Calcium/Calmodulin Kinase Inhibitors and Immunosuppressant Macrolides Rapamycin and FK506 Inhibit Progestin- and Glucocorticosteroid Receptor-Mediated Transcription in Human Breast Cancer T47D Cells
Stéphane Le Bihan,
Véronique Marsaud,
Christine Mercier-Bodard,
Etienne-Emile Baulieu,
Sylvie Mader,
John H. White and
Jack-Michel Renoir
URA 1218 Centre Nationale de la Recherche Scientifique (V.M.,
J.-M.R.) Pharmacologie Cellulaire 92296 Chatenay-Malabry
Cédex, France
INSERM U33 (S.L.B., E.-E.B.) and IFR 21
(C.M.-B.) 94276 Le Kremlin-Bicêtre Cedex, France
Departments of Physiology and Medicine (J.H.W.) McGill
University Montréal, Quebec, H361Y6 Canada
Department of Biochemistry (S.M.) Faculty of Medicine
University of Montreal Montréal, Québec, H3TIJ4,
Canada
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ABSTRACT
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The effects of immunosuppressants and inhibitors
of specific calcium/calmodulin kinase (CaMK) of types II and IV on
progestin/glucocorticosteroid-induced transcription were studied in two
human stably transfected breast cancer T47D cell lines. The lines
contain the chloramphenicol acetyl transferase (CAT) gene under control
either of the mouse mammary tumor virus promoter (T47D-MMTV-CAT), or
the minimal promoter containing five glucocorticosteroid/progestin
hormone response elements [T47D-(GRE)5-CAT].
Progestin- and triamcinolone acetonide (TA)-induced CAT gene expression
was inhibited in a dose-dependent manner in both lines by preincubation
with rapamycin (Rap) and, to a lesser extent, with FK506, but not with
cyclosporin A. CaMK II and/or IV inhibitors KN62 and KN93 also
inhibited progestin- and TA-stimulated transcription in both lines.
None of these drugs had any effect on basal transcription. The
antagonist RU486 inhibited all the effects of both progestin and TA,
suggesting that progesterone receptor (PR)-, as well as
glucocorticosteroid receptor (GR)- mediated transactivation are targets
of immunosuppressants and CaMKs in T47D cells. Indeed, Northern
analysis showed that Rap, KN62, and, to a lesser degree, FK506
inhibited progestin stimulation of Cyclin D1 mRNA levels, but not those
of the non-steroid-regulated glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) gene. Addition of Rap or KN62 after exposure of cells to
progesterone agonist Org 2058 had no effect on induction of CAT
activity. Taken together, these data indicate that Rap and FK506, as
well as CaMK inhibitors, inhibit steroid-induced activities of
exogenous, as well as of some endogenous, steroid receptor-regulated
genes by a mechanism preceding hormone-induced receptor activation.
Rap appeared to stabilize a 9S form of
[3H]Org 2058-PR complexes isolated from T47D
(GRE)5CAT cell nuclei. By contrast, the
progesterone receptor (PR) was isolated from cells treated with KN62 as
a 5S entity, undistinguishable from the 5S PR species extracted from
cells treated with progestin only. The nuclear
9S-[3H]Org2058-PR resulting from cells
exposed to Rap, contained, in addition to the heat shock proteins of 90
kDa and 70 kDa (hsp90 and hsp70), the FK506-binding immunophilin FKBP52
but not FKBP51, although the latter was part of unliganded PR
heterocomplex associated with hsp90. These results suggest that Rap and
KN62 act upon the PR by distinct mechanisms, with only Rap impeding
progestin-induced PR transformation. FKBP51 appeared to dissociate from
the receptor heterocomplex, but not from hsp90, after hormone binding
to PR in vitro and in vivo, whether in the
presence or not of Rap and KN62. Immunoprecipitation experiments
distinguished two PR- and glucocorticosteroid (GR)-associated
molecular chaperone complexes, containing hsp90 and hsp70 and FKBP52 or
FKBP51. Another complex identified in T47D cytosol contained hsp90 and
the cyclosporin A-binding cyclophilin of 40 kDa, CYP40, but not hsp70,
PR, or GR. These observations support the concept that FKBP51 and
FKBP52 can act as regulators of Rap and FK506 activity upon PR and
GR-mediated transcription, a mechanism that could be also regulated by
type II and/or type IV CaMKs.
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INTRODUCTION
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In their unliganded form, steroid receptors from cytosolic
extracts are complexed in large inactive heterooligomeric structures
with several receptor-associated proteins (see Ref. 1 for a review).
Among these proteins, two have been identified as heat shock proteins
(hsps) namely hsp901 (2, 3, 4),
and hsp70 (5, 6). Other receptor-associated proteins belong to a class
of peptidyl-prolyl isomerases, termed immunophilins (7): two
FK506-binding proteins, FKBP52 (8, 9, 10, 11), which migrates at 59 kDa in
denaturing gel electrophoresis and therefore was initially called p59
(12), and FKBP51 (13, 14), previously named FKBP54 (15). In addition to
these FKBPs, a cyclosporin A (CsA)-binding protein of 40 kDa,
cyclophilin 40 (CYP40) (16, 17) was characterized. These immunophilins,
FKBP51 and FKBP52, as well as CYP40, bind at the same site on hsp90
(Refs. 14, 18 ; see Ref. 1 for a review). CYP40, FKBP51, and FKBP52 were
the first of the cyclophilin and FKBP classes of immunophilins found to
possess C-terminal amino acid sequence identities (10, 11, 13, 14, 15, 16, 17) in
the form of three tetratricopeptide repeat units required for binding
to hsp90 (19). The association of FKBP52, FKBP51, and CYP40 with hsp90
was demonstrated by coimmunoprecipitation (8, 12, 14, 15, 16, 20, 21, 22, 23),
native gel electrophoresis (19), cross-linking (20, 21), and
immunosuppressant-affinity chromatography (14, 15, 16, 17, 18, 22, 23, 24) experiments.
Therefore, FKBP52, FKBP51, and CYP40 may represent the first members of
a new class of peptidyl-prolyl isomerases, the heat shock protein
binding immunophilins (25). They all exist in independent
heterocomplexes with hsp 90 (14, 18, 24), in association with other
proteins thought to participate in the folding process of the receptor
(see Ref. 1 for a review).
Hsp70 has also been shown to be part of heterooligomeric
complexes containing hsp90 and FKBP52 that are associated with the PR
in human breast cancer T47D cells (26, 27). In fact, large molecular
chaperone complexes including FKBP52, FKBP51, hsp90 and, in some cases,
hsp70 seem to exist in many cells. Up to now, these receptor-associated
immunophilins are thought to be involved in a molecular chaperone
mechanism, and although it has been recently proposed that FKBP 52
could serve as a mediator of dexamethasone (Dex)-induced
glucocorticosteroid (GR) trafficking (28), their targets in the
receptor complex(es) are still not well identified.
Recent studies have suggested that FKBPs and cyclophilins modulate
Dex-induced transcription in mouse fibroblasts since immunosuppressants
potentiate steroid-induced transcription in these cells (24, 29).
Similarly, FK506 potentiates progestin-induced transcription in yeast
strains transfected with human PR B form (30). This potentiation was
shown recently to be the consequence of the reversal activity of a
membrane efflux mechanism resembling, but different, from that of
phospho-glycoprotein, P-gp (31, 32, 33). Then, at least in mouse
fibroblasts and in yeast, it is obvious that the targets of the effects
of immunosuppressants on steroid actions could not explain the observed
cellular responses.
In this work, we used two lines derived from T47D cells, known to
express high levels of PR (34), to study the effects of
immunosuppressants on steroid-induced transcription. The lines
contained stably transfected plasmids encoding CAT reporter genes under
control of the mouse mammary tumor virus long terminal repeat
(MMTV-CAT) (35) or the synthetic steroid-inducible minimal promoter
GRE5 (GRE5tkCAT) (36). In the two T47D cell
lines, Rap and FK506, but not CsA, inhibited progestin-, as well as
triamcinolone acetonide (TA)-, induced transcription. This occurred
at a step preceding steroid-induced receptor
activation.2 Similar inhibiting
activity by Rap was also observed on progestin-induced Cyclin D1 mRNA
levels, a progestin-regulated gene (37, 38).
Numerous works have established that immunophilins play a role in
cell-signaling pathways involving phosphorylation (Ref. 39 for a
review). Since we had previously found that rabbit uterus FKBP52
copurifies with either a Mg2+ or a calcium-calmodulin
dependent kinase (CaMK) (40), and since two CaMK consensus sequences
are present in FKBP52 (10, 11) and FKBP51 (14, 41), we tested the
effect of CaMK inhibitors on steroid-induced transcription. The two
CaMK inhibitors, KN62 and KN93, inhibited the steroid-induced
transcription in both cell lines, at a step upstream to
steroid-induced receptor activation; in addition, Rap and KN62
inhibited progestin-induced expression of Cyclin D1 mRNA. This suggests
that a CaMK phosphorylation/dephosphorylation-mediated mechanism
regulates steroid-induced transactivation before the steroid-induced
dissociation of receptor-associated hsps/FKBP complexes from the
receptor, that immunosuppressant ligands for FKBPs 51 and 52 act at a
step preceeding such a dissociation process, and that two exogenous and
endogenous genes are similarly regulated by Rap and by CaMKs.
