Effect of immunosuppressive agents on glucocorticoid receptor
function in A6 cells
Robert S.
Edinger1,
Simon C.
Watkins2,
David
Pearce3, and
John P.
Johnson1
1 Renal-Electrolyte Division, Department of
Medicine, and 2 Department of Cell Biology and
Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213;
and 3 Department of Medicine, University of
California, San Francisco, California 94143
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ABSTRACT |
Immunosuppressive agents such as FK-506
and rapamycin inhibit aldosterone- stimulated Na+ transport
in A6 cells. Concentration dependence is consistent with the known
affinities of these agents for immunophilins. The inhibition was also
dependent on time, requiring preincubation with FK-506 or rapamycin
before inhibition was seen. The present studies were designed to
determine whether this inhibition was pretranscriptional and whether it
was due to an effect on either receptor translocation or nuclear
accumulation. Because transport effects of steroids in A6 cells are
mediated by glucocorticoid receptors (GRs), we examined the
transcriptional response of GR-regulated reporters transfected into
these cells. Preincubation of cells with FK-506 and rapamycin
completely blocked reporter gene activation, whereas preincubation with
cyclosporin A partially inhibited this activation. A minimum of 8 h of preincubation was required before the effect was seen. Using a
transiently transfected green fluorescent protein-GR construct, we
examined the effect of FK-506 and rapamycin on GR translocation. GR
translocation induced by dexamethasone was extremely rapid (<5 min)
and was largely unaffected by FK-506 or rapamycin but was completely
blocked by geldanamycin. Digital deconvolutions revealed a punctate
nuclear accumulation of GR, which was still seen after preincubation
with immunosuppressive agents. These agents clearly inhibit steroid
action by blocking GR-stimulated gene transcription, but this effect is
not mediated by altered translocation or nuclear accumulation of
receptors. Inhibition of steroid-regulated gene transcription by
immunosuppressive agents may explain the electrolyte abnormalities seen
in patients receiving these drugs.
FK-506; rapamycin; transcriptional inhibition
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INTRODUCTION |
GLUCOCORTICOID RECEPTORS
(GRs) are localized predominantly in the cytoplasm until bound by
their cognate ligand. These unbound receptors exist as heterooligomeric
protein complexes in association with several heat shock proteins,
HSP90 and HSP70, as well as immunophilins such as FKBP-52, and
potentially other cyclophilins (for a review, see Ref. 2;
16, 17, 20). The exact functions of these associated proteins are not
known, and some parts of the complex may dissociate from the receptor
on hormone binding (2). It has been suggested that the
receptor-associated proteins may serve to retain the unbound receptor
in the cytoplasm (16) or act as part of a mechanism for
vectorial translocation of the bound receptor to the nucleus
(17). In support of this latter possibility are
observations that agents that bind to HSP90 such as geldanamycin alter
the kinetics of receptor translocation (21, 27) and the
suggestion that the immunophilin component of the complex may be
required for translocation (16), possibly because it binds
both HSP90 and cytoplasmic dynein (6, 22). However, initial studies using FK-506, which binds to immunophilins and inhibits
their peptidylprolyl isomerase activity, either failed to show an
effect on transcriptional activation by ligand-bound glucocorticoid
receptor (GR) or suggested an enhancement of activity (9,
14).
Our laboratory has previously examined the effect of the
immunosuppressive agents FK-506, rapamycin, and cyclosporin on basal and aldosterone-stimulated ion transport in A6 cells (18).
Aldosterone is known to stimulate Na+ transport in A6 cells
via interaction with GR (18). Consistent with observations
that coincubation of FK-506 with glucocorticoids has no effect on
receptor function (9), we observed no effect of this agent
on steroid-stimulated Na+ transport when coincubated with
hormone. We did, however, note a time-dependent effect: a complete
inhibition of steroid-stimulated transport by both FK-506 and rapamycin
was seen after preincubation with these agents before hormone addition.
