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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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). beta -Galactosidase activity in the same samples was measured by using the method of Sambrook et al. (19). Luciferase activity was normalized to beta -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 [gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -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 beta -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.

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.

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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Table 1.   Time course of nuclear translocation

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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Table 2.   Effect of FK-506 and RU-486 on punctate accumulation of GR in the nucleus

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    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).


    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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 283(2):F254-F261
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society




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