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RESULTS
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Inhibition by Rap and FK506 of Progestin-Induced Transcription in
T47D(MMTV-CAT) Cells
The maximal induction of CAT activity in T47D (MMTV-CAT) cells
(
200 fold that of the basal level) occurred at 100 nM
R5020 (Fig. 1A
). When cells were
preincubated with a single concentration (10 µM) of Rap,
before addition of a range of R5020 concentrations, an inhibition of
CAT activity was observed (Fig. 1A
). FK506 was a weaker inhibitor than
Rap, and CsA did not seem to affect CAT gene expression. At low R5020
concentrations, a dose-dependent inhibition by Rap and FK506 was
obtained (Fig. 1B
). The inhibitory effect of Rap appeared more
pronounced than that obtained with FK506. CsA had no significant
inhibitory effect on CAT activity even when used at 10 µM
(Fig. 1B
). None of the drugs had any effect on CAT activity when used
alone (not shown). A similar inhibitory effect of Rap was obtained,
whether the immunosuppressant was added before or at the same time as
the progestin (Fig. 1B
). All these effects were abolished by a 100-fold
excess of the antiprogestin RU486 (not shown), indicating that
induction of CAT activity is mediated by the PR.

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Figure 1. Effects of Immunosuppresssants Rap, FK506, and CSA
on R5020-Induced CAT Expression in T47D MMTV-CAT Cells
A, Cells were exposed as indicated for 2 h to 10 µM
of immunosuppressants, followed by the indicated concentrations of
R5020 for 16 h. CAT activity was calculated relative to the basal
activity measured in the absence of steroid. The results are the mean
values for at least three determinations in duplicate. B, Cells were
exposed to the indicated concentrations of immunosuppressants then to
0.1 nM (filled bars) or 1 nM
(open bars) R5020. CAT activity was measured as in Fig.
lA. The CAT activity calculated for each drug concentration is the
result of a decreased activated level of CAT and not that of an
increased background. Control experiments with only 0.1 and 1
nM R5020 alone are shown (bars, C). Similar
results were obtained when drugs and steroid were added at the same
time.
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Effect of Immunosuppressants on Progestin-Induced Transcription in
T47D (GRE5-CAT) Cells
In T47D-GRE5-CAT cells, the maximum CAT activity
(
200 times that of the basal level) was obtained for
3
nM R5020 (36) or 0.3 nM Org 2058 (not shown).
Figure 2
shows that, similar to T47D MMTV
CAT cells, Rap strongly inhibited progestin-induced transcription, in a
dose-dependent manner. Once again, Rap was more efficient than FK506
and CsA had no effect. Addition of a 100-fold molar excess of RU486
blocked progestin-inducible CAT activity (not shown), strongly
suggesting that the observed gene expression in both cell lines is
mediated by the PR.

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Figure 2. Inhibition by Rap and FK506 of R5020-Induced CAT
Expression in T47D (GRE)5-CAT Cells
T47D cells stably transfected with the reporter gene
GRE5-CAT were exposed for 16 h to 0.1 or 10
nM R5020 before (C) or after exposure to different
concentrations (0.1 to 10 nM) of Rap, FK506, or CsA. CAT
activity was measured as described in the legend of Fig. 1 .
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Effect of Immunosuppressants on Glucocorticosteroid-Induced
Transcription
In the cell line T47D (GRE5)-CAT, TA induced
transcription in a dose-dependent manner (Fig. 3A
). The maximal induction of CAT
activity occurred at 10 nM. This maximum reached almost
150-fold that of the basal level and was inhibited by RU486 (not
shown). Figure 3B
shows that exposure of cells to Rap or FK506 provoked
a decreased response to either 1 or 10 nM TA. CsA, on the
other hand, did not affect TA-induced CAT activity.

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Figure 3. Effect of Immunosuppressants on
Glucocorticosteroid-Induced CAT Expression in T47D
(GRE)5-CAT Cells
A, Cells were exposed for 16 h to different concentrations (10
pM to 1 µM) of TA, and CAT activity was
measured for 50 µg of protein as described in Fig. 1 . B, Cells were
treated for 2 h with 5 µM of Rap or FK506 or CSA
before exposure for 16 h to 1 nM (filled
bars) or 10 nM (open bars). CAT
activity was measured and compared with that of control experiments
performed without immunosuppressants (C).
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Effect of Ca2+/Calmodulin-Dependent Protein
Kinase Inhibitors on Progestin- and TA-Induced Transcription in Both
T47D Cell Lines
The immunosuppressants FK506 and CsA bind to a variety of
immunophilins in cells and act on Calcineurin (CN) by inhibiting its
phosphatase activity (for a review, see Ref. 39). However, CN is not a
target of Rap, which has been shown to modify the signaling pathways
mediated by the expanding family of lipid/protein kinases (review in
Ref. 42). In addition, we showed that FKBP52 from rabbit uterus is
phosphorylated in vitro by a copurified
Ca2+/calmodulin-like dependent protein kinase (CaMk)
activity (40). We have therefore tested the effects of KN62 and KN93,
two specific inhibitors of both types II and IV CaMks (43, 44), on
steroid-inducible CAT activity in both cell lines. Figure 4A
shows that KN62 decreased
R5020-induced transcription in a dose-dependent manner in both T47D
cell lines. Similarly, KN62 also inhibited TA-induced transcription in
T47D (GRE5)-CAT (Fig. 4B
), as well as in T47D (MMTV)-CAT
(not shown). KN93 inhibited both R5020- and TA-induced CAT activity in
both cell lines but at a 2- to 3-fold lower concentration than KN62
iself (Fig. 4C
). This difference may reflect the greater water
solubility of KN93 (44). These results suggest that a CaMK activity is
involved in the steroid-induced activation of either PR and GR and that
its inhibition decreases steroid-induced transcription in T47D
cells.

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Figure 4. Effect of CaMK Inhibitors on R5020- and TA-Induced
CAT Gene Expression in T47D Cells
Different concentrations of KN62 were added for 2 h to the culture
medium of both cell lines before exposure for 16 h to 1
nM R5020 (panel A), or TA (panel B). In panel C, different
concentrations (0.1 to 1 µM) of KN93 were incorporated as
indicated for 2 h in the culture medium of
T47D-(GRE5)-CAT cells, followed by addition for 16 h
of either 1 nM R5020 (left) or TA
(right). CAT activity was measured as in Fig. 1 .
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Effects of Rap and KN62 after Exposure of Cells to Steroids
All the experiments described above were performed by adding drugs
(immunosuppressants or CaMK inhibitors) before steroids in cell culture
medium. Figure 5
shows that if Org 2058
(0.33 or 1 nM) was added to T47D (GRE5)-CAT
cells before addition of either KN62 or Rap, no decrease of CAT gene
expression was observed. It has been established that hormone binding
induces a conformational change in receptor structure, which leads to
dissociation of molecular chaperones from the hormone binding unit(s)
(see Ref. 1 for a review). Thus, the data from Fig. 5
may indicate that
the drugs Rap and KN62 act before this transformation process. Similar
observations were made in T47D MMTV-CAT cells, as well as with TA
instead of Org 2058 in both cell lines (not shown).

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Figure 5. Effect of Immunosuppressive Drugs after Steroid
Incorporation in Cell Culture Medium
T47D (GRE5) CAT cells were grown as indicated for 4 h
in culture medium in the presence of 0.33 or 1 nM Org 2058.
Then the medium was replaced for 2 h by steroid-free medium (2 h)
or medium containing either 10 µM KN62 (KN62 2 h) or
10 µM Rap (Rap 2 h). Control experiments in which 10
µM KN62 and 10 µM Rap were added 2 h
before 4 h of incubation of cells with 0.33 and 1 nM
Org 2058 are shown (black bars). CAT activity was
measured as in Fig. 1 .
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Effects of Immunosuppressants and CaMK Inhibitors on
Progestin-Induced mRNA Synthesis
The mRNA encoding cyclin D1 is one of many transcripts whose
levels are increased in T47D cells in the presence of progestin (37, 38). We analyzed the influence of immunosuppressants and KN62 on cyclin
D1 mRNA level. Rap, FK506, or KN62 had no individual effect on the
abundance of Cyclin D1 transcripts. By contrast, Rap and KN62 stongly
inhibited the R5020-induced increase of Cyclin D1 mRNA, while FK506 had
a weaker effect (Fig. 6
).

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Figure 6. Northern Blot of GAPDH and CYCLIN D1
T47D (GRE5) CAT cells were grown to 50% confluence in T150 flasks in
DMEM supplemented with 10% FCS. The medium was then replaced by DMEM
without serum for 48 h, followed by DMEM supplemented with 10%
stripped and heat-inactivated serum containing or not (control) 10
µM of the indicated drugs for 2 h; then 10
nM R5020 was added for 16 h to the culture medium. RNA
was isolated with Trizol reagent, and Northern analysis was performed
to determine Cyclin D1 and GAPDH expression as described in
Materials and Methods. Typical GAPDH signals were
achieved after 24 h of autoradiography, and Cyclin D1 signals were
achieved after 72 h of autoradiography (panel A). Using
densitometric analysis, GAPDH was used to normalize for variations in
signal intensity. The autoradiogram shown was typical of three Northern
blots.
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In a control experiment, the mRNA level of the ubiquitous
non-progestin-regulated GAPDH was not modified by any of the drugs
tested under these conditions. These results would indicate that Rap,
FK506, and KN62, but not CsA, down-regulate the progestin-induced
transcription of the endogenous Cyclin D1 gene.
