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INTRODUCTION |
Glucocorticoids are known to modulate a variety of cellular
processes and play an important role in homeostasis and development. Furthermore, glucocorticoids are potent suppressors of the immune system and are therefore used as therapeutic treatment in a broad range
of autoimmune and in- flammatory diseases. Glucocorticoid action is
mediated via binding to the glucocorticoid receptor (GR).1 The inactive receptor
is bound to chaperone proteins, including two heat shock protein 90 (hsp90) subunits, and is located in the cytoplasm (1, 2). Upon ligand
binding, the activated GR dissociates from the chaperone complex and
translocates to the nucleus to activate or repress transcription of
glucocorticoid target genes. Stimulation of gene transcription is
mediated via binding of GR to glucocorticoid responsive elements (GREs)
in the promoter region of glucocorticoid responsive genes (1-3).
Renal tubular epithelial cells (TEC) are an important source of
cytokines and chemokines and are thought to play an active role in the
progression of inflammatory processes in the kidney (4). Although
glucocorticoids are known to have a profound inhibitory effect on
cytokine production by a variety of cell types (5-7), we have
previously shown that production of inflammatory mediators by renal
tubular epithelial cells is insensitive to the inhibitory action of
glucocorticoids (8). Inhibition of cytokine production by
glucocorticoids has been attributed to transrepression of NF-
B, a
pro-inflammatory transcription factor that regulates the expression of
many inflammatory mediators, including IL-6 and IL-8 (9, 10). The
mechanism of NF-
B suppression by glucocorticoids has been studied
extensively. Initially it was postulated that corticosteroids inhibit
NF-
B activation by increasing the transcription of I
B-
, the
endogenous inhibitor of NF-
B (11, 12), but this occurs only in
certain T-cell lines. Subsequent studies have shown that the activated
glucocorticoid receptor can bind to the p65 subunit of NF-
B, thus
interfering in the binding of NF-
B to DNA (13, 14). The currently
accepted view is that corticosteroids interfere with the
transactivating potential of NF-
B via alterations in cofactor
recruitment (15-17).
In the present study, we have investigated modulation of gene
transcription by dexamethasone in the renal epithelial cell line HK-2
to obtain more insight in the observed steroid insensitivity. We show
that dexamethasone is unable to prevent activation of the NF-
B
pathway. Furthermore, we demonstrate that glucocorticoid receptor
translocation to the nucleus in renal epithelial cells is functional
and comparable to the glucocorticoid-sensitive airway epithelial cell
line A549. In renal epithelial cells, dexamethasone stimulated the
expression of two glucocorticoid-responsive genes, angiotensinogen
(AGT) and
2-adrenoreceptors, but had no effect on
cytokine production, clearly demonstrating dissociation of the positive
and negative regulatory function of glucocorticoids in this cell type.
These results emphasize the cell type-specific characteristics of
glucocorticoid action.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The renal epithelial cell line HK-2 was kindly
provided by M. Ryan, University College Dublin, Ireland (18). Cells
were cultured in RPMI 1640 medium (Invitrogen) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal
calf serum (
FCS) and 100 units/ml penicillin, 100 µg/ml
streptomycin (all from Sigma Chemical Co.). The human airway
epithelial cell line A549 was cultured in Dulbecco's modified Eagle's
medium (Invitrogen) supplemented with L-glutamine and 10%
FCS. Prior to stimulation, cells were cultured in serum-free medium
for 48 h.
IL-6 and IL-8 ELISA--
Prior to stimulation, cells were
transferred to 48-well plates (Costar, Corning, NY) at a density of
0.5 × 105 cells per well and serum-starved. Cells
were stimulated with 5 ng/ml IL-1
(Preprotech, Rocky Hill, NJ). For
inhibition experiments, cells were pretreated for 2 h with
dexamethasone (Sigma) before addition of IL-1. Production of IL-6 and
IL-8 in culture supernatants was measured by specific ELISA as
described previously (19). Cytokine production is presented as mean
concentration ± S.D. from representative experiments. Experiments
were repeated at least three times.
