A Dual Mechanism Mediates
Repression of NF-kB 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
Repression of nuclear factor (NF)-kB-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-kB and the glucocorticoid receptor,
possibly explaining their mutual repression of transcriptional activity. Furthermore, glucocorticoidinduced transcription of IkBa 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 IkBa gene
transcription in human pulmonary epithelial A549
cells. However, this enhanced IkBa synthesis did
not cause repression of NF-kB DNA-binding activity. In addition, dexamethasone was still able
to inhibit the expression of NF-kB target genes
(cyclooxygenase-2, intercellular adhesion molecule-1) in the absence of protein synthesis. Furthermore, we show that the antihormone RU486
did not induce IkBa expression. However, RU486
was still able to induce, albeit less efficiently, both
glucocorticoid- and progesterone receptor-mediated repression of endogenous NF-kB target gene
expression in A549 cells and the breast cancer cell
line T47D, respectively. Taken together, these results indicate that induced IkBa expression accounts for only part of the repression of NF-kB
activity by glucocorticoids and progestins. In addition, protein-protein interactions between NF-kB
and the glucocorticoid or progesterone receptor,
resulting in repression of NF-kB activity, seem also
to be involved. We therefore conclude that NF-kB
activity is repressed via a dual mechanism involving both protein-protein interactions and induction
of IkBa. (Molecular Endocrinology 12: 355–363,
1998)
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)-kB (3–5).
The NF-kB/Rel family of transcription factors regulates the expression of many genes involved in immune and inflammatory responses. NF-kB was originally identified as a heterodimer of NF-kB1 and RelA
(6), but a variety of other kB/Rel homo- and heterodimers have now been described. NF-kB is present
in an inactive state in the cytoplasm, sequestered by
an inhibitor protein, designated IkB. After stimulation
of the cells, IkB becomes phosphorylated, ubiquitinated, and subsequently degraded (7). As a result,
NF-kB is free to translocate to the nucleus and activate transcription of target genes. In the nucleus,
NF-kB can also induce the synthesis of IkBa, which
terminates the NF-kB response, explaining its transient nature (8).
Glucocorticoids were shown to be potent inhibitors
of NF-kB activation. In addition, the NF-kB subunit,
RelA, has been shown to physically interact with GR in
vitro (9–12) as well as with other steroid receptors,
such as the estrogen receptor (ER; Ref. 13), the pro-
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Molecular Endocrinology
Copyright © 1998 by The Endocrine Society
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gesterone receptor (PR; Ref. 14), and the androgen
receptor (AR; Ref. 15). Since it has been demonstrated
that NF-kB 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 (9–15).
A second independent mechanism through which
glucocorticoids could repress NF-kB activity has been
described (16, 17). Glucocorticoids were shown to
induce transcription of the IkBa gene in HeLa cells,
monocytic cells, and T-lymphocytes. This induction
resulted in increased synthesis of IkBa protein, which
is able to interact with activated NF-kB, thereby terminating the NF-kB response. However, Brostjan et al.
(18) reported that glucocorticoid-mediated repression
of NF-kB activity did not involve induction of IkBa
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 IkBa in human pulmonary epithelial A549 cells. Furthermore, we show that Dex is able to inhibit the
expression of two endogenous NF-kB target genes,
cyclooxygenase-2 (COX-2) and intercellular adhesion
molecule-1 (ICAM-1) partially independent of newly
synthesized IkBa. On the basis of these results, we
conclude that glucocorticoids repress NF-kB activity
in A549 cells via a dual mechanism, which involves
both protein-protein interaction and induction of IkBa.
RESULTS
Glucocorticoids Induce IkBa in A549 Cells
Glucocorticoids have been described to induce IkBa
synthesis in monocytic and lymphocytic cells (16, 17),
but not in endothelial cells (18). To determine whether
glucocorticoids increased IkBa 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 IkBa
mRNA in these cells, which peaked by 2–8 h (3- to
4-fold) and remained elevated to 24 h (2-fold).