Effects of Rap and KN62 on the Heterooligomeric Forms of PR and
GR
Given the drug effects reported above, we next sought to
investigate whether stabilization of an inactive non-DNA-binding form
of steroid receptors could be responsible for these effects. In the
absence of hormone, PR is located in the cell nuclei, from which it can
be recovered in the low-salt cytosoluble fraction of cellular
homogenates. Under these conditions, PR can be postlabeled with
3H-labeled ligands, and ligand/PR complexes migrate at 9S
in density gradients. They contain PR hormone-binding units, hsp(s),
and immunophilin(s). In vivo, after exposure to hormone, the
PR is tightly bound to nuclei and can be extracted by high
salt-containing buffers (>0.4 M NaCl), giving rise to
45S complexes that are free of receptor-associated proteins (see Ref.
1 for a review). Molybdate ions have been widely shown to protect 9S
species from the in vitro transformation into 45S species
induced by high salt. We have found that tungstate oxyanions are more
efficient than molybdate in preventing such a salt-induced
transformation (20, 45). In the presence of tungstate, 9S nuclear
[3H]RU 486-PR species were recovered in which hsp90 and
FKBP52 still remained associated with the PR units (45). Here, we
analyzed tungstate-stabilized complexes extracted from cells treated
with Rap. T47D cells were exposed to 10 µM Rap in the
presence of 10 nM [3H]Org 2058. The
tungstate-stabilized nuclear form of [3H]Org 2058-PR
complexes extracted in high salt separated in two unequal species in
density gradients (Fig. 7C
), one
sedimenting at approximately 9S, the other at approximately 45S. The
former represents approximately 40% of the total [3H]Org
2058-PR complexes. In contrast, in the absence of steroid and of any
drug, identical 9S sedimentation profiles for both cytosolic and
nuclear PR were obtained after postlabeling with the
3H-labeled ligand (Fig. 7A
). However, a dramatic loss of
hormone binding capacity occurred, as a consequence of the instability
of unoccupied PR during cellular extraction and homogenization
procedures. In the absence of Rap, and in the presence of
[3H]Org 2058, only the
5S form of nuclear PR was
obtained with a small shoulder at 9S position (Fig. 7B
). In
contrast to treatment with Rap, treatment with KN93 or CsA (Fig. 7
, D
and E) had no effect on the density gradient profile of
PR-[3H]Org 2058 complexes extracted by high salt. Under
these conditions, there was still a small portion of the PR that was
recovered in the cytosolic extracts (Fig. 7
, B, D, and E). Identical
results were obtained with [3H]TA-GR complexes extracted
from nuclei of T47D(GRE5)-CAT cells exposed to the same
drugs (not shown).
These results indicate that Rap would act as a stabilizer of the 9S
species (Fig. 7C
). Numerous studies from many laboratories have
established that the 9S form of PR represents a combination of
complexes of the hormone binding B and A PR forms associated with the
molecular chaperone hsp90 and FKBPs (review in Ref. 1). In addition,
the unliganded rabbit uterus 9S-PR has been shown to be stabilized
in vitro by FK506 and Rap (22). Similarly, the untransformed
GR complex from S49 lymphocytes was also stabilized in vitro
by FK506 (46) and did not interact with nonspecific DNA, consistent
with the behavior of the GR compatible with the presence of GR in
complexes with associated hsp(s) and immunophilin(s). We conclude from
experiments of Fig. 7
that Rap may act in T47D cells by impairing the
agonist-induced release of molecular chaperones from the PR. In
contrast, KN93 itself does not stabilize the PR and seems to act
differently than Rap (Fig. 7D
).
Protein Composition of PR Heterocomplexes from T47D Cells
Immunoprecipitation experiments were used to identify the
different proteins present in PR heterocomplexes. Cytosol from
T47D-(GRE5) CAT cells was incubated with
[3H]Org 2058. The [3H]Org2058-PR B
complexes were immunoprecipitated with the MPR1 antibody, and the
radioactivity in the immunoprecipitate was measured by the
dextran-coated charcoal technique described previously (47).
Specifically bound [3H]Org 2058 was found in the
immunopellet (Fig. 8A
). When
immunoprecipitation was performed with the anti-FKBP52 EC1 antibody,
20% of the initial PR binding was immunoprecipitated. No radioactivity
was recovered in immunoprecipitates of similar experiments performed
with either the anti-CYP40 638W antibody or with the anti-FKBP51 HI51
antibody, or with control nonimmune rabbit or mouse antibodies. This
suggested that PR-B was absent from immunopellets. This was confirmed
by Western blot experiments (Fig. 8B
). The PR-B form was detected in
the mPRI and in the EC1 immunopellets, along with hsp90 and hsp70,
arguing for the presence of cytosolic complexes including PR-B, hsp90,
hsp70, and FKBP52 in T47D cells. Neither the PR-B form nor hsp70 was
detected in the 638W immunopellets, whereas CYP40 and hsp90 were
readily detected. This indicates that CYP40 is neither associated with
PR-B nor with hsp70 in T47D cells cytosol. Western blot analysis with
MA1140 antibody of 638 W immunopellets did not detect PR-B or PR-A
(not shown), confirming that no PR is associated with CYP40 in cytosol
of T47D cells. Similarly, FKBP51 was not associated with PR either in
mPRI or HI51 immunopellets, but it coprecipitated with hsp90.

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Figure 8. Immunoprecipitation of PR-Associated Proteins with
anti PR-B and anti- Immunophilin Antibodies
Aliquots (500 µl) of [3H]Org2058-PR complexes were
incubated with pellets of preformed protein G-Sepharose IgG complexes
as described in Materials and Methods: mPR1, protein
G-Sepharose, mPR1 (10 µg/100 µl); mouse n.i., protein G-Sepharose
mouse nonimmune IgGs (10 µg/100 µl); 638W, protein G-Sepharose
anti-CYP40 638W antibody (20 µg/100 µl); rabbit n.i., protein
G-Sepharose rabbit nonimmune IgGs (10 µg/100 µl); EC1, protein
G-Sepharose anti-FKBP52 EC1 antibody (10 µg/100 µl); HI51, 205
protein G-Sepharose anti-FKBP51 antibody (20 µg/100 µl). After
washing of the immunopellets according to the protocol described in
Materials and Methods, immunopellets were counted for
determining the amount of bound [3H]Org2058 (panel A),
boiled in Laemmli sample buffer, and analyzed on 10% or 12% SDS-PAGE
(panel B) before Western blotting. A, Aliquots of initial labeled
cytosol, supernatants (1 ), high salt (2 ), low salt (3 ) washes of the
immunopellets and immunopellets (4 ) were treated with dextran-coated
charcoal for measurement of [3H]Org2058 binding. The bars
represent the total amount of bound [3H]Org2058 in each
sample. B, Western blotting of PR B and coimmunoprecipitated proteins
after 12% SDS-PAGE. PR B was revealed by mPR1 (1 µl/ml); hsp90 and
hsp70 were revealed by 174 (1:6000); FKBP52 was revealed by 173
(1:10000); the rabbit anti C-terminal anti-FKBP52 was used to avoid
recognition of the heavy chains of mouse IgGs that occurs when MPRI,
mouse nonimmune (mouse n.i.), and EC1 antibodies are used.
FF1 (2 µg/ml) was used to detect FKBP51, and 638W (1:6000) was used
to detect CYP40. The ECL chemiluminescent technique was employed.
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FK506- and CsA-Affigel Affinity Chromatography
Data from Fig. 8
suggested that PR from T47D associates with only
one immunophilin-hsp complex. This may appear to contrast with early
published results (14), indicating a preferential association of PR
with FKBP51 in a cell-free reconstitution system (14). Therefore, we
checked for the protein composition of PR from T47D cells exposed to
immunosuppressants or to PR ligands or to both, by FK506 and CsA
affinity chromatography, performed as described in
Materials and Methods with T47D cytosolic extracts in
which the PR had been labeled with 10 nM
[3H]RU486. Specific [3H] RU486 binding was
determined by the dextran-coated charcoal technique (47). Complexes of
[3H]RU486-PR were retained by and eluted from the
FK506-Affigel column (Fig. 9
, upper
panel), but not from the CsA-Affigel or the non-grafted control
Affigel 10 columns. Western blot experiments (Fig. 9
, AE) confirmed
these results, as no PR-A and PR-B forms were detected in the CsA
eluate from the CsA-Affigel column. In contrast, both PR forms were
detectable in the FK506 eluate from FK506-Affigel (see lane 4 in Fig. 9A
). In addition, FKBP52 was almost totally retained on the
FK506-Affigel resin and eluted as a doublet, concomitantly with hsp90
and hsp70, as revealed by Western blot using EC1 and 174 antibodies,
respectively (Fig. 9
, A and C, left panels, lane 4).

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Figure 9. Identification of Immunophilins and
Immunophilin-Associated Proteins Eluted from Immunosuppressant-Affinity
Chromatography by Western Blot
Aliquots (500 µl) of [3H]RU486-labeled cytosolic
PR from T47D MMTV-CAT cells, in the presence of 1 µM
cortisol, were incubated with 0.2 ml FK506-Affigel, or 0.2 ml
CsA-Affigel, or 0.2 ml Affigel control; the chromatography was
performed as described in Materials and Methods. In the
diagram of the figure, the total [3H]RU486 binding was
measured with the dextran-coated charcoal technique. C, Control of
initial cytosol: 1, flow through of each column; 2, high-salt wash; 3,
final low-salt wash; and 4, pooled FK506 or CsA-eluted fractions.