Preparation of Cell Extracts--
I
B-
and
2-adrenoreceptor protein levels were detected in whole
cell extracts. Cells were harvested at different time points after
stimulation and incubated in lysis buffer containing 20 mM
Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100,
2 mM EDTA, and a protease inhibitor mix consisting of 5 units/ml Trasylol (Bayer, Leverkusen, Germany), 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml antipain, 2 µg/ml
chymostatin, and 2 µg/ml leupeptin (all from Roche Molecular
Biochemicals) for 10 min on ice. Supernatants were collected after
centrifugation for 15 min at 13,000 rpm.
For detection of GR translocation, nuclear and cytosolic extracts were
prepared. After stimulation with dexamethasone (10
10 to
10
6 M), cells were harvested by scraping in 2 ml of ice-cold Hanks' Balanced Salt Solution (HBSS; Sigma) and
centrifuged for 5 min. Cytosolic extracts were prepared by addition of
buffer A (10 mM Hepes pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, complete protease inhibitor mixture) for
10 min on ice and centrifugation. The pellets containing the nuclear proteins were extracted in buffer B (20 mM Hepes, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% glycerol, 1 mM dithiothreitol,
complete protease inhibitor mixture) for 30 min on ice. After
centrifugation, the supernatant was mixed with buffer C (20 mM Hepes pH 7.9, 50 mM KCl, 0.2 mM
EDTA, 1 mM dithiothreitol, complete protease inhibitor mixture). Protein concentration was determined with a Bradford protein
assay kit (Bio-Rad, München, Germany).
Western Blot Analysis--
Cell extracts were size-fractionated
by SDS-PAGE using NuPAGE precast gradient gels (Invitrogen) and
transferred to Hybond ECL membranes (Amersham Biosciences). The
membrane was blocked in Tris-buffered saline, 0.1% Tween 20 containing
5% nonfat dry milk. Primary antibodies used were rabbit anti-human
I
B-
(C-21) antibody, rabbit anti-human
2-adrenoreceptor (H-73) antibody (both from Santa Cruz
Biotechnology, Santa Cruz, CA), and mouse anti-human GR antibody
(Signal Transduction Laboratories, Lexington, KY). Horseradish
peroxidase-conjugated secondary antibodies were obtained from DAKO,
Glostrup, Denmark. Blots were developed with ECL chemiluminescence
substrate (Amersham Biosciences). Densitometry was performed using a
Stratagene Eagle sight analysis program. Efficiency of transfer was
verified by staining with Ponceau Red (Sigma).
RT-PCR--
Total RNA was isolated using RNAzolB (Campro,
Veenendaal, The Netherlands) according to the manufacturer's
instructions. A260/A280 ratios were measured to determine the quantity and purity of RNA preparations. Fixed amounts of total cellular RNA (1 µg) were reverse-transcribed into cDNA by oligo(dT) priming using M-MLV reverse transcriptase (Invitrogen). Expression of I
B-
and AGT mRNA was determined by RT-PCR with
-actin as a control. Primer sequences are shown in Table I. PCR was performed under standard conditions (50 mM KCl, 10 mM Tris-HCl, pH 8.4, 0.06 mg/ml bovine serum albumin, 0.25 mM dNTPs, 25 pmol of
each primer, 1 unit of Taq polymerase; PerkinElmer Life
Sciences) with MgCl2 2.5 mM for I
B-
and
1.5 mM for AGT and
-actin. The following scheme was used: 5 min 95 °C, I
B-
/
-actin: 35 cycles of 1 min 95 °C,
1 min 60 °C (I
B-
) or 55 °C (
-actin) and 1 min 72 °C;
AGT: 40 cycles of 1.5 min 95 °C, 2.5 min 60 °C, 1.5 min 72 °C;
7 min 72 °C. PCR products were analyzed on a 1% agarose gel
containing ethidium bromide.