FK-506 also inhibited basal as well as steroid-stimulated
Na+-K+-ATPase activity. Preincubation with
cyclosporin did inhibit basal Na+-K+- ATPase
activity and maximal transport rates after apical membrane permeabilization with nystatin but did not have the complete inhibitory effect on steroid-stimulated Na+ transport that was seen
with FK-506 and rapamycin. We speculated that the inhibition of both
basal Na+-K+-ATPase activity and
steroid-stimulated transport by FK-506 might contribute to the
hyperkalemia seen after organ transplantation, which is seen with both
FK-506 and cyclosporin but is more common with FK-506 than with
cyclosporin (18). Our data suggested, but did not prove,
that the inhibition of steroid-stimulated transport was at a
pretranscriptional site (18). Recently, LeBihan and colleagues (10) have reported an inhibition of both
progestin- and GR-mediated transcription in breast cancer cells by
FK-506 and rapamycin, but not cyclosporin. These observations led us to
reexamine whether the dose- and time-related inhibition of GR-induced
transport we have described was due to inhibition of steroid-regulated
transcription. We also sought to determine whether this phenomenon was
related to altered nuclear translocation or accumulation of GR.
 |
MATERIALS AND METHODS |
Plasmids.
The green fluorescent protein (GFP)-GR, GFP-mineralocorticoid (MR)
plasmids were previously described (3, 5). The
chloramphenicol acetyltransferase and luciferase reporter genes contain
three copies of a glucocorticoid response element (GRE) sequence
derived from the rat TAT gene linked upstream of the
Drosophila melanoganser alcohol dehydrogenase
gene minimal promoter sequence (23) driving the expression
of the chloramphenicol acetyltransferase or luciferase (12) genes.
Cell culture and transfection.
A6 cells obtained from American Type Culture Collection were cloned by
limiting dilution and selected for high-amiloride-selected short-circuit current. Cells were maintained in amphibian medium with
10% fetal bovine serum in an atmosphere of humidified air-4% CO2 at room temperature. For transfections, A6 cells were
plated at ~40% confluency on 25-mm circular glass coverslips (Fisher Scientific) in 60-mm dishes. Cells were transfected the next day using
Lipofectamine Plus (GIBCO BRL) according to the manufacturer's directions. DNA was incubated on the cells for 5 h and washed, and
the medium containing charcoal-stripped serum was added. Reporter gene
analysis or translocation/accumulation was performed within 24 h.
Reporter gene analysis.
The hormonally inducible luciferase reporter gene was transfected as
above, and the cells were pretreated with either 1 µM FK-506, 1 µM
rapamycin, 1 µM cyclosporin A (CyA), or vehicle for 18 h in
stripped media. Aldosterone (1 µM) was then added and incubated for
4 h. Cells were then harvested, and luciferase activity was
measured using a TD-20/20 luminometer (Turner-Designs).
-Galactosidase activity in the same samples was measured by using
the method of Sambrook et al. (19). Luciferase activity
was normalized to
-galactosidase activity.
Nuclear translocation.
A6 cells were transfected with GFP-GR as described above. The cells
were grown on coverslips and pretreated with 1 mM FK-506, 1 mM
rapamycin, or vehicle in stripped media for 18 h. The coverslip was mounted in a chamber and perfused with media. All photographs were
obtained using a multimode microscope (Zeiss Axiovert 135) and
Metamorph Software (UIC, West Chester, PA) using a
high-numerical-aperture (1.0) ×40 oil-immersion objective. ImageSpace
(Molecular Dynamics) software was used to measure the kinetics of
translocation. This measurement was based on relative increases in
fluorescence concentration within the nucleus over time. All
translocations occurred at room temperature. At time
0, 100 nM dexamethasone (Dex) was added. Time-lapse
photography was taken every minute. For cotreatment, the cells were
mounted in the chamber and covered with media and either 1 mM
FK-506/100 nM Dex or 1 µM rapamycin/100 nM Dex was added at
time 0. Geldenamycin was used at 0.3 µg/ml, and RU-486 was
used at 1 µM.
Nuclear accumulation.
After translocation was complete, the objective was switched to a
×100, 1.3 numerical aperture to study the accumulation within the
nucleus. Using iterative deconvolution algorithms (Huygens, Bitplane)
to process Z-axis stacks, collected at 2× axial Nyquist separation (0.17 µm), focused deconvolved images were prepared. The
images were quantitated using stereological measurements as described
(26). Briefly a 1 × 1-mm grid was overlaid on the nucleus. All punctuate structures overlapping a grid point were counted
and divided by the total number of points available in the nucleus. The
results for all conditions are reported.