Panels AE, After 10% SDS-PAGE and blotting, fractions (20 µl) 1 to
4 from FK506- and CsA-Affigel columns were probed (panels A and B) with
the mixed mouse monoclonal antibodies EC1 (to identify FKBP52) and
MA1140 for PR B and A identification. In panels C and D, the rabbit
antibodies 638W (to identify CYP40) and 174 antibody (to identify hsp90
and hsp70) were used. In panels B and D, samples from the control
experiment performed with non-grafted Affigel 10 were analyzed. In
panel E, [3H]RU486-PR containing cytosol (left
part) and nonlabeled T47D cell cytosol (right
part) were chromatographed onto FK506-Affigel; both PR B and A
forms were probed, concomitantly with FKBP51, in each fraction, with
MA1 140 and FF1 mouse antibodies, respectively. The procedure described
for detection of antigen-antibody complexes in the Vectastain
manufacturer protocol was used. Positions of Mr markers are indicated
on the left of panels A, C, and E and on
the right of panels B and D.
|
|
Similarly, CYP40 and hsp90 coeluted from the CsA-Affigel column (Fig. 9C
, right panel, lane 4). Since no radioactivity or
immunoreactive PR were found in the CsA eluates, we concluded that PR
from T47D cells is not associated with CYP40, or that high-salt
exposure provoked dissociation of CYP40 from the PR-hsp90-CYP40
complex. However, both FKBP52 and CYP40 were found associated with
hsp90, and exposure to immunosuppressant did not disrupt this
association. Confirming the results of Fig. 8
, hsp70 was not found to
be associated with CYP40, (Fig. 9
, panel C, right, lane 4).
FKBP52, PR-A, PR-B, hsp90, and CYP40 were all found in the flowthrough
of the control Affigel-10 column (Fig. 9
, B and D, lane 1), none of
them being detectable in the eluates (Fig. 9
, B and D, lane 4).
FKBP51 was eluted from FK506-Affigel resin, loaded with unlabeled-PR
containing cytosol concomitantly with both PR-A and PR-B (Fig. 9
, right part of panel E). Similar experiment performed with
cytosol containing [3H]RU486-PR complexes, revealed that
FKBP51 did not coeluted with PR from the FK506 column; both forms of PR
were found in the flow-through fraction (Fig. 9
, left panel). Some
FKBP51 did not bind to the resin possibly due to overloading of the
column and/or to loading conditions using buffer containing low salt
concentration; FKBP51 binds in fact, better to FK506 in high salt
rather than in low salt buffers (15), but to maintain as much as
possible the integrity of the heterocomplex(es) which dissociate in
high salt (1, 5, 6, 20), we chose to add 0.15 M NaCl in
loading buffer as a compromise. These data, along with those shown in
Fig. 7
, support the concept that hormone-binding to PR induces FKBP51
release from the heterocomplex, in agreement with previous report from
Smith et al. (15).
Identical experiments were performed with cytosol from T47D
(GRE5) CAT cells, in which the GR had been labeled with
[3H]TA. In this case, [3H]TAGR-hsp90-FKBP52
and unlabeled GR-hsp90-FKBP51 complexes were characterized after the
same western blot technique as that described in Fig. 9
. TA binding to
the GR, like progestin binding to the PR, induced a release of FKBP51
from the receptor heterocomplex (not shown). Similar to the PR, the GR
was not found associated with the CYP40-hs90 complex (not shown).
Altogether, these results indicate the presence of PR and GR in
different molecular chaperone complexes, including always hsps plus
cochaperones such as FKBP51 and FKBP52 but not CYP40.
Stabilization of FKBP52-(hsp90-PR) Complex by Rapamycin in Cells
Exposed to PR Ligands
[3H]Org2058-PR complexes extracted from T47D cells
exposed or not to Rap and KN62 (instead of KN93), were
immunoprecipitated with the anti PR antibody MA1 140. After treatment
of cells with Rap and steroid, FKBP52 coimmunoprecipitated with PR B
form (Fig. 10
, panels A and C), along
with hsp90 and hsp70 (panel B), but not with FKBP51 (panel D) or CYP40
(panel E). By contrast, no FKBP and no hsp coimmunoprecipitated with PR
when cells were exposed to progestin alone, to CsA plus progestin or to
KN62 plus progestin. These results supported the results obtained in
Fig. 7
, which suggested that Rap, but not KN62, may stabilize the
FKBP52-hsp90-PR complex. In contrast, Rap did not stabilize a
FKBP51-hsp90-PR complex which appeared to dissociate after steroid
binding to the PR.

View larger version (47K):
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|
Figure 10. Protein Composition of PR from T47D Cells Exposed
to Immunosuppressants or CaMK Inhibitors
An experiment identical to that described in Fig. 7 was performed, and
whole cell extracts (250 µl) were immunoprecipitated with 100 µl
protein G Sepharose to which 20 µg MA1 140 anti-PR antibody had been
covalently cross-linked by the method described in Ref. 74;
immunoprecipitated proteins were eluted at elevated pH as described in
Materials and Methods. After 10% SDS-PAGE, antigens
were probed with the following antibodies: mPR1, for detection of PR B
(panel A), 174 for detection of hsp90 and hsp70 (panel B), EC1 for
detection of FKBP52 (panel C), FFI for detection of FKBP51 (panel D),
and 638W for detection of CYP40 (panel E). Each lane in each panel
contains 40 µl of eluted proteins: 1, immunoprecipitate from control
cells not exposed to steroid and drug; 2, immunoprecipitate from cells
exposed to 10 nM [3H]Org2058; 3,
immunoprecipitate from cells exposed to 10 µM Rap and
then to radioactive progestin; 4, immunoprecipitate from cells exposed
to 10 µM KN62 and then to radioactive progestin; 5,
immunoprecipitate from cells exposed to 10 µM CsA and
then to 10 nM [3H]Org 2058. Antigen-antibody
complexes were detected with the biotinylated second antibody according
to the Vectastain protocol. The figure represents a summary of two
typical experiments.
|
|
 |
DISCUSSION
|
---|
In previous reports, immunosuppressive drugs such as FKBPs
(FK506 and rapamycin) and cyclophilin ligands (CsA and analogs) were
shown to potentiate the Dex-induced expression of the MMTV-CAT reporter
gene (24, 29) in a stably transfected mouse fibroblast LMCAT cell line,
(29). This potentiation was demonstrated to be the consequence of the
reversal by immunosuppressants of an efflux membrane mechanism
resembling P-gp(s) which led to increased intracellular steroid
concentrations in yeast (31, 32), and in mouse fibroblasts (33). These
observations called into question any role of receptor-bound
immunophilins in steroid-induced gene transactivation. In this
study, we observed that two human T47D breast cancer cell lines, stably
transfected with the same promoter/reporter construct MMTV-CAT (35)
or a GRE5-CAT reporter (36), respond differently from
LMCAT cells when exposed to immunosuppressants. In fact, instead of a
potentiation of the steroid-induced transcription, an inhibition of
glucocorticosteroid and progestin-induced CAT activity was observed
after exposure of T47D cells to Rap or FK506 (
Figs. 13

). This effect
was also observed with the endogenous Cyclin D1 gene. All
glucocorticosteroid- and progestin-inducible gene expression observed
in these lines was inhibited by RU486, supporting a direct role of the
GR and the PR. By contrast, no change in the expression level of GAPDH
transcripts, a non-steroid-regulated gene was observed in the presence
of Rap, FK506 and KN62. The effect of Rap correlated with a
stabilization of 9S receptor-associated complex(es), strong suggesting
the observed inhibition is not due to modulation of P-gp activity. In
addition, results obtained with T47D GRE5CAT cells led us
to conclude that the inhibition of steroid-induced transcription by
immunosuppressants involves recruitment of transcription factors
specific for GRE/PRE sequences and not those specific to other
sequences present in the MMTV promoter. Moreover, no changes in levels
of PR or GR, or their affinities for hormone was observed in
drug-treated cells (S. LeBihan, unpublished results).
In T lymphocytes, a number of genes was reported to be inhibited by
immunosuppressants (39, 42). In contrast, in non lymphoid cells, only a
few genes were affected, such as a cAMP-responsive element
(CRE)-regulated gene (48), the PRL promoter (49) and the gene coding
for the orphan steroid receptor NUR77 (50). In these cases, an
inhibition of transactivation by immunosuppressive drugs was observed
which was suggested to involve the smaller forms of immunophilins,
FKBP12 and CYP18 (51, 52). The inhibition occurred at drug
concentration at least 100-fold smaller than those necessary to inhibit
CAT activity in the studies presented here. In all cells, the common
feature of CYP18-CsA and FKBP12-FK506 complexes is binding and
inhibiting CN protein phosphatase activity (53, 54). In contrast, the
FKBP12-rapamycin complex does not block CN activity (Ref. 42 , for a
review) but interrupts, although indirectly, the activation of the
70-kDa S6 kinase (55, 56) and p34cdc2 kinase activity
in vivo (57). These observations provide a mechanism for
cell cycle arrest by Rap. More recently, the mammalian target of
Rap-FKBP12 complexes was identified as a 280-kDa molecular mass protein
(58, 59, 60) homologous to the yeast product of TOR2 gene, which has
phosphatidylinositol kinase activity (61). However, this kinase
activity is not directly inhibited by binding of Rap to FKBP12. Rather,
it was suggested that Rap interferes with the association or
phosphorylation of a G1 effector (62). All these findings indicate that
immunosuppressant-immunophilin complexes play a role in cell-signaling
pathways involving phosphorylation of a number of substrates. It is
therefore tempting to speculate that, by analogy, receptor-associated
immunophilins could influence the phophorylation of one or several
proteins of the receptor heterocomplexes or may act upon effectors of
some phosphorylation/dephosphorylarion process involved in steroid
receptor-mediated signaling.