Repression of NF-kB-Regulated Genes Is Not
Only Mediated by IkBa Induction
To investigate the mechanism(s) involved in repression
of NF-kB activity by glucocorticoids, we determined
the repression by Dex of NF-kB-regulated genes in the
absence of IkBa protein synthesis. Treatment of A549
cells with interleukin (IL)-1b resulted in a 5-fold increase in IkBa mRNA expression, and a 3-fold induc-
Fig. 1. Dex-Induced IkBa Gene Expression in A549 Cells
A549 cells were treated for increasing periods of time with
1 mM Dex. Total RNA was isolated and Northern blotting
analysis was performed. Upper panel shows blot sequentially
hybridized with a probe containing IkBa and GAPDH cDNA,
which serves as a control for the amount of RNA loaded in
each lane. The positions of the transcripts of IkBa and
GAPDH are indicated. Lower panel shows PhosphoImager
(Molecular Dynamics, Sunnyvale, CA) quantification of IkBa
hybridization signal. Fold induction indicates hybridization
signal for cells treated with Dex over untreated cells. Error
bars indicate SE.
tion was observed after treatment with Dex. The combination of IL-1b and Dex showed a similar induction
as IL-1b treatment alone (Fig. 2A, lanes 1–4). Dex-
Glucocorticoid Repression of NF-kB Activity
mediated IkBa induction and IkBa resynthesis after
IL-1b-induced degradation can be observed for IkBa
protein (Fig. 2B, lanes 1–4), indicating that IL-1b and
Dex can induce both IkBa transcription and protein
synthesis in A549 cells. Cycloheximide (CHX), an inhibitor of protein synthesis, also induced IkBa mRNA
expression (5-fold; Fig. 2A, lane 5), as has been observed for other NF-kB target genes, e.g. ICAM-1 (19).
CHX together with IL-1b superinduced IkBa 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 IkBa protein could
be observed in the presence of CHX (Fig. 2B, lanes 6
and 8).
To study whether protein synthesis was necessary
for the repressive effect of Dex on endogenous NF-kB
target gene expression, COX-2 (20) and ICAM-1 (21)
mRNA expression was investigated. As shown in Fig.
2C, IL-1b 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 IkBa protein induction, Dex was still
able to repress IL-1b-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 IkBa plays a
role, but is obviously not the only mechanism mediating the repressive effect of Dex.
NF-kB DNA-Binding Activity Is Not Inhibited by
Dex
It has been shown that Dex-induced IkBa was able
to inhibit NF-kB activity by preventing nuclear translocation and DNA binding (16, 17). To determine
whether Dex-induced IkBa could block NF-kB DNAbinding activity in A549 cells, the cells were pretreated with Dex for 15 h and subsequently treated
with IL-1b for 1 h. NF-kB 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-1b (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 kB probe but not with a mutant
kB probe (lanes 6 and 7). The kB-binding activity
was composed mostly of NF-kB1 and RelA heterodimer (NF-kB) as determined by supershift analysis (lanes 8 and 9). The faster migrating complexes
appeared to contain NF-kB1 protein in other
combinations.
These results show that, in A549 cells, Dex-induced
IkBa is not able to prevent nuclear translocation or
DNA binding of NF-kB, suggesting a minor contribution of IkBa in the mechanism of repression.
357
Antihormones Repress NF-kB Activity without
Induction of IkBa
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-kB target genes by RU486-occupied
GR, we transiently transfected COS-1 cells with a
reporter construct containing four NF-kB 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).
In addition to being antagonistic, RU486 has also
partial agonistic activity with respect to PR- and GRmediated 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-1b-induced COX-2 mRNA expression, although the anti-hormone was less efficient (Fig. 5A,
lanes 1–4). As expected, the antagonist RU486 was
unable to induce IkBa mRNA (Fig. 5A, lane 6) or IkBa
protein (Fig. 5C, lanes 4 and 6) in these cells, indicating
that IkBa synthesis is not necessary for repression of
NF-kB activity by RU486.
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-kB 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-1b-induced
ICAM-1 expression (Fig. 5B, lanes 3 and 4). Whereas
Org2058 induced IkBa mRNA expression, RU486 was
unable to induce IkBa mRNA in these cells (Fig. 5B,
lanes 5 and 6), although a small increase in IkBa
protein could be observed (Fig. 5D, lanes 4 and 6). The
fact that RU486-occupied receptors are able to repress NF-kB target gene expression in the absence of
induced IkBa expression indicates that the repression
of endogenous NF-kB target genes by GR and PR is at
least partially mediated by an IkBa-independent
mechanism.
MOL ENDO · 1998
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Fig. 2. Repression of IL-1b-Induced COX-2 and ICAM-1 Expression by Dex in the Absence of Protein Synthesis
A549 cells were treated with IL-1b (I; 100 U/ml) and Dex (D; 1 mM) in the absence or presence of CHX (10 mg/ml) for 6 h. A,
Total RNA was isolated and Northern blotting analysis was performed. Left panel shows blot sequentially hybridized with a probe
Glucocorticoid Repression of NF-kB Activity
Fig. 3. No Effect of Dex on DNA-Binding Activity of NF-kB
A549 cells were pretreated with Dex (1 mM) for 15 h and
stimulated with Il-1b (100 U/ml) for 1 h. Subsequently, nuclear
extracts were analyzed by electrophoretic mobility shift assay
with 32P-labeled probe containing the kB site from the HIV
LTR. Specificity of binding was demonstrated by competition
with 100-fold molar excess of unlabeled probe containing the
kB site from the HIV LTR (lane 6) or a mutant kB site (lane 7).
kB-Binding complexes were characterized by supershift
analysis using antisera to NF-kB1 (lane 8) and RelA (lane 9).
