(Received for publication, May 22, 1995; and in revised form, October 16, 1995)
From the
The glucocorticoid dexamethasone inhibited the production of the
rat cytokine-induced neutrophil chemoattractant CINC/gro, a counterpart
of human melanoma growth-stimulating activity that belongs to the
interleukin-8 (IL-8) family, in the normal rat kidney epithelial cell
line NRK-52E stimulated with interleukin-1 (IL-1
),
lipopolysaccharide, or tumor necrosis factor
. The accumulation of
CINC/gro mRNA induced by these activators was also decreased comparably
by dexamethasone. A nuclear run-on assay revealed that dexamethasone
decreased the IL-1
-induced transcription of the CINC/gro gene. The
half-life of CINC/gro mRNA transcripts did not change significantly
after exposure to dexamethasone, suggesting that this glucocorticoid
acts mainly at the transcriptional level. Transfection with luciferase
expression vectors containing 5`-deleted and mutated CINC/gro gene
sequences demonstrated that the 5`-flanking region containing the
NF-
B binding site is involved in the IL-1
- and
dexamethasone-induced activation and repression of the CINC/gro gene
expression, respectively. Furthermore, a tandem repeat of the NF-
B
sequence in the CINC/gro gene conferred the inducibility by IL-1
and suppression of luciferase activity by dexamethasone. In an
electrophoretic mobility shift assay, dexamethasone diminished the
IL-1
-induced formation of NF-
B complexes, which consisted of
p65 and p50. Western blotting revealed that dexamethasone inhibited the
IL-1
-induced translocation of p65 from the cytoplasm into the
nucleus, while the nuclear level of NF-
B p50 remained almost
unchanged. In addition, the degradation of I
B-
induced by
IL-1
was not inhibited by dexamethasone. These results indicated
that the suppression of the CINC/gro gene transcription by
glucocorticoid occurs through the impairment of NF-
B activation,
possibly by interference with the translocation of NF-
B p65 from
the cytoplasm into the nucleus, thereby suppressing transactivation of
the CINC/gro gene.
Neutrophil accumulation at sites of inflammation is induced by
chemoattractants, including C5a (1) and leukotriene
B(2) . In addition to these, we found a novel
polypeptide neutrophil chemotactic factor, cytokine-induced neutrophil
chemoattractant (CINC), (
)which is produced by the normal
rat kidney epithelial cell line NRK-52E(3, 4) .
According to its amino acid sequence, CINC appears to be a member of
the human cytokine family that includes interleukin-8 (IL-8) and three
closely related gro gene products, GRO
, GRO
, and
GRO
(melanoma growth-stimulating activity
,
, and
,
respectively), all of which have chemotactic activities for human
neutrophils(5, 6, 7, 8) . Since rat
CINC has closer sequence homology to GRO than to IL-8(4) , CINC
is the rat equivalent of human GRO but not of IL-8 and hereafter will
be referred to as CINC/gro.
The IL-8 family acts as a functional chemoattractant for neutrophils in vivo(6, 9, 10, 11, 12) . In our previous studies(13, 14) , CINC/gro is shown to contribute to neutrophil infiltration into inflammatory sites in lipopolysaccharide (LPS)-induced inflammation models in rats. Moreover, several lines of evidence have implicated IL-8 in several types of inflammation, such as synovitis(15) , LPS-induced acute dermatitis(6) , and reperfusion injury in the ischemic lung(16) . These findings are suggestive of pathophysiological roles of CINC/gro and IL-8 in inflammatory reactions. Thus, the regulation of CINC/gro and/or IL-8 production is critically involved in the control of inflammatory reactions associated with neutrophil infiltration.
The production of CINC/gro is not constitutive, but
can be induced by several inflammatory stimuli, such as
interleukin-1 (IL-1
), LPS, and tumor necrosis factor
(TNF
)(3, 17, 18) , although little is
known about the intracellular regulatory mechanisms that trigger
CINC/gro up-regulation. The production of human IL-8 and GRO can also
be induced by stimulation with inflammatory cytokines at the
transcriptional level in a wide variety of
cells(5, 19, 20) . In addition, the NF-
B
binding site in the promoter region of human IL-8 and GRO is
indispensable for gene expression in response to cytokine
stimulation(10, 21, 22, 23, 24) .