To test this hypothesis, we analyzed the effects of different kinase
inhibitors on receptor signaling. We found that wortmannin, an
inhibitor of PI3 and PI4 kinases, as well as the protein kinase A
inhibitor H89, did not modify either progestin- and
glucocorticosteroid-induced CAT gene activity in the two cell lines
studied (not shown). However, KN62 and KN93, specific inhibitors of
Ca2+/CaM-dependent kinases of type II and of type IV,
inhibited both progestin- and glucocorticosteroid-induced transcription
in a dose-dependent manner (Fig. 4
). This may indicate that
phosphorylation by a CaMK is a process that can modulate steroid
receptor-mediated gene expression. This is compatible with the observed
generation of GR-hsp 90 complexes incapable of binding hormone after
in vivo treatment of LMCAT cells with Calmodulin antagonists
(63). It is not known whether Rap or FK506, which also inhibits
steroid-induced transcription in T47D cells, block the activity of
CaMKs of type II and of type IV.
It is interesting to note that these CaMK inhibitors potentiated
Dex-induced expression of the CAT gene in LMCAT cells similarly to the
effects of immunosuppressants in these cells (64). Thus, the effects of
CaMK inhibitors and immunosuppressants with regard to steroid-induced
transcription appeared strictly parallel in different cell types. CaMK
inhibitors were shown to affect the Ca2+ regulation of
several immediate early genes such as NUR77 (50), supporting the
concept of a broad role for CaMKs in transcriptional regulation. We
have also shown that FKBP52 is a delayed early gene (65). It is
therefore possible that a decrease of FKBP52 gene expression, which
could result from CaMK inhibition, could induce a repression of
steroid-induced gene transcription. Whether steroid-induced gene
expression inhibited by Rap or other immunosuppressants or whether
effects of CaMK inhibitors are interrelated remains to be established.
But it is likely that both kinds of drugs affect pathways modulating
the activity of (a) common factor(s). Such a hypothesis is consistent
with the experiments of Figs. 5
and 7
, which indicated that
immunosuppressants and CaMK inhibitors act 1) before steroid-induced
receptor activation and 2) by apparently two different pathways, since
only Rap but not KN62 stabilizes the PR (and the GR)
heterooligomer.
In fact, unliganded PR and GR from T47D cells are contained in
different heterocomplexes including immunophilins and hsps like PR and
GR from other cells and/or tissues (Ref. 1 for review). Obviously, from
the data of Fig. 9
, the PR from T47D cells eluted from FK506-affinity
chromatography and coimmunoprecipitated with FKBP51 anf FKBP52, both
associated with hsp90 in separate complexes, but not with CYP40.
However,CYP40 is present in T47D cell cytosol, associated with hsp90
similar to other cell types such as mouse fibroblasts (24) and
lymphoids (63). Such an observation had been already made by Milad
et al. (66) in T47D B11 subline stably transfected with the
MMTV-CAT construct (67). In this subline, CYP40 did not coimmunopurify
with PR, whereas CsA potentiated R5020-induced CAT activity.
Surprisingly, Rap and FK506 had no effect and FKBP52
coimmunoprecipitated with PR (66), results that are the opposite of
those obtained here. Thus, it is possible that specific cellular
factors are recruited in hormone-induced transactivation of
transfected exogenous genes, the level of expression of which may be
quite different, as well as their integration at the chromatin level
from one cell line to another one.
The targets of CaMK inhibitors in steroid-regulated gene transcription
are not known. Like immunosuppressants Rap and FK506, they act before
the ligand binding-induced conformational change of the receptors,
which conducts to the release of receptor-associated
hsp-90/immunophilins complexes. PR- and GR-FKBP52-hsp90 complexes have
been shown to be stabilized by immunosuppressant binding (22, 46). In
addition, the phosphorylation of FKBP52 by casein kinase II inhibits
its binding to hsp90 (68). This suggests that interactions between the
components of molecular chaperone complexes involved in steroid
receptor regulation are likely controlled by a
phosphorylation/dephosphorylation mechanism. It is quite possible that
both CKII and CaMKs II or IV may intervene directly or not, in both the
maturation of steroid receptors and the steroid receptor-mediated
transactivation processes. Steroid receptors are known to be influenced
by different signaling pathways such as cAMP, insulin-like growth
factor-I, and kinase(s) and/or phosphatase(s) inhibitors (69, 70, 71, 72). In
addition to their immunosuppressive activity, FK506, Rap, and CsA (as
well as some of their analogs), cause a variety of effects not only at
the membrane level but also in calcium mobilization and cell-signaling
cascades (41, 42, 51, 52). Their effects are different from one cell
type to another and likely depend on the expression level of certain
type(s) of immunophilin(s) and membrane transporter(s). The data from
the present work indicate that CaMK inhibitors and immunosuppressants
may serve as useful tools to elucidate some of the steps by which
steroid receptors modulate gene transcription.
 |
MATERIALS AND METHODS
|
---|
Drug Treatment of Cells
Two T47D human breast carcinoma cell lines were used. One cell
line was stably transfected with a MMTV-CAT reporter gene (35), while
the other contained a synthetic steroid-inducible promoter controlling
CAT gene expression (GRE5tkCAT) (36). Cells were grown in
10-cm Petri dishes in DMEM containing 10% charcoal-treated and
heat-inactivated (30 min at 55 C) FBS and 0.2 mg/ml geneticin (G418,
Sigma), 24 h before drug and steroid treatment. Hygromycin B (75
µg/ml, Boehringer Mannheim, Indianapolis, IN) was used for
T47D-GRE5 cells (36). Immunosuppressive drugs or kinase
inhibitors were added for 2 h to the culture medium before steroid
exposure when cells were at 50% confluence. Cells were harvested
16 h later, except when noted. CAT enzyme activity was measured in
triplicate samples of the cell extracts containing 50 µg total
protein, as previously described (24, 29) using
[14C]chloramphenicol (98 mCi/mmol, ICN, Irvine, CA) as
substrate and acetyl coenzyme A as cofactor. All experiments were
repeated at least three times. In some experiments, steroids were added
4 h before or at the same time as immunosuppressants or
inhibitors. Specific calcium/calmodulin kinase inhibitors KN62 and KN93
as well as H89 and wortmannin (Calbiochem, La Jolla, CA), inhibitors of
protein kinase A, and PI3 kinase, respectively, were also
used in other experiments.
Immunosuppressant-Affinity Chromatography
Cytosoluble fractions of T47D cells were prepared by disrupting
cells (5.109 cells) at 2 C with a glass/glass homogenizer
in 1.75 ml buffer A (10 mM HEPES, 1 mM EDTA,
10% glycerol, 1 mM dithiothreitol, 20 mM
Na2MoO4, pH 7.5), followed by centrifugation at
2 C in a Ti50 rotor [45 min at 45,000 rpm in a Beckman (Fullerton, CA)
L2 68B ultracentrifuge]. The supernatant was incubated or not
overnight at 2 C with 10 nM of the antagonist
[3H]RU486 (38.4 Ci/mmol, ROUSSEL-UCLAF, Romainville,
France) or [3H]TA (48 Ci/mmol, Amersham, Bucks, England),
or [3H]Org 2058 (51.9 Ci/mmol, Amersham). A 100-fold
excess of radioinert cortisol or Org2058 was added to the incubates to
block either glucocorticosteroid or progestin receptor binding in the
case of progestins or glucocorticoids labeled cytosol samples,
respectively. Portions of cytosol (500 µl) were chromatographed on
200 µl of packed dihydro-FK506-Affigel 10 or CsA-Affigel 10 columns
(1 mg drug/ml gel). To improve FKBP51 binding to FK506-Affigel, 0.15
M NaCl was added to the cytosol sample. Control experiments
used non-grafted Affigel-10 resins (Bio-Rad, Richmond, CA). The
purification protocol described in Ref. 24 was employed and
affinity-bound proteins were eluted by exchange (3 x 30 min at 4
C) with 100 µl of the appropriate immunosuppressant solution (1
mM) in buffer A.
Immunoprecipitation Experiments
Cytosol from T47D cells in buffer A, incubated with
3H-labeled steroid as indicated above was treated with
dextran-coated (vol/vol) charcoal (0.5, 5 g/100 ml) for measurement of
ligand binding, and 0.5 ml aliquots were incubated (4 h, 4 C) with 10
µg of anti-human PR monoclonal antibody mPR1 also named LET126 (73)
(Transbio, Paris, France) or MAI140 (Affinity Bioreagents, Neshanic
Station, NJ), covalently cross-linked to protein G-Sepharose beads
(Pharmacia, Piscataway, NJ), according to the method described in Ref.
74 . Immunopellets containing the same amount of nonimmune IgGs were
used as control. For CYP40 and FKBP51 immunoprecipitation, 500 µl of
cytosol were incubated under the same conditions with 25 µl of 638W,
a rabbit antiserum against the C-terminal peptide of human CYP40 bound
to protein G-Sepharose immunoadsorbent, or 20 µg of HI51, a mouse
monoclonal anti-FKBP51 efficient in native conditions (14).
FKBP52 from T47D cell cytosol was immunoprecipitated by the same
technique with EC1, a mouse monoclonal antibody specific for mammalian
FKBP52 (75).
After centrifugation (700 x g for 5 min at 0 C), the
pellets were washed by two successive centrifugations with 250 µl of
buffer B (0.1 M potassium phosphate, 10% glycerol, 20
mM sodium tungstate, pH 7.5) and then three times with 250
µl of buffer A. The pellets were resuspended in 200 µl of Laemmli
(76) sample buffer and boiled before denaturing SDS-PAGE and Western
blotting experiments. In some experiments, immunoadsorbed proteins were
eluted from the immunomatrix by a pH 10.2 buffer two times for 1 h
and immediately neutralized by NaH2PO4.