Electrophoretic mobility shift assay.
Isolation of the nuclear extract was performed as described
(1). The following oligonucleotides containing a
palindromic GRE sequence (underlined) were synthesized (Invitrogen):
5'-GCAGGTCCTCTGCAGAACACAGTGTTCTAGCTACAAGG-3' and
5'-ATCCT- TGTAGCTAGAACACTGTGTTCTGCAGAGGACCT-3'
(GRE bottom). Equal molar amounts of each oligonucleotide were heated
to 95°C and cooled to 25°C in 3 h. Thirty picomoles of
annealed primer were end-labeled with [
-32P]ATP (ICN)
using T4-polynucleotide kinase (New England Biolabs). For 20-µl
binding reactions, 15 mg of nuclear extract were mixed with 100,000 counts/min of labeled probe in 40 mM Tris · Cl (pH 7.5), 0.2 mM
EDTA, 80 mM NaCl, 8 mM dithiothreitol, 20% glycerol, 0.1% BSA, 0.25%
Nonidet P-40, and 20 mg poly dI-dC. For competition reactions, 50× M
extract of unlabeled probe was included in the incubation. The reaction
was incubated for 30 min at room temperature. The samples were
separated on a 5% nondenaturing polyacrylamide gel and analyzed on a
PhosphorImager screen.
 |
RESULTS |
Our laboratory has previously demonstrated that FK-506 and
rapamycin, but not cyclosporin, inhibit aldosterone-stimulated Na+ transport in A6 cells. To determine whether inhibition
of steroid action in A6 cells by FK-506 or rapamycin was due to
inhibition of steroid-induced transcription, we transiently
cotransfected a hormone-inducible luciferase reporter gene into the A6
cell line along with a
-galactosidase reporter gene to normalize for transfection efficiency. The transfected cells were pretreated with
either 1 µM FK-506, rapamycin, or CyA for 18 h before the addition of 1 µM aldosterone or the immunosuppressive agents were added simultaneously with the steroid. Luciferase and
-galactosidase activity were measured 4 h later. As shown in Fig.
1, both FK-506 and rapamycin completely
inhibited steroid-induced luciferase activity in the 18-h
preincubation. There was no effect on transcriptional activation when these agents were incubated simultaneously with the
steroid (Fig. 1, A and B). CyA had no effect on
luciferase activity when added concurrently with steroid hormone but
did partially inhibit reporter gene activation after the 18-h
preincubation (Fig. 1C). Similar results were obtained using
a hormone-inducible chloramphenicol acetyltransferase reporter gene
(Fig. 2). To determine the time of
preincubation with FK-506 required for inhibition of the steroid, the
hormonally induced luciferase reporter gene activity normalized to
-galactosidase activity was monitored over several hours. At least
8 h of preincubation were required to inhibit steroid hormone
activation (Fig. 1D). Thus FK-506 and rapamycin and, to a
lesser extent, CyA, inhibited transcriptional activation in a
time-dependent manner, requiring at least an 8-h preincubation period.

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Fig. 1.
Immunosuppressant agents cause a time-dependent inhibition of the
steroid-responsive luciferase reporter gene. A6 cells transiently
transfected with both a hormonally inducible luciferase and
-galactosidase reporter genes were either coincubated (co) with 1 µM aldosterone or preincubated (pre) with 1 µM each of FK-506
(A), rapamycin (RAP; B), or cyclosporin A (CyA;
C) for 18 h before the addition of aldosterone. Values
are means ± SD of reporter gene activity. The minimum time for
preincubation of FK-506 before the addition of steroid is shown in
D. * Significantly different from similar condition
without aldosterone; n = 6 for all conditions.
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Fig. 2.
FK-506 causes time-dependent inhibition of the
chloramphenicol acetyltransferase (CAT) reporter gene. The conditions
described in Fig. 1 were repeated using the Maloney murine leukemia
virus (MMLTV)-CAT reporter gene. Aldo, aldosterone. Values are
means ± SD. * Significantly different from similar condition
without aldosterone; n = 6.
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Because one of the components of the hormone receptor complex has been
implicated in translocation to the nucleus, we next examined the
possibility that immunophilin ligands might affect the kinetics of
nuclear translocation or alter nuclear accumulation. To examine this
possibility, A6 cells were transiently transfected with constructs
containing GFP fused to the COOH terminus of either the GR or the MR.
