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* Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206; and
Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GC action is mediated through the GC receptor (GCR), which is found in
the cytosol of most human cells. As the result of alternative splicing
of the GCR pre-mRNA, there are two homologous mRNAs and protein
isoforms, termed GCR
and GCR
(6). GCR differs
from GCR only in its C terminus, with replacement of the last 50 aa
of GCR with a unique 15-aa sequence lacking a steroid binding
domain. These differences render GCR unable to bind GCs, reduce its
binding affinity for DNA recognition sites, abolish its ability to
trans-activate GC-sensitive genes, and make it function as a
dominant inhibitor of GCR. Although GCR has been reported to be
increased in steroid-resistant diseases, its role in steroid resistance
has been controversial (7, 8).
In steroid-resistant asthmatics, GCs fail to inhibit T cell
proliferation and to reduce secretion of proinflammatory cytokines
(5). Their airways have abnormally high levels of IL-2 and
IL-4 expression (5, 9). Recent studies indicate that the
combination of IL-2 and IL-4 induces T cell resistance to GCs and
increased GCR expression in the T cells of normal subjects
(10). However, in contrast to neutrophils
(11), the increased GCR expression in T cells is not
sufficiently high to solely account for steroid resistance.
Interestingly, in mice IL-2 alone can induce T cell resistance. The
mouse genome does not contain the GCR gene (12).
Therefore, the study of mouse T cells provides an opportunity to
examine mechanisms of steroid resistance in the absence of GCR. The
aim of this work was to characterize the molecular basis for the
functional interaction between IL-2 and the GCR signal transduction
pathways to establish the mechanisms by which IL-2 can induce steroid
insensitivity in lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant murine IL-2 and IL-4 were purchased from R&D (Minneapolis, MN), Janus-associated kinase 3 (JAK3) inhibitor 1 (IC50 = 78 µM) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (IC50 = 39 nM) were purchased from Calbiochem (San Diego, CA); dexamethasone (DEX) and cycloheximide were purchased from Sigma-Aldrich (St. Louis, MO).
Cell culture
Murine IL-2-dependent HT-2 cell line was cultured in complete medium, IMDM (Sigma-Aldrich) containing 7.5% heat-inactivated FCS (Gemini Bio Products, Calabasas, CA) with supplements as previously described (13) in the presence of 50 U/ml murine IL-2. For the studies of GCR nuclear translocation in response to steroids, charcoal-filtered GC-free FCS (Gemini Bio Products, Calabasas, CA) was used in the growth medium.
Murine spleen cells from wild-type and knockout mice were grown at a concentration 106 cells/ml in IMDM (Cellgro; Mediatech, Herndon, VA) containing 10% heat-inactivated FCS, 400 µM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 20 mM HEPES (Mediatech) with 5 µg/ml PHA (Sigma-Aldrich) for 48 h.
Mice
C57BL6/129 mice were purchased from Harlan Sprague Dawley (Indianapolis, IN), and STAT5a/b knockout mice were provided by Dr. Ihle (St. Jude Medical and Research Hospital, Memphis, TN).
Lymphocyte proliferation assays
HT-2 cells were plated in 96-well round-bottom culture plates (Nalge Nunc International, Rochester, NY) at 1 x 104 cells/well in growth medium in the absence or the presence of 10-8–10-5 M DEX. HT-2 cells were stimulated with different concentrations of murine IL-2 (1, 5, 10, and 50 U/ml) or IL-4 (50, 100, 250, and 500 U/ml). Cultures were incubated for 24 h at 37°C in humidified, 7% carbon dioxide-containing incubator. Six hours before harvest 1 µCi/well of [3H]thymidine (ICN Biomedicals, Costa Mesa, CA) was added. Cells were then harvested onto glass-fiber filters (Tomtec Harvester 96, Orange, CT), placed into a liquid scintillation cocktail (Wallac, Milton Keynes, U.K.), and incorporated tritium was counted in a liquid scintillation counter (1450 Microbeta Plus Counter, Wallac, OH). Results were presented as mean counts per minute of triplicate cultures.