SDS-PAGE and Western Blotting
Samples were resolved on either 10% or 12% SDS-PAGE, according
to Laemmli (76). Western blots were performed as previously described
(24). The nitrocellulose papers were probed with the following
antibodies: the mouse monoclonal mPR1 for the detection of the human B
form of PR (73), the mouse monoclonal MA1140 for the detection of
PR-A and PR-B isoforms (77). EC1 (75) and/or 173, a rabbit polyclonal
antipeptide antibody (10), were both used for the detection of FKBP52,
638W (24), a rabbit polyclonal antipeptide
(Asp356-Asp370) antibody, for the detection of
CYP 40. Antibody 174 (24), a rabbit polyclonal antipeptide antibody
raised against the C-terminal sequence
(Met807-Glu823) of human hsp90. (78),
recognizes both human hsp90 and hsp70 in Western blots due to sequence
homologies (24). FKBP51 was identified with FF1 (a mouse monoclonal
antibody working in denaturing conditions). All the antibodies were
used at 10 µg Ig/ml, 174 and 638W antisera were used at 1:500
dilution. The detection of the antigen-antibody complexes was performed
with a second biotinylated antibody (Vectastain ABC Kit, Vector
Laboratories, Burlingame, CA).
When the chemiluminescent detection system (ECL, Amersham Bucks,
England) was employed, antibodies were used at 2 µg/ml or 1:6000
serum dilution.
Nuclear Receptor Extraction and Sucrose Gradient Analysis
The effects of immunosuppressants upon the size of the
heterooligomeric PR were tested after exposure of T47D cells to Rap (10
µM) for 2 h before addition of 10 nM
[3H]Org 2058. Cells were then harvested and washed three
times in PBS, before being homogenized in buffer A (1 vol of buffer A
for 1 vol of pelleted cells). The cytosol was obtained as mentioned
above. The pellet remaining after cytosol preparation was washed three
times with 5 vol of buffer A, before being homogenized in buffer A
containing 0.4 M KCl plus 20 mM
Na2WO4 (buffer B), as described previously
(45). In these conditions no cytosol contamination is obtained since no
lactate dihydrogenase activity can be detected (45). In particular
experiments, whole cells extracts were obtained in buffer B after four
freezing steps in liquid nitrogen and subsequent thawing at 20 C. The
samples containing [3H]Org 2058-PR complexes were
centrifuged for 2 h at 70, 000 rpm in 520% sucrose gradients in
buffer A containing 20 mM Na2WO4 in
a Vti 80 rotor (Beckman) with internal markers GO (glucose oxidase,
7.9S) and PO (peroxidase, 3.6S).
Northern Blot Analysis
RNA from T47D cells exposed to the different drugs was extracted
using Trizol reagent (Life Technologies, Gaithersburg, MD) according to
the manufacturers instructions. Total RNA (20 µg) of each sample
was electrophoresed through a 1% denaturing agarose-formaldehyde gel
and transferred to a nylon membrane (Hybond-NX, Amersham). RNAs were
cross-linked to the membrane using a X-link apparatus (Bio-Rad,
Ilkirch, France) at 0.7 J/cm2. The membrane was
prehybridized for 6 h in 50% formamide and then hybridized with
probes labeled to high specific activity (
2.108
cpm/µg) using [
32P]dCTP by the random
primer-labeling procedure (Amersham). Hybridization was carried out at
42 C in 50% formamide containing 5x Denhardts, 1% (wt/vol) SDS, 2x
SSPE (225 mM NaCl, 16 mM
NaH2PO4, 1.5 mM EDTA, pH 7.4) 0.25
mg/ml salmon sperm DNA (Sigma, St. Louis, MO). The membranes were
washed two times in 1x SSC (15 mM NaCl, 1.5 mM
sodium citrate, pH 7.0), 0.1% (wt/vol) SDS at room temperature for 20
min, followed by two 20-min washes in 0.1x SSC, 0.1% SDS at 55 C. The
dry blot were exposed for 13 days at -80 C to Kodak X-OMAT AR-5
films, using intensifying screens. mRNA was quantitated by densitometry
scanning using a Storm apparatus (Molecular Dynamics, Sunnyvale, CA).
For Northern blots using Cyclin D1 probe, T47D cells were grown in
serum-free DMEM for 48 h, to synchronize them after which the
medium was replaced by DMEM containing stripped serum to which drugs
had been added.
Radioactivity Counting and Protein Determination
Radioactivity was measured in a Minaxi tricarb (Packard, Downers
Grove, IL) spectrometer, and proteins were determined with the BIORAD
Kit using BSA as the standard.
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to Fujisawa, Wyeth Ayerst and Sandoz
Laboratories for the gift of immunosuppressives drugs. We thank Dr. A
Cato for (MMTV-CAT)-T47D cells and Drs. D. O. Toft, D. Edwards and
S. Nordeen for helpful discussion and communication of manuscripts
before publication. We thank also Drs. D. Smith and R. Rimmerman for
the gift of HI51 and FF1 antibodies, and L. Faber, R. Handshumacher,
and K. Hoffman for the gift of antibodies EC1 and 63 8W, respectively.
Dr. D. Philibert provided us with R5020 and RU486 and is acknowledged
as well as Dr. D. Chalbos for the gift of the GADPH probe, and Dr E.
Wientjens for the gift of the Cyclin D1 probe. P. Delangle, R.
Villerot, and S. Oboukhoff are acknowledged for art work.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jack-Michel Renoir, faculté de Pharmacie URA 1218 CNRS, Pharmacologie Cellulaire, 5 Rue Jean-Baptiste Clement, Chatenay-Malabry France 92296. E-mail:
Michel.Renoir{at}cep.u-psud.fr
This work was supported by INSERM, Centre National de la Recherche
Scientifique, and Association pour la Recherche sur le Cancer, Grant
6510 (to J.-M.R.) and Grants MT-11704 and MT-13147 from the Medical
Research Council of Canada (to J.H.W. and S.M.), respectively. Partial
financial support was obtained from the DGA Grant 94/064 (to
J.-M.R.). S.L.B. and V.M. had fellowships from the Direction
Generale aux Armées and from the Ligue Nationale contre le
Cancer.
1 Abbreviations used are: hsp90, heat shock
protein of molecular mass 90 kDa; hsp70, heat shock protein of
molecular mass 70 kDa; FKBP, FK506 binding protein; FKBP51,
FK506-binding protein of apparent molecular mass 54 kDa; FKBP52,
FK506-binding protein of apparent molecular mass 59 kDa; CYP40,
cyclophilin of molecular mass 40 kDa; R5020, promegestone,
17
,21-dimethyl-19-norpregna-4,9-diene-3,20 one [17
methyl-3H]; [3H] Org2058,
16a-ethyl-21-hydroxy-19-nor
[6,7-3H]-pregn-4-ene-3,20-dione; RU486, mifepristone,
11ß-(4-dimethyl-aminophenyl)-17b-hydroxy-17a-(prog-1-ynyl)
estra-4,9-dien-3-one; dexamethasone, (11ß, 16
)-9-fluoro-11, 17,
21, trihydroxy-16-methylpregna-1,4-diene 3,20 dione; triamcinolone
acetonide, 9
-fluoro-11ß,16
,17,21-tetrahydroxypregna-1,4 dienee,
3,20 dione cyclic 16,17-acetal with acetone; KN62,
1-[N,O-bis(5-isoquinoline
sulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine.
KN93,
(2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N(-chlorocinnamyl)-N-methylbenzylamine). 
2 The term "activation" relates to the ligand
binding-induced process by which a receptor converts from a non-DNA
binding form to a DNA-binding form, while the term "transformation"
identifies the dissociation process of molecular chaperone proteins
from the receptor heterocomplexes. 
Received for publication June 9, 1997.
Revision received February 11, 1998.
Accepted for publication March 13, 1998.
 |
REFERENCES
|
---|
-
Pratt WB, Toft DO 1997 Steroid receptor interactions with
heat shock protein and immunophilin chaperones. Endocr Rev 18:306360[Abstract/Free Full Text]
-
Joab I, Radanyi C, Renoir M, Buchou T, Catelli MG, Binart N,
Mester J, Baulieu EE 1984 Common non-hormone binding component in
non-transformed chick oviduct receptors of four steroid hormones.