Glucocorticoids are used as anti-inflammatory and immunomodulatory
agents in a wide variety of diseases. Their physiological effects may
be accomplished largely by modulating the expression of many cytokine
genes, such as IL-1(25, 26) , IL-2(27) ,
TNF(28) , interferon-
(29) ,
interferon-
(27) , and monocyte chemotactic and activating
factor(30) . Glucocorticoids also inhibit the production of
human IL-8 (31) , and the mechanisms of this have been examined
by several groups. Mukaida et al. reported that dexamethasone
decreases human IL-8 mRNA expression at the transcriptional level in
human fibrosarcoma (32) and in human glioblastoma cell
lines(33) . In normal human embryonic lung fibroblasts,
dexamethasone decreases IL-8 gene expression by reducing the stability
of its mRNA(34) . However, a different mechanism may be
responsible in primary cultured human airway epithelial
cells(35) . On the other hand, although glucocorticoids inhibit
GRO production in mouse (36) and rat (18) cell lines,
little is known about the molecular mechanisms by which this is
achieved.
As inhibition of GRO production by glucocorticoids must
also have important implications for their actions as anti-inflammatory
agents, we analyzed the effects of dexamethasone on CINC/gro expression
at the molecular level in the normal rat kidney epithelial cell line
NRK-52E. Evidence is presented here that this steroid hormone
significantly decreases the transcription rate of the CINC/gro gene
without affecting the stability of its mRNA in NRK-52E cells activated
with IL-1. We identified the NF-
B binding site on the
CINC/gro gene as the cis-element responsible for repression by
dexamethasone as well as IL-1
-induced CINC/gro gene activation.
Moreover, an electrophoretic mobility shift assay revealed that
dexamethasone decreased IL-1
-induced NF-
B-binding site
complexes, which were recognized using antibodies against p50 and p65.
Furthermore, we found that this inhibition resulted from the prevention
of NF-
B p65 translocation from the cytoplasm into the nucleus
after degradation of the NF-
B inhibitor I
B-
in the
cytoplasm. Thus, glucocorticoids can inhibit NF-
B activity by a
novel mechanism involving a blockage of the cytokine-induced nuclear
translocation of NF-
B.
The luciferase activity in cell extracts was determined using either a luciferase assay system (Promega) or PicaGene(TM) (Toyo Ink Co., Tokyo, Japan), following the supplier's instructions. The light intensity was measured with a Lumat model LB953 luminometer (Berthold, Germany). The protein concentration was measured by the Bradford method (41) (Bio-Rad protein assay) with bovine serum albumin as a standard. To analysis stably transfected cells, the luciferase assay was performed at least three times with three different transfectants. For transiently transfected cells, results were confirmed by at least two independent transfection experiments with triplicate dishes and are expressed as means ± S.E.
Figure 1:
Dexamethasone
inhibits CINC/gro transcription in NRK-52E cells. A and D, effects of dexamethasone (Dex) on CINC/gro
production. NRK-52E cells were cultured in 24-well plastic dishes in
0.5 ml of Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum until reaching subconfluence. The cells were
then incubated with the indicated concentrations of dexamethasone for 3
h, then stimulated with the indicated stimuli for 5 h. CINC/gro
contents in the culture media were determined by ELISA as described
under ``Experimental Procedures.'' The data were normalized
to the IL-1-treated control (100%) (B). Experiments were
performed three times and representative results are shown. B and E, effects of dexamethasone on CINC/gro mRNA
induction. NRK-52E cells were incubated with the indicated
concentrations of dexamethasone for 3 h, then stimulated with the
indicated stimuli for 3 h. Slot blotting proceeded using 10 µg of
total RNA extracted from NRK-52E cells. The quantity of mRNA was
determined using a bioimage analyzer (Fujix BAS2000). The data are
presented as the -fold increase over the resting value (B) or
were normalized to the IL-1
-treated control (100%) (E)
from three separate experiments. C and F, effects of
dexamethasone on CINC/gro promoter-driven transcription. NRK-52E cells
were permanently transfected with the CINC/gro promoter (nt -1034
to +7) linked to the luciferase gene as described under
``Experimental Procedures.'' The cells were incubated with
the indicated concentrations of dexamethasone for 3 h, then stimulated
with the indicated stimuli for 5 h. Thereafter, the cells were
harvested to determine the luciferase activity. The data are presented
as the -fold increase over resting value (C) or were
normalized to IL-1
-treated control (100%) (F) from three
separate experiments. The stimulants were 100 units/ml IL-1
, 10
µg/ml LPS, 100 units/ml TNF
, or 20 ng/ml
12-O-tetradecanoylphorbol-13-acetate (TPA).