Similar proliferation assays were performed with primary murine splenocytes. Murine spleen cells were aseptically isolated and put into culture as described above. After 48 h the cells were washed twice, resuspended in fresh medium, plated in 96-well, round-bottom culture plates at 1 x 105 cells/well, and stimulated by different concentrations of murine IL-2 or IL-4. DEX (10-8–10-5 M) was used to examine the steroid sensitivity of the cells. Cultures were incubated for 24 h at 37°C in humidified, 7% carbon dioxide-containing incubator. IC50, defined as the concentration of DEX that inhibits the proliferation of cytokine-stimulated lymphocytes to 50% of the level seen in the absence of DEX, was used as the parameter to measure the steroid sensitivity of the cells.
Immunofluorescence staining of HT-2 cells for GCR
Number 1 coverslips (22 x 22 mm; thickness range, 0.13–0.17 mm; Fisher Scientific, Pittsburgh, PA) were precoated by soaking with poly-D-lysine (Sigma-Aldrich) at a concentration of 1 mg/ml for 10 min, then washed twice in distilled water. Cells were resuspended in growth medium with IL-2 or IL-4 at a final concentration of 1 x 106 cells/ml. Five hundred microliters of each cell suspension was then applied to a separate coverslip, and the cells were allowed to settle and adhere for 5 min at room temperature, fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich) in PBS for 5 min at room temperature, then washed twice in PBS. DEX was added to the remaining cells to a final concentration of 1 x 10-6 M. Cell suspensions were incubated at 37°C for an additional 30 min. After the incubation period cells were applied to a separate coverslips, allowed to settle and adhere, fixed, and washed as described above.
In some sets of experiments cells were grown in the medium with 500 U/ml of murine IL-4 for 18–20 h and then treated with 50 U/ml murine IL-2 for 30 min. For GCR nuclear translocation experiments with inhibitors the cells were treated with cycloheximide (50 µg/ml), p38 MAPK inhibitor SB203580 (10 µM) or JAK3 inhibitor 1 (312 µM) for 1 h before IL-2 stimulation. Then the cells were treated as described above.
The cells fixed on coverslips were incubated for 15 min at room
temperature with permeabilization solution (PBS containing 0.1% (v/v)
Tween 20, 0.1% (w/v) BSA (Sigma-Aldrich), and 0.01% (w/v) saponin
(Sigma-Aldrich)). The permeabilization solution was then tipped off,
and cells were blocked with a commercial blocking solution
(Superblock; Scytek, Logan, UT) for 15 min at room temperature. After
the incubation period, the blocking solution was aspirated off and
discarded. Cells were then incubated with an affinity purified
polyclonal Ab to GCR (Affinity Bio-Reagents, Golden, CO) diluted
in permeabilization solution (1/250) overnight at 4°C. Purified
nonimmune rabbit IgG (Southern Biotechnology Associates, Birmingham,
AL) was used as an isotype control. After the incubation period the
coverslips were washed in PBS/0.1% Tween 20 for 15 min at room
temperature with gentle agitation, then incubated with a donkey
anti-rabbit IgG, F(ab')2-Cy3 conjugate
secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA;
1/200), and the nucleus was counterstained with 300 nm
4',6'-diamidino-2-phenylindole (DAPI) (Sigma) for 45 min at room
temperature and washed in PBS/0.1% Tween 20 for 15 min at room
temperature with gentle agitation.
Coverslips were then mounted on slides (SuperFrost Plus Microscope
Slides; Fisher Scientific) in mounting solution (2% (w/v)
o-phenylenediamine dihydrochloride, 0.1 M Tris-HCl, and 90%
(v/v) glycerol (Sigma), pH 8.5) and examined by confocal fluorescence
microscopy (Leica, Wetzlar, Germany) using a x40 objective.
The intensity of GCR staining was assessed using image analysis
software (Slidebook; Intelligent Imaging Innovations, Denver, CO) and
was expressed as the mean fluorescence intensity (MFI). In brief, a
series of 1 µm z-frames were taken through groups of cells in both
the DAPI (nuclear) and Cy3 (GCR) channels. Each stack of images then
underwent nearest neighbors deconvolution. This allowed for exact
localization of the nucleus within the cell and removed background
fluorescence from above and below the z-frame that could affect the
reliability of the results. A single computer-generated mask was then
defined based on the DAPI staining. The intensity of Cy3 staining (GCR)
was assessed by the analysis software purely within the DAPI mask and,
hence, the nucleus. In this way increases in GCR within the nucleus
could be detected.