These fusion proteins have previously been shown to remain active and
functional compared with the wild-type receptor (5, 8).
Both the GFP-GR and GFP-MR constructs were tested for nuclear
translocation in A6 cells. The experiments shown here were performed
using the GFP-GR construct. GFP-GR was transiently transfected into A6
cells, which were then treated with 100 nM Dex. Sequential images were
recorded every minute for 30 min after the addition of the hormone. As
can be seen in Fig. 3, control cells
transfected with GFP-GR underwent complete translocation within 15 min.
No nuclear translocation occurred in the absence of Dex (data not
shown). Before hormone addition, there was minimal fluorescence within
the nucleus, suggesting that the majority of the GFP-GR is cytoplasmic,
as expected. With normalization to time 0 fluorescence, it
is then possible to measure an increase in the signal within the
nucleus or decrease in the signal within the cytoplasm after hormone
addition. This allows a more quantitative assessment of GR
translocation. Such an experiment is shown in Fig.
4. After addition of steroid
ligand to the bath, there was a lag period of 2-3 min, after which
the GFP-GR rapidly translocated to the nucleus and the process is well
fit by a single sigmoid function. The actual period of rapid
translocation was highly reproducible and was just under 2 min under
control conditions, somewhat more rapid than previously reported in
other cell systems (8).

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Fig. 3.
Control translocation of the glucocorticoid receptor (GR) to the
nucleus. A6 cells transiently transfected with enhanced green
fluorescent protein (EGFP)-GR are shown. At time 0,
dexamethasone (1 µM) was added and time-lapse photography was
performed over the time course indicated.
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Fig. 4.
Kinetics of GR translocation. The appearance and
disappearance of EGFP-GR in the nucleus and cytoplasm were measured as
described in MATERIALS AND METHODS. Representative receptor
translocation induced by addition of dexamethasone is shown. Time from
ligand addition to maximal nuclear accumulation is indicated
(t). Time from initial increase in nuclear signal to maximal
accumulation is indicated as the rapid accumulation phase
(tRA); n = 6 for both
conditions.
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The kinetics of nuclear translocation of GFP-GR were examined under the
conditions described to examine the effect of FK-506 on reporter gene
activation. In addition, we examined the effects of two other agents,
geldanamycin and RU-486, on GFP-GR translocation. Geldanamycin binds to
another component of the steroid receptor complex, HSP90, and has been
reported to both slow nuclear translocation and inhibit
receptor-mediated transactivation under various conditions (7,
21, 24, 27). RU-486, a GR antagonist, readily binds to the
receptor and induces translocation but not transactivation (8,
13). As shown in Fig. 5,
translocation of the GFP-GR to the nucleus after hormone addition
occurred readily in the presence of FK-506 with or without
preincubation. Incubation of the cells with 0.3 ug/ml geldanamycin for
3 h resulted in total inhibition of translocation (Fig.
6). Treatment of A6 cells with geldanamycin at this concentration also completely blocked steroid stimulation of transport as measured by the short-circuit current in
these cells over a 6-h period (data not shown). The effects of various
agents on the time course of GR translocation are shown in Table
1. Preincubation with FK-506 did result
in a modest slowing of the rapid nuclear accumulation of GFP-GR, and a
further slowing was seen with RU-486, but under all conditions
translocation was complete and equal within 20 min. In additional
experiments, preincubation with FK-506 had no effect on the time course
of nuclear translocation measured with a GFP-MR construct (data not shown).

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Fig. 5.
Effects of FK-506 on nuclear translocation. Cells were coincubated
with FK-506 and dexamethasone (A) or preincubated for
18 h with FK-506 before the addition of dexamethasone
(B). Time-lapse photography was performed over the time
course indicated.
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Fig. 6.
Effects of geldanamycin on nuclear translocation. Cells transiently
transfected with EGFP-GR were preincubated with 0.3 µg/ml
geldanamycin for 2 h before the addition of dexamthasone at
time 0. Time-lapse photography was performed over the time
course indicated.