Figure 2:
Effects of dexamethasone (Dex) on
CINC/gro mRNA induction in NRK-52E cells. NRK-52E cells were incubated
with (filled triangles and filled circles) or without (unfilled triangles and unfilled circles) 1
µM dexamethasone (Dex) for 3 h, then stimulated
with (circles) or without (triangles) 100 units/ml
IL-1 for 3 h. Slot blotting proceeded using 10 µg of total RNA
extracted from NRK-52E cells. The quantities of CINC/gro (A),
-actin (B), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (C) mRNAs were determined using a bioimage
analyzer (Fujix BAS2000). The data are presented as the -fold increase
over the resting value at time 0 from three separate
experiments.
Figure 3:
Effects of dexamethasone on the
transcription of CINC/gro gene in NRK-52E cells. NRK-52E cells were
incubated with or without 1 µM dexamethasone (Dex) for 3 h, then stimulated with or without 100 units/ml
IL-1 for 1 h. Nuclei were isolated and nuclear run-on assays were
performed as described under ``Experimental Procedures.'' The
quantity of the CINC/gro or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was determined using a bioimage analyzer
(Fujix BAS2000). Data were normalized to IL-1
-treated CINC/gro
transcriptional rate (100) and are shown as means ± S.E. of
three separate experiments.
Figure 4:
Effects of dexamethasone on stability of
CINC/gro mRNA in NRK-52E cells. NRK-52E cells were incubated with (filled symbols) or without (unfilled symbols) 1
µM dexamethasone (Dex) for 3 h, then stimulated
with 100 units/ml IL-1 for 3 h. Actinomycin D (10 µg/ml) was
added at time 0, and total RNA was isolated at the indicated times.
Slot blotting proceeded using 10 µg of total RNA extracted from
NRK-52E cells. The quantities of CINC/gro (circles) and
-actin (triangles) mRNAs were determined using a bioimage
analyzer (Fujix BAS2000). The amounts of mRNA at various times are
expressed as a function of the mRNA level at time 0. The data presented
are representative of two independent experiments with two
cultures.
Figure 5:
Identification of the responsive elements
for dexamethasone-mediated transcriptional repression of CINC/gro gene
in NRK-52E cells. A, schematic structure of the 5`-flanking
region of the CINC/gro gene. B, delineation of dexamethasone (Dex)-responsive elements in the CINC/gro promoter. Various
lengths of wild-type CINC/gro promoter, or mutants carrying the
internal deletions or point mutations linked to the luciferase gene,
were transiently transfected into NRK-52E cells. The cells were then
incubated with or without 1 µM dexamethasone for 3 h then
stimulated with or without 100 units/ml IL-1 for 5 h, and the
luciferase activity was measured. The promoter activity of each test
plasmid is indicated as luciferase activity relative to that of the nt
-1034/+7-luciferase construct in the presence of IL-1
.
Values are means ± S.E. for at least three separate
experiments.
Northern blotting analysis of
total RNA from the stable transformants revealed that dexamethasone
suppressed the expression of luciferase mRNA induced by IL-1 (data
not shown), indicating that suppression of luciferase activities by
dexamethasone was caused by transcriptional repression of the
introduced fusion gene. These results provide evidence for the presence
of sufficient information within the 5`-flanking sequences between nt
-1034 and +7 of the CINC/gro gene for transactivation, as
well as for suppression by these cytokines and dexamethasone,
respectively.