Western blot analysis
HT-2 cells were harvested in PBS and lysed in TENT buffer (20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100 containing protease inhibitors (0.1 mM PMSF, 1 µg/ml aprotinin, 1 µM pepstatin, and 1 µM leupeptin)). After 30-min incubation on ice, cell lysates were centrifuged at 13,000 x g for 10 min at 4°C, the supernatant was collected, and the protein concentration was determined by the method of Bradford using the Bio-Rad (Hercules, CA) protein assay. Proteins (400 µg for HT-2 cells) were incubated with the appropriate precipitating Ab, 2 µg of anti-GCR total (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) and 2 µg anti-pSTAT5 Ab (Tyr694; Santa Cruz Biotechnology), for 1 h at 4°C with rotation.
For binding immune complexes, 20 µl protein A-agarose or protein G-agarose beads (Santa Cruz Biotechnology) were added, and the incubation was continued overnight at 4°C with rotation. The protein A- or G-agarose immune complexes were washed four times with 200 µl TENT buffer and then resuspended in loading buffer (10% glycerol, 2% SDS, 0.2 mg/ml bromophenol blue, 62.5 mM Tris-HCl (pH 6.8), and 5% 2-ME). Immunoprecipitated proteins were eluted from the protein A- or G-agarose by boiling for 3 min. Proteins were resolved on 10% Tris-HCl gels (Bio-Rad) and electrophoretically transferred to nitrocellulose in Tris/CAPS buffer (Bio-Rad) containing 15% methanol. Membranes were stained with Ponceau S (0.5% in 1% acetic acid) to evaluate loading equivalency and transfer efficiency and were blocked for 30 min at room temperature in PBS (5 mM Na2HPO4, 0.15 M NaCl, and 0.05% Tween 20 containing 5% nonfat dry milk (w/v)). The blot was then incubated overnight with the appropriate primary Ab in PBS with 0.25% BSA and 0.05% Tween 20 (1/250 anti-GCR total (M-20) Ab (Santa Cruz Biotechnology), and 1/250 anti-p-STAT5 (Tyr694) Ab (Santa Cruz Biotechnology)). Subsequently, membranes were washed in PBS with 0.05% Tween 20; incubated for 1 h at room temperature with HRP-labeled protein A (Amersham Pharmacia Biotech, Piscataway, NJ; 1/10,000) or HRP-labeled protein G (1/5000; Sigma) diluted in PBS with 0.05% Tween 20, 0.25% BSA and 5% dry milk; washed in PBS with 0.05% Tween 20; incubated with chemiluminescent reagents (Western Blot Chemiluminescence Reagent Plus; Perkin-Elmer, Boston, MA); and then processed for autoradiography.
Statistical methods
Immunofluorescence and cell proliferation data were analyzed using Student’s t test. A value of p < 0.05 was considered significant. Data are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proliferation of the murine T cell line HT-2 can be stimulated
with either IL-2 or IL-4. IL-4 induced proliferation of the murine T
cell line CTLL-2 has previously been shown to be sensitive to
inhibition by DEX, while IL-2-induced proliferation was insensitive to
DEX (14). We have extended these observations by
determining the effects of DEX on IL-2- and IL-4-stimulated
proliferation of the HT-2 cell line. The amounts of IL-4 (500 U/ml) and
IL-2 (1 U/ml) used were titrated to stimulate similar levels of
proliferation in the HT-2 cultures. Fig. 1
shows the results of a representative
dose-response experiment (of six total experiments performed)
demonstrating that IL-4-induced proliferation was inhibited by >50%
at 10-8 M DEX, while IL-2-induced proliferation
was unaffected even at 10-5 M DEX
(p < 0.05).
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To investigate the mechanism by which IL-2 induced steroid
insensitivity in HT-2 cells, we initially compared the ability of GCR
to translocate into the nuclei of IL-2- or IL-4-stimulated cells after
treatment with 10-6 M DEX for 30 min at 37°C.