Detection of NF-
B DNA Binding Activity--
For
electrophoretic mobility shift assay (EMSA), nuclear extracts were
prepared as previously described (23). Cells were stimulated with IL-1
for 1 h with or without preincubation with dexamethasone. A small
fraction (3 µg) of nuclear extracts was used to establish DNA binding
as described previously (24). The probe used for detection of DNA
binding was a 32P-labeled oligonucleotide containing the
B site from HLA-A (5'-GTG GGG ATT CCC CAC TGC A-3') (23). For
supershift assays, anti-p50 (sc-114) and anti-p65 (sc-109) antibodies
(Santa Cruz Biotechnology) were added to the nuclear extract and probe
mixture and incubated for 1 h at 4 °C. Samples were run on
a 6% polyacrylamide gel in 0.5× Tris borate/EDTA buffer and
analyzed by autoradiography.
NF-
B DNA binding activity was also assessed with Trans-AM NF-
B
p65 and p50 transcription factor assay kits (Active Motif Europe,
Rixensart, Belgium) according to the manufacturer's instructions. Cells were incubated with 10
6 M dexamethasone
for 30 min and stimulated with 5 ng/ml IL-1 for 30 min. Whole cell
lysates were prepared and 2-µg extracts were added to 96-well plates
coated with an oligonucleotide containing the NF-
B consensus site.
Binding of NF-
B to the DNA was visualized by anti-p50 and anti-p65
antibodies that specifically recognize activated NF-
B. Antibody
binding was measured at 450 nm. Specificity of NF-
B activation was
determined by competition experiments using NF-
B wild-type and
mutant consensus oligonucleotides that were provided with the kit.
Plasmids--
The reporter plasmids pGL-3
B-Luc and pMMf-Luc
were a generous gift from J. W. Bloom, Dept. of Pharmacology, Tucson,
AZ. Plasmid pGL-3
B-Luc contains 3 NF-
B sites linked to the
luciferase gene inserted into the pGL3-basic vector. Plasmid pMMf-Luc
is a derivative of pGL3-basic containing the MTVGRE
promoter of the mouse mammary tumor virus (MMTV) linked to the
luciferase gene.
Transient Transfection and Luciferase Assay--
Before
transfection, cells were plated at a density of 0.5 × 106 cells/well in 6-well plates (Costar) and serum-starved
for 24 h. On the day of transfection, culture medium was switched
to serum-free Dulbecco's modified Eagle's medium. For inhibition studies, cells were pretreated with 10
6 M
dexamethasone or 10 µg/ml caffeic acid phenethyl ester (CAPE; Sigma) for 1 h prior to transfection. Cells were transfected with LipofectAMINE/Plus Reagent (Invitrogen) following the manufacturer's instructions using 1 µg of DNA per well. After a 1-h incubation, 1 ml
of Dulbecco's modified Eagle's medium culture medium containing the
appropriate stimulus was added to the cells. 6 h after
stimulation, cells were lysed via addition of 500 µl of reporter
lysis buffer (Promega, Madison, WI) and overnight incubation at
20 °C. Lysates were harvested, and luciferase activity was
assessed using a luciferase assay substrate (Promega) according to the
manufacturer's instructions.
Immunocytochemistry--
A549 and HK-2 cells were cultured in
8-well chamberslides (Falcon, BD PharMingen, Franklin Lakes, NJ) at a
density of 0.5 × 105 cells per well. After incubation
with dexamethasone, slides were harvested and fixed in ice-cold acetone
for 10 min. Cells were permeabilized with 0.8% Nonidet P-40 and
blocked for 20 min in phosphate-buffered saline, 20% normal swine
serum (DAKO). Primary rabbit anti-human GR (E-20, Santa Cruz
Biotechnology) was diluted 1:25 in phosphate-buffered saline, 0.1%
bovine serum albumin and incubated for 60 min. After extensive washing
in phosphate-buffered saline, biotin-conjugated swine anti-rabbit Ig
(DAKO) was incubated for 45 min. After another wash, slides were
incubated with streptavidin-fluorescein isothiocyanate (DAKO) for 45 min. Slides were counterstained with 10
6 M
DAPI (4',6-diamidino-2-phenylindole) (Sigma) for 4 min and washed and
mounted in 50% phosphate-buffered saline, 50% glycerol. Slides were
analyzed with a Leica TCS-SP confocal laser-scanning microscope.