DISCUSSION
NF-kB plays a pivotal role in the regulation of a variety
of genes involved in immune and inflammatory re-
359
sponses. Therefore, inhibition of NF-kB 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-kB activity via a dual mechanism.
First, Dex was shown to induce IkBa 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 IkBa
gene transcription directly. For these cells it has been
shown that Dex-induced IkBa was able to inhibit
NF-kB activity by preventing nuclear translocation and
DNA binding of NF-kB (16, 17). However, in A549 cells
we observed no inhibition of NF-kB DNA-binding activity by Dex, suggesting that in this case the Dexinduced IkBa was not able to efficiently sequester
NF-kB in the cytoplasm and to prevent DNA binding.
Similar results have been described for endothelial
cells (18). Nevertheless, repression of NF-kB activity
by protein-protein interaction can occur via tethering
of GR to NF-kB in its DNA-bound form, without affecting DNA binding.
Second, we showed that in the absence of IkBa
protein synthesis, Dex was still able to repress IL-1binduced expression of the NF-kB 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-kB
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-kB DNA binding
activity could no longer be observed, suggesting a
requirement of IkBa for repression of NF-kB activity.
However, we showed that in A549 cells, IkBa was
unable to prevent NF-kB DNA binding, suggesting that
inhibition of NF-kB DNA binding is not essential for
repression of NF-kB 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-1b-induced expression of COX-2 in
A549 cells, albeit less efficiently than the agonist, Dex.
Furthermore, RU486 was able to induce PR-mediated
containing IkBa and GAPDH cDNA, which serves as a control for the amount of RNA loaded in each lane. The positions of the
transcripts of IkBa and GAPDH are indicated. Right panel shows quantification of IkBa hybridization signal. Fold induction
indicates hybridization signal for cells treated with IL-1b, 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 IkBa. IkBa 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-1b over untreated cells, in the absence (black bars) and presence (white bars) of CHX is set at 100%. Error bars
indicate SE.
MOL ENDO · 1998
360
Fig. 4. Effects of the Antiglucocorticoid RU486 on GR-RelA
Interaction
A, COS-1 cells were transiently transfected with 0.4 mg
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 mM (black
bars) or 0.1 mM (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 mg 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 mM (black
bars) or 0.1 mM (hatched bars). Fold induction indicates reporter activity in cells treated with Dex or RU486 over untreated cells. Error bars indicate SE.
Vol 12 No. 3
repression of the IL-1b-induced expression of the
NF-kB target gene, ICAM-1, in T47D cells. In contrast to the agonists, Dex and Org2058, RU486 was
not able to induce IkBa synthesis in both cell lines.
Taken together, these findings demonstrate that in
addition to Dex- and Org2058-induced IkBa synthesis, a second mechanism must be involved in the
repression of NF-kB activity by both glucocorticoids
and progestins. Furthermore, Dex-mediated repression of NF-kB activity has been shown to be independent of IkBa synthesis in endothelial cells (18)
and in rat kidney epithelial cells (23), which again
suggests that the induction of IkBa is not a universal
mechanism explaining NF-kB repression by glucocorticoids in all cell types. In addition to the induction of IkBa 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-kB (9–12). In this way, GR can interfere with
the transcriptional activity of NF-kB by 1) forming a
complex with NF-kB and inhibiting its DNA-binding
activity or by 2) forming a complex with NF-kB in its
DNA-bound form without affecting DNA binding, or
by 3) contacting a cofactor that is bound to NF-kB
and thereby inhibiting the transactivation potential
of NF-kB. 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-kB 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-kB 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-kB in vitro and inhibit its transcriptional
activity, suggesting an important role for proteinprotein interactions in repression of NF-kB activity
by steroids.
In contrast to the previously described mechanism,
which indicates that inhibition of NF-kB activity does
not rely on interaction between GR and NF-kB but is
predominantly based on induction of IkBa expression,
we provide evidence that both mechanisms, resulting
in repression of NF-kB activity, contribute to the antiinflammatory action of glucocorticoids. The involvement of both these mechanisms emphasizes the importance of multiple levels of regulation of NF-kB
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.