In these experiments the nuclei were defined by staining with the blue
fluorescent DNA dye DAPI. GCR localization was examined by indirect
immunofluorescence using anti-GCR rabbit polyclonal antiserum
detected by Cy3-conjugated secondary Ab (red). In the absence of DEX
stimulation GCR was primarily in the cell cytoplasm (Fig. 2, A and B). The
Cy3 MFI values for the nuclear region of the cells were 355.27 ±
20.50 (IL-2 stimulated cells) and 451.46 ± 18.80 (IL-4 stimulated
cells). After DEX treatment GCR translocated to the cell nucleus in
IL-4-stimulated cells, but failed to translocate in IL-2-stimulated
cells (Fig. 2). The Cy3 MFI values for the nuclear region of the cells
were 449.55 ± 30.58 (IL-2-stimulated cells treated with
10-6 M DEX) and 857.60 ± 66.20 (IL-4
stimulated cells treated with 10-6 M DEX;
p < 0.01 compared with all other groups). These data
suggest that the resistance of IL-2-stimulated HT-2 cells to DEX is due
to the failure of GCR to translocate from cytoplasm to nucleus.
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Since HT-2 cells treated with IL-4 were steroid sensitive and effectively translocated their GCR to the cell nucleus, we chose them as the model comparison for studies of IL-2-induced steroid resistance. HT-2 cells were grown in medium with 500 U/ml of IL-4 for 18–20 h, and then the medium was changed to medium containing 50 U/ml of IL-2 for 30 min. Cells were then treated with 10-6 M DEX for 30 min. The cells were fixed and stained, and GCR localization was examined by confocal microscopy.
HT-2 cells stimulated with IL-4 and treated with DEX for 30 min
revealed effective nuclear translocation of GCR (GCR staining
nuclear/cytoplasm ratio, 1.63 ± 0.09; Fig. 3A), which was blocked by 30
min of IL-2 exposure (GCR staining nuclear/cytoplasm ratio, 1.07
± 0.07; Fig. 3C; p < 0.01 between groups A
and C). Fig. 3, B and D, show that when protein
synthesis was blocked with 50 µg/ml of cycloheximide, neither nuclear
translocation nor IL-2-mediated inhibition of translocation was
affected (GCR staining nuclear/cytoplasm ratio, 1.45 ± 0.10 and
1.18 ± 0.06, respectively; p < 0.01 between
groups A and D). The rapidity with which IL-2 induced steroid
resistance coupled with the insensitivity of this effect to
cycloheximide suggested that the synthesis of new proteins was not
required for IL-2 to induce steroid resistance.
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Since IL-2-induced steroid insensitivity of HT-2 cells was not
associated with the synthesis of new proteins, we sought to determine
whether the prevention of signaling through IL-2R 
-chain could block
the induction of steroid insensitivity by IL-2. Indeed, JAK3 inhibitor
1, which blocks JAK3 kinase activity (15, 16), was able to
prevent the induction of steroid insensitivity of HT-2 cells at a
concentration of 7.8 µM in a dose-dependent manner after
24 h of incubation (Fig. 4A). Incubation of HT-2 cells
with 312 µM JAK3 inhibitor 1 (IC50 = 78 µM)
for 1 h before treatment with IL-2 also restored DEX-induced
nuclear translocation of GCR (Fig. 5B). Incubation with JAK3
inhibitor for this period of time was not toxic to the cells, as
determined by exclusion of trypan blue.
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According to proliferation assay results, the p38 MAPK-specific
inhibitor SB203580 partially shifted the steroid sensitivity of
IL-2-induced proliferation of HT-2 cells (Fig. 4
B). Fig. 5, B and C, illustrate that DEX-induced nuclear
translocation of the GCR occurred in the presence of JAK3 inhibitor and
the p38 MAPK inhibitor SB203580 even in the presence of IL-2 (GCR
staining nuclear/cytoplasm ratio, 1.38 ± 0.05 and 1.81 ±
0.26, respectively; p < 0.01 compared with GCR
staining nuclear/cytoplasm ratio of 0.98 ± 0.06 for
IL-2-stimulated cells). JAK3 inhibitor and p38 MAPK inhibitor did not
alter the DEX sensitivity of IL-4-treated cells (data not shown). This
suggested that the signal transduction pathway leading through JAK3 to
STAT5 was directly involved in the genesis of IL-2-induced steroid
resistance in HT-2 cells. p38 MAPK-dependent phosphorylation also
restores nuclear translocation of GCR.