The regulatory roles of the
NF-IL6 and NF-B binding sites were examined by site-directed
mutagenesis of each element in the -164 luciferase plasmid (Fig. 5). The deletion of the NF-IL6 binding site had little
effects on CINC/gro gene activation and repression by IL-1
and
dexamethasone, respectively, indicating that this site was not
essential for gene regulation by these agents. Deletion or mutation of
the NF-
B binding site abolished IL-1
-induced luciferase
activity, indicating the essential role of the NF-
B binding site
for CINC/gro gene activation by IL-1
.
To further examine the
effect of dexamethasone on the transcriptional activity through the
NF-B binding site of the CINC/gro gene, two copies of the CINC/gro
NF-
B binding site were linked to the adenovirus 2 major late
promoter, which was inserted upstream of the luciferase gene. As shown
in Fig. 6, luciferase gene expression driven from the adenovirus
promoter was not affected by treatment with IL-1
and/or
dexamethasone. Dexamethasone reduced the nt -164 to +7
region of the CINC/gro promoter-driven transcription induced by
IL-1
(Fig. 6). Mutation of the NF-
B target sequence of
the CINC/gro gene in the same luciferase construct abolished the
induction of luciferase activity by IL-1
. Furthermore, the
transcriptional activity of the NF-
B motif, but not of the NF-IL6
motif, was significantly enhanced by IL-1
, and the increased
activity was inhibited by dexamethasone (Fig. 6). These findings
suggested that the NF-
B binding site is the target for gene
repression by dexamethasone.
Figure 6:
Requirement of NF-B binding site for
repression of transcription of CINC/gro gene by dexamethasone. One copy
of the wild-type or NF-
B site-mutated CINC/gro promoter from nt
-164 to +7 as shown in Fig. 5was inserted upstream
of the adenovirus promoter in the antisense orientation with respect to
the luciferase gene. Two copies of the CINC/gro NF-
B site
(5`-GGGAATTTCC-3`) or two copies of the CINC/gro NF-IL6 site
(5`-TGGAGCAAG-3`) were also inserted upstream of the adenovirus
promoter linked to the luciferase gene. Transfection proceeded as
described under ``Experimental Procedures.'' After
transfection, the cells were incubated with or without 1 µM dexamethasone (Dex) for 3 h, then stimulated with or
without 100 units/ml IL-1
for 5 h and the luciferase activity was
measured. The promoter activity of each test plasmid is indicated as
luciferase activity relative to that of adenovirus promoter construct
in the absence of both dexamethasone and IL-1
. These values are
means ± S.E. for at least three separate
experiments.
Figure 7:
Effects of dexamethasone on the formation
of NF-B complexes induced by IL-1
stimulation. NRK-52E cells
were incubated with or without 1 µM dexamethasone for 3 h,
then stimulated with or without 100 U/ml IL-1
for 1 h. Nuclear
extracts were prepared as described under ``Experimental
Procedures.'' A, detection of NF-
B binding activity
by IL-1
stimulation. An electrophoretic mobility shift assay was
performed with no nuclear extracts (lane 1) or with 1 µg
of nuclear extract from either unstimulated (lane 2) or
IL-1
-stimulated (lanes 3-7) NRK-52E cells. Either a
4- (lanes 4 and 6) or 16-fold (lanes 5 and 7) excess of wild-type (lanes 4 and 5) or
mutant (lanes 6 and 7) NF-
B oligonucleotides
were added to the binding reactions as competitors. B,
dexamethasone suppressed the NF-
B complex formation induced by
IL-1
. An electrophoretic mobility shift assay was performed with
no nuclear extract (lane 1), with 1 µg of nuclear extract
from unstimulated cells (lane 2), with those incubated with
IL-1
(lane 3), IL-1
plus dexamethasone (lane
4), or dexamethasone (lane 5). The positions of the
IL-1
-induced bands are indicated by arrows.