Chromatin Immunoprecipitation (ChIP) Assay--
HK-2 cells
(1 × 106) were stimulated with dexamethasone
(10
8 M) for 4 h. ChIP assay was
performed according to the manufacturer's instructions (Upstate
Biotechnology, Lake Placid, NY). Briefly, protein-DNA complexes were
fixed in a final concentration of 1% formaldehyde. Cell pellets were
resuspended in 5 mM Pipes, pH 8.0, 85 mM KCl,
0.5% Nonidet P-40 containing protease inhibitors (1 µg/ml aprotinin,
1 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 10 min. After centrifugation, pellets were resuspended in SDS lysis buffer (50 mM Tris,
pH 8.1, 1% SDS, 5 mM EDTA, protease inhibitors) and
sonicated (nine 10-s pulses). Sonicated samples were incubated
overnight with 5 µg of rabbit anti-histone H4 (ac 5) antibody
(Serotec, Oxford, UK). A null-antibody immunoprecipitation served as
negative control. Of each sample, 20 µl was kept as input/starting
material. The soluble chromatin was immunoprecipitated and washed, and
histone-DNA complexes were eluted from the antibody by adding elution
buffer (1% SDS, 0.1 M NaHCO3). Histone-DNA
cross-links were reversed in 0.2 M NaCl by heating at
65 °C for 4 h. The DNA sample was further purified by 1-h
incubation at 45 °C in 0.01 M EDTA, 0.04 M
Tris-HCl, pH 6.5 and 40 µg/ml proteinase K (Sigma). DNA was extracted
with phenol/chloroform, precipitated with ice-cold 100% ethanol, and
resuspended in 50 µl of Tris/EDTA.
The promoter sequence of the human angiotensinogen gene was obtained
from GenBankTM (AF424741). We used the TRANSFAC data base
(transfac.gbf.de/TRANSFAC/) (25) to predict GR-binding sites in the
AGT promoter sequence. Two PCR primer pairs were designed to
cover the first (AGT1) or second and third (AGT2) GR-binding sites
(Table I). PCR was performed under
standard conditions as described for RT-PCR using input samples as
positive control. The following scheme was used: 94 °C, 4 min; 35 cycles of 94 °C, 45 s; 60 °C, 45 s; 72 °C, 45 s; and 10 min, 72 °C. PCR products were run on a 2% agarose gel and visualized with ethidium bromide.
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RESULTS |
Differential Effect of Dexamethasone on HK-2 and A549
Cells--
Stimulation of the renal epithelial cell line HK-2 with
IL-1 (5 ng/ml) for 6 h resulted in increased production of
inflammatory mediators, IL-6 and IL-8. To examine the effect of
glucocorticoids on cytokine production, cells were preincubated for
2 h with dexamethasone and stimulated with IL-1 in the continuous
presence of dexamethasone. No effect of dexamethasone
(10
6 M) was observed on either constitutive
or IL-1-induced production of IL-6 and IL-8 by HK-2 cells (Fig.
1, A and C). In
contrast, dexamethasone completely abolished IL-8 production in the
airway epithelial cell line A549 (Fig. 1B). Thus, already at
6 h after stimulation there is a clear discrimination in steroid
responsiveness between renal and airway epithelial cells, which is more
pronounced after 48 h (Fig. 1C).

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Fig. 1.
Differential effect of dexamethasone on HK-2
and A549 cells. Dexamethasone (Dex) does not affect
IL-1-induced IL-6 and IL-8 production by HK-2 cells (A and
C) but completely inhibits IL-8 production by A549 cells
(B and C). Cells were incubated with
dexamethasone for 2 h before stimulation with IL-1 (5 ng/ml) for
6 h (A and B) or 48 h (C).
IL-6 and IL-8 production in culture supernatants was measured by
specific ELISA. Results are expressed as mean ± S.D. of one
representative of six independent experiments.
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I
B-
Degradation Is Not Inhibited by Dexamethasone--
To
identify the mechanism of steroid insensitivity in HK-2, we examined
the ability of dexamethasone to repress the activity of NF-
B. Since
inhibition of the NF-
B pathway by glucocorticoids has been suggested
to be mediated via induction of I
B-
expression (11, 12), this
pathway was first investigated. Western blot analysis showed that IL-1
induced degradation of I
B-
15-60 min after stimulation, whereas
protein levels of I
B-
were unaltered after treatment with
dexamethasone for 0.5-24 h (Fig.