STAT5-GCR heterodimer formation is associated with the steroid insensitivity of IL-2-induced proliferation in mice
Since activation of the post-JAK3 elements of the IL-2 signal transduction pathway was required for inhibition, we examined whether there was evidence of direct interaction between GCR and IL-2-induced signal transduction factors. STAT5 was our first protein of interest, because this molecule is known to be activated by IL-2. Furthermore, our previous experiments indicated that incubation with JAK3 inhibitor 1 prevented induction of steroid insensitivity in IL-2-treated HT-2 cells.
To determine whether STAT5 is involved in induction of steroid
insensitivity by IL-2, several approaches were used. Whole cell lysates
were prepared from IL-2-stimulated HT-2 cells and immunoprecipitated
with anti-GCR or anti-phospho-STAT5 Abs. Western blot analysis
was performed using a specific anti-phospho-STAT5 antiserum to
determine whether immunoprecipitation of GCR also captured
phospho-STAT5. Alternatively, specific anti-GCR antiserum was used
to show the association of immunoprecipitated STAT5 with GCR, revealing
physical interaction between them. Fig. 6
illustrates that immunoprecipitation of STAT5 does indeed capture GCR,
although it appears that only a small percentage of the total STAT5 is
associated with GCR. Conversely, Fig. 6 also shows that
immunoprecipitation of GCR captures STAT5. In this case, a higher
percentage of the precipitable GCR is associated with STAT5. When we
compared cytoplasmic to nuclear extracts for the relative proportion of
STAT5 remaining in the cytoplasm, we found a 1:1 nuclear/cytoplasmic
ratio of STAT5 in IL-2-stimulated HT-2 cells. This indicates that there
is no defect in STAT5 nuclear translocation in HT-2 cells.
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If GCR-STAT5 protein-protein interactions were important, we
hypothesized that IL-2 would be unable to induce steroid insensitivity
in STAT5 knockout mice. For this purpose murine splenocytes from
STAT5a/b knockout mice were stimulated in culture with PHA. After
48 h cells were stimulated with different concentrations of IL-2
or IL-4 in the presence of DEX for 24 h to determine their steroid
sensitivity. We found that IL-2-stimulated spleen cells from STAT5a/b
knockout mice were steroid sensitive, while spleen cells from wild-type
mice of the same background (C57BL6/129) were steroid insensitive after
IL-2 treatment (Table I). We observed
that IL-2-induced proliferation of splenocytes from STAT5 knockout mice
was up to 100-fold more sensitive to inhibition by DEX
(IC50 = 10-8 to 5 x
10-8) than were control mice
(IC50 = 10-7 to 5 x
10-6; p < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that human neutrophils have high constitutive
expression of GCR and a 73-fold greater ratio of GCR/GCR
compared with human PBMC (11). Moreover, proinflammatory
cytokines such as IL-8 further up-regulate GCR levels in
neutrophils, which enhances their survival in the presence of GCs
during inflammation. Several additional pathways that may lead to
steroid resistance have been described in PBMC. It has been established
that IL-2 and IL-4 can potentiate GCR expression in T cells as well
(18). Whether increased GCR can completely account for
GC insensitivity in T cells, however, remains controversial. It was
shown that viral transduction of GCR cDNA into the mouse DO11.10
cell line leads to 2.5-fold GCR overexpression and results in GC
insensitivity of these cells (19). On the other hand,
Irusen et al. (17) were unable to detect increased GCR
expression in IL-2/IL-4-stimulated human PBMC. Thus, GCR is unlikely
to completely account for steroid resistance in PBMC (17, 20). Recent studies indicate that GCR does not act as a
dominant negative modulator of GR for repression of AP-1 and NF-
B
activities, and overexpression of either GCR or GCR may have an
anti-inflammatory effect (20). The study of mouse T
cells provides an opportunity to examine mechanisms of steroid
resistance in the absence of GCR. This makes murine T cells a good
model for examining the effects of proinflammatory cytokines on the
response to steroids.