To examine whether the induced complex contained any of the
known forms of the Rel family proteins, we performed electrophoretic
mobility shift assays using antibodies against p50, p52, p65, c-Rel,
and RelB. Anti-p50 and anti-p65 antibodies supershifted the complex
induced by IL-1, whereas the other antibodies did not (Fig. 8), indicating that the induced NF-
B complexes were
composed of p50-p65 heterodimers. Dexamethasone inhibited the formation
of the NF-
B complexes induced by IL-1
(Fig. 7B). The radioactivity of the bands was
quantified using a bioimage analyzer (Fujix BAS2000), and we found that
dexamethasone inhibited the complex formation by 30-40% (seven
separate experiments), which were similar to the values of the CINC/gro
production inhibited by dexamethasone. In addition, we found that the
inhibited NF-
B complexes were also composed of p50 and p65
heterodimers according to the results of the supershift assay (data not
shown). These results indicated that dexamethasone inhibits the binding
of the NF-
B transcription factor to the NF-
B binding site of
the CINC/gro promoter, leading to the suppression of CINC/gro mRNA and
protein synthesis.
Figure 8:
The factor induced by IL-1
stimulation is NF-
B composed of p50 and p65. NRK-52E cells were
stimulated with or without 100 units/ml of IL-1
for 1 h, and
nuclear extracts were prepared as described under ``Experimental
Procedures.'' Electrophoretic mobility shift assays were performed
with no nuclear extracts (lane 1) or with 1 µg of nuclear
extracts from either unstimulated (lane 2) or
IL-1
-stimulated (lanes 3-17) NRK-52E cells.
Supershift analysis was performed using 0.5 (lanes 4, 6, 8, 10, 12,
14, and 16) and 1 µg (lanes 5, 7, 9, 11, 13,
15, and 17) of rabbit IgG (lanes 16 and 17) or antibodies against p50 (lanes 4 and 5), p52 (lanes 6 and 7), p65 (lane 8 and 9), c-Rel (lane 10 and 11), RelB (lane 12 and 13), or the glucocorticoid receptor (lanes 14 and 15). The positions of the
IL-1
-induced bands and supershifted bands are indicated by arrows A and B,
respectively.
Figure 9:
Effect of dexamethasone on the levels of
the p65 or p50 subunits of NF-B. NRK-52E cells were incubated with
or without 1 µM dexamethasone for 3 h, then stimulated
with or without 100 units/ml IL-1
for 1 h. SDS-polyacrylamide gel
electrophoresis was performed using 25 µg of nuclear (A and D), 30 µg of cytoplasmic (B), or 30
µg of whole cell (C and E) extracts from
unstimulated cells (lane 1) or those treated with IL-1
(lane 2), IL-1
plus dexamethasone (lane 3), or
dexamethasone (lane 4). After electrophoresis, the proteins
were electroblotted onto Immobilon-P
polyvinylidene
difluoride membranes. Transblotted membranes were incubated with either
anti-p65 (A-C) or anti-p50 (D and E)
antibody, followed by a reaction with horseradish peroxidase-conjugated
anti-rabbit IgG. Proteins detected by the primary antibody were
visualized using an ECL assay kit (Amersham) and by exposure to x-ray
film.
Figure 10:
Effect of dexamethasone on the
degradation of IB-
induced by IL-1
. A, effect
of IL-1
on cytoplasmic I
B-
and nuclear p65 levels in
NRK-52E cells. Cells were stimulated with 100 units/ml IL-1
for
the indicated periods of time. Cytoplasmic (30 µg) and nuclear (25
µg) extracts were analyzed by Western blotting using
anti-I
B-
and anti-p65 antibodies, respectively, followed by
densitometric quantitation of the respective bands in fluorograms. The
amounts of I
B-
and p65 at various times are expressed as a
function of the I
B-
and p65 levels at time 0 and 60 min,
respectively. B, effect of dexamethasone on the
IL-1
-induced degradation of I
B-
. NRK-52E cells were
incubated with or without 1 µM dexamethasone for 3 h, then
stimulated with or without 100 units/ml IL-1
for 1 h. Western
blotting was performed using anti-I
B-
antibody on 30 µg
of cytoplasmic extracts from unstimulated cells (lane 1),
IL-1
(lane 2), or those incubated with IL-1
plus
dexamethasone (lane 3) or dexamethasone (lane
4).