2A). Furthermore, dexamethasone was unable to prevent IL-1-induced degradation of I
B-
(Fig. 2B). After I
B-
degradation, prolonged
stimulation of cells with IL-1 (4-24 h) increased I
B-
protein
levels, suggesting the presence of an autoregulatory feedback mechanism
(Fig. 2A). In accordance, IL-1 increased mRNA expression
of I
B-
, which peaked at 1 h after stimulation (Fig.
2C). Dexamethasone showed no effect on I
B-
mRNA
levels, which is compatible with our protein data.

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Fig. 2.
Expression and degradation of
I B- is not altered in
HK-2 cells treated with dexamethasone. A, HK-2 cells
were incubated with 10 6 M dexamethasone
(Dex) or 5 ng/ml IL-1 for up to 24 h. Data were
analyzed with densitometry. One representative experiment is shown.
B, cells were incubated with dexamethasone for 2 h and
stimulated with IL-1 for 30 min. C, I B- mRNA
expression was analyzed by RT-PCR as described under "Experimental
Procedures." Top panel, I B- and -actin
mRNA expression levels 1 h after stimulation with IL-1 or
dexamethasone. Bottom panel, I B- / -actin ratio
was calculated after densitometry.
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DNA Binding Activity of NF-
B Is Not Affected by
Dexamethasone--
To study the effect of dexamethasone on DNA binding
activity of NF-
B we performed EMSA. In non-stimulated HK-2 cells we
could already detect NF-
B binding (Fig.
3A). Previously we have shown that primary renal epithelial cells expressed two NF-
B complexes, a
p50-p65 heterodimer and a more abundant p50-p50 homodimer (24). Supershift analysis showed that also in HK-2 this p50-p50 complex was
the most abundant complex, while the p50-p65 complex was expressed only
very weakly. Stimulation of HK-2 for 1 h with IL-1 increased DNA
binding activity, which was not inhibited when cells were stimulated in
the presence of 1 µM dexamethasone (Fig. 3A,
lane 4).

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Fig. 3.
Dexamethasone has no effect on DNA binding of
NF- B. A, HK-2 cells were
incubated for 2 h with 10 6 M
dexamethasone (Dex) and stimulated with 5 ng/ml IL-1 for
1 h. Nuclear extracts were prepared as described under
"Experimental Procedures" and analyzed with electrophoretic
mobility shift assay using the B site of HLA-A as a probe.
B, assessment of NF- B p65 and p50 DNA binding activity
using TransAm transcription factor assay kits as described under
"Experimental Procedures." IL-1-induced p65 and p50 DNA binding
activity in HK-2 cells was not blocked by dexamethasone.
A450 values were corrected for background
levels. Experiments were performed three times.
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The inhibitory effect of dexamethasone on NF-
B has been specifically
attributed to interaction with the p65 subunit of NF-
B (13, 14). To
investigate the effect of dexamethasone on p65-mediated DNA binding, we
used p65 and p50 transcription factor assay kits, which allow specific
detection of activated p65 and p50. As shown in Fig. 3B, a
high level of constitutive binding of p65 and p50 to the NF-
B
consensus site could be detected in non-stimulated HK-2 cells compared
with the steroid-sensitive cell line A549. Stimulation of HK-2 cells
with IL-1 induced an increase in p65- and p50-mediated DNA binding
activity, which was not blocked by pretreatment with dexamethasone.
High Constitutive NF-
B Activation in HK-2 Cells--
The high
constitutive level of DNA binding prompted us to study transcriptional
activation by NF-
B in HK-2 cells using a NF-
B-responsive
luciferase reporter gene, pGL-3
B-Luc, which contains three NF-
B
consensus sites. In non-stimulated HK-2 cells, a high constitutive
activity of the reporter gene was observed, indicating a high level of
constitutively active NF-
B in this cell type (Fig.
4A). In contrast, constitutive
levels of reporter gene activity were low in transfected A549 cells.