GCs interact with the GCR in the cytoplasm. This causes the receptor to
undergo an allosteric change that enables the hormone-receptor complex
to translocate into the cell nucleus, bind to specific DNA-response
elements, and modulate transcription (6). There are
several signal transduction pathways and transcription factors that
have been shown to interact with and modulate the function of
GCR-ligand complexes, including AP-1, NF-B, and STAT
(21, 22, 23).
JAK-STAT signaling by IL-2 is critically important in the generation of
immune and inflammatory responses and in T cell expansion and
differentiation (24). Protein tyrosine kinases JAK1 and
JAK3 that are associated with cytoplasmic subunits of the - and
-chains of IL-2R, respectively, are activated after IL-2 binding
(25). The - and -subunits of the IL-2 receptor
become phosphorylated and provide docking sites for monomeric STAT5
proteins (26). STAT5 becomes phosphorylated, dimerizes,
and translocates to the cell nucleus (26, 27).
STAT5 was first characterized as a mammary gland transcription factor
induced by prolactin (28). Most previous investigations
have been focused on STAT5 inhibition of GCR
trans-activation activity induced by prolactin (29, 30). In addition to prolactin it has been established that both
STAT5a and STAT5b proteins, which share 
95% homology in amino acid
structure, are activated by a number of cytokines (IL-2, IL-3, IL-5,
IL-7, IL-9, IL-15, GM-CSF, erythropoietin, and thrombopoietin)
(26, 27). It has been suggested that GCR can act as a
transcriptional coactivator for STAT5 and enhance STAT5-dependent
transcription in response to prolactin. However, complex formation
between STAT5 and GCR diminishes the GC responsiveness of GC response
element-containing promoters (31).
Our studies confirm that heterodimers form between GCR and STAT5. However, we have further demonstrated that this leads to inhibition of the nuclear import of GCR, thus leading to the induction of steroid insensitivity. We suggest a novel role for STAT5 in IL-2-induced steroid insensitivity: the prevention of efficient nuclear translocation of GCR due to heterodimer formation between GCR and STAT5 proteins.
Recent studies indicate that GCR can interact with several STAT
proteins (21, 29). Despite the high degree of homology
existing between different STAT proteins, functional interaction with
GCR does not necessarily imply a common mechanism. For instance, it has
been demonstrated that GCR and STAT6 physically and functionally
interact in CTLL-2 cells (32), and that DEX was able to
block the IL-4-induced proliferation of these cells (14).
Transcriptional cooperation between STAT-1, the GCR, and the Ets family
transcription factor PU.1 has been described in monocytes. Such
interaction enhanced IFN--induced expression of FcR1
(33).
Our results suggest that exposure of the murine T cell line HT-2 to IL-2 blocks both translocation of the GCR to the nucleus and steroid responsiveness. These data are consistent with previous observations obtained with CTLL-2 cells, in which it has been established that IL-2 strongly inhibits GC-induced transcription from the mouse mammary tumor virus promoter due to STAT5-GCR interaction (34).
The mechanism by which IL-2 can induce these defects in steroid responsiveness is revealed by our experiments in which prevention of STAT5 phosphorylation by JAK3 inhibitor 1 prevented the effect. Therefore, phosphorylation of STAT5 must be required to enable its association with GCR.
Our studies have extended previous work from cell lines to primary
murine lymphocytes, in which we observed induction of steroid
insensitivity by IL-2, while IL-4-induced proliferation was steroid
sensitive (Table I). The hypothesis that nuclear translocation of GCR
is inhibited by interaction with STAT5 is further supported by the
observation that lymphocytes from mice deficient in STAT5 do not
develop steroid resistance in response to IL-2 stimulation, while
lymphocytes from control mice readily develop resistance.
The precise nature of the interaction between STAT5 and GCR remains unclear. However, Wang et al. (35) recently demonstrated that phosphorylation of serine 211 of human GCR was associated with nuclear localization. In addition, nuclear localization sequences adjacent to the DNA binding domain and in the ligand binding domain of rat GCR have been shown to regulate translocation of GCR into the nucleus (36). GCR is phosphorylated on multiple serine residues in addition to serine 211, which is mediated in part by p38 MAPK (17).