Nature 308:850853[Medline]
-
Catelli MG, Binart N, Jung-Testas I, Renoir JM, Baulieu EE,
Feramisco JR, Welch WJ 1985 The common 90-kd protein component of
non-transformed 8S steroid receptors is a heat-shock protein. EMBO
J 4:31313135[Abstract]
-
Onate SA, Estes PA, Welsh WJ, Nordeen SK, Edwards DP 1991 Evidence that heat shock protein-70 associated with progesterone
receptors is not involved in receptor-DNA binding. Mol Endocrinol 5:19932004[Abstract]
-
Sanchez ER, Hirst M, Scherrer LC, Tang HY, Welsh MJ, Harmon
JM, Simons SS, Ringold GM, Pratt WB 1990 Hormone-free mouse
glucocorticoid receptors overexpressed in Chinese hamster ovary cells
are localized to the nucleus and are associated with both Hsp70 and Hsp
90. J Biol Chem 265:2012320130[Abstract/Free Full Text]
-
Rehberger P, Rexin M, Gehring U 1992 Heterotetrameric
structure of the human progesterone receptor. Proc Natl Acad Sci USA 89:80018005[Abstract]
-
Galat AM, Metcalfe SM 1995 Peptidyl prolyl cis/trans
isomerase. Int Prog Biophys Mol Biol 63:67118[CrossRef]
-
Tai PKK, Albers MN, Chang H, Faber LE, Schreiber SL 1992 Association of a 59-kilodalton immunophilin with the glucocorticoid
receptor complex. Science 256:13151318[Medline]
-
Yem AW, Tomasselli AG, Heinrikson RL, Zurcher-Neely H, Ruff
VA, Johnson RA, Deibel MR 1992 The hsp56 component of steroid receptor
complexes bind to immobilized FK506 and shows homology to FKBP12 and
FKBP13. J Biol Chem 267:28682871[Abstract/Free Full Text]
-
Lebeau MC, Massol N, Herrick J, Faber LE, Renoir JM, Radanyi
C, Baulieu EE 1992 P59, an hsp 90-binding protein. Cloning and
sequencing of its cDNA and preparation of a peptide-directed polyclonal
antibody. J Biol Chem 267:42814284[Abstract/Free Full Text]
-
Peattie DA, Harding MW, Fleming MA, De Cenzo MT, Lippke JA,
Livingston DJ, Benasutti M 1992 Expression and characterization of
human FKBP52, an immunophilin that associates with the 90 kDa heat
shock protein and is a component of steroid receptor complexes. Proc
Natl Acad Sci USA 89:1097410978[Abstract]
-
Tai PKK, Maeda Y, Nakao K, Wakim NG, Duhring JL, Faber LE 1985 A 59-kilodalton protein associated with progestin, estrogen, androgen
and glucocorticoid-receptor. Biochemistry 25:52695275
-
Baughman G, Wiederrecht GJ, Campbell NF, Martin MM, Bourgeois
S 1995 FKBP51, a novel T-cell specific immunophilin capable of
calcineurin inhibition. Mol Cell Biol 15:43954402[Abstract]
-
Nair SC, Rimerman RA, Toran EJ, Chen S, Prapapanich V, Butts
RN, Smith DF 1997 Molecular cloning of human FKB51 and comparisons of
immunophilin interactions with Hsp90 and progesterone receptor. Mol
Cell Biol 17:594603[Abstract]
-
Smith DF, Albers MW, Schreiber SL, Leach KL, Deibel MR 1993 FKBP54, a novel FK506 binding protein in avian progesterone receptor
complexes and HeLa extracts. J Biol Chem 268:2427024273[Abstract/Free Full Text]
-
Kieffer LJ, Seng TW, Li W, Osterman DG, Handschumacher RE,
Bayney RM 1993 Cyclophilin 40, a protein with homology to the p59
component of the steroid receptor complex. Cloning of the cDNA and
further characterization. J Biol Chem 268:1230312310[Abstract/Free Full Text]
-
Ratajczak T, Carello A, Mark PJ, Warner BJ, Simpson RJ, Moritz
RL, House AK 1993 The cyclophilin component of the unactivated estrogen
receptor contains a tetratricopeptide repeat domain and shares identity
with p59 (FKBP59). J Biol Chem 268:1318713192[Abstract/Free Full Text]
-
Owens-Grillo JK, Hoffman K, Hutchison KA, Yem AW, Deibel MR,
Handschumacher RE, Pratt WB 1995 The cyclosporin A binding immunophilin
CYP40 and the FK506-binding immunophilin hsp56 bind to a common site on
hsp90 and exist in independent cytosolic heterocomplexes with the
untransformed glucocorticoid receptor. J Biol Chem 270:2047920484[Abstract/Free Full Text]
-
Radanyi C, Chambraud B, Baulieu EE 1994 The ability of the
immunophilin FKBP59-HBI to interact with the 90-kDa heat shock protein
is encoded by its tetratricopeptide repeat domain. Proc Natl Acad
Sci USA 91:1119711201[Abstract/Free Full Text]
-
Renoir JM, Radanyi C, Faber LE, Baulieu EE 1990 The
non-DNA-binding heterooligomeric form of mammalian steroid hormone
receptors contains a hsp90-bound 59-kilodalton protein. J Biol
Chem 265:1074010745[Abstract/Free Full Text]
-
Rexin M, Busch W, Segnitz B, Gehring U 1992 Structure of the
glucocorticoid receptor in intact cells in the absence of hormone.
J Biol Chem 267:96199621[Abstract/Free Full Text]
-
Renoir JM, Le Bihan S, Mercier-Bodard C, Gold A, Arjomandi M,
Radanyi C, Baulieu EE 1994 Effects of immunosuppressants FK506 and
rapamycin on the heterooligomeric form of the progesterone receptor. J
Steroid Biochem Mol Biol 48:101110[CrossRef][Medline]
-
Hoffmann K, Handschumacher RE 1995 Cyclophilin 40: evidence
for a dimeric complex with hsp90. Biochem J 307:58[Medline]
-
Renoir JM, Mercier-Bodard C, Hoffmann K, Le Bihan S, Ning YM,
Sanchez ER, Handschumacher RE, Baulieu EE 1995 Cyclosporin A
potentiates the dexamethasone-induced mouse mammary tumor
virus-chloramphenicol acetyltransferase activity in LMCAT cells: a
possible role for different heat shock protein-binding immunophilins in
glucocorticosteroid receptor-mediated gene expression. Proc Natl
Acad Sci USA 92:49774981[Abstract]
-
Callebaut I, Renoir JM, Lebeau MC, Massol N, Burny A, Baulieu
EE, Mornon JP 1992 An immunophilin that binds Mr 90,000 heat shock
protein: main structural features of a mammalian p59 protein. Proc Natl
Acad Sci USA 89:62706274[Abstract]
-
Estes PA, Suba EJ, Lauber-Heavner J, Elashry-Stowers D, Wei
LL, Toft DO, Horwitz KB, Edwards DP 1987 Immunologic analysis of human
breast cancer progesterone receptors. 1. Immunoaffinity purification of
transformed receptors and production of monoclonal antibodies.
Biochemistry 26:62506262[Medline]
-
Wei LL, Sheridan PL, Krett NL, Francis MD, Toft DO, Edwards
DP, Horwitz KB 1987 Immunological analysis of human breast cancer
progesterone receptors. 2. Structure, phosphorylation and processing.
Biochemistry 26:62626272[Medline]
-
Czar M, Lyons R, Welsh M, Renoir J-M, Pratt W 1995 Evidence
that the FK506-binding immunophilin heat shock protein 56 is required
for trafficking of the glucocorticoid receptor from the cytoplasm to
the nucleus. Mol Endocrinol 9:15491560[Abstract]
-
Ning YM, Sanchez ER 1993 Potentiation of
glucocorticoid-mediated gene expression by the immunophilin ligands
FK506 and rapamycin. J Biol Chem 268:60736076[Abstract/Free Full Text]
-
Tai PKK, Albers MW, McDonnell DP, Chang H, Schreiber SL, Faber
LE 1994 Potentiation of progesterone receptor-mediated transcription by
the immunosuppressant FK506. Biochemistry 33:1066610671[Medline]
-
Kralli A, Bohen SP, Yamamoto KR 1995 LEM1, an
ATP-binding-cassette transporter, selectively modulates the biological
potency of steroid hormones. Proc Natl Acad Sci USA 92:47014705[Abstract]
-
Kralli A, Yamamoto KR 1996 An FK506-sensitive transporter
selectively decreases intracellular levels and potency of steroid
hormones. J Biol Chem 271:1715217156[Abstract/Free Full Text]
-
Marsaud V, Mercier-Bodard C, Fortin D, Le Bihan S, Renoir J-M,
Dexamethasone, triamcinolone acetonide accumulation in mouse
fibroblasts is differently modulated by the immunosuppressants
Cyclosporin A, FK506, Rapamycin, their analogues, as well as by other
P-Glycoprotein ligands. J Steroid Biochem Mol Biol, in press
-
Horwitz KB, Mockus MB, Lessey BA 1982 Variant T47D human
breast cancer cells with high progesterone-receptor levels despite
estrogen and antiestrogen resistance. Cell 28:633642[Medline]
-
Cato ACB, Henderson D, Ponta H 1987 The hormone response
element of the mouse mammary tumor virus mediates the progestin and
androgen induction of transcription in the proviral long terminal
repeat region. EMBO J 6:363368[Abstract]
-
White JH, McCuaig KA, Mader S 1994 A simple and sensitive
high-throughput assay for steroid agonists and antagonists.