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Because nuclear translocation was not altered in the presence of
FK-506, we next examined the specific location of the receptor within
the nucleus after translocation. Previous work has shown that RU-486, a
glucocorticoid antagonist, binds to the receptor and activates nuclear
translocation but does not activate hormone-responsive genes
(8). However, RU-486 is associated with a marked change in
the pattern of receptor accumulation within the nucleus.
Transcription-competent agonists are associated with a punctate
appearance of receptor accumulation within the nucleus
(8), whereas antagonists such as RU-486 produce a diffuse
pattern of nuclear accumulation. It appears that the punctate
accumulation may be necessary for or indicative of transcriptional
activation (5, 8, 13). If nuclear accumulation of the
hormone receptor is critical for activation of hormone-responsive
genes, it remained possible that FK-506 affected this aspect of the
hormone-response cascade. To examine this possibility,
three-dimensional digital deconvolutions were utilized to determine the
accumulation of the activated GFP-fused receptors within the nucleus.
Cells were pretreated or cotreated with the immunosuppressant drugs as
described above, and then translocation of the GFP-GR was initiated by
the addition of 100 nM Dex. The accumulation of the GFP-GR within the
nucleus was determined. As shown in Fig.
7A, the three-dimensional
digital deconvolutions of A6 cells transfected with GFP-GR treated only with Dex demonstrated GFP fluorescence in a punctate pattern. These
punctate structures were observed in all control cells. As seen in Fig.
7B, no punctate structures were observed in the presence of
RU-486, as previously described (8). Cells exposed to
FK-506 demonstrated normal accumulation of the punctate structures within the nucleus, suggesting that this agent has no effect on the
accumulation of the GR after translocation to the nucleus. The relative
abundance of punctate structures in the nucleus after translocation of
the GFP-GR was scored under each condition examined, and the results
are shown in Table 2. FK-506 does not
appear to alter the accumulation of the GFP-GR within the nucleus.

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Fig. 7.
Nuclear sublocalization of EGFP-GR. Cells transfected
with EGFP-GR were either induced with dexamethasone (A) or
RU-486 (B) to initiate nuclear translocation. After
translocation was complete, the nucleus was subjected to digital
deconvolution to examine the sublocalization of the receptor.
FK-506/dexamethasone coincubation (C) and FK-506
preincubation (18 h) then dexamethasone (D) are also
shown.
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Because nuclear translocation or accumulation was not altered by
FK-506, we next examined what effect FK-506 has on GR-DNA interactions.
Nuclear extracts isolated from cells treated with 1 µM aldosterone
for 30 min (Fig. 8, lanes 1 and 2) or pretreated with 1 µM FK-506 for 18 h then
with 1 µM aldosterone (lanes 3 and 4) were
incubated with a radiolabeled DNA fragment containing a palindromic GRE
sequence. Sequence specificity was determined by adding 50× M extract
of unlabeled probe (lanes 2 and 4). The results
show that preincubation of cells with FK-506, which inhibits transcription, does not alter the binding of the GR to DNA.

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Fig. 8.
Effects of FK-506 on GR-glucocorticoid response element
(GRE) interactions (GR:GRE). Nuclear extracts from cells treated with 1 µM aldosterone for 1 h (lanes 1 and 2) or
pretreated for 18 h with 1 µM FK-506 and then 1 µM aldosterone
for 1 h (lanes 3 and 4) were isolated. A
radiolabeled DNA fragment containing a consensus GRE was subjected to
the nuclear extract (lanes 1 and 3) or nuclear
extract and 50× M extract of unlabeled probe (lanes 2 and
4) were resolved on a nondenaturing gel. The positions of
free probe and GR:GRE are shown.
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 |
DISCUSSION |
Our laboratory has previously described the inhibition of
GR-mediated Na+ transport in A6 cells by the immunophilin
ligands FK-506 and rapamycin (18). At the time, we
speculated that this might be mediated through a common interaction of
these agents with the steroid receptor-associated HSP56. LeBihan and
colleagues (10) described FK-506 and rapamycin inhibition
of GR and progestin receptor-mediated transcription in a line of breast
cancer cells. We therefore decided to revisit the question of
inhibition of GR-mediated effects in A6 cells to determine whether this
was in fact due to transcriptional inhibition by these agents. Earlier reports had suggested variable effects of FK-506 on GR-mediated transcription but not complete inhibition (9, 14). We have examined this issue with the use of GR-reporter constructs. Results of
these experiments indicate that FK-506 and rapamycin do indeed inhibit
GR-mediated gene activation but only after a preincubation period of
8 h. This result is compatible with both the results of
LeBihan et al. (10), who examined the effects of these
agents under similar conditions, as well as the results noted earlier (9), which found, as we did, no inhibition of steroid
action with simultaneous addition of the agents.