We have observed that the induction of steroid resistance by IL-2 can
be inhibited not only via the STAT5-dependent pathway, but also via a
p38 MAPK-dependent pathway (Fig. 4). Inhibition of p38 MAPK activity
also led to rescue of nuclear translocation of GCR in IL-2-treated
cells (Fig. 5). This suggests that the interaction between STAT5 and
GCR occurs via STAT5 binding to one of the phosphoserine residues of
GCR. This may prevent translocation of GCR to the nucleus either by
blocking access of importin- to the nuclear localization-1
domain (36) or by masking other domains needed for nuclear
import. Doppler et al. (37) have determined that
functional interaction between GCR and STAT5 requires residues 77–262
of the human GCR, which includes serine 211, previously shown to be
associated with nuclear localization.
In summary, we have shown that steroid resistance induced in T cell lines by IL-2 is associated with a defect in nuclear translocation of the GCR in response to DEX. This defect was dependent upon phosphorylation of STAT5 by JAK3 and was independent of the synthesis of new proteins. IL-2-induced steroid resistance was also inhibited by an inhibitor of p38 MAPK, suggesting that phosphorylation of the GCR was necessary in addition to STAT5 activation. This study has identified both STAT5 and JAK3 as potential targets for pharmaceutical intervention and treatment of inflammatory diseases associated with steroid insensitivity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Donald Y. M. Leung, National Jewish Medical Research Center 1400 Jackson Street, Room K926I Denver, CO 80206. E-mail address: leungd{at}njc.org
3 Abbreviations used in this paper: GC, glucocorticoid; DAPI, 4, 6'-diamidino-2-phenylindole; DEX, dexamethasone; GCR, glucocorticoid receptor; JAK3, Janus-associated kinase 3; MFI, mean fluorescence intensity; MAPK, mitogen-activated protein kinase.
Received for publication July 30, 2002. Accepted for publication September 9, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 D. M. Presman, E. Hoijman, N. R. Ceballos, M. D. Galigniana, and A. Pecci Melatonin Inhibits Glucocorticoid Receptor Nuclear Translocation in Mouse Thymocytes Endocrinology, November 1, 2006; 147(11): 5452 - 5459. [Abstract] [Full Text] [PDF]
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 E. Goleva, L.-b. Li, P. T. Eves, M. J. Strand, R. J. Martin, and D. Y. M. Leung Increased Glucocorticoid Receptor beta Alters Steroid Response in Glucocorticoid-insensitive Asthma Am. J. Respir. Crit. Care Med., March 15, 2006; 173(6): 607 - 616. [Abstract] [Full Text] [PDF]
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 O. Tliba, J. A. Cidlowski, and Y. Amrani CD38 Expression Is Insensitive to Steroid Action in Cells Treated with Tumor Necrosis Factor-{alpha} and Interferon-{gamma} by a Mechanism Involving the Up-Regulation of the Glucocorticoid Receptor beta Isoform Mol. Pharmacol., February 1, 2006; 69(2): 588 - 596. [Abstract] [Full Text] [PDF]
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 B. M. Necela and J. A. Cidlowski Mechanisms of Glucocorticoid Receptor Action in Noninflammatory and Inflammatory Cells Proceedings of the ATS, November 1, 2004; 1(3): 239 - 246. [Full Text] [PDF]
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 I. C. Chikanza and D. L. Kozaci Corticosteroid resistance in rheumatoid arthritis: molecular and cellular perspectives Rheumatology, November 1, 2004; 43(11): 1337 - 1345. [Full Text] [PDF]
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Q. Xu, E. Goleva, L.-S. Ou, L.-B. Li, and D. Y. M. Leung CD56+ Cells Induce Steroid Resistance in B Cells Exposed to IL-15 J. Immunol., June 1, 2004; 172(11): 7110 - 7115. [Abstract] [Full Text] [PDF]
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 I. Remy and S. W. Michnick Regulation of Apoptosis by the Ft1 Protein, a New Modulator of Protein Kinase B/Akt Mol. Cell. Biol., February 15, 2004; 24(4): 1493 - 1504. [Abstract] [Full Text] [PDF]
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