Glucocorticoid Repression of NF-kB Activity
361
Fig. 5. Repression of Il-1b-Induced NF-kB Target Gene Expression by RU486
A, A549 cells were treated with Il-1b (I; 100 U/ml) and Dex (D; 1 mM) or RU486 (R; 1 mM) 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 IkBa
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, IkBa, and GAPDH are indicated. Right panel shows quantification of COX-2 (black bars) and IkBa (white bars)
hybridization signal. Fold induction indicates hybridization signal for cells treated with IL-1b, Dex, and/or RU486 over untreated
cells. B, T47D cells were treated with Il-1b (I; 100 U/ml) and Org2058 (O; 10 nM) or RU486 (R; 1 mM) 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
IkBa 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, IkBa, and GAPDH are indicated. Right panel shows PhosphoImager quantification of ICAM-1 (black bars) and IkBa
(white bars) hybridization signal. Fold induction indicates hybridization signal for cells treated with IL-1b, 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 IkBa. IkBa 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.
MOL ENDO · 1998
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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-1b was obtained from NCI Biological Resources
Branch (Frederick, MD). RU486 was obtained form RousselUclaf (Romainville, France). Polyclonal antibody against IkBa
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-kB1 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 Ham’s 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 dextrancoated charcoal to remove steroids, as described previously
(26).
Plasmids and Transient Transfections
The luciferase reporter plasmid (43NF-kB(IC)tkluc) containing four NF-kB sites from the ICAM-1 promoter
was constructed by ligating two copies of the annealed
oligonucleotides (59-AGCTTATGGAAATTCCGAGATCATGGAAATTCCGAC-39) and (59-AGCTGTCGGAATTT-CCATGATCTCGGAATTTCCATA-39), containing two NF-kB 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 mg luciferase reporter, 0.6 mg PDMlacZ, and the
indicated amount of expression plasmids. pBluescript was
added to obtain a total amount of 2 mg 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 manufacturer’s protocol and the Topcount liquid scintillation
counter (Packard Instruments). Values were corrected
for transfection efficiency by measuring b-galactosidase
activity (28).
Vol 12 No. 3
hybridized to cDNA probes overnight at 42 C in hybridization
buffer. Subsequently, blots were washed with 23SSC/
0.1%SDS, 13SSC/0.1%SDS, 0.23SSC/0.1%SDS, and
0.13SSC/0.1%SDS when necessary. cDNA probes were labeled with [a32P]dCTP by random priming according to the
manufacturer’s protocol (Amersham Pharmacia Biotech.,
Rainham, Essex, UK). As probes for Northern blotting, a 1-kb
HindIII fragment of the IkBa 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 mg/ml aprotinin, and 1 mg/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 BioRad (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 IkBa (Upstate Biotechnology Inc.), all incubations were carried out
according to the manufacturer’s 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 mM) for 15 h and with IL-1b (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 kB site from the HIV LTR (59-agcttcagaGGGGACTTTCCgagagg-39) 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 mg per assay) were
incubated with 10.000 cpm of probe (0.1 to 0.5 ng) and 1 mg
poly(dI-dC), respectively, for 30 min at room temperature in a
total reaction mixture of 20 ml containing 20 mM HEPES, pH
7.5, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 1 mg/ml BSA. Samples were loaded on a 5% polyacrylamide (29:1) gel, containing 0.25 3 TBE as running
buffer, and the gel was run at room temperature at 150 V for
2–2.5 h. Excess unlabeled competitor oligonucleotide, containing the HIV kB site or a mutant kB site (59-AGCTTGTAAATTGTGGAGC-39) or antisera to NF-kB1 and RelA, was
added to the reaction mixture before addition of labeled
probe. After electrophoresis, gels were dried and autoradiographed for 1 day at 280 C.
Acknowledgments
Northern Blotting Analysis
We thank J. Heinen and F. Vervoordeldonk for photographic
reproductions.
A549 cells were cultured in 10-cm dishes, treated as indicated, and harvested. T47D cells were cultured in DF1, 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 103standard sodium citrate (SSC). The blots
were baked under vacuum for 2 h at 80 C. The blots were
Received June 2, 1997. Re-revision received November
11, 1997. Accepted December 18, 1997.
Address requests for reprints to: Dr. P. T. van der Saag,
Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, The
Netherlands. E-mail: [email protected].
This research was supported by grants from the Netherlands Asthma Foundation (92.96), the European Community
(BIOMED. 2, PL 95–1358), and Boehringer Ingelheim GmbH.
Glucocorticoid Repression of NF-kB Activity
363
Note added in Proof. Recently two papers have appeared
reporting findings similar to those reported here: Heck et al.
(1997) EMBO J 16:4698–4707; de Bosscher et al. (1997) Proc
Natl Acad Sci USA 94:13504–13509.
17.
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