This was not due to a decreased transfection efficiency of A549 cells,
which was measured with a control plasmid expressing green fluorescent protein (GFP; data not shown). These results are compatible with the
DNA binding studies shown in Fig. 3.

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Fig. 4.
High constitutive
NF- B activation in HK-2 cells.
A, cells were transfected with 0.1 or 1 µg of a
NF- B-inducible reporter gene (pGL-3 B-Luc) and assessed for
luciferase activity. B, for inhibition studies, cells were
pretreated with 10 6 M dexamethasone
(Dex) or 10 µg/ml CAPE for 1 h. Shown is the
stimulation index of five independent experiments.
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Next, we studied the capability of dexamethasone to transrepress
NF-
B-induced transcription of the reporter gene. Fig. 4B shows that in HK-2 cells, reporter gene activity was only minimally blocked by dexamethasone but was strongly repressed (>95%) by CAPE, a
known inhibitor of NF-
B activation. In A549 cells, dexamethasone and
CAPE suppressed
B-reporter gene activity by, respectively, 65 and 85%.
Dexamethasone Induces Glucocorticoid Receptor
Translocation--
To further analyze the mechanism of steroid action
in renal epithelial cells we compared the glucocorticoid receptor
signaling pathway in HK-2 and A549 cells. One of the early steps in
glucocorticoid receptor signaling is translocation of GR from the
cytosol to the nucleus upon activation of the receptor through ligand
binding (1, 2). In both cell types, GR expression in control cells was
mainly cytosolic as visualized with immunocytochemistry (Fig. 5, A and C).
Already at 30 min after stimulation with 10
6
M dexamethasone an increase in nuclear staining was
observed, which was more pronounced at 4 h (Fig. 5, B
and D).

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Fig. 5.
Nuclear translocation of GR is comparable in
HK-2 and A549 cells. A-D, immunocytochemistry of GR
localization. Cells were incubated with (B and D)
or without (A and C) 10 6
M dexamethasone (Dex) for 4 h. GR
localization in A549 (A and B) and HK-2 cells
(C and D) was analyzed by immunocytochemistry.
Results are representative of four independent experiments.
E, representative Western blot showing the effect of
dexamethasone on GR localization. Cells were stimulated with
dexamethasone (10 10 to 10 6 M)
for 2 h. Cytosolic and nuclear extracts were prepared and analyzed
for GR protein levels. F, Western blots were analyzed with
densitometry. Shown are mean ± S.D. from three independent
experiments. *, p < 0.05 versus control
cytosolic extract; **, p < 0.01 versus
control cytosolic extract; #, p < 0.05 versus control nuclear extract; ##, p < 0.01 versus control nuclear extract.
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The effect of dexamethasone on GR translocation was also assessed
quantitatively with Western blotting. Cytosolic and nuclear extracts
were prepared after stimulation with increasing doses of dexamethasone
(10
10 to 10
8 to 10
6
M) for 2 h. Glucocorticoid receptor expression in HK-2
cells was comparable to the expression levels in A549 (Fig.
5E). Stimulation of HK-2 cells with 10
8 and
10
6 M dexamethasone induced an increase in
nuclear GR, which was accompanied by a decrease in cytosolic GR (Fig.
5, E and F). Nuclear translocation induced by
dexamethasone was similar in both cell types, suggesting that the first
part of the GR signaling pathway in HK-2 cells is functional.
Dexamethasone Stimulates the Expression of
Glucocorticoid-responsive Genes--
In A549 cells it has been
demonstrated that dexamethasone treatment induces an increase in
2-adrenoreceptor mRNA levels and receptor number
(26). Therefore, we analyzed the effect of dexamethasone on the
expression of
2-adrenoreceptor in HK-2 cells with
Western blotting. In both cell types, dexamethasone (10
6
M) induced an increase in
2-adrenoreceptor
protein levels (Fig. 6).

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Fig. 6.
Increased expression of
2-adrenoreceptors after stimulation
with dexamethasone. Cells were stimulated with dexamethasone
(10 6 M) for 4 h. Expression levels of
2-adrenoreceptors ( 2-AR) in whole cell
lysates were analyzed by Western blot. One representative blot of three
independent experiments is shown.