To determine whether
dexamethasone prevents the release of NF-B from I
B-
after exposure to IL-1
, we examined the amount of I
B-
protein in cytoplasmic extracts prepared 1 h after exposure to
IL-1
in the presence or absence of dexamethasone. As shown in Fig. 10B, the amount of I
B-
in
IL-1
-activated cells was markedly reduced to 21% of that in
resting cells. Dexamethasone, however, had no effect on the
IL-1
-induced degradation of I
B-
(Fig. 10B). These results suggested that dexamethasone
inhibits the activation of NF-
B by interfering with a pathway
after the degradation of I
B-
.
Glucocorticoid hormones are highly immunosuppressive and
reportedly inhibit the gene expression of several cytokines,
particularly those with proinflammatory actions, such as
IL-1(25, 26) , IL-2(27) ,
TNF(28) , interferon-
(29) ,
interferon-
(27) , IL-8 (31) , and monocyte
chemotactic and activating factor(30) . The results of this
study demonstrated the inhibitory effect of the synthetic
glucocorticoid dexamethasone on CINC/gro production in rat NRK-52E
cells and its mechanism. We found that dexamethasone suppressed
IL-1
-induced CINC/gro gene expression ( Fig. 1and Fig. 2) by inhibiting the transcriptional rate of the CINC/gro
gene (Fig. 3) without affecting the stability of CINC/gro mRNA (Fig. 4). The inhibition of CINC/gro gene expression by
dexamethasone was reversed by the specific glucocorticoid receptor
antagonist RU486 (data not shown), suggesting the involvement of the
glucocorticoid receptor in transcriptional repression. Furthermore, we
showed that the 5`-flanking region of the CINC/gro gene (extending from
nt -1034 through +7) is sufficient to confer responsiveness
to IL-1
and dexamethasone, since dexamethasone inhibited the
IL-1
-induced expression of the CINC/gro promoter-driven luciferase
vector stably transfected into NRK-52E cells (Fig. 1, C and F).
Functional analysis of the regulatory
sequences of the CINC/gro gene demonstrated that the minimally
essential elements for the induction by IL-1 and repression by
dexamethasone of the CINC/gro gene were present within the 3` region
downstream of nt -164, which contains the two known cis-elements, NF-IL6 (nt -119 to -111) and
NF-
B (nt -62 to -53) binding sites (Fig. 5).
Deletion of the NF-IL6 binding site did not abolish the inhibition of
IL-1
-induced luciferase activity by dexamethasone (Fig. 5B), suggesting that dexamethasone does not
inhibit CINC/gro gene transcription through interference with the
NF-IL6 binding site. Although either a deletion or mutation of the
NF-
B binding site abolished luciferase activity induced by
IL-1
(Fig. 5B), a tandem repeat of the NF-
B
sequence in the CINC/gro gene conferred inducibility by IL-1
and
suppression of luciferase activity by dexamethasone (Fig. 6),
suggesting that this site is the element responsible for
dexamethasone-mediated gene repression. In addition, an electrophoretic
mobility shift assay demonstrated that dexamethasone significantly
diminished the IL-1
-induced formation of NF-
B complexes (Fig. 7B), which were identified immunochemically to
consist of p50 and p65 (Fig. 8). Our results suggested that the
NF-
B binding site is responsible for CINC/gro gene repression by
dexamethasone.
Western blotting of cytoplasmic and nuclear extracts
from NRK-52E cells demonstrated that IL-1 treatment induced the
translocation of p65 from the cytoplasm into the nucleus (Fig. 9, A and B). Dexamethasone diminished
the amount of p65 translocated to the nuclear extracts from cells
exposed to IL-1
(Fig. 9A). We also found that the
loss of nuclear p65 was paralleled by an increase in cytoplasmic p65 (Fig. 9, A and B) without affecting the total
amount of p65 in the cells (Fig. 9C), suggesting that
dexamethasone interfered with its translocation from the cytoplasm into
the nucleus. Under our experimental conditions for Western blotting
however, we detected neither the IL-1
-induced translocation of p50
protein into the nucleus nor the inhibition of translocation by
dexamethasone (Fig. 9D). It is possible that the amount
of p50 protein translocated from the cytoplasm into the nucleus was
much less than that of the nuclear p50 protein constitutively present
even in unstimulated cells.