The immunosuppressive agents FK-506 and CyA are known to act, at least
partly, through inhibition of transcription mediated by the
calcium-dependent phosphatase calcineurin (11). We did not
feel that inhibition of GR-mediated transcription was mediated by this
pathway for several reasons. First, the time lag is not typical of
FK-506 or CyA inhibition mediated via calcineurin. Second, rapamycin,
which does not act via calcineurin (18), gave similar
inhibition of GR activation of reporter constructs to that seen with
FK-506. Finally, cyclosporin appears to be less potent than either
FK-506 or rapamycin in blocking steroid-stimulated Na+
transport or reporter gene activation. When examining the mechanism of
this inhibition of GR activity in A6 cells, we focused on the presence
of binding proteins for these immunosuppressive drugs as components of
the untransformed GR heterocomplex. Immunophilin ligands are known to
bind to HSP56 even when it is incorporated in the untransformed GR
complex (4, 9, 15, 25). A cyclophilin is also known to be
part of the untransformed GR heterocomplex (15, 16). The
role of chaperone proteins in the receptor complex is the subject of
debate, but a number of lines of evidence suggest that they may be
related to nuclear translocation or accumulation after ligand binding
(7, 16, 17, 22). The GR heterocomplex is thought to be a
dynamic structure (17). We hypothesized that disruption of
this complex by immunophilin ligands in a time-dependent manner might
result in alterations of the rate of nuclear translocation or in
subnuclear accumulation. We examined this possibility using a GFP-GR
construct. In our experiments, GR translocation to the nucleus was
quite rapid. Within 5 min of addition of ligand to the bath,
translocation of the GFP-GR construct could be detected. When viewed in
serial images, this appears as a wave of directed movement into the
nucleus. Under control conditions, translocation was virtually
completed within 2 min of initial vectorial movement. This is somewhat
faster than previously reported (8), but we used a higher
concentration of Dex than other investigators, and rate of
translocation does appear to be dependent on ligand concentration (8). Preincubation with FK-506 or rapamycin at
concentrations that inhibited reporter gene activation had no
significant effect on the rate of nuclear translocation. In contrast,
geldenamycin, a naturally occurring benzoquinone antibiotic which is
known to bind to the HSP90 component of the GR heterocomplex,
completely blocked GR translocation over the time course studied here
and completely inhibited steroid-stimulated Na+ transport.
Previously, geldanamycin has been shown to inhibit GR-mediated gene
induction (21, 25) and to slow nuclear translocation of GR
in the presence of an intact cytoskeleton but not after cytoskeletal
disruption (7).
We next considered the possibility that agents binding the chaperone
components of the receptor complex might interfere with nuclear
accumulation. Here again, there was no significant effect of either FK
or rapamycin on nuclear accumulation and the GFP-GR construct appeared
in the nucleus in punctate structures similar to those seen under
control conditions. This pattern of nuclear accumulation has been
proposed as essential to gene activation, as the
translocation-competent antagonist RU-486 inhibits both gene activation
and nuclear accumulation (8). Although accumulation into
such punctate structures may be necessary for transcriptional activation, it is clearly not sufficient for such activation. FK-506
and rapamycin inhibit GR activation of transcription after nuclear
accumulation and binding. The mechanism remains unknown. It seems
likely that this inhibitory action that blocks epithelial transport
responses to aldosterone in A6 cells may play a role in the
hyperkalemia commonly seen in transplant recipients who receive these
agents as immunosuppressive drugs.
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ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-47874 (to J. P. Johnson) and DK-51151 (to D. Pearce).
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FOOTNOTES |
Address for reprint requests and other correspondence:
J. P. Johnson, 935 Scaife Hall, 3550 Terrace St.,
Pittsburgh, PA 15213 (E-mail
johnson{at}msx.dept-med.pitt.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 5, 2002;10.1152/ajprenal.00337.2001
Received 9 November 2001; accepted in final form 27 February 2002.
 |
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