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Next, we performed semiquantitative ChIP assays to study
AGT expression, which is known to be stimulated by glucocorticoids in vitro and in vivo (27-29). Sequence analysis
of the AGT promoter (
1223 to +35) identified the
presence of three putative GR-binding sites. Two different primer pairs
were designed to amplify the first GR-binding site (AGT1) or the second
and third GR-binding site (AGT2) as shown in Fig.
7A.

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Fig. 7.
Dexamethasone stimulates expression of
angiotensinogen. A, angiotensinogen promoter regions
containing GRE consensus sites (boxed text). Sequences
containing the first (AGT1) or second and third (AGT2) GRE consensus
site were amplified by PCR primer pairs. The start of the coding region
(CR) is shown with an arrow. B,
association of acetylated histone 4 (Lys-5) with the angiotensinogen
promoter. Cells were stimulated with 10 8 M
dexamethasone (Dex) for 4 h. Proteins and DNA were
cross-linked with formaldehyde, sonicated, and acetylated histone 4 (Lys-5) was precipitated. Associated DNA was amplified with PCR primer
pairs shown in A. Results shown are representative of four
independent experiments. C, effect of dexamethasone on
angiotensinogen mRNA expression. Total RNA was isolated from
dexamethasone-treated cells and expression of angiotensinogen mRNA
was determined by specific RT-PCR. Expression levels of -actin were
used as a control. One representative RT-PCR of three independent
experiments is shown.
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Immunoprecipitation with an antibody against histone H4 (ac 5) showed
that in non-stimulated cells, a low level of constitutive histone H4
(Lys-5) acetylation was present (Fig. 7B).
Stimulation of HK-2 cells with 10
8 M
dexamethasone resulted in a marked enrichment of AGT
promoter DNA (Fig. 7B). The enrichment for AGT
promoter segments was observed for both AGT1 and AGT2, suggesting that
more than one GR-binding site within the AGT promoter is
acetylated by dexamethasone-activated GR.
In addition we investigated whether the increase in histone acetylation
upon stimulation with dexamethasone correlated with an increase in AGT
mRNA expression. In accordance with the histone acetylation data,
semiquantitative RT-PCR showed that angiotensinogen mRNA levels
were increased in cells stimulated with dexamethasone for 4 h
compared with non-stimulated cells (Fig. 7C).
GR-mediated Transactivation Is Functional--
To compare the
transactivating capacity of GR in HK-2 and A549, cells were transfected
with a luciferase reporter gene, which is regulated by a promoter
containing glucocorticoid-responsive elements (pMMf-Luc). Constitutive
GR activity in HK-2 cells was very low compared with A549 cells (Fig.
8A). This was not due to
altered transfection rates as confirmed by co-transfection with
GFP-expressing control plasmids. At high concentrations of dexamethasone (10
5 M), stimulation of the
reporter gene was comparable in both cell types (35-fold), indicating
that the positive regulatory pathway of glucocorticoids is intact (Fig.
8B). However, transactivation of the reporter gene in HK-2
cells was less sensitive than in A549 cells with a log shift in
EC50 for dexamethasone between the cell lines (Fig. 8,
A and B).

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Fig. 8.
Transactivation capacity of GR in HK-2 cells
is functional but less sensitive than in A549 cells. A,
cells were transfected with a GR-inducible reporter gene (pMMf-Luc) and
stimulated with increasing concentrations of dexamethasone for 6 h. B, stimulation index of the experiment described in
A. n = 3.
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 |
DISCUSSION |
In the present study we demonstrate dissociation of
transrepression and transactivation by glucocorticoids in renal
tubular epithelial cells. Glucocorticoids are potent suppressors of the immune system and are known to inhibit cytokine production by a variety
of cell types (5-7). However, we have shown previously that cytokine
and chemokine production by activated renal tubular epithelial cells is
insensitive to the inhibitory action of dexamethasone (8). The present
study was undertaken to investigate the mechanism of steroid action
in renal epithelial cells. We compared the effect of dexamethasone on
cytokine production by the renal epithelial cell line HK-2 with the
airway epithelial cell line A549 and found that already at 6 h
after stimulation with IL-1 there is a clear discrimination in steroid
responsiveness between renal and airway epithelial cells, suggesting
that an early signaling pathway might be involved. Suppression of
cytokine production by glucocorticoids is mainly attributed to
inhibition of NF-
B (3, 10), for which several mechanisms have been
proposed (11-17). In our study, no effect of dexamethasone on
I
B-
synthesis and DNA binding activity of NF-
B was observed.