We surmised that dexamethasone prevented
the release of NF-B from I
B after exposure to IL-1
.
Since the phosphorylation and degradation of I
B is necessary for
the activation of NF-
B (46, 47, 48, 49) , we examined the
amount of I
B protein by Western blotting. Time course experiments
showed that the activation of NF-
B by IL-1
in NRK-52E cells
is correlated closely with the degradation of I
B-
, a member
of the I
B family (Fig. 10A). We found that
dexamethasone did not prevent the IL-1
-induced degradation of
I
B-
(Fig. 10B), suggesting that it interfered
with the translocation of NF-
B into the nucleus after dissociation
of NF-
B/I
B-
complexes in the cytoplasm. However, we
cannot exclude the possibility that dexamethasone has some effects on
the synthesis and stability of other members of the I
B family,
including I
B-
(50) , as well as the NF-
B
precursors p105 (51, 52) and p100(53) .
Mechanisms of glucocorticoid receptor-mediated repression of
transcription have been proposed involving the physical interaction of
the glucocorticoid receptor and
NF-B(33, 54, 55) . This interaction may
result in the glucocorticoid-mediated blockage of nuclear NF-
B
binding to NF-
B binding sites. We showed that glucocorticoid
decreased the levels of nuclear NF-
B of IL-1
-stimulated cells (Fig. 9A) while the glucocorticoid receptor level in
the nucleus was markedly increased (
)under the conditions
where dexamethasone inhibited the NF-
B binding to DNA in the
nucleus (Fig. 7B). Thus, although we could not
immunochemically detect the glucocorticoid receptor in the NF-
B
complexes formed in the presence of IL-1
and dexamethasone by
means of an electrophoretic mobility shift assay,
it is
likely that a combination of decreasing the nuclear NF-
B protein
level and blocking the binding of nuclear NF-
B to DNA by
protein-protein interaction accounts for the total repression of
CINC/gro gene expression by dexamethasone. The interaction of a
glucocorticoid receptor with NF-
B complexes in the nucleus is now
under study to determine whether the interaction also involves
suppression of NF-
B activation in NRK-52E cells.
The AUUUA
sequence in the 3`-untranslated region may be involved in the rapid
degradation of mRNAs for some inflammatory cytokines and
proto-oncogenes(56, 57) . Moreover, several reports
have shown that dexamethasone decreases the stability of mRNAs
containing the AUUUA sequence in the 3`-untranslated
region(25, 29) . A specific protein binding to RNAs
containing AUUUA has been identified(58, 59) , and it
is thought that formation of this complex may target susceptible mRNAs
for rapid cytoplasmic degradation. CINC/gro mRNA contains similar AUUUA
sequences in the 3`-untranslated region(39) . However, we found
that dexamethasone does not significantly decrease the half-life of
entire CINC/gro mRNA, although its half-life was much shorter than that
of the -actin transcript which does not contain this sequence (Fig. 4). Thus, the present findings suggest that the AUUUA
sequence is responsible for destabilization of CINC/gro mRNA, but not
for CINC/gro gene repression by dexamethasone in NRK-52E cells.
Early studies showed that glucocorticoid inhibits the transcription
of human IL-8 gene, either by binding of the glucocorticoid receptor to
the glucocorticoid responsive element in the 5`-flanking region of the
gene (32) or by interference with the binding of NF-B to
DNA without inhibiting the nuclear translocation of the
factor(33) . We demonstrated here that dexamethasone interfered
with the binding of NF-
B to its cis-element on the rat
CINC/gro gene that lacks a glucocorticoid responsive element in its
promoter region. Unlike human IL-8, dexamethasone decreased the nuclear
level of p65, perhaps by sequestering p65 in the cytoplasm after
dissociation from I
B-
in NRK-52E cells. Thus, it is likely
that glucocorticoids can inhibit NF-
B activity by two novel
mechanisms involving blocks of cytokine-induced nuclear translocation
and DNA binding to NF-
B. Given the role of NF-
B in the
transcriptional activation of many inflammatory cytokine genes, we
propose that these types of inhibition of NF-
B activation
represents an important mechanism for the immunosuppressive properties
of glucocorticoids.