Biotechnology 12:10031007[Medline]
-
Said TK, Conneely OM, Medina D, OMalley, Lydon J 1997 Progesterone, in addition to estrogen, induces Cyclin D1 expression in
the murine mammary epithelial cell, in vivo. Endocrinology 138:39333939[Abstract/Free Full Text]
-
Musgrove EA, Hamilton JA, Lee CSL, Sweeney KJE, Watts CKW,
Sutherland RL 1993 Growth factor, Steroid, and steroid antagonist
regulation of cyclin gene expression associated with changes in T-47D
human breast cancer cell cycle progression. Mol Cell Biol 13:35773587[Abstract]
-
Wiederrecht G, Etzkorn F 1994 The immunophilins. Perspect Drug
Discovery Design 2:5784
-
Le Bihan S, Renoir JM, Radanyi C, Chambraud B, Joulin V,
Catelli MG, Baulieu EE 1993 The mammalian heat shock protein binding
immunophilin (p59/HBI) is an ATP and GTP binding protein. Biochem
Biophys Res Commun 195:600607[CrossRef][Medline]
-
Baughman G, Wiederrecht GJ, Chang F, Martin MM, Bourgeois S 1997 Tissue distribution and abundance of human FKBP51, an
FK506-binding protein that can mediate calcineurin inhibition. Biochem
Biophys Res Commun 232:437443[CrossRef][Medline]
-
Dumont FJ, Su Q 1996 Mechanism of action of the
immunosuppressant rapamycin. Life Sci 58:373395[CrossRef][Medline]
-
Tokimitsu H, Chijiwa T, Hagiuwara M, Mitzutani A, Terasawa K,
Hidaka H 1990 KN62, 1-[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-1
tyrosyl] 4-phenylpiperazine, a specific inhibitor of
Ca++/calmodulin-dependant protein kinase II. J Biol
Chem 265:43154323[Abstract/Free Full Text]
-
Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T,
Hidaka H 1991 The newly synthetized selective
Ca++/calmodulin dependent protein kinase II inhibitor KN93
reduces dopamine content in PC12h cells. Biochem Biophys Res Commun 181:968975[Medline]
-
Renoir JM, Radanyi C, Jung-Testas I, Faber LE, Baulieu EE 1990 The nonactivated progesterone receptor is a nuclear heterooligomer.
J Biol Chem 265:1440214406[Abstract/Free Full Text]
-
Ning YM, Sanchez ER 1995 Stabilization in vitro of
the untransformed glucocorticoid-receptor complex of S49 lymphocytes by
the immunophilin ligand FK506. J Steroid Biochem Mol Biol 52:187194[CrossRef][Medline]
-
Moudgil VK, Eessalu TE, Buchou T, Renoir JM, Mester J, Baulieu
EE 1985 Transformation of chick oviduct progesterone receptor in
vitro: effects of hormone, salt, heat, and adenosine triphosphate.
Endocrinology 116:12671274[Abstract]
-
Schwaninger M, Blume R, Oetjen E, Lux G, Knepel W 1993 Inhibition of cAMP-responsive element-mediated gene transcription by
cyclosporin A and FK506 after membrane depolarization. J Biol Chem 268:2311123115[Abstract/Free Full Text]
-
Wera S, Belayew A, Martial JA 1995 Rapamycin, FK506 and
cyclosporin A inhibit human prolactin gene expression. FEBS Lett 358:158160[CrossRef][Medline]
-
Enslen H, Soderling TR 1994 Roles of calmodulin-dependent
protein kinase and phosphatase in calcium-dependent transcription of
immediate early genes. J Biol Chem 269:2087220877[Abstract/Free Full Text]
-
Kunz J, Hall MN 1993 Cylosporin A, FK506 and rapamycin: more
than just immunosuppression. Trends Pharmacol Sci 18:334338
-
Liu J 1993 FK506 and cyclosporine: molecular probes for
studying intracellular signal transduction. Trends Pharmacol Sci 14:182188[CrossRef][Medline]
-
Liu J, Farmer JJD, Lane WS, Friedman J, Weissman I, Schreiber
SL 1991 Calcineurin is a common target of cyclophilin-cyclosporin A and
FKBP-FK506 complexes. Cell 66:807815[Medline]
-
OKeefe SJ, Tamura J, Kincaid RL, Tocci MJ, ONeill EA 1992 FK506- and CsA-sensitive activation of the interleukin-2 promoter by
calcineurin. Nature 357:692694[CrossRef][Medline]
-
Chung J, Kuo CJ, Crabtree GR, Blenis J 1997 Rapamycin-FKBP
specifically blocks growth-dependent activation of and signalling by
the 70 kDa S6 protein kinase. Cell 69:12271236
-
Calvo V, Crews CM, Vik TA, Bierer BE 1992 Interleukin-2
stimulation of p70 S6 kinase activity is inhibited by the
immunosuppressant rapamycin. Proc Natl Acad Sci USA 89:75717575[Abstract]
-
Morice WG, Brunn GJ, Wiederrecht G, Siekerka JJ, Abraham RT 1993 Rapamycin-induced inhibition of p34cdc2 kinase
activation is associated with G2/S-phase growth arrest in T
lymphocytes. J Biol Chem 268:37343738[Abstract/Free Full Text]
-
Brown EJ, Albers MW, Shiu TB, Ishikawa K, Keith CT, Lane WS,
Schreiber SL 1994 A mammalian protein targeted by G1-arresting
rapamycin-receptor complex. Nature 369:756758[CrossRef][Medline]
-
Sabatini DM, Pierchala BA, Barrow RK, Schell MJ, Snyder SH 1995 The rapamycin and FKBP12 target (RAFT) displays
phosphatidylinositol 4-kinase activity. J Biol Chem 270:2087520878[Abstract/Free Full Text]
-
Sabers CJ, Martin MM, Brunn GJ, Williams JM, Dumont FJ,
Wiederrecht G, Abraham RT 1995 Isolation of a protein target of the
FKBP12-rapamycin complex in mammalian cells. J Biol Chem 270:815819[Abstract/Free Full Text]
-
Kunz I, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR,
Hall MN 1995 Target of rapamycin in yeast, TOR2 is an essential
phosphatidylinositol kinase homolog required for G1 progression. Cell 82:121130[Medline]
-
Zhen XF, Fiorentino D, Chen J, Crabree GR, Schreiber SL 1995 TOR kinase domains are required for two distinct functions, only one of
which is inhibited by rapamycin. Cell 82:121130[Medline]
-
Ning YM, Sanchez ER 1996 In vivo evidence for the
generation of a glucocorticoid-receptor-heat shock protein-90 complex
incapable of binding hormone by the calmodulin antagonist
phenoxybenzamine. Mol Endocrinol 10:1423[Abstract]
-
Renoir JM, Marsaud V, Mercier-Bodard C, Le Bihan S, Radanyi C,
Baulieu EE 1996 Modulation of steroid-induced transcription by
cyclophilin and FKBPs. In Workshop on Cyclophilin Structure and
Function, Oct 1012 1996, Lille, France, Centre National de la
Recherche Scientifique and Université des Sciences et
Technologies de Lille, France, pp 117127
-
Doucet-Brutin S, Renoir M, Le Gallic L, Vincent S, Marty L,
Fort P 1995 Growth-regulated expression of FKBP-59 immunophilin in
normal and transformed fibroblastic cells. Exp Cell Res 220:152160[CrossRef][Medline]
-
Milad M, Sullivan W, Diehl E, Altman M, Nordeen S, Edwards DP,
Toft DO 1995 Interaction of the progesterone receptor with binding
proteins for FK506 and cyclosporin A. Mol Endocrinol 9:838847[Abstract]
-
El-Ashry D, Onate S, Nordeen SK, Edwards DP 1987 Human
progesterone receptor complexed with the antagonist RU486 binds to
hormone response element in a structurally altered form. Mol Endocrinol 3:15451548[Abstract]
-
Miyata Y, Chambraud B, Radanyi C, Lebeau M-C, Renoir J-M,
Leclerc J, Shirai R, Catelli M-G, Yahara I, Baulieu EE (1997)
Phosphorylation of immunosuppressant FK 506 binding protein, FKBP52 by
casein kinase II (CKII): regulation of hsp90-binding activity of
FKBP-52. Proc Natl Acad Sci USA 94:1450014505
-
Aronica SM, Katzenellenbogen BS 1991 Progesterone receptor
regulation in uterine cells: stimulation by estrogen, cyclic adenosine
3',5'-monophosphate and insulin-like growth factor 1 and suppression by
antiestrogen and protein kinase inhibitors. Endocrinology 128:20452052[Abstract]
-
Beck CA, Weigel NL, Moyer ML, Nordeen SK, Edwards DP 1993 The
progesterone antagonist RU486 acquire agonist activity upon stimulation
of cAMP signalling pathways. Proc Natl Acad Sci USA 90:44414445[Abstract]
-
Moyer ML, Borror KC, Bona ZJ, DeFranco DB, Nordeen SK 1993 Modulation of cell signalling pathways can enhance or impair
glucocorticoid-induced gene expression without altering the
phosphorylation state of the receptor. J Biol Chem 268:2293322940[Abstract/Free Full Text]
-
Zhang Y, Bai W, Allgood VE Weigel NL 1994 Multiple signalling
pathways activate the chicken progesterone receptor. Mol Endocrinol 8:577584[Abstract]
-
Lorenzo F, Jolivet A, Loosfelt H, Vu Hai MT, Brailly S,
Perrot-Applanat M, Milgrom E 1988 A rapid method of epitope mapping.
Application to the study of immunogenic domains and to the
characterization of various forms of rabbit progesterone receptors. Eur
J Biochem 176:5360[Abstract]
-
Schneider C, Newman RA, Sutherland DR, Asser U, Greaves MF 1982 A one step purification of membrane proteins using a high
efficiency immunomatrix. J Biol Chem 257:1076610769[Abstract]
-
Laemmli UK 1970 Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680685[Medline]
-
Nakao K, Myers JE, Faber LE 1985 Development of a monoclonal
antibody to the rabbit 8.5S uterine progestine receptor. Can J
Biochem Cell Biol 63:3340[Medline]
-
Traish A, Wotiz H 1990 Monocloncal and polyclonal antibodies
to human progesterone receptor peptide (533547) recognize a specific
site in unactivated (8S) and activated (4S) progesterone receptor and
distinguish between intact and proteolysed receptors. Endocrinology 127:11671175[Abstract]
-
Nover L, Sharf KD, Nover L (eds) 1991 Heat Shock Response. CRC
Press, Boca Raton, FL, pp 41128