Furthermore, transactivation of an NF-
B-inducible reporter gene was
not inhibited.
The absence of transrepression by dexamethasone prompted us to
investigate the glucocorticoid signaling pathway in HK-2. We demonstrate that dexamethasone induced translocation of GR from the
cytosol to the nucleus, suggesting that the first part of the signaling
pathway is intact. Next, we examined the transactivation capacity of
dexamethasone on the expression of AGT, a gene that is
positively regulated by glucocorticoids in various cell types, including rat proximal tubular epithelial cells (27-29). Stimulation of gene transcription is thought to be mediated via acetylation of core
histones on highly conserved lysine residues (2, 30). Co-activator
molecules including the CREB-binding protein (CBP) are able to induce
histone acetylation via intrinsic histone acetyl transferase activity
(HAT) and have been shown to bind to activated GR, resulting in a
dexamethasone-specific pattern of histone acetylation (15, 31, 32), as
was demonstrated previously for the SLPI gene (32). In the
present study we show that stimulation of HK-2 cells with dexamethasone
induced an increase in histone 4 (Lys-5) acetylation on different GR
binding sites in the AGT promoter, indicating increased
transcription of the AGT gene. The increase in histone
acetylation was accompanied by an increase in angiotensinogen mRNA
levels. Nuclear translocation and transcriptional activation in HK-2
cells was increased after dexamethasone concentrations that did not
inhibit cytokine production, suggesting that the steroid insensitivity
of renal epithelial cells is specific for the inhibitory function of
glucocorticoids, whereas the stimulatory function is intact.
At high doses of dexamethasone, transactivation of a GRE-inducible
reporter gene in HK-2 cells was comparable to the steroid-sensitive cell line A549. However, at low doses activation of the reporter gene
appeared to be less sensitive in HK-2 cells. In parallel, we observed a
high level of constitutive NF-
B activation in non-stimulated HK-2
cells compared with A549 cells. Interaction between GR and NF-
B has
been shown to result in functional antagonism (13, 33). The mechanism
underlying this antagonism might be competition for cofactors,
including CBP, as has been proposed in some studies (15, 33). Based on
this model of functional antagonism, we would like to propose that a
high level of constitutive NF-
B activation contributes to the low
level of GR reporter gene activity we observed in HK-2 cells or
vice versa. Other possibilities include a failure of GR to
repress p50-mediated transactivation through the
B site since p50
levels are high under basal levels and are further increased following
IL-1 stimulation. Despite the high level of constitutively active
NF-
B, cytokine production by non-stimulated HK-2 is low, albeit
higher than in A549 cells. This suggests either that it is unlikely
that NF-
B is the sole transcription factor involved in increased
cytokine production by HK-2 cells, but rather functions in conjunction
with other signaling pathways, including mitogen-activated protein
kinases (34, 35) or that a repressor must be removed to enable cytokine
transcription to occur efficiently.
Alternatively, it has been shown that inhibition of transcription
factors involves only a single GR monomer and that DNA binding is not
required (36, 37), whereas activation of gene transcription by
glucocorticoids is mediated via binding of GR homodimers to GRE in the
promoter of glucocorticoid-responsive genes. Defective interaction of
GR monomers with NF-
B might also explain the lack of transrepression
by glucocorticoids. Inhibition of gene transcription by glucocorticoids
can also occur via negative GRE or via transrepression of other
transcription factors like AP-1. Whether these inhibitory functions of
GR are also hampered in renal epithelial cells still has to be determined.
In conclusion, we have demonstrated that renal epithelial cells are
responsive to positive but not to negative modulation by
glucocorticoids. Decreased GR activity or increased constitutive activation of the NF-
B signaling pathway might contribute to the
lack of suppression by glucocorticoids. These results emphasize the
cell type-specific characteristics of glucocorticoid action.