of I
B
in mediation of
immunosuppression by glucocorticoids. Science 270:283286[Abstract]
-
Brostjan C, Anrather J, Csizmadia V, Stroka D, Soares M, Bach
FH, Winkler H 1996 Glucocorticoid-mediated repression of NF-
B
activity in endothelial cells does not involve induction of I
B
synthesis. J Biol Chem 271:1961219616[Abstract/Free Full Text]
-
Van de Stolpe A, Caldenhoven E, Raaijmakers JAM, Van der Saag
PT, Koenderman L 1993 Glucocorticoid-mediated repression of
intercellular adhesion molecule-1 expression in human monocytic and
bronchial epithelial cell lines. Am J Respir Cell Mol Biol 8:340347[Medline]
-
Appleby SB, Ristimäki A, Neilson K, Narko K, Hla T 1994 Structure of the human cyclooxygenase-2 gene. Biochem J 302:723727[Medline]
-
Van de Stolpe A, Caldenhoven E, Stade BG, Koenderman L,
Raaijmakers JAM, Johnson JP, Van der Saag PT 1994 12-O-tetradecanoylphorbol-13-acetate and tumor necrosis
factor
-mediated induction of intercellular adhesion molecule-1 is
inhibited by dexamethasone. J Biol Chem 269:61856192[Abstract/Free Full Text]
-
Meyer ME, Pornon A, Ji JW, Bocquel MT, Chambon P, Gronemeyer H 1990 Agonistic and antagonistic activities of RU486 on the functions of
the human progesterone receptor. EMBO J 9:39233932[Abstract]
-
Ohtsuka T, Kubota A, Hirano T, Watanabe K, Yoshida H,
Tsurufuji M, Iizuka Y, Konishi K, Tsurufuji S 1996 Glucocorticoid-mediated gene suppression of rat cytokine-induced
neutrophil chemoattractant CINC/gro, a member of the interleukin-8
family, through impairment of NF-
B activation. J Biol Chem 271:16511659[Abstract/Free Full Text]
-
Yang-Yen H-F, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin
J, Karin M 1990 transcriptional interference between c-Jun and the
glucocorticoid receptor: mutual inhibition of DNA binding due to direct
protein-protein interaction. Cell 62:12051215[Medline]
-
König H, Ponta H, Rahmsdorf HJ, Herrlich P 1992 Interference between pathway-specific transcription factors:
glucocorticoids antagonize phorbol ester-induced AP-1 activity without
altering AP-1 site occupation in vivo. EMBO J 11:22412246[Abstract]
-
Van der Burg B, Rutteman GR, Blankenstijn MA, de Laat SW, Van
Zoelen EJJ 1988 Mitogenic stimulation of human breast cancer cells in a
growth factor-defined medium: synergistic action of insulin and
estrogen. J Cell Physiol 123:101108
-
Schüle R, Muller M, Kaltschmidt C Renkawitz R 1988 Many
transcription factors interact synergistically with steroid receptors.
Science 242:14181421[Medline]
-
Pfahl M, Tzukerman M, Zhang X-K, Lehmann JM, Hermann T,
Wills KN, Graupner G 1990 Nuclear retinoic acid receptors: cloning,
analysis, and function. Methods Enzymol 189:256270[Medline]
-
Chomczynski P, Sacchi N 1987 Single step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Lee KA, Bindereif AS, Green MR 1988 A small-scale procedure
for preparation of nuclear extracts that support efficient
transcription and pre-mRNA splicing. Gene Anal Technol 5:2231[